BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.","More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).","2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.","Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).","The inability to send and receive information when opening TCP communications into the private network.","Each of the below described references teach the method of firewalls in general.","However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).","U.S. Pat.","No.","5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.","Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.","A time limit is set and so long as the set time limit does not run out, the communication is allowed.","Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.","A connection type session does not appear to be initiated for the UDP transport.","U.S. Pat.","No.","5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.","The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.","The firewall then binds a port and notes it in a link table.","The packet is passed to a stack, on the private side, which forwards the packet to the server.","Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.","U.S. Pat.","No.","5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.","A client on the public network connects to the external machine.","A private channel is opened between the external machine and a machine internal to the private network.","The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.","U.S. Pat.","No.","5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).","When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.","The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.","It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.","Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.","Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention."],[" SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).","Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.","Connections are established between each machine and a proxy server sitting on a public network.","The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address."],["RELATED APPLICATIONS The present application claims the benefit of provisional patent application “Traversing Firewalls and NATs”, Ser.","No.","60/255,422, filed Dec. 14, 2000.In addition, this application incorporates by reference, co-pending U.S. patent application Ser.","No.","09/867,371, filed May 29, 2001.BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.","More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).","2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.","Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).","The inability to send and receive information when opening TCP communications into the private network.","Each of the below described references teach the method of firewalls in general.","However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).","U.S. Pat.","No.","5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.","Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.","A time limit is set and so long as the set time limit does not run out, the communication is allowed.","Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.","A connection type session does not appear to be initiated for the UDP transport.","U.S. Pat.","No.","5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.","The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.","The firewall then binds a port and notes it in a link table.","The packet is passed to a stack, on the private side, which forwards the packet to the server.","Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.","U.S. Pat.","No.","5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.","A client on the public network connects to the external machine.","A private channel is opened between the external machine and a machine internal to the private network.","The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.","U.S. Pat.","No.","5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).","When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.","The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.","It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.","Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.","Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.","SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).","Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.","Connections are established between each machine and a proxy server sitting on a public network.","The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 illustrates the Intranet to Internet data transfer scenario.","FIG.","2 illustrates the Internet to Intranet data transfer scenario.","FIG.","3 illustrates the Intranet to Intranet data transfer scenario.","FIG.","4 illustrates a bi-directional connection, using TCP and HTTP, communicating indirectly with the proxy.","FIG.","5 illustrates TCP spoofing of the present invention.","FIG.","6 illustrates TCP spoofing of the present invention in the presence of a packet forwarder.","FIG.","7 illustrates the methodology associated with the present invention.","DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is illustrated and described in a preferred embodiment, the invention may be produced in many different configurations, forms and materials.","There is depicted in the drawings, and will herein be described in detail, a preferred embodiment of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and the associated functional specifications for its construction and is not intended to limit the invention to the embodiment illustrated.","Those skilled in the art will envision many other possible variations within the scope of the present invention.","When a communicating device, such as the Internet phone or a Voice-over-IP Gateway or an IETF MGCP Gateway or an ITU-T H.248 Gateway or a PacketCable Residential Gateway or a CPE Gateway (Customer premises equipment Gateway), opens a signaling connection from a private network to a public network, the TCP channel is bi-directional, and therefore the signaling protocol can execute in both directions.","This also allows HTTP to work behind network address translators (NATs) and Firewalls.","Connections are always opened from the private network to the public network; taking advantage of the fact that TCP data communications are bi-directional.","NATs present an additional translation step when communicating.","NATs map the source addresses (in the private network) of the originating computer into a public address and a port number on the public interface of the NAT.","As long as TCP is used, the translation can be done in reverse, as the TCP channel is bi-directional.","Multimedia signaling and media streaming is usually UDP-based for better efficiency, which introduces the problem—the ingress system sends UDP packets to the public interface on the NAT, and the NAT has no automatic method to map this UDP data-gram to the actual computer that is supposed to receive that data-gram.","The solution provided by the present invention is to stream audio and video (and other time-sensitive data) over TCP, but TCP streaming and windowing mechanizing hurts the real-time performance.","The present invention opens a TCP connection as usual (using TCP), and then switches to a Raw-IP interface that sends Raw-IP data-grams that are legal TCP messages using just opened TCP channel parameters (e.g., session number, port, etc.)","To an intermediate system, these messages will look like standard TCP messages, but as they are sent using Raw-IP, the usual timing issues that TCP introduces to real-time media streaming are not in place.","Thus, the present invention uses the protocol software to “spoof” the TCP channel to enable real-time TCP communications.","It is impossible with NATs and Firewalls to open ingress connections (e.g., it is impossible to open a TCP connection to a computer behind a Firewall or the NAT.","Thus: 1.It is impossible to originate communications from the public network into the private network.","2.It is impossible to originate communications from a private network (to a public network) to another private network.","Thus, the present invention uses a server proxy that both communication parties open their TCP channels to (using the previous procedure).","Then, the proxy communicates to each party the other party's source address/port (of the TCP channel).","Finally, each communication element sends information to the other party using the server proxy source address/port.","It should be noted that packets are sent directly between the communicating entities, as the proxy is only used to hold the TCP state to “spoof” the NATs and Firewalls.","In the preferred embodiment of the present invention, full communication is possible between all types of private and public networks, as long as outgoing TCP channel establishment is allowed.","In another embodiment, the server proxy and originating clients use TCP/HTTP, which is universally supported, and in this instance, all information is tunneled over the simulated TCP/HTTP channel.","In the Internet as it exists today, using the small address space provided by IPv4, many networks deploy NAT (network address translation) devices to enlarge the internal address space.","In addition, many networks deploy firewall devices to block intrusions and hacking.","Many firewalls also support integrated NAT capabilities.","The end-result of both types of devices is that ingress traffic (one originating outside the network and destined into the network) is usually blocked, as incoming connections are usually blocked for firewalls and are impossible to complete on NAT devices, and as the originating (outside the NAT) IP host is unaware of the destination internal IP address.","Thus, users cannot place audio/video calls from NAT protected networks (as the audio and video will not penetrate back into the network from the remote called host), and in many cases users behind corporate firewalls are blocked from using such services.","A communications protocol such as the TrulyGlobal™ Protocol (TGP), (as described in the related application, “Communication Protocol”) can be used in conjunction with the present invention to operate over standard HTTP and remote TGP servers to use the HTTP back-channel to send information to the client; and ensuring that all actions carried by TGP traverse both NATs and firewalls.","Intranet, as defined in this application, is a network that is protected by a NAT or a firewall device, and blocks all incoming traffic into the protected network (e.g., TCP connections cannot be initiated into the network, and UDP traffic will be blocked at the entry-point into the network).","Similarly, Internet is defined as a public addressed, unprotected network, where fill IP communication is possible.","In the Intranet to Internet scenario, as illustrated in FIG.","1, a user inside an Intranet is attempting to call a user that is outside the Intranet, and the remote user is in the public Intranet.","The problem encountered is that while the call will be successfully set-up (as the originating host is allowed to open connection to the outside network), audio and/or video data will not be able to get back into the Intranet, hence the caller will not be able to hear and/or see the called device.","Similarly, in an Internet to Intranet scenario, as illustrated in FIG.","2, the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet are not allowed.","Lastly, in the Intranet to Intranet scenario, as illustrated in FIG.","3, the same end-effect as the previous scenario happens, e.g., the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet from the Internet are not allowed.","The solution provided for by the present invention uses TCP and potentially HTTP, and a service is provided outside the Intranet (in the public Internet) to help both end-points to complete calls.","The first assumption made is that the clients inside the Intranet can initiate TCP or at least TCP/HTTP specifically to the public Internet, so some form of communications is possible.","HTTP can be used to insure safe traversal via HTTP proxies.","Once a TCP/HTTP connection is available, bi-directional communications are possible.","Outwards messages use standard HTTP commands to request resources (using URLs), and incoming information flow returns using the HTTP reply channel (as TCP/HTTP is full-duplex).","Furthermore, at any time, as illustrated by FIG.","4, the caller can initiate a TCP/HTTP connection (or a plain TCP connection) to a service that resides in the public Internet, and that service is responsible to “proxy” the request (using the reply leg of the remote HTTP session) to the called-device.","When both devices have bi-directional connections to the proxy, they can communicate indirectly via the proxy.","While the present invention shows how proxy-based communication can work, the problem when the communication between the two hosts has to flow via the proxy, which adds delay and has limited scalability.","The solution as per the present invention is to spoof the TCP session to allow direct TCP communications between the two machines.","This scenario is illustrated in FIG.","5.When a machine behind a proxy, NAT or firewall establishes a session with the outside world, the session is mapped on the outside of Intranet 1 and 2 on the public interface address(s) to an internal connection between Host 1 and 2 and their gateways to the Internet.","Sending correctly formed TCP packets to that interface will result in the gateway forwarding these packets to the correct host inside the private network.","The sequence is as follows: 1.A session is established from Host 1 in Intranet 1 to the Proxy (AB session).","2.A session is established from Host 2 in Intranet 2 to the Proxy (DC session).","3.The Address BP (public side of session A) is found by inspecting the source address/port of B.","4.The Address of CP (public side of session D) is found by inspecting the source address/port of C. 5.The external mapped addresses are provided to the other hosts, i.e., Host 1 is provided with address/pair CP and Host 2 is provided with address/port pair BP.","TCP session B parameters are provided to host 2.TCP session C parameters are provided to host 1.6.Hosts 1 and 2 will spoof TCP packets for sessions B and C, sent to target address/port pairs BP and CP.","This traffic will go directly between the two networks and not via the Proxy.","In effect, a virtual TCP session C1/B1 is created by combining the two existing TCP sessions C and B.","Once the spoofed TCP session is handed over to Hosts 1 and 2, the Proxy should not send any information on that session, as session parameters may be out-of-date.","The session is kept open for the duration Hosts 1 and 2 requires it, and will be closed by either Host when required.","The proxy is only used for establishing session, and does not use the session for anything else once it is “handed over”.","In some cases the internal network will filter spoofed packets (for security, e.g., hack prevention) and therefore will not let the packets with the spoofed source address leave the internal network.","In such cases, as illustrated by FIG.","6, the TCP connections will be handed over to a packet forwarder (that resides in the same server or a separate server) that handles the packet interchange.","One of the side effects of spoofing the TCP session between Hosts 1 and 2 is that the TCP session parameters can be changed or completely ignored, as long as packets are synthetically correct (as per TCP), they can be sent without consideration to window-sizes, exponential back-off algorithms or slow-start mechanisms.","When such a spoofed TCP session is in place, it can be used to transmit both audio and video with the same time of performance that is expected from UDP.","The session-establishment procedures described above allow any session to be established between any two computers.","This is done as a result of Host 1 calling Host 2 (or the reverse).","The calling host will send a Call-Establishment message to the Proxy, which will (pending, any policy decision) forward the request to the called Host.","The called host will receive the Call Answer transaction over the back-channel of the session it already has with the Proxy, requesting it to answer the call.","If the called host responses positively, one or more media channel(s) will be established between Host 1 and 2, with the help of the proxy as required by the session's parameters (audio only, audio and video, etc).","It should be noted that both IETF SIP and ITU-T H.323 signaling can be used, but are not required.","In one embodiment, the Proxy contains all the required functionality (e.g., signaling a RTP:Address:Port destination instead of a H323:Address:Port destination).","Furthermore, one skilled in the art can recognize that IETF SIP (by manipulating IETF Session Description Protocol (SDP) parameters) and ITU H.323 (by Manipulating ITU-T H.245 OpenLogicalChannel or FastStart parameters) can be used, with minor changes, to accomplish the required signaling.","The present invention is implemented using a raw-IP interfaces that spoofs the TCP sessions.","A limited TCP stack is implemented that creates synthetically correct TCP packets, to insure the packets are interpreted and forwarded correctly by the NATs, proxies and firewalls in the way.","Such a spoofed-TCP stack does not need to support any reliable transmission, as it is only used for real-time sensitive transmission purposes.","FIG.","7 summarizes the methodology 700 associated with the present invention.","In step 702, both hosts establish a connection (e.g., TCP connection or TCP/HTTP connection) with a TCP proxy server.","Next, in step 704, external mapped addresses BP and CP associated with the firewalls of both hosts are identified.","Subsequently, in step 706, the identified external mapped addresses are exchanged between the two hosts.","Lastly, the TCP packets are spoofed to transmit the data (e.g., streaming multimedia data) between the hosts.","Furthermore, the present invention includes a computer program code based product, which is a storage medium having program code stored therein, which can be used to instruct a computer to perform any of the methods associated with the present invention.","The computer storage medium includes any of, but not limited to, the following: CD-ROM, DVD, magnetic tape, optical disc, hard drive, floppy disk, ferroelectric memory, flash memory, ferromagnetic memory, optical storage, charge coupled devices, magnetic or optical cards, smart cards, EEPROM, EPROM, RAM, ROM, DRAM, SRAM, SDRAM, or any other appropriate static or dynamic memory, or data storage devices.","Implemented in computer program code based products are software modules for: aiding in establishing a communication link with a proxy server over a network, wherein a first and second device can access the network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said first device regarding said identified external mapped address CP and notifying said second device regarding said identified external mapped address BP; and aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.","Also implemented in computer program based products are software modules for: aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.","CONCLUSION A system and method has been shown in the above embodiments for the effective implementation of a method and a system for traversing firewalls and network address translations (NATs).","While various preferred embodiments have been shown and described, it will be understood that there is no intent to limit the invention by such disclosure, but rather, it is intended to cover all modifications and alternate constructions falling within the spirit and scope of the invention, as defined in the appended claims.","For example, the present invention should not be limited by type of firewall, type of network address translation device, location of packet forwarder, software/program, computing environment, or specific computing hardware.","The above enhancements are implemented in various computing environments.","For example, the present invention may be implemented on a multi-nodal system (e.g., LAN) or networking system (e.g., Internet, WWW, wireless web).","All programming, and data related thereto are stored in computer memory, static or dynamic, and may be retrieved by the user in any of: conventional computer storage, display (i.e., CRT) and/or hardcopy (i.e., printed) formats.","The programming of the present invention may be implemented by one of skill in the art of network communications."]],"string":"[\n [\n \"Traversing firewalls and nats\",\n \"An incoming UDP packet is allowed to traverse a network address translation (NAT) device or a firewall, wherein first, a TCP connection is opened and a Raw-IP interface is utilized to build the UDP-like packet using the parameters of the TCP connection (e.g., session number, port, etc.)\",\n \"Furthermore, when one of two communicating machines is behind a firewall, a connection is established between each of the machines and a proxy server located in a public network.\",\n \"The proxy then communicates the port and address information while using the proxy server's port and address information as the source port and address, or provides both with an address of an appropriate (potentially based on network proximity) packet forwarder.\"\n ],\n [\n \"1.A method for transmitting data between a first and second device by traversing firewalls, said method comprising the steps of: a. said first and second device establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. said proxy server inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. said proxy server notifying said first device regarding said identified external mapped address CP and said proxy server notifying said second device regarding said identified external mapped address BP, and d. said first or second device spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.\",\n \"2.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said step of spoofing TCP packets is done via Raw-IP datagrams.\",\n \"3.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said communication link is established via TCP.\",\n \"4.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said communication link is established via TCP/HTTP.\",\n \"5.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"6.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"7.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said data is streaming multimedia data.\",\n \"8.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, said method comprising the steps of: a. said first and second device establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. said proxy server inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. said proxy server notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and d. said first device forwarding TCP packets via transmitting data with said packet forwarder as said destination address and said packet forwarder forwarding said data with CP as the destination address, or said second device forwarding TCP packets via transmitting data with said packet forwarder as said destination address and said packet forwarder forwarding said data with BP as the destination address.\",\n \"9.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said step of forwarding TCP packets is done via Raw-IP datagrams.\",\n \"10.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per.\",\n \"Claim 8, wherein said communication link is established via TCP.\",\n \"11.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said communication link is established via TCP/HTTP.\",\n \"12.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"13.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"14.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said data is streaming multimedia data.\",\n \"15.A system for transmitting data between a first and second device by traversing firewalls, said system comprising: a. a first host and an associated first firewall; b. a second host and an associated second firewall; c. a proxy server that establishes a communication link with said first and second host, identifies from said first and second firewalls external mapped addresses BP and CP respectively, and forwards said CP to said first device and forwards said BP to said second device, whereupon said first and second host utilize said forwarded external mapped addresses to spoof TCP packets and forward data directly between each other.\",\n \"16.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said proxy server forwards TCP packets via Raw-IP datagrams.\",\n \"17.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said communication link is established via TCP.\",\n \"18.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said communication link is established via TCP/HTTP.\",\n \"19.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"20.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"21.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said data is streaming multimedia data.\",\n \"22.A system for transmitting data between a first and second device by traversing firewalls, said system comprising: a. a first host and an associated first firewall; b. a second host and an associated second firewall; c. a proxy server that establishes a communication link with said first and second host, identifies from said first and second firewalls external mapped addresses BP and CP respectively; d. a packet forwarder receiving BP and CP from said proxy server and using BP and CP to forward incoming communications from said first device to second device with CP as destination address, or forwarding incoming communications from said second device to first device with BP as the destination address.\",\n \"23.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said packet forwarder forwards TCP packets via Raw-IP datagrams.\",\n \"24.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said communication link is established via TCP.\",\n \"25.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said communication link is established via TCP/HTTP.\",\n \"26.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said firewall is equipped with a network address translation device (NAT).\",\n \"27.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.\",\n \"28.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said data is streaming multimedia data.\",\n \"29.An article of manufacture comprising a computer usable medium having computer readable program code embodied therein for assisting in the transmission of data between a first and second device by traversing firewalls, said article further comprising: a. computer readable program code aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. computer readable program code inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. computer readable program code notifying said first device regarding said identified external mapped address CP and computer readable program code notifying said second device regarding said identified external mapped address BP, and d. computer readable program code aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.\",\n \"30.An article of manufacture comprising a computer usable medium having computer readable program code embodied therein for aiding in forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, said medium further comprising: a. computer readable program code aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. computer readable program code inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. computer readable program code notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and d. computer readable program code forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or computer readable program code forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.\",\n \"More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).\",\n \"2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.\",\n \"Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).\",\n \"The inability to send and receive information when opening TCP communications into the private network.\",\n \"Each of the below described references teach the method of firewalls in general.\",\n \"However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.\",\n \"Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.\",\n \"A time limit is set and so long as the set time limit does not run out, the communication is allowed.\",\n \"Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.\",\n \"A connection type session does not appear to be initiated for the UDP transport.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.\",\n \"The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.\",\n \"The firewall then binds a port and notes it in a link table.\",\n \"The packet is passed to a stack, on the private side, which forwards the packet to the server.\",\n \"Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.\",\n \"A client on the public network connects to the external machine.\",\n \"A private channel is opened between the external machine and a machine internal to the private network.\",\n \"The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).\",\n \"When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.\",\n \"The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.\",\n \"It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.\",\n \"Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.\",\n \"Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).\",\n \"Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.\",\n \"Connections are established between each machine and a proxy server sitting on a public network.\",\n \"The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.\"\n ],\n [\n \"RELATED APPLICATIONS The present application claims the benefit of provisional patent application “Traversing Firewalls and NATs”, Ser.\",\n \"No.\",\n \"60/255,422, filed Dec. 14, 2000.In addition, this application incorporates by reference, co-pending U.S. patent application Ser.\",\n \"No.\",\n \"09/867,371, filed May 29, 2001.BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.\",\n \"More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).\",\n \"2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.\",\n \"Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).\",\n \"The inability to send and receive information when opening TCP communications into the private network.\",\n \"Each of the below described references teach the method of firewalls in general.\",\n \"However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.\",\n \"Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.\",\n \"A time limit is set and so long as the set time limit does not run out, the communication is allowed.\",\n \"Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.\",\n \"A connection type session does not appear to be initiated for the UDP transport.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.\",\n \"The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.\",\n \"The firewall then binds a port and notes it in a link table.\",\n \"The packet is passed to a stack, on the private side, which forwards the packet to the server.\",\n \"Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.\",\n \"A client on the public network connects to the external machine.\",\n \"A private channel is opened between the external machine and a machine internal to the private network.\",\n \"The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).\",\n \"When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.\",\n \"The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.\",\n \"It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.\",\n \"Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.\",\n \"Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.\",\n \"SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).\",\n \"Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.\",\n \"Connections are established between each machine and a proxy server sitting on a public network.\",\n \"The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 illustrates the Intranet to Internet data transfer scenario.\",\n \"FIG.\",\n \"2 illustrates the Internet to Intranet data transfer scenario.\",\n \"FIG.\",\n \"3 illustrates the Intranet to Intranet data transfer scenario.\",\n \"FIG.\",\n \"4 illustrates a bi-directional connection, using TCP and HTTP, communicating indirectly with the proxy.\",\n \"FIG.\",\n \"5 illustrates TCP spoofing of the present invention.\",\n \"FIG.\",\n \"6 illustrates TCP spoofing of the present invention in the presence of a packet forwarder.\",\n \"FIG.\",\n \"7 illustrates the methodology associated with the present invention.\",\n \"DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is illustrated and described in a preferred embodiment, the invention may be produced in many different configurations, forms and materials.\",\n \"There is depicted in the drawings, and will herein be described in detail, a preferred embodiment of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and the associated functional specifications for its construction and is not intended to limit the invention to the embodiment illustrated.\",\n \"Those skilled in the art will envision many other possible variations within the scope of the present invention.\",\n \"When a communicating device, such as the Internet phone or a Voice-over-IP Gateway or an IETF MGCP Gateway or an ITU-T H.248 Gateway or a PacketCable Residential Gateway or a CPE Gateway (Customer premises equipment Gateway), opens a signaling connection from a private network to a public network, the TCP channel is bi-directional, and therefore the signaling protocol can execute in both directions.\",\n \"This also allows HTTP to work behind network address translators (NATs) and Firewalls.\",\n \"Connections are always opened from the private network to the public network; taking advantage of the fact that TCP data communications are bi-directional.\",\n \"NATs present an additional translation step when communicating.\",\n \"NATs map the source addresses (in the private network) of the originating computer into a public address and a port number on the public interface of the NAT.\",\n \"As long as TCP is used, the translation can be done in reverse, as the TCP channel is bi-directional.\",\n \"Multimedia signaling and media streaming is usually UDP-based for better efficiency, which introduces the problem—the ingress system sends UDP packets to the public interface on the NAT, and the NAT has no automatic method to map this UDP data-gram to the actual computer that is supposed to receive that data-gram.\",\n \"The solution provided by the present invention is to stream audio and video (and other time-sensitive data) over TCP, but TCP streaming and windowing mechanizing hurts the real-time performance.\",\n \"The present invention opens a TCP connection as usual (using TCP), and then switches to a Raw-IP interface that sends Raw-IP data-grams that are legal TCP messages using just opened TCP channel parameters (e.g., session number, port, etc.)\",\n \"To an intermediate system, these messages will look like standard TCP messages, but as they are sent using Raw-IP, the usual timing issues that TCP introduces to real-time media streaming are not in place.\",\n \"Thus, the present invention uses the protocol software to “spoof” the TCP channel to enable real-time TCP communications.\",\n \"It is impossible with NATs and Firewalls to open ingress connections (e.g., it is impossible to open a TCP connection to a computer behind a Firewall or the NAT.\",\n \"Thus: 1.It is impossible to originate communications from the public network into the private network.\",\n \"2.It is impossible to originate communications from a private network (to a public network) to another private network.\",\n \"Thus, the present invention uses a server proxy that both communication parties open their TCP channels to (using the previous procedure).\",\n \"Then, the proxy communicates to each party the other party's source address/port (of the TCP channel).\",\n \"Finally, each communication element sends information to the other party using the server proxy source address/port.\",\n \"It should be noted that packets are sent directly between the communicating entities, as the proxy is only used to hold the TCP state to “spoof” the NATs and Firewalls.\",\n \"In the preferred embodiment of the present invention, full communication is possible between all types of private and public networks, as long as outgoing TCP channel establishment is allowed.\",\n \"In another embodiment, the server proxy and originating clients use TCP/HTTP, which is universally supported, and in this instance, all information is tunneled over the simulated TCP/HTTP channel.\",\n \"In the Internet as it exists today, using the small address space provided by IPv4, many networks deploy NAT (network address translation) devices to enlarge the internal address space.\",\n \"In addition, many networks deploy firewall devices to block intrusions and hacking.\",\n \"Many firewalls also support integrated NAT capabilities.\",\n \"The end-result of both types of devices is that ingress traffic (one originating outside the network and destined into the network) is usually blocked, as incoming connections are usually blocked for firewalls and are impossible to complete on NAT devices, and as the originating (outside the NAT) IP host is unaware of the destination internal IP address.\",\n \"Thus, users cannot place audio/video calls from NAT protected networks (as the audio and video will not penetrate back into the network from the remote called host), and in many cases users behind corporate firewalls are blocked from using such services.\",\n \"A communications protocol such as the TrulyGlobal™ Protocol (TGP), (as described in the related application, “Communication Protocol”) can be used in conjunction with the present invention to operate over standard HTTP and remote TGP servers to use the HTTP back-channel to send information to the client; and ensuring that all actions carried by TGP traverse both NATs and firewalls.\",\n \"Intranet, as defined in this application, is a network that is protected by a NAT or a firewall device, and blocks all incoming traffic into the protected network (e.g., TCP connections cannot be initiated into the network, and UDP traffic will be blocked at the entry-point into the network).\",\n \"Similarly, Internet is defined as a public addressed, unprotected network, where fill IP communication is possible.\",\n \"In the Intranet to Internet scenario, as illustrated in FIG.\",\n \"1, a user inside an Intranet is attempting to call a user that is outside the Intranet, and the remote user is in the public Intranet.\",\n \"The problem encountered is that while the call will be successfully set-up (as the originating host is allowed to open connection to the outside network), audio and/or video data will not be able to get back into the Intranet, hence the caller will not be able to hear and/or see the called device.\",\n \"Similarly, in an Internet to Intranet scenario, as illustrated in FIG.\",\n \"2, the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet are not allowed.\",\n \"Lastly, in the Intranet to Intranet scenario, as illustrated in FIG.\",\n \"3, the same end-effect as the previous scenario happens, e.g., the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet from the Internet are not allowed.\",\n \"The solution provided for by the present invention uses TCP and potentially HTTP, and a service is provided outside the Intranet (in the public Internet) to help both end-points to complete calls.\",\n \"The first assumption made is that the clients inside the Intranet can initiate TCP or at least TCP/HTTP specifically to the public Internet, so some form of communications is possible.\",\n \"HTTP can be used to insure safe traversal via HTTP proxies.\",\n \"Once a TCP/HTTP connection is available, bi-directional communications are possible.\",\n \"Outwards messages use standard HTTP commands to request resources (using URLs), and incoming information flow returns using the HTTP reply channel (as TCP/HTTP is full-duplex).\",\n \"Furthermore, at any time, as illustrated by FIG.\",\n \"4, the caller can initiate a TCP/HTTP connection (or a plain TCP connection) to a service that resides in the public Internet, and that service is responsible to “proxy” the request (using the reply leg of the remote HTTP session) to the called-device.\",\n \"When both devices have bi-directional connections to the proxy, they can communicate indirectly via the proxy.\",\n \"While the present invention shows how proxy-based communication can work, the problem when the communication between the two hosts has to flow via the proxy, which adds delay and has limited scalability.\",\n \"The solution as per the present invention is to spoof the TCP session to allow direct TCP communications between the two machines.\",\n \"This scenario is illustrated in FIG.\",\n \"5.When a machine behind a proxy, NAT or firewall establishes a session with the outside world, the session is mapped on the outside of Intranet 1 and 2 on the public interface address(s) to an internal connection between Host 1 and 2 and their gateways to the Internet.\",\n \"Sending correctly formed TCP packets to that interface will result in the gateway forwarding these packets to the correct host inside the private network.\",\n \"The sequence is as follows: 1.A session is established from Host 1 in Intranet 1 to the Proxy (AB session).\",\n \"2.A session is established from Host 2 in Intranet 2 to the Proxy (DC session).\",\n \"3.The Address BP (public side of session A) is found by inspecting the source address/port of B.\",\n \"4.The Address of CP (public side of session D) is found by inspecting the source address/port of C. 5.The external mapped addresses are provided to the other hosts, i.e., Host 1 is provided with address/pair CP and Host 2 is provided with address/port pair BP.\",\n \"TCP session B parameters are provided to host 2.TCP session C parameters are provided to host 1.6.Hosts 1 and 2 will spoof TCP packets for sessions B and C, sent to target address/port pairs BP and CP.\",\n \"This traffic will go directly between the two networks and not via the Proxy.\",\n \"In effect, a virtual TCP session C1/B1 is created by combining the two existing TCP sessions C and B.\",\n \"Once the spoofed TCP session is handed over to Hosts 1 and 2, the Proxy should not send any information on that session, as session parameters may be out-of-date.\",\n \"The session is kept open for the duration Hosts 1 and 2 requires it, and will be closed by either Host when required.\",\n \"The proxy is only used for establishing session, and does not use the session for anything else once it is “handed over”.\",\n \"In some cases the internal network will filter spoofed packets (for security, e.g., hack prevention) and therefore will not let the packets with the spoofed source address leave the internal network.\",\n \"In such cases, as illustrated by FIG.\",\n \"6, the TCP connections will be handed over to a packet forwarder (that resides in the same server or a separate server) that handles the packet interchange.\",\n \"One of the side effects of spoofing the TCP session between Hosts 1 and 2 is that the TCP session parameters can be changed or completely ignored, as long as packets are synthetically correct (as per TCP), they can be sent without consideration to window-sizes, exponential back-off algorithms or slow-start mechanisms.\",\n \"When such a spoofed TCP session is in place, it can be used to transmit both audio and video with the same time of performance that is expected from UDP.\",\n \"The session-establishment procedures described above allow any session to be established between any two computers.\",\n \"This is done as a result of Host 1 calling Host 2 (or the reverse).\",\n \"The calling host will send a Call-Establishment message to the Proxy, which will (pending, any policy decision) forward the request to the called Host.\",\n \"The called host will receive the Call Answer transaction over the back-channel of the session it already has with the Proxy, requesting it to answer the call.\",\n \"If the called host responses positively, one or more media channel(s) will be established between Host 1 and 2, with the help of the proxy as required by the session's parameters (audio only, audio and video, etc).\",\n \"It should be noted that both IETF SIP and ITU-T H.323 signaling can be used, but are not required.\",\n \"In one embodiment, the Proxy contains all the required functionality (e.g., signaling a RTP:Address:Port destination instead of a H323:Address:Port destination).\",\n \"Furthermore, one skilled in the art can recognize that IETF SIP (by manipulating IETF Session Description Protocol (SDP) parameters) and ITU H.323 (by Manipulating ITU-T H.245 OpenLogicalChannel or FastStart parameters) can be used, with minor changes, to accomplish the required signaling.\",\n \"The present invention is implemented using a raw-IP interfaces that spoofs the TCP sessions.\",\n \"A limited TCP stack is implemented that creates synthetically correct TCP packets, to insure the packets are interpreted and forwarded correctly by the NATs, proxies and firewalls in the way.\",\n \"Such a spoofed-TCP stack does not need to support any reliable transmission, as it is only used for real-time sensitive transmission purposes.\",\n \"FIG.\",\n \"7 summarizes the methodology 700 associated with the present invention.\",\n \"In step 702, both hosts establish a connection (e.g., TCP connection or TCP/HTTP connection) with a TCP proxy server.\",\n \"Next, in step 704, external mapped addresses BP and CP associated with the firewalls of both hosts are identified.\",\n \"Subsequently, in step 706, the identified external mapped addresses are exchanged between the two hosts.\",\n \"Lastly, the TCP packets are spoofed to transmit the data (e.g., streaming multimedia data) between the hosts.\",\n \"Furthermore, the present invention includes a computer program code based product, which is a storage medium having program code stored therein, which can be used to instruct a computer to perform any of the methods associated with the present invention.\",\n \"The computer storage medium includes any of, but not limited to, the following: CD-ROM, DVD, magnetic tape, optical disc, hard drive, floppy disk, ferroelectric memory, flash memory, ferromagnetic memory, optical storage, charge coupled devices, magnetic or optical cards, smart cards, EEPROM, EPROM, RAM, ROM, DRAM, SRAM, SDRAM, or any other appropriate static or dynamic memory, or data storage devices.\",\n \"Implemented in computer program code based products are software modules for: aiding in establishing a communication link with a proxy server over a network, wherein a first and second device can access the network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said first device regarding said identified external mapped address CP and notifying said second device regarding said identified external mapped address BP; and aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.\",\n \"Also implemented in computer program based products are software modules for: aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.\",\n \"CONCLUSION A system and method has been shown in the above embodiments for the effective implementation of a method and a system for traversing firewalls and network address translations (NATs).\",\n \"While various preferred embodiments have been shown and described, it will be understood that there is no intent to limit the invention by such disclosure, but rather, it is intended to cover all modifications and alternate constructions falling within the spirit and scope of the invention, as defined in the appended claims.\",\n \"For example, the present invention should not be limited by type of firewall, type of network address translation device, location of packet forwarder, software/program, computing environment, or specific computing hardware.\",\n \"The above enhancements are implemented in various computing environments.\",\n \"For example, the present invention may be implemented on a multi-nodal system (e.g., LAN) or networking system (e.g., Internet, WWW, wireless web).\",\n \"All programming, and data related thereto are stored in computer memory, static or dynamic, and may be retrieved by the user in any of: conventional computer storage, display (i.e., CRT) and/or hardcopy (i.e., printed) formats.\",\n \"The programming of the present invention may be implemented by one of skill in the art of network communications.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450751"}}},{"rowIdx":282,"cells":{"text":{"kind":"list like","value":[["Novel nucleic acids and polypeptides","The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof."],["1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, and complementary sequences thereof.","2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.","3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.","5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.","6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.","10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-30368.11.A composition comprising the polypeptide of claim 10 and a carrier.","12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.","14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.","15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.","16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.","17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.","18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.","19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-30368, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).","20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30369-60736, the mature protein portion thereof, or the active domain thereof.","21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.","22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-30368.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.","24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.","25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.","26.The collection of claim 22, wherein the collection is provided in a computer-readable format.","27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.","28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier."],[" 2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.","cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.","The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).","More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.","Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences."],[" 3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.","The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.","The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.","The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.","These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.","In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.","In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.","The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.","The identifying sequence can be 100 base pairs in length.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.","The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.","In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.","The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.","The collection can also be provided in a computer-readable format.","This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.","Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.","In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.","orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.","The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.","Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.","The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.","host cells) of the invention.","The invention also provides compositions comprising a polypeptide of the invention.","Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The invention also provides host cells transformed or transfected with a polynucleotide of the invention.","The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.","Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.","Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.","These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.","For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.","In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.","For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.","Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.","The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.","Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.","In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.","The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.","Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.","The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.","The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.","The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.","Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.","The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.","Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.","Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.","The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.","The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.","In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.","Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.","The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).","If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these-polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.","2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.","cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.","The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).","More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.","Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.","3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.","The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.","The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.","The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.","These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.","In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.","In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.","The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.","The identifying sequence can be 100 base pairs in length.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.","The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.","In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.","The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.","The collection can also be provided in a computer-readable format.","This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.","Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.","In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.","orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.","The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.","Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.","The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.","host cells) of the invention.","The invention also provides compositions comprising a polypeptide of the invention.","Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The invention also provides host cells transformed or transfected with a polynucleotide of the invention.","The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.","Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.","Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.","These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.","For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.","In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.","For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.","Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.","The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.","Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.","In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.","The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.","Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.","The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.","The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.","The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.","Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.","The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.","Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.","Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.","The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.","The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.","In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.","Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.","The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).","If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.","4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.","The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.","According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.","Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.","The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.","The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.","For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.","Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.","The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.","The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.","The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.","The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.","PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.","Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.","The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.","As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.","EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).","One class of EMEs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.","The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.","These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.","In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).","It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).","Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.","The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.","The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.","Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.","Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.","A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.","Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-30368.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.","(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).","They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.","Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-30368.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.","Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.","Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.","The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.","Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.","The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).","The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.","The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.","The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.","The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.","For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.","While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.","repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.","The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.","A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.","The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.","A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.","The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.","Preferably the peptide is from about 5 to about 200 amino acids.","To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.","The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.","The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.","The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.","The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.","The peptide may have been produced by processing in the cell which removes any leader/signal sequence.","The mature protein portion may or may not include an initial methionine residue.","The methionine residue may be removed from the protein during processing in the cell.","The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.","The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.","The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.","Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.","Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.","Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.","Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.","Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.","“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.","For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.","“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.","The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.","Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.","Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.","For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.","Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.","For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.","The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.","In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).","The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.","In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.","The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.","The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.","“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.","As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.","Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.","The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.","An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.","Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.","Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.","This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.","The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.","Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.","This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.","Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.","The cells can be prokaryotic or eukaryotic.","The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.","“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.","“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.","“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.","Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.","Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.","(1998) Annu.","Rev.","Immunol.","16:27-55).","Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.","Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.","The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.","Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).","Other exemplary hybridization conditions are described herein in the examples.","In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).","As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.","Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).","Such a sequence is said to have 65% sequence identity to the listed sequence.","In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).","Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.","Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.","Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.","For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.","For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.","Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.","(1990) Methods Enzymol.","183:626-645).","Identity between sequences can also be determined by other methods known in the art, e.g.","by varying hybridization conditions.","The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.","The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.","The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.","The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.","As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.","UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.","The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.","The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.","As described above, a UMF will increase the frequency of uptake of a linked marker sequence.","Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.","4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.","The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-30368; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 30369-60736; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 30369-60736.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-30368; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 30369-60736.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.","The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.","The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.","The present invention also provides genes corresponding to the cDNA sequences disclosed herein.","The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.","Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.","Further 5′ and 3′ sequence can be obtained using methods known in the art.","For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-30368 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-30368 or a portion there of as a probe.","Alternatively, the polynucleotides of SEQ ID NO: 1-30368 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.","The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.","The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.","The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.","Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.","Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-30368, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.","Fragments of, e.g.","15, 17, or 20 nucleotides or more that are selective for (i.e.","specifically hybridize to any one of the polynucleotides of the invention) are contemplated.","Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.","The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.","Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-30368, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-30368 with a sequence from another isolate of the same species.","Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.","In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.","The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-30368 can be obtained by searching a database using an algorithm or a program.","Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.","Evol.","36 290-300 (1993) and Altschul S. F. et al.","J. Mol.","Biol.","21:403-410(1990)).","Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.","Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.","Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.","The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.","The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.","These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.","There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.","Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.","These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).","Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.","Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.","Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.","Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.","Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.","In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.","This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.","In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).","A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.","10:6487-6500 (1982).","PCR may also be used to create amino acid sequence variants of the novel nucleic acids.","When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.","PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.","The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.","A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.","Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.","Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.","Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.","The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.","The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.","Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.","In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-30368, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.","Also included are the cDNA inserts of any of the clones identified herein.","A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.","(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).","Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.","Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.","In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.","Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.","A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.","The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof or any other polynucleotides of the invention.","In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof is inserted, in a forward or reverse orientation.","In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.","Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.","The following vectors are provided by way of example.","Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).","Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).","The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.","19, 44854490 (1991), in order to produce the protein recombinantly.","Many suitable expression control sequences are known in the art.","General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).","As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.","Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.","Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.","Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.","Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.","Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TP1gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.","Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.","The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.","Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.","Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.","The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.","Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.","As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).","Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).","These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.","Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.","Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.","Polynucleotides of the invention can also be used to induce immune responses.","For example, as described in Fan et al, Nat.","Biotech.","17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.","The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.","4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-30368, or fragments, analogs or derivatives thereof.","An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.","In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.","Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 30369-60736 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-30368 are additionally provided.","In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.","The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.","In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.","The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).","Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-30368), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.","The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.","For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.","An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.","An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.","For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.","Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.","Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).","The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.","The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.","An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.","Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.","For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.","The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.","To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.","In yet another embodiment, the antisense nucleic acid molecule of the invention is an-anomeric nucleic acid molecule.","An-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual-units, the strands run parallel to each other (Gaultier et al.","(1987) Nucleic Acids Res 15: 6625-6641).","The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.","(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.","(1987) FEBS Lett 215: 327-330).","4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.","Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.","Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.","A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-30368).","For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-30368 (see, e.g., Cech et al.","U.S. Pat.","No.","4,987,071; and Cech et al.","U.S. Pat.","No.","5,116,742).","Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.","See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.","See generally, Helene.","(1991) Anticancer Drug Des.","6: 569-84; Helene.","et al.","(1992) Ann.","N.Y. Acad.","Sci.","660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.","For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.","(1996) Bioorg Med Chem 4: 5-23).","As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.","The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.","The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.","(1996) above; Perry-O'Keefe et al.","(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.","For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.","PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymnes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.","(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.","(1996), above; Perry-OKeefe (1996), above).","In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.","For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.","Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.","PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).","The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.","(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.","(1989) Nucl Acid Res 17: 5973-88).","PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.","(1996) above).","Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.","See, Petersen et al.","(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.","Natl.","Acad.","Sci.","U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.","Natl.","Acad.","Sci.","84:648-652; PCT Publication No.","WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No.","WO89/10134).","In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.","Res.","5:539-549).","To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.","4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.","For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.","The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.","Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.","Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.","See, for example, PCT International Publication No.","WO94/12650, PCT International Publication No.","WO92/20808, and PCT International Publication No.","WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.","The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.","Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).","The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.","Any host/vector system can be used to express one or more of the ORFs of the present invention.","These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.","The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.","Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.","Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.","Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.","Various mammalian cell culture systems can also be employed to express recombinant protein.","Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).","Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.","Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.","DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.","Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.","Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.","Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.","Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.","Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.","Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.","Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.","If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.","Such covalent attachments may be accomplished using known chemical or enzymatic methods.","In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.","As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.","Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.","Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.","These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.","The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.","Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.","Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.","Here, the naturally occurring sequences are deleted and new sequences are added.","In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.","The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative, selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.","Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.","The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.","No.","5,272,071 to Chappel; U.S. Pat.","No.","5,578,461 to Sherwin et al.","; International Application No.","PCT/US92/09627 (WO93/09222) by Selden et al.","; and International Application No.","PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.","4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 30369-60736 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-30368 or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.","The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.","Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 30369-60736.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.","Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.","Chem.","Soc.","114, 9245-9253 (1992), both of which are incorporated herein by reference.","Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.","The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.","The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.","The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.","The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.","Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.","In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.","Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.","By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.","Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.","A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.","At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.","The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.","This technique is particularly useful in producing small peptides and fragments of larger polypeptides.","Fragments are useful, for example, in generating antibodies against the native polypeptide.","Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.","The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.","As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.","One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.","The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.","For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.","The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.","Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.","In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.","One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.","These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.","See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.","Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.","The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.","These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.","The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.","In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.","In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.","The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 30369-60736.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.","The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.","For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.","Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.","For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.","Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.","No.","4,518,584).","Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.","Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.","This type of analysis determines the importance of the substituted amino acid(s) in biological activity.","Regions of the protein that are important for protein function may be determined by the eMATRIX program.","Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.","Such modifications are encompassed by the present invention.","The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.","Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.","1555 (1987), incorporated herein by reference.","As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.","The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.","The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.","Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.","For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX), or as a His-tag.","Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.","), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.","The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.","One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).","Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.","Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.","The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).","This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.","Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.","Such analogs may exhibit improved properties such as activity and/or stability.","Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.","Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.","Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.","4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.","Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.","Biol.","215:403410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.","vol.","25, pp.","3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.","Biol., Vol.","6, pp.","219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.","4, pp.","202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.","26(1), pp.","320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.","Biol, 157, pp.","105-31 (1982), incorporated herein by reference).","The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.","NCB NLM NIH Bethesda, Md.","20894; Altschul, S., et al., J. Mol.","Biol.","215:403-410 (1990).","4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.","As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.","Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.","In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.","In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.","Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.","The polypeptide can be fused to the N-terminus or C-terminus.","For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.","In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.","In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.","The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.","The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.","Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g.","promoting or inhibiting) cell survival.","Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.","A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.","For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.","In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.","Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.","(eds.)","CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).","Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).","A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.","4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.","The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.","Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).","See, for example, Anderson, Nature, supplement to vol.","392, no.","6679, pp.","25-20 (1998).","For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).","Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).","Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.","Treated cells can then be introduced in vivo for therapeutic purposes.","Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.","It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.","Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.","Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.","The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.","These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.","Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.","Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.","See, for example, PCT International Publication No.","WO 94/12650, PCT International Publication No.","WO 92/20808, and PCT International Publication No.","WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplificationof the desired protein coding sequences in the cells.","In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.","As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.","Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.","Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.","These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.","The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.","Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.","Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.","Here, the naturally occurring sequences are deleted and new sequences are added.","In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.","The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.","Markers useful for this purpose include the Herpes Sinplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphonbosyl-transferase (gpt) gene.","The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.","No.","5,272,071 to Chappel; U.S. Pat.","No.","5,578,461 to Sherwin et al.","; International Application No.","PCT/US92/09627 (WO93/09222) by Selden et al.","; and International Application No.","PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.","4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].","Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.","Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.","Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.","No.","5,557,032, incorporated herein by reference.","Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.","Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.","Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.","No.","5,489,743 and PCT Publication No.","WO94/28122, incorporated herein by reference.","Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.","Inactivation can be carried out using homologous recombination methods described above.","Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.","The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.","The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.","Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.","In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)).","Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.","Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.","Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.","No.","5,557,032, incorporated herein by reference.","Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.","Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.","Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.","No.","5,489,743 and PCT Publication No.","WO94/28122, incorporated herein by reference.","Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.","Inactivation can be carried out using homologous recombination methods described above.","Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.","The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.","4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.","Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).","The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.","Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.","Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.","The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.","4.10.1 Research uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.","The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.","Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.","The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.","Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.","Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.","Methods for performing the uses listed above are well known to those skilled in the art.","References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.","Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.","In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.","In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.","4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.","The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.","Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.","137:3494-3500, 1986; Bertagnolli et al., J. Immunol.","145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., 1.Immunol.","149:3778-3783, 1992; Bowman et al., I. Immunol.","152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","3.12.1-3.12.14, John Wiley and Sons, Toronto.","1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.","J. E. e.a Coligan eds.","Vol 1 pp.","6.8.1-6.8.8, John Wiley and Sons, Toronto.","1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.","J. E. e.a Coligan eds.","Vol 1 pp.","6.3.1-6.3.12, John Wiley and Sons, Toronto.","1991; deVries et al., J. Exp.","Med.","173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.","Natl.","Acad.","Sci.","U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.6.1-6.6.5, John Wiley and Sons, Toronto.","1991; Smith et al., Proc.","Natl.","Aced.","Sci.","U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.","In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.15.1 John Wiley and Sons, Toronto.","1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.","In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.13.1, John Wiley and Sons, Toronto.","1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.","Natl.","Acad.","Sci.","USA 77:6091-6095, 1980; Weinberger et al., Eur.","J. Immun.","11:405-411, 1981; Takai et al., J. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.","Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.","The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.","It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).","Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.","Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.","This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.","Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.","Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.","No.","5,690,926).","Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.","This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.","These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.","These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.","Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.","For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.","The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.","for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.","In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.","Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.","A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.","The selectable marker allows only cells of the desired type to survive.","For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.","Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.","Lanza et al., Academic Press (1997)).","Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.","In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.","Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.","Proc.","Natl.","Acad.","Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.","The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.","as described by Bernstein et al., Blood, 77: 2316-2321 (1991).","4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.","Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.","in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (ie., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.","Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.","Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.","Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.","Natl.","Acad.","Sci.","USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.","In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.","In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.","A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.","Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.","De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.","A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.","Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)","mediated by inflammatory processes may also be possible using the composition of the invention.","Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.","Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.","Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.","De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.","The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.","The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.","The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.","The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.","for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.","More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.","Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.","Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.","Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.","Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.","Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.","A polypeptide of the present invention may also exhibit angiogenic activity.","A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.","A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.","Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.","WO95/16035 (bone, cartilage, tendon); International Patent Publication No.","WO95/05846 (nerve, neuronal); International Patent Publication No.","WO91/07491 (skin, endothelium).","Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.","71-112 (Maibach, H. I. and Rovee, D. T., eds.","), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.","Invest.","Dermatol 71:382-84 (1978).","4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.","A polynucleotide of the invention can encode a polypeptide exhibiting such activities.","A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.","These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.","More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.","and various fungal infections such as candidiasis.","Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.","Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.","Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.","Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.","The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig slin sensitization test (Vohr et al., Arch.","Toxocol.","73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.","Environ.","Health 53: 563-79).","Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.","Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.","The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.","Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.","Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.","Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.","Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).","For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.","Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.","The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.","Moreover, a lack of costinulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.","Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.","To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.","The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.","Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4I g fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.","Natl.","Acad.","Sci USA, 89:11102-11105 (1992).","In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.","846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.","Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.","Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.","Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.","Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.","Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.","The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.","Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.","840-856).","Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.","Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.","For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.","Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.","Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.","The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.","A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.","In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class 1 alpha chain protein and P2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.","Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.","Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.","Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.","The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.","Natl.","Acad.","Sci.","USA 78:2488-2492, 1981; Herrmnann et al., J. Immunol.","128:1968-1974, 1982; Handa et al., J. Immunol.","135:1564-1572, 1985; Takai et al., I. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.","153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.","144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","3.8.1-3.8.16, John Wiley and Sons, Toronto.","1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988; Bertagnolli et al., J. Immunol.","149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.","134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260,1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:40374045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.","Nat.","Acad.","Sci.","USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.","A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.","Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).","Thus, a polypeptide of the present invention, alone or in heterodirners with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.","Administration of sufficient amounts of other inhibins can induce infertility in these mammals.","Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.","See, for example, U.S. Pat.","No.","4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.","The activity of a polypeptide of the invention may, among other means, be measured by the following methods.","Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.","Natl.","Acad.","Sci.","USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Chemotactic and chemolinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.","Chemotactic or chemokinetic compositions (e.g.","proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.","For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.","A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.","Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.","Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.","Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.","Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.","J. Clin.","Invest.","95:1370-1376, 1995; Lind et al.","APMIS 103:140-146, 1995; Muller et al Eur.","J. Immunol.","25:1744-1748; Gruber et al.","J. of Immunol.","152:5860-5867, 1994; Johnston et al.","J. of Immunol.","153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Compositions may be usefull in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.","A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).","Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.","Pharmacol.","26:131-140, 1986; Burdick et al., Thrombosis Res.","45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79(1991); Schaub, Prostaglandins 35:467-474,1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.","Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.","For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.","Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.","Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.","Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.","Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.","Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.","Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.","reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.","The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.","An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.","The use of anti-cancer cocktails as a cancer treatment is routine.","Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.","In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.","There are hereditary conditions and/or environmental situations (e.g.","exposure to carcinogens) known in the art that predispose an individual to developing cancers.","Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.","In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.","These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.","Can.","Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.","J. Dev.","Biol., 40: 1189-97 (1999) and Li et al., Clin.","Exp.","Metastasis, 17:423-9 (1999), respectively.","Suitable tumor cells lines are available, e.g.","from American Type Tissue Culture Collection catalogs.","4.10.12 RECEPTOR/LIGAND ACTIVITY A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.","A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.","Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.","Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.","A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.","The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.","Natl.","Acad.","Sci.","USA 84:6864-6868, 1987; Bierer et al., J. Exp.","Med.","168:1145-1156, 1988; Rosenstein et al., J. Exp.","Med.","169:149-160 1989; Stoltenborg et al., J. Immunol.","Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).","Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.","Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.","The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.","(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.","182 (1990) Academic Press, Inc. San Diego).","Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.","Examples of toxins include, but are not limited, to ricin.","4.10.13 DRUG SCREENING This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.","The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.","One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.","Drugs are screened against such transformed cells in competitive binding assays.","Such cells, either in viable or fixed form, can be used for standard binding assays.","One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.","Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.","Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.","The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.","Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.","For a review, see Science 282:63-68 (1998).","Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.","Of particular interest are peptide and oligonucleotide combinatorial libraries.","Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.","For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.","Opin.","Biotechnol.","8:701-707 (1997).","For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.","Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).","Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.","The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.","In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.","The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.","The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.","Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.","4.10.14 ASSAY FOR RECEPTOR ACTIVITY The invention also provides methods to detect specific binding of a polypeptide e.g.","a ligand or a receptor.","The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.","For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.","As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.","There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.","Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.","The responses of the two cell populations to the addition of ligands(s) are then compared.","Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).","As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.","The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.","For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.","The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.","Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.","phosphorylation.","Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.","4.10.15 Anti-Inflammtory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.","The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.","Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.","Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.","4.10.16 LEUKEMIAS Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.","Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.","B. Lippincott Co., Philadelphia).","4.10.17 NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.","Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vi) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (vii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.","Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.","For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.","Such effects may be measured by any method known in the art.","In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.","(1990, J. Neurosci.","10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.","(1980, Exp.","Neurol.","70:65-82) or Brown et al.","(1981, Ann.","Rev.","Neurosci.","4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.","In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).","4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fingi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.","4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.","Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.","For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.","Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.","For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.","Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).","In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.","Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.","The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.","In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.","Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.","4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.","The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.","Arch.","Allergy Appl.","Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).","The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.","The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.","The control consists of administering PBS only.","The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.","An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.","4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.","Examples of therapeutic applications include, but are not limited to, those exemplified herein.","4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.","While the mode of administration is not particularly important, parenteral administration is preferred.","An exemplary mode of administration is to deliver an intravenous bolus.","The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.","It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.","Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.","For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.","Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.","The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.","The preparation of such solutions is within the skill of the art.","4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.","Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.","The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).","The characteristics of the carrier will depend on the route of administration.","The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, ThF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.","In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.","These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.","The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.","Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.","Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).","A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.","As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.","As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).","Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.","A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.","When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.","When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.","In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.","Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.","When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.","If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.","4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.","Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.","Intravenous administration to the patient is preferred.","Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.","In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.","Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.","The liposomes will be targeted to and taken up selectively by the afflicted tissue.","The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.","The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.","Preferably for wound treatment, one administers the therapeutic compound directly to the site.","Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.","Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.","4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.","These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.","Proper formulation is dependent upon the route of administration chosen.","When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.","When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.","The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.","When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.","The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.","When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.","When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.","The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.","A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.","The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.","For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.","Such penetrants are generally known in the art.","For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.","Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.","Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.","Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).","If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.","Dragee cores are provided with suitable coatings.","For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.","Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.","Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.","The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.","In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.","In addition, stabilizers may be added.","All formulations for oral administration should be in dosages suitable for such administration.","For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.","For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.","In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.","Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.","The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.","Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.","The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.","Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.","Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.","Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.","Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.","Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.","Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.","The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.","In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.","Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.","Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.","A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.","The co-solvent system may be the VPD co-solvent system.","VPD is a solution of 3%/o w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.","The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.","This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.","Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.","Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.","polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.","Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.","Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.","Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.","Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.","Various types of sustained-release materials have been established and are well known by those skilled in the art.","Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.","Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.","The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.","Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.","Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.","Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.","The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.","The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.","B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.","T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.","MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.","The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.","Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.","The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.","Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.","Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.","Nos.","4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.","The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.","Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.","Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.","Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.","It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.","For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.","When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.","Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.","Topical administration may be suitable for wound healing and tissue repair.","Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.","Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.","Such matrices may be formed of materials presently in use for other implanted medical applications.","The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.","The particular application of the compositions will define the appropriate formulation.","Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.","Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.","Further matrices are comprised of pure proteins or extracellular matrix components.","Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.","Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.","The biocermics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.","Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.","In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.","A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).","Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).","The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.","In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.","These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).","The therapeutic compositions are also presently valuable for veterinary applications.","Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.","The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.","The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.","For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.","Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.","Polynucleotides of the present invention can also be used for gene therapy.","Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.","Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).","Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.","Treated cells can then be introduced in vivo for therapeutic purposes.","4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.","More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.","Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.","For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity).","Such information can be used to more accurately determine useful doses in humans.","A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.","Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).","The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.","The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.","The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity.","The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.","The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.","See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.","1 p.1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).","The MEC will vary for each compound but can be estimated from in vitro data.","Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.","However, HPLC assays or bioassays can be used to determine plasma concentrations.","Dosage intervals can also be determined using MEC value.","Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.","In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.","An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.","Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.","The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.","4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.","The pack may, for example, comprise metal or plastic foil, such as a blister pack.","The pack or dispenser device may be accompanied by instructions for administration.","Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.","4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.","The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.","Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab′)2 fragments, and an Fab expression library.","In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.","Certain classes have subclasses as well, such as IgG1, IgG2, and others.","Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.","Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.","An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.","The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.","An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, (for example the amino acid sequence shown in SEQ ID NO: 30369), and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.","Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.","Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.","In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention that is located on the surface of the protein, e.g., a hydrophilic region.","A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.","As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.","See, e.g., Hopp and Woods, 1981, Proc.","Nat.","Acad.","Sci.","USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.","Biol.","157: 105-142, each of which is incorporated herein by reference in its entirety.","Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.","A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.","Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).","Some of these antibodies are discussed below.","5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.","An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.","Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.","Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.","The preparation can further include an adjuvant.","Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.","), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.","Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).","The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.","Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.","Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.","14, No.","8 (Apr.","17, 2000), pp.","25-28).","5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.","In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.","MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.","Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).","In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.","Alternatively, the lymphocytes can be immunized in vitro.","The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.","Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.","The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.","59-103).","Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.","Usually, rat or mouse myeloma cell lines are employed.","The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.","For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.","Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.","More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.","Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.","51-63).","The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.","Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).","Such techniques and assays are known in the art.","The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.","Biochem.","107:220 (1980).","Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.","After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.","Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.","Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.","The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.","The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.","No.","4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).","The hybridoma cells of the invention serve as a preferred source of such DNA.","Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.","The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.","No.","4,816,567; Morrison, Nature 368 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.","Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.","5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.","These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.","Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.","Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.","(See also U.S. Pat.","No.","5,225,539.)","In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.","Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.","In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.","The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.","Op.","Struct.","Biol., 2:593-596 (1992)).","5.13.3 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.","Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.","Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.","77-96).","Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.","77-96).","In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.","Biol., 227:381 (1991); Marks et al., J. Mol.","Biol., 222:581 (1991)).","Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.","Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.","This approach is described, for example, in U.S. Pat.","Nos.","5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.","(Bio/Technology 10, 779-783 (1992)); Lonberg et al.","(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.","Rev.","Immunol.","13 65-93 (1995)).","Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.","(See PCT publication WO94/02602).","The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.","The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.","An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.","The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.","The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.","Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.","An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.","No.","5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.","A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.","No.","5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.","The hybrid cell expresses an antibody containing the heavy chain and the light chain.","In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.","No.","4,946,778).","In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.","Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) FV fragments.","5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.","In the present case, one of the binding specificities is for an antigenic protein of the invention.","The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.","Methods for making bispecific antibodies are known in the art.","Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).","Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.","The purification of the correct molecule is usually accomplished by affinity chromatography steps.","Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.","The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.","It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.","DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.","For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).","According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.","The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.","In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.","tyrosine or tryptophan).","Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.","alanine or threonine).","This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.","Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.","F(ab′)2 bispecific antibodies).","Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.","For example, bispecific antibodies can be prepared using chemical linkage.","Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.","These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.","The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.","One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.","The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.","Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.","Shalaby et al., J. Exp.","Med.","175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.","Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.","The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.","Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.","For example, bispecific antibodies have been produced using leucine zippers.","Kostelny et al., J. Immunol.","148(5):1547-1553 (1992).","The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.","The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.","This method can also be utilized for the production of antibody homodimers.","The “diabody” technology described by Hollinger et al., Proc.","Natl.","Acad.","Sci.","USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.","The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.","Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.","Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported.","See, Gruber et al., J. Immunol.","152:5368 (1994).","Antibodies with more than two valencies are contemplated.","For example, trispecific antibodies can be prepared.","Tutt et al., J. Immunol.","147:60 (1991).","Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.","Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.","CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.","Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.","These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.","Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (° F.).","5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.","Heteroconjugate antibodies are composed of two covalently joined antibodies.","Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.","No.","4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).","It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.","For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.","Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.","No.","4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.","For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.","The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).","See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.","Immunol., 148: 2918-2922 (1992).","Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.","Cancer Research, 53: 2560-2565 (1993).","Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.","See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).","5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).","Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.","Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.","A variety of radionuclides are available for the production of radioconjugated antibodies.","Examples include 212Bi, 131I, 90Y, 90Y, and 186Re.","Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).","For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).","Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.","See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.","4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.","As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.","Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.","As used herein, “recorded” refers to a process for storing information on computer readable medium.","A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.","A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.","The choice of the data storage structure will generally be based on the means chosen to access the stored information.","In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.","The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.","A skilled artisan can readily adapt any number of data processor structuring formats (e.g.","text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.","By providing any of the nucleotide sequences SEQ ID NO: 1-30368 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-30368 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.","Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.","The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.","Biol.","215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.","Chem.","17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.","Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.","As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.","The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.","A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.","As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.","As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.","As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.","Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.","A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.","Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASIN and BLASTA (NPOLYPEPTIDEIA).","A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.","As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.","A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.","The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.","However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.","As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.","There are a variety of target motifs known in the art.","Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.","Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).","4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.","Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.","Acids Res.","6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olnno, J. Neurochem.","56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).","Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.","Both techniques have been demonstrated to be effective in model systems.","Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.","4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.","In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.","Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.","In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.","In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.","Conditions for incubating a nucleic acid probe or antibody with a test sample vary.","Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.","One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.","Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.","1 (1982), Vol.","2 (1983), Vol.","3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).","The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.","The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.","Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.","In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.","Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.","In detail, a compartment kit includes any kit in which reagents are contained in separate containers.","Such containers include small glass containers, plastic containers or strips of plastic or paper.","Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.","Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.","), and containers which contain the reagents used to detect the bound antibody or probe.","Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.","One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.","4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).","See, e.g., Kunkel et al., U.S. Pat.","No.","5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.","4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-30368, or bind to a specific domain of the polypeptide encoded by the nucleic acid.","In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.","In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.","Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.","Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.","Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).","Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).","Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.","The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.","The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.","For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.","Alternatively, agents may be rationally selected or designed.","As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.","For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.","Freeman, N.Y. (1992), pp.","289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.","In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.","As described above, such agents can be randomly screened or rationally designed/selected.","Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.","One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.","Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.","Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.","Acids Res.","6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.","56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).","Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.","Both techniques have been demonstrated to be effective in model systems.","Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.","Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.","Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.","4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.","The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-30368.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-30368 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.","Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.","PCR as described in U.S. Pat.","Nos.","4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.","Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.","The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.","Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.","Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.","The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.","The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.","These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.","The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.","Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f).","Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.","The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.","4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.","Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.","One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.","Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.","Microbiol.","28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey& Collins, (1989) Mol.","Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.","Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.","For example, Broude et al.","(1994) Proc.","Natl.","Acad.","Sci.","USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.","Streptavidin-coated beads may be purchased from Dynal, Oslo.","Of course, this same linking chemistry is applicableto coating any surface with streptavidin Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).","Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.","Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.","CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.","CovaLink Modules may be purchased from Nunc Laboratories.","DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.","Biochem.","198(1) 138-42).","The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).","In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.","1 (8) 6513-29).","This is beneficial as immobilization using only a single covalent bond is preferred.","The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.","To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.","It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.","More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.","at 95° C. and cooling on ice for 10 min.","Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.","Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well.","The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).","It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.","This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.","The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.","Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.","An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.","For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al.","(1991) Science 251(4995) 767-73, incorporated herein by reference.","Probes may also be immobilized on nylon supports as described by Van Ness et al.","(1991) Nucleic Acids Res.","19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.","Biochem.","169(1) 104-8; all references being specifically incorporated herein.","To link an oligonucleotide to a nylon support, as described by Van Ness et at (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.","One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference).","These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).","These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.","A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.","4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.","For example, Sambrook et al.","(1989) describes three protocols for the isolation ofhigh molecular weight DNA from mammalian cells (p. 9.14-9.23).","DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.","Samples may be prepared or dispensed in multiwell plates.","About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.","The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.","(1989), shearing by ultrasound and NaOH treatment.","Low pressure shearing is also appropriate, as described by Schriefer et at (1.990) Nucleic Acids Res.","18(24) 7455-6, incorporated herein by reference).","In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.","A lever device allows controlled application of low to intermediate pressures to the cell.","The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.","One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.","(1992) Nucleic Acids Res.","20(14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.","The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.","A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).","Fitzgerald et al.","(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest ofpUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.","Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.","As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.","Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.","This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.","Phosphate groups must also be removed from genomic DNA by methods known in the art.","4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.","Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.","By offset printing, a density of dots higher than the density of the wells is achieved.","One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.","By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.","Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.","Each of the subarrays may represent replica spotting of the same samples.","In one example, a selected gene segment may be amplified from 64 patients.","For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).","A plate for each of the 64 patients is prepared.","By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.","Subarrays may contain 64 samples, one from each patient.","Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.","Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.","a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.","A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.","The present invention is illustrated in the following examples.","Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.","Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.","The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.","Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.","Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.","All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.","5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.","The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.","Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.","The clones were clustered into groups of similar or identical sequences.","Representative clones were selected for sequencing.","In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.","PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.","Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.","In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.","5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.","The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-30368 and are provided in the attached Sequence Listing.","The contigs were assembled using an EST sequence as a seed.","Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.","The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.","Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.","The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-30368) of the present invention are incorporated in the attached Sequence Listing.","A subset the predicted polypeptide sequences contain an unknown amino acid, a stop codon, a possible nucleotide deletion or a possible nucleotide insertion.","These sequences have been shown in their entirety with the special characters in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-30368.Table 2 also indicates the method by which the polypeptide was predicted.","Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fastabioch.virginia.edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).","Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.","Biol., 268:78-94(1997), incorporated herein by reference).","Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.","The nearest neighbor results for SEQ ID NO: 1-30368 were obtained by a BLASTP version 2.0al 19 MP-WashU search against Genpept release 121 and Geneseq release 200103 (Derwent), using BLAST algorithm.","The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-30368.The nearest neighbor results for SEQ ID NO: 1-30368 are incorporated in the attached Sequence Listing.","Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.","Biol., Vol.","6 pp.","219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions.","The attached Sequence Listing provodes the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.","Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.","26(1) pp.","320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.","The attached Sequence Listing provides the results obtained by PFAM analysis for each peptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.","Tables 1 and 2 follow.","Table I shows the various tissue sources of SEQ ID NO: 1-30368.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.","Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.","No.","09/540,217"]],"string":"[\n [\n \"Novel nucleic acids and polypeptides\",\n \"The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof.\"\n ],\n [\n \"1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, and complementary sequences thereof.\",\n \"2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.\",\n \"3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.\",\n \"5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.\",\n \"6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.\",\n \"10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-30368.11.A composition comprising the polypeptide of claim 10 and a carrier.\",\n \"12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.\",\n \"14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.\",\n \"15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.\",\n \"16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.\",\n \"17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.\",\n \"18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.\",\n \"19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-30368, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).\",\n \"20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30369-60736, the mature protein portion thereof, or the active domain thereof.\",\n \"21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.\",\n \"22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-30368.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.\",\n \"24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.\",\n \"25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.\",\n \"26.The collection of claim 22, wherein the collection is provided in a computer-readable format.\",\n \"27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.\",\n \"28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.\"\n ],\n [\n \" 2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.\",\n \"cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.\",\n \"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).\",\n \"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.\",\n \"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.\"\n ],\n [\n \" 3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.\",\n \"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.\",\n \"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.\",\n \"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.\",\n \"These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.\",\n \"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.\",\n \"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.\",\n \"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.\",\n \"The identifying sequence can be 100 base pairs in length.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.\",\n \"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.\",\n \"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.\",\n \"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.\",\n \"The collection can also be provided in a computer-readable format.\",\n \"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.\",\n \"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.\",\n \"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.\",\n \"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.\",\n \"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.\",\n \"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.\",\n \"host cells) of the invention.\",\n \"The invention also provides compositions comprising a polypeptide of the invention.\",\n \"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.\",\n \"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.\",\n \"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.\",\n \"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.\",\n \"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.\",\n \"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.\",\n \"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.\",\n \"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.\",\n \"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.\",\n \"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.\",\n \"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.\",\n \"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.\",\n \"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.\",\n \"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.\",\n \"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.\",\n \"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.\",\n \"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.\",\n \"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.\",\n \"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.\",\n \"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.\",\n \"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.\",\n \"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.\",\n \"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.\",\n \"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.\",\n \"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).\",\n \"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these-polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.\",\n \"2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.\",\n \"cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.\",\n \"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).\",\n \"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.\",\n \"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.\",\n \"3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.\",\n \"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.\",\n \"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.\",\n \"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.\",\n \"These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.\",\n \"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.\",\n \"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.\",\n \"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.\",\n \"The identifying sequence can be 100 base pairs in length.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.\",\n \"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.\",\n \"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.\",\n \"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.\",\n \"The collection can also be provided in a computer-readable format.\",\n \"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.\",\n \"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.\",\n \"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.\",\n \"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.\",\n \"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.\",\n \"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.\",\n \"host cells) of the invention.\",\n \"The invention also provides compositions comprising a polypeptide of the invention.\",\n \"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.\",\n \"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.\",\n \"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.\",\n \"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.\",\n \"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.\",\n \"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.\",\n \"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.\",\n \"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.\",\n \"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.\",\n \"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.\",\n \"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.\",\n \"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.\",\n \"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.\",\n \"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.\",\n \"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.\",\n \"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.\",\n \"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.\",\n \"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.\",\n \"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.\",\n \"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.\",\n \"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.\",\n \"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.\",\n \"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.\",\n \"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.\",\n \"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).\",\n \"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.\",\n \"4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.\",\n \"The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.\",\n \"According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.\",\n \"Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.\",\n \"The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.\",\n \"The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.\",\n \"For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.\",\n \"Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.\",\n \"The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.\",\n \"The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.\",\n \"The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.\",\n \"The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.\",\n \"PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.\",\n \"Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.\",\n \"The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.\",\n \"As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.\",\n \"EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).\",\n \"One class of EMEs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.\",\n \"The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.\",\n \"These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.\",\n \"In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).\",\n \"It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).\",\n \"Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.\",\n \"The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.\",\n \"The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.\",\n \"Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.\",\n \"Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.\",\n \"A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.\",\n \"Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-30368.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.\",\n \"(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).\",\n \"They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.\",\n \"Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-30368.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.\",\n \"Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.\",\n \"Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.\",\n \"The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.\",\n \"Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.\",\n \"The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).\",\n \"The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.\",\n \"The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.\",\n \"The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.\",\n \"The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.\",\n \"For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.\",\n \"While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.\",\n \"repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.\",\n \"The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.\",\n \"A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.\",\n \"The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.\",\n \"A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.\",\n \"The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.\",\n \"Preferably the peptide is from about 5 to about 200 amino acids.\",\n \"To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.\",\n \"The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.\",\n \"The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.\",\n \"The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.\",\n \"The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.\",\n \"The peptide may have been produced by processing in the cell which removes any leader/signal sequence.\",\n \"The mature protein portion may or may not include an initial methionine residue.\",\n \"The methionine residue may be removed from the protein during processing in the cell.\",\n \"The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.\",\n \"The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.\",\n \"The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.\",\n \"Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.\",\n \"Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.\",\n \"Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.\",\n \"Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.\",\n \"Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.\",\n \"“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.\",\n \"For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.\",\n \"“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.\",\n \"The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.\",\n \"Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.\",\n \"Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.\",\n \"For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.\",\n \"Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.\",\n \"For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.\",\n \"The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.\",\n \"In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).\",\n \"The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.\",\n \"In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.\",\n \"The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.\",\n \"The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.\",\n \"“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.\",\n \"As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.\",\n \"Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.\",\n \"The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.\",\n \"An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.\",\n \"Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.\",\n \"Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.\",\n \"This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.\",\n \"The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.\",\n \"Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.\",\n \"This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.\",\n \"Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.\",\n \"The cells can be prokaryotic or eukaryotic.\",\n \"The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.\",\n \"“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.\",\n \"“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.\",\n \"“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.\",\n \"Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.\",\n \"Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.\",\n \"(1998) Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"16:27-55).\",\n \"Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.\",\n \"Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.\",\n \"The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.\",\n \"Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).\",\n \"Other exemplary hybridization conditions are described herein in the examples.\",\n \"In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).\",\n \"As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.\",\n \"Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).\",\n \"Such a sequence is said to have 65% sequence identity to the listed sequence.\",\n \"In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).\",\n \"Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.\",\n \"Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.\",\n \"Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.\",\n \"For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.\",\n \"For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.\",\n \"Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.\",\n \"(1990) Methods Enzymol.\",\n \"183:626-645).\",\n \"Identity between sequences can also be determined by other methods known in the art, e.g.\",\n \"by varying hybridization conditions.\",\n \"The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.\",\n \"The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.\",\n \"The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.\",\n \"The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.\",\n \"As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.\",\n \"UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.\",\n \"The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.\",\n \"The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.\",\n \"As described above, a UMF will increase the frequency of uptake of a linked marker sequence.\",\n \"Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.\",\n \"4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.\",\n \"The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-30368; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 30369-60736; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 30369-60736.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-30368; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 30369-60736.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.\",\n \"The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.\",\n \"The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.\",\n \"The present invention also provides genes corresponding to the cDNA sequences disclosed herein.\",\n \"The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.\",\n \"Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.\",\n \"Further 5′ and 3′ sequence can be obtained using methods known in the art.\",\n \"For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-30368 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-30368 or a portion there of as a probe.\",\n \"Alternatively, the polynucleotides of SEQ ID NO: 1-30368 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.\",\n \"The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.\",\n \"The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.\",\n \"The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.\",\n \"Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.\",\n \"Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-30368, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.\",\n \"Fragments of, e.g.\",\n \"15, 17, or 20 nucleotides or more that are selective for (i.e.\",\n \"specifically hybridize to any one of the polynucleotides of the invention) are contemplated.\",\n \"Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.\",\n \"The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.\",\n \"Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-30368, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-30368 with a sequence from another isolate of the same species.\",\n \"Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.\",\n \"In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.\",\n \"The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-30368 can be obtained by searching a database using an algorithm or a program.\",\n \"Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.\",\n \"Evol.\",\n \"36 290-300 (1993) and Altschul S. F. et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"21:403-410(1990)).\",\n \"Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.\",\n \"Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.\",\n \"Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.\",\n \"The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.\",\n \"The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.\",\n \"These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.\",\n \"There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.\",\n \"Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.\",\n \"These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).\",\n \"Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.\",\n \"Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.\",\n \"Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.\",\n \"Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.\",\n \"Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.\",\n \"In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.\",\n \"This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.\",\n \"In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).\",\n \"A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.\",\n \"10:6487-6500 (1982).\",\n \"PCR may also be used to create amino acid sequence variants of the novel nucleic acids.\",\n \"When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.\",\n \"PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.\",\n \"The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.\",\n \"A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.\",\n \"Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.\",\n \"Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.\",\n \"Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.\",\n \"The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.\",\n \"The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.\",\n \"Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.\",\n \"In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-30368, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.\",\n \"Also included are the cDNA inserts of any of the clones identified herein.\",\n \"A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.\",\n \"(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).\",\n \"Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.\",\n \"Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.\",\n \"In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.\",\n \"Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.\",\n \"A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.\",\n \"The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof or any other polynucleotides of the invention.\",\n \"In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof is inserted, in a forward or reverse orientation.\",\n \"In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.\",\n \"Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.\",\n \"The following vectors are provided by way of example.\",\n \"Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).\",\n \"Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).\",\n \"The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.\",\n \"19, 44854490 (1991), in order to produce the protein recombinantly.\",\n \"Many suitable expression control sequences are known in the art.\",\n \"General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).\",\n \"As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.\",\n \"Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.\",\n \"Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.\",\n \"Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.\",\n \"Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.\",\n \"Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TP1gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.\",\n \"Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.\",\n \"The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.\",\n \"Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.\",\n \"Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.\",\n \"The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.\",\n \"Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.\",\n \"As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).\",\n \"Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).\",\n \"These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.\",\n \"Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.\",\n \"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.\",\n \"Polynucleotides of the invention can also be used to induce immune responses.\",\n \"For example, as described in Fan et al, Nat.\",\n \"Biotech.\",\n \"17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.\",\n \"The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.\",\n \"4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-30368, or fragments, analogs or derivatives thereof.\",\n \"An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.\",\n \"In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.\",\n \"Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 30369-60736 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-30368 are additionally provided.\",\n \"In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.\",\n \"The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.\",\n \"In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.\",\n \"The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).\",\n \"Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-30368), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.\",\n \"The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.\",\n \"For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.\",\n \"An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.\",\n \"An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.\",\n \"For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.\",\n \"Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.\",\n \"Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).\",\n \"The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.\",\n \"The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.\",\n \"An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.\",\n \"Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.\",\n \"For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.\",\n \"The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.\",\n \"To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.\",\n \"In yet another embodiment, the antisense nucleic acid molecule of the invention is an-anomeric nucleic acid molecule.\",\n \"An-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual-units, the strands run parallel to each other (Gaultier et al.\",\n \"(1987) Nucleic Acids Res 15: 6625-6641).\",\n \"The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.\",\n \"(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.\",\n \"(1987) FEBS Lett 215: 327-330).\",\n \"4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.\",\n \"Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.\",\n \"Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.\",\n \"A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-30368).\",\n \"For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-30368 (see, e.g., Cech et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,987,071; and Cech et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,116,742).\",\n \"Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.\",\n \"See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.\",\n \"See generally, Helene.\",\n \"(1991) Anticancer Drug Des.\",\n \"6: 569-84; Helene.\",\n \"et al.\",\n \"(1992) Ann.\",\n \"N.Y. Acad.\",\n \"Sci.\",\n \"660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.\",\n \"For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.\",\n \"(1996) Bioorg Med Chem 4: 5-23).\",\n \"As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.\",\n \"The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.\",\n \"The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.\",\n \"(1996) above; Perry-O'Keefe et al.\",\n \"(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.\",\n \"For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.\",\n \"PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymnes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.\",\n \"(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.\",\n \"(1996), above; Perry-OKeefe (1996), above).\",\n \"In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.\",\n \"For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.\",\n \"Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.\",\n \"PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).\",\n \"The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.\",\n \"(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.\",\n \"(1989) Nucl Acid Res 17: 5973-88).\",\n \"PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.\",\n \"(1996) above).\",\n \"Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.\",\n \"See, Petersen et al.\",\n \"(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"84:648-652; PCT Publication No.\",\n \"WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No.\",\n \"WO89/10134).\",\n \"In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.\",\n \"Res.\",\n \"5:539-549).\",\n \"To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.\",\n \"4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.\",\n \"For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.\",\n \"The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.\",\n \"Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.\",\n \"See, for example, PCT International Publication No.\",\n \"WO94/12650, PCT International Publication No.\",\n \"WO92/20808, and PCT International Publication No.\",\n \"WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.\",\n \"The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.\",\n \"Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).\",\n \"The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.\",\n \"Any host/vector system can be used to express one or more of the ORFs of the present invention.\",\n \"These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.\",\n \"The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.\",\n \"Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.\",\n \"Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.\",\n \"Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.\",\n \"Various mammalian cell culture systems can also be employed to express recombinant protein.\",\n \"Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).\",\n \"Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.\",\n \"Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.\",\n \"DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.\",\n \"Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.\",\n \"Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.\",\n \"Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.\",\n \"Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.\",\n \"Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.\",\n \"Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.\",\n \"Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.\",\n \"If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.\",\n \"Such covalent attachments may be accomplished using known chemical or enzymatic methods.\",\n \"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.\",\n \"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.\",\n \"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.\",\n \"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.\",\n \"These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.\",\n \"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.\",\n \"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.\",\n \"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.\",\n \"Here, the naturally occurring sequences are deleted and new sequences are added.\",\n \"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.\",\n \"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative, selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.\",\n \"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.\",\n \"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.\",\n \"No.\",\n \"5,272,071 to Chappel; U.S. Pat.\",\n \"No.\",\n \"5,578,461 to Sherwin et al.\",\n \"; International Application No.\",\n \"PCT/US92/09627 (WO93/09222) by Selden et al.\",\n \"; and International Application No.\",\n \"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.\",\n \"4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 30369-60736 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-30368 or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.\",\n \"The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.\",\n \"Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 30369-60736.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.\",\n \"Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.\",\n \"Chem.\",\n \"Soc.\",\n \"114, 9245-9253 (1992), both of which are incorporated herein by reference.\",\n \"Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.\",\n \"The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.\",\n \"The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.\",\n \"The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.\",\n \"The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.\",\n \"Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.\",\n \"In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.\",\n \"Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.\",\n \"By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.\",\n \"Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.\",\n \"A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.\",\n \"At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.\",\n \"The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.\",\n \"This technique is particularly useful in producing small peptides and fragments of larger polypeptides.\",\n \"Fragments are useful, for example, in generating antibodies against the native polypeptide.\",\n \"Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.\",\n \"The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.\",\n \"As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.\",\n \"One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.\",\n \"The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.\",\n \"For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.\",\n \"The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.\",\n \"Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.\",\n \"In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.\",\n \"One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.\",\n \"These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.\",\n \"See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.\",\n \"Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.\",\n \"The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.\",\n \"These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.\",\n \"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.\",\n \"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.\",\n \"In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.\",\n \"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 30369-60736.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.\",\n \"The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.\",\n \"For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.\",\n \"Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.\",\n \"For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.\",\n \"Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"4,518,584).\",\n \"Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.\",\n \"Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.\",\n \"This type of analysis determines the importance of the substituted amino acid(s) in biological activity.\",\n \"Regions of the protein that are important for protein function may be determined by the eMATRIX program.\",\n \"Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.\",\n \"Such modifications are encompassed by the present invention.\",\n \"The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.\",\n \"Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.\",\n \"1555 (1987), incorporated herein by reference.\",\n \"As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.\",\n \"The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.\",\n \"The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.\",\n \"Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.\",\n \"For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX), or as a His-tag.\",\n \"Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.\",\n \"), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.\",\n \"The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.\",\n \"One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).\",\n \"Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.\",\n \"Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.\",\n \"The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).\",\n \"This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.\",\n \"Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.\",\n \"Such analogs may exhibit improved properties such as activity and/or stability.\",\n \"Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.\",\n \"Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.\",\n \"Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.\",\n \"4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.\",\n \"Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.\",\n \"Biol.\",\n \"215:403410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.\",\n \"vol.\",\n \"25, pp.\",\n \"3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.\",\n \"Biol., Vol.\",\n \"6, pp.\",\n \"219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.\",\n \"4, pp.\",\n \"202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.\",\n \"26(1), pp.\",\n \"320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.\",\n \"Biol, 157, pp.\",\n \"105-31 (1982), incorporated herein by reference).\",\n \"The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.\",\n \"NCB NLM NIH Bethesda, Md.\",\n \"20894; Altschul, S., et al., J. Mol.\",\n \"Biol.\",\n \"215:403-410 (1990).\",\n \"4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.\",\n \"As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.\",\n \"Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.\",\n \"In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.\",\n \"In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.\",\n \"Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.\",\n \"The polypeptide can be fused to the N-terminus or C-terminus.\",\n \"For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.\",\n \"In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.\",\n \"In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.\",\n \"The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.\",\n \"The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.\",\n \"Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g.\",\n \"promoting or inhibiting) cell survival.\",\n \"Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.\",\n \"A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.\",\n \"For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.\",\n \"In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.\",\n \"Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.\",\n \"(eds.)\",\n \"CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).\",\n \"Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).\",\n \"A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.\",\n \"4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.\",\n \"The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.\",\n \"Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).\",\n \"See, for example, Anderson, Nature, supplement to vol.\",\n \"392, no.\",\n \"6679, pp.\",\n \"25-20 (1998).\",\n \"For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).\",\n \"Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).\",\n \"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.\",\n \"Treated cells can then be introduced in vivo for therapeutic purposes.\",\n \"Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.\",\n \"It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.\",\n \"Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.\",\n \"Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.\",\n \"The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.\",\n \"These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.\",\n \"Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.\",\n \"See, for example, PCT International Publication No.\",\n \"WO 94/12650, PCT International Publication No.\",\n \"WO 92/20808, and PCT International Publication No.\",\n \"WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplificationof the desired protein coding sequences in the cells.\",\n \"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.\",\n \"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.\",\n \"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.\",\n \"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.\",\n \"These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.\",\n \"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.\",\n \"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.\",\n \"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.\",\n \"Here, the naturally occurring sequences are deleted and new sequences are added.\",\n \"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.\",\n \"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.\",\n \"Markers useful for this purpose include the Herpes Sinplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphonbosyl-transferase (gpt) gene.\",\n \"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.\",\n \"No.\",\n \"5,272,071 to Chappel; U.S. Pat.\",\n \"No.\",\n \"5,578,461 to Sherwin et al.\",\n \"; International Application No.\",\n \"PCT/US92/09627 (WO93/09222) by Selden et al.\",\n \"; and International Application No.\",\n \"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.\",\n \"4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].\",\n \"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.\",\n \"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.\",\n \"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.\",\n \"No.\",\n \"5,557,032, incorporated herein by reference.\",\n \"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.\",\n \"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.\",\n \"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.\",\n \"No.\",\n \"5,489,743 and PCT Publication No.\",\n \"WO94/28122, incorporated herein by reference.\",\n \"Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.\",\n \"Inactivation can be carried out using homologous recombination methods described above.\",\n \"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.\",\n \"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.\",\n \"The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.\",\n \"Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.\",\n \"In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)).\",\n \"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.\",\n \"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.\",\n \"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.\",\n \"No.\",\n \"5,557,032, incorporated herein by reference.\",\n \"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.\",\n \"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.\",\n \"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.\",\n \"No.\",\n \"5,489,743 and PCT Publication No.\",\n \"WO94/28122, incorporated herein by reference.\",\n \"Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.\",\n \"Inactivation can be carried out using homologous recombination methods described above.\",\n \"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.\",\n \"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.\",\n \"4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.\",\n \"Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).\",\n \"The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.\",\n \"Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.\",\n \"Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.\",\n \"The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.\",\n \"4.10.1 Research uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.\",\n \"The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.\",\n \"Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.\",\n \"The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.\",\n \"Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.\",\n \"Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.\",\n \"Methods for performing the uses listed above are well known to those skilled in the art.\",\n \"References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.\",\n \"Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.\",\n \"In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.\",\n \"In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.\",\n \"4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.\",\n \"The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Bertagnolli et al., J. Immunol.\",\n \"145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., 1.Immunol.\",\n \"149:3778-3783, 1992; Bowman et al., I. Immunol.\",\n \"152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"3.12.1-3.12.14, John Wiley and Sons, Toronto.\",\n \"1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.\",\n \"J. E. e.a Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.8.1-6.8.8, John Wiley and Sons, Toronto.\",\n \"1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.\",\n \"J. E. e.a Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.3.1-6.3.12, John Wiley and Sons, Toronto.\",\n \"1991; deVries et al., J. Exp.\",\n \"Med.\",\n \"173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.6.1-6.6.5, John Wiley and Sons, Toronto.\",\n \"1991; Smith et al., Proc.\",\n \"Natl.\",\n \"Aced.\",\n \"Sci.\",\n \"U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.\",\n \"In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.15.1 John Wiley and Sons, Toronto.\",\n \"1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.\",\n \"In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.13.1, John Wiley and Sons, Toronto.\",\n \"1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 77:6091-6095, 1980; Weinberger et al., Eur.\",\n \"J. Immun.\",\n \"11:405-411, 1981; Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.\",\n \"Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.\",\n \"The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.\",\n \"It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).\",\n \"Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.\",\n \"Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.\",\n \"This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.\",\n \"Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.\",\n \"Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.\",\n \"No.\",\n \"5,690,926).\",\n \"Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.\",\n \"This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.\",\n \"These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.\",\n \"These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.\",\n \"Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.\",\n \"For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.\",\n \"The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.\",\n \"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.\",\n \"In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.\",\n \"Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.\",\n \"A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.\",\n \"The selectable marker allows only cells of the desired type to survive.\",\n \"For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.\",\n \"Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.\",\n \"Lanza et al., Academic Press (1997)).\",\n \"Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.\",\n \"In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.\",\n \"Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.\",\n \"Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.\",\n \"The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.\",\n \"as described by Bernstein et al., Blood, 77: 2316-2321 (1991).\",\n \"4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.\",\n \"Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.\",\n \"in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (ie., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.\",\n \"Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.\",\n \"Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.\",\n \"Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.\",\n \"In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.\",\n \"In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.\",\n \"A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.\",\n \"Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.\",\n \"De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.\",\n \"A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.\",\n \"Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)\",\n \"mediated by inflammatory processes may also be possible using the composition of the invention.\",\n \"Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.\",\n \"Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.\",\n \"Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.\",\n \"De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.\",\n \"The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.\",\n \"The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.\",\n \"The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.\",\n \"The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.\",\n \"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.\",\n \"More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.\",\n \"Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.\",\n \"Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.\",\n \"Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.\",\n \"Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.\",\n \"Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.\",\n \"A polypeptide of the present invention may also exhibit angiogenic activity.\",\n \"A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.\",\n \"A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.\",\n \"WO95/16035 (bone, cartilage, tendon); International Patent Publication No.\",\n \"WO95/05846 (nerve, neuronal); International Patent Publication No.\",\n \"WO91/07491 (skin, endothelium).\",\n \"Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.\",\n \"71-112 (Maibach, H. I. and Rovee, D. T., eds.\",\n \"), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.\",\n \"Invest.\",\n \"Dermatol 71:382-84 (1978).\",\n \"4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such activities.\",\n \"A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.\",\n \"These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.\",\n \"More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.\",\n \"and various fungal infections such as candidiasis.\",\n \"Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.\",\n \"Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.\",\n \"Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.\",\n \"Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.\",\n \"The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig slin sensitization test (Vohr et al., Arch.\",\n \"Toxocol.\",\n \"73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.\",\n \"Environ.\",\n \"Health 53: 563-79).\",\n \"Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.\",\n \"Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.\",\n \"The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.\",\n \"Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.\",\n \"Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.\",\n \"Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.\",\n \"Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).\",\n \"For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.\",\n \"Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.\",\n \"The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.\",\n \"Moreover, a lack of costinulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.\",\n \"Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.\",\n \"To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.\",\n \"The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.\",\n \"Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4I g fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 89:11102-11105 (1992).\",\n \"In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.\",\n \"846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.\",\n \"Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.\",\n \"Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.\",\n \"Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.\",\n \"Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.\",\n \"Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.\",\n \"The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.\",\n \"Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.\",\n \"840-856).\",\n \"Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.\",\n \"Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.\",\n \"For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.\",\n \"Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.\",\n \"Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.\",\n \"The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.\",\n \"A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.\",\n \"In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class 1 alpha chain protein and P2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.\",\n \"Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.\",\n \"Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.\",\n \"Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.\",\n \"The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 78:2488-2492, 1981; Herrmnann et al., J. Immunol.\",\n \"128:1968-1974, 1982; Handa et al., J. Immunol.\",\n \"135:1564-1572, 1985; Takai et al., I. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.\",\n \"153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.\",\n \"144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"3.8.1-3.8.16, John Wiley and Sons, Toronto.\",\n \"1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988; Bertagnolli et al., J. Immunol.\",\n \"149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.\",\n \"134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260,1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:40374045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.\",\n \"A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.\",\n \"Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).\",\n \"Thus, a polypeptide of the present invention, alone or in heterodirners with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.\",\n \"Administration of sufficient amounts of other inhibins can induce infertility in these mammals.\",\n \"Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.\",\n \"See, for example, U.S. Pat.\",\n \"No.\",\n \"4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.\",\n \"The activity of a polypeptide of the invention may, among other means, be measured by the following methods.\",\n \"Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Chemotactic and chemolinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.\",\n \"Chemotactic or chemokinetic compositions (e.g.\",\n \"proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.\",\n \"For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.\",\n \"A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.\",\n \"Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.\",\n \"Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.\",\n \"Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.\",\n \"J. Clin.\",\n \"Invest.\",\n \"95:1370-1376, 1995; Lind et al.\",\n \"APMIS 103:140-146, 1995; Muller et al Eur.\",\n \"J. Immunol.\",\n \"25:1744-1748; Gruber et al.\",\n \"J. of Immunol.\",\n \"152:5860-5867, 1994; Johnston et al.\",\n \"J. of Immunol.\",\n \"153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Compositions may be usefull in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.\",\n \"A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).\",\n \"Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.\",\n \"Pharmacol.\",\n \"26:131-140, 1986; Burdick et al., Thrombosis Res.\",\n \"45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79(1991); Schaub, Prostaglandins 35:467-474,1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.\",\n \"Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.\",\n \"For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.\",\n \"Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.\",\n \"Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.\",\n \"Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.\",\n \"Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.\",\n \"Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.\",\n \"Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.\",\n \"reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.\",\n \"The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.\",\n \"An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.\",\n \"The use of anti-cancer cocktails as a cancer treatment is routine.\",\n \"Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.\",\n \"In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.\",\n \"There are hereditary conditions and/or environmental situations (e.g.\",\n \"exposure to carcinogens) known in the art that predispose an individual to developing cancers.\",\n \"Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.\",\n \"In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.\",\n \"These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.\",\n \"Can.\",\n \"Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.\",\n \"J. Dev.\",\n \"Biol., 40: 1189-97 (1999) and Li et al., Clin.\",\n \"Exp.\",\n \"Metastasis, 17:423-9 (1999), respectively.\",\n \"Suitable tumor cells lines are available, e.g.\",\n \"from American Type Tissue Culture Collection catalogs.\",\n \"4.10.12 RECEPTOR/LIGAND ACTIVITY A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.\",\n \"Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.\",\n \"Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.\",\n \"A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.\",\n \"The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84:6864-6868, 1987; Bierer et al., J. Exp.\",\n \"Med.\",\n \"168:1145-1156, 1988; Rosenstein et al., J. Exp.\",\n \"Med.\",\n \"169:149-160 1989; Stoltenborg et al., J. Immunol.\",\n \"Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).\",\n \"Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.\",\n \"Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.\",\n \"The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.\",\n \"(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.\",\n \"182 (1990) Academic Press, Inc. San Diego).\",\n \"Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.\",\n \"Examples of toxins include, but are not limited, to ricin.\",\n \"4.10.13 DRUG SCREENING This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.\",\n \"The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.\",\n \"One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.\",\n \"Drugs are screened against such transformed cells in competitive binding assays.\",\n \"Such cells, either in viable or fixed form, can be used for standard binding assays.\",\n \"One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.\",\n \"Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.\",\n \"Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.\",\n \"The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.\",\n \"Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.\",\n \"For a review, see Science 282:63-68 (1998).\",\n \"Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.\",\n \"Of particular interest are peptide and oligonucleotide combinatorial libraries.\",\n \"Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.\",\n \"For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.\",\n \"Opin.\",\n \"Biotechnol.\",\n \"8:701-707 (1997).\",\n \"For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.\",\n \"Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).\",\n \"Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.\",\n \"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.\",\n \"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.\",\n \"The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.\",\n \"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.\",\n \"Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.\",\n \"4.10.14 ASSAY FOR RECEPTOR ACTIVITY The invention also provides methods to detect specific binding of a polypeptide e.g.\",\n \"a ligand or a receptor.\",\n \"The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.\",\n \"For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.\",\n \"As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.\",\n \"There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.\",\n \"Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.\",\n \"The responses of the two cell populations to the addition of ligands(s) are then compared.\",\n \"Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).\",\n \"As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.\",\n \"The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.\",\n \"For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.\",\n \"The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.\",\n \"Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.\",\n \"phosphorylation.\",\n \"Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.\",\n \"4.10.15 Anti-Inflammtory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.\",\n \"The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.\",\n \"Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.\",\n \"Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.\",\n \"4.10.16 LEUKEMIAS Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.\",\n \"Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.\",\n \"B. Lippincott Co., Philadelphia).\",\n \"4.10.17 NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.\",\n \"Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vi) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (vii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.\",\n \"Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.\",\n \"For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.\",\n \"Such effects may be measured by any method known in the art.\",\n \"In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.\",\n \"(1990, J. Neurosci.\",\n \"10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.\",\n \"(1980, Exp.\",\n \"Neurol.\",\n \"70:65-82) or Brown et al.\",\n \"(1981, Ann.\",\n \"Rev.\",\n \"Neurosci.\",\n \"4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.\",\n \"In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).\",\n \"4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fingi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.\",\n \"4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.\",\n \"Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.\",\n \"For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.\",\n \"Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.\",\n \"For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.\",\n \"Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).\",\n \"In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.\",\n \"Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.\",\n \"The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.\",\n \"In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.\",\n \"Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.\",\n \"4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.\",\n \"The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.\",\n \"Arch.\",\n \"Allergy Appl.\",\n \"Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).\",\n \"The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.\",\n \"The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.\",\n \"The control consists of administering PBS only.\",\n \"The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.\",\n \"An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.\",\n \"4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.\",\n \"Examples of therapeutic applications include, but are not limited to, those exemplified herein.\",\n \"4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.\",\n \"While the mode of administration is not particularly important, parenteral administration is preferred.\",\n \"An exemplary mode of administration is to deliver an intravenous bolus.\",\n \"The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.\",\n \"It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.\",\n \"Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.\",\n \"For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.\",\n \"Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.\",\n \"The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.\",\n \"The preparation of such solutions is within the skill of the art.\",\n \"4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.\",\n \"Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.\",\n \"The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).\",\n \"The characteristics of the carrier will depend on the route of administration.\",\n \"The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, ThF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.\",\n \"In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.\",\n \"These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.\",\n \"The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.\",\n \"Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.\",\n \"Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).\",\n \"A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.\",\n \"As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.\",\n \"As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).\",\n \"Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.\",\n \"A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.\",\n \"When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.\",\n \"When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.\",\n \"In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.\",\n \"Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.\",\n \"When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.\",\n \"If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.\",\n \"4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.\",\n \"Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.\",\n \"Intravenous administration to the patient is preferred.\",\n \"Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.\",\n \"In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.\",\n \"Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.\",\n \"The liposomes will be targeted to and taken up selectively by the afflicted tissue.\",\n \"The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.\",\n \"The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.\",\n \"Preferably for wound treatment, one administers the therapeutic compound directly to the site.\",\n \"Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.\",\n \"Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.\",\n \"4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.\",\n \"These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.\",\n \"Proper formulation is dependent upon the route of administration chosen.\",\n \"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.\",\n \"When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.\",\n \"The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.\",\n \"When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.\",\n \"The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.\",\n \"When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.\",\n \"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.\",\n \"The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.\",\n \"A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.\",\n \"The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.\",\n \"For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.\",\n \"Such penetrants are generally known in the art.\",\n \"For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.\",\n \"Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.\",\n \"Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.\",\n \"Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).\",\n \"If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.\",\n \"Dragee cores are provided with suitable coatings.\",\n \"For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.\",\n \"Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.\",\n \"Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.\",\n \"The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.\",\n \"In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.\",\n \"In addition, stabilizers may be added.\",\n \"All formulations for oral administration should be in dosages suitable for such administration.\",\n \"For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.\",\n \"For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.\",\n \"In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.\",\n \"Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.\",\n \"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.\",\n \"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.\",\n \"The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.\",\n \"Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.\",\n \"Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.\",\n \"Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.\",\n \"Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.\",\n \"Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.\",\n \"Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.\",\n \"The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.\",\n \"In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.\",\n \"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.\",\n \"Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.\",\n \"A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.\",\n \"The co-solvent system may be the VPD co-solvent system.\",\n \"VPD is a solution of 3%/o w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.\",\n \"The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.\",\n \"This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.\",\n \"Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.\",\n \"Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.\",\n \"polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.\",\n \"Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.\",\n \"Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.\",\n \"Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.\",\n \"Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.\",\n \"Various types of sustained-release materials have been established and are well known by those skilled in the art.\",\n \"Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.\",\n \"Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.\",\n \"The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.\",\n \"Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.\",\n \"Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.\",\n \"Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.\",\n \"The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.\",\n \"The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.\",\n \"B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.\",\n \"T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.\",\n \"MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.\",\n \"The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.\",\n \"Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.\",\n \"The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.\",\n \"Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.\",\n \"Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.\",\n \"Nos.\",\n \"4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.\",\n \"The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.\",\n \"Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.\",\n \"Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.\",\n \"Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.\",\n \"It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.\",\n \"For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.\",\n \"When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.\",\n \"Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.\",\n \"Topical administration may be suitable for wound healing and tissue repair.\",\n \"Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.\",\n \"Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.\",\n \"Such matrices may be formed of materials presently in use for other implanted medical applications.\",\n \"The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.\",\n \"The particular application of the compositions will define the appropriate formulation.\",\n \"Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.\",\n \"Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.\",\n \"Further matrices are comprised of pure proteins or extracellular matrix components.\",\n \"Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.\",\n \"Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.\",\n \"The biocermics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.\",\n \"Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.\",\n \"In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.\",\n \"A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).\",\n \"Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).\",\n \"The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.\",\n \"In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.\",\n \"These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).\",\n \"The therapeutic compositions are also presently valuable for veterinary applications.\",\n \"Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.\",\n \"The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.\",\n \"The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.\",\n \"For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.\",\n \"Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.\",\n \"Polynucleotides of the present invention can also be used for gene therapy.\",\n \"Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.\",\n \"Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).\",\n \"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.\",\n \"Treated cells can then be introduced in vivo for therapeutic purposes.\",\n \"4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.\",\n \"More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.\",\n \"Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.\",\n \"For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity).\",\n \"Such information can be used to more accurately determine useful doses in humans.\",\n \"A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.\",\n \"Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).\",\n \"The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.\",\n \"The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.\",\n \"The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity.\",\n \"The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.\",\n \"The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.\",\n \"See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.\",\n \"1 p.1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).\",\n \"The MEC will vary for each compound but can be estimated from in vitro data.\",\n \"Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.\",\n \"However, HPLC assays or bioassays can be used to determine plasma concentrations.\",\n \"Dosage intervals can also be determined using MEC value.\",\n \"Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.\",\n \"In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.\",\n \"An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.\",\n \"Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.\",\n \"The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.\",\n \"4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.\",\n \"The pack may, for example, comprise metal or plastic foil, such as a blister pack.\",\n \"The pack or dispenser device may be accompanied by instructions for administration.\",\n \"Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.\",\n \"4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.\",\n \"The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.\",\n \"Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab′)2 fragments, and an Fab expression library.\",\n \"In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.\",\n \"Certain classes have subclasses as well, such as IgG1, IgG2, and others.\",\n \"Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.\",\n \"Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.\",\n \"An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.\",\n \"The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.\",\n \"An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, (for example the amino acid sequence shown in SEQ ID NO: 30369), and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.\",\n \"Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.\",\n \"Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.\",\n \"In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention that is located on the surface of the protein, e.g., a hydrophilic region.\",\n \"A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.\",\n \"As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.\",\n \"See, e.g., Hopp and Woods, 1981, Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.\",\n \"Biol.\",\n \"157: 105-142, each of which is incorporated herein by reference in its entirety.\",\n \"Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.\",\n \"A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.\",\n \"Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).\",\n \"Some of these antibodies are discussed below.\",\n \"5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.\",\n \"An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.\",\n \"Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.\",\n \"Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.\",\n \"The preparation can further include an adjuvant.\",\n \"Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.\",\n \"), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.\",\n \"Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).\",\n \"The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.\",\n \"Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.\",\n \"Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.\",\n \"14, No.\",\n \"8 (Apr.\",\n \"17, 2000), pp.\",\n \"25-28).\",\n \"5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.\",\n \"In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.\",\n \"MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.\",\n \"Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).\",\n \"In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.\",\n \"Alternatively, the lymphocytes can be immunized in vitro.\",\n \"The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.\",\n \"Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.\",\n \"The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.\",\n \"59-103).\",\n \"Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.\",\n \"Usually, rat or mouse myeloma cell lines are employed.\",\n \"The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.\",\n \"For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.\",\n \"Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.\",\n \"More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.\",\n \"Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.\",\n \"51-63).\",\n \"The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.\",\n \"Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).\",\n \"Such techniques and assays are known in the art.\",\n \"The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.\",\n \"Biochem.\",\n \"107:220 (1980).\",\n \"Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.\",\n \"After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.\",\n \"Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.\",\n \"Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.\",\n \"The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.\",\n \"The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.\",\n \"No.\",\n \"4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).\",\n \"The hybridoma cells of the invention serve as a preferred source of such DNA.\",\n \"Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.\",\n \"The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.\",\n \"No.\",\n \"4,816,567; Morrison, Nature 368 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.\",\n \"Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.\",\n \"5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.\",\n \"These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.\",\n \"Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.\",\n \"Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.\",\n \"(See also U.S. Pat.\",\n \"No.\",\n \"5,225,539.)\",\n \"In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.\",\n \"Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.\",\n \"In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.\",\n \"The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.\",\n \"Op.\",\n \"Struct.\",\n \"Biol., 2:593-596 (1992)).\",\n \"5.13.3 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.\",\n \"Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.\",\n \"Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.\",\n \"77-96).\",\n \"Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.\",\n \"77-96).\",\n \"In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.\",\n \"Biol., 227:381 (1991); Marks et al., J. Mol.\",\n \"Biol., 222:581 (1991)).\",\n \"Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.\",\n \"Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.\",\n \"This approach is described, for example, in U.S. Pat.\",\n \"Nos.\",\n \"5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.\",\n \"(Bio/Technology 10, 779-783 (1992)); Lonberg et al.\",\n \"(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.\",\n \"Rev.\",\n \"Immunol.\",\n \"13 65-93 (1995)).\",\n \"Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.\",\n \"(See PCT publication WO94/02602).\",\n \"The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.\",\n \"The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.\",\n \"An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.\",\n \"The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.\",\n \"The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.\",\n \"Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.\",\n \"An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.\",\n \"A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.\",\n \"The hybrid cell expresses an antibody containing the heavy chain and the light chain.\",\n \"In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.\",\n \"No.\",\n \"4,946,778).\",\n \"In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.\",\n \"Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) FV fragments.\",\n \"5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.\",\n \"In the present case, one of the binding specificities is for an antigenic protein of the invention.\",\n \"The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.\",\n \"Methods for making bispecific antibodies are known in the art.\",\n \"Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).\",\n \"Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.\",\n \"The purification of the correct molecule is usually accomplished by affinity chromatography steps.\",\n \"Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.\",\n \"The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.\",\n \"It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.\",\n \"DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.\",\n \"For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).\",\n \"According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.\",\n \"The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.\",\n \"In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.\",\n \"tyrosine or tryptophan).\",\n \"Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.\",\n \"alanine or threonine).\",\n \"This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.\",\n \"Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.\",\n \"F(ab′)2 bispecific antibodies).\",\n \"Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.\",\n \"For example, bispecific antibodies can be prepared using chemical linkage.\",\n \"Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.\",\n \"These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.\",\n \"The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.\",\n \"One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.\",\n \"The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.\",\n \"Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.\",\n \"Shalaby et al., J. Exp.\",\n \"Med.\",\n \"175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.\",\n \"Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.\",\n \"The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.\",\n \"Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.\",\n \"For example, bispecific antibodies have been produced using leucine zippers.\",\n \"Kostelny et al., J. Immunol.\",\n \"148(5):1547-1553 (1992).\",\n \"The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.\",\n \"The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.\",\n \"This method can also be utilized for the production of antibody homodimers.\",\n \"The “diabody” technology described by Hollinger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.\",\n \"The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.\",\n \"Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.\",\n \"Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported.\",\n \"See, Gruber et al., J. Immunol.\",\n \"152:5368 (1994).\",\n \"Antibodies with more than two valencies are contemplated.\",\n \"For example, trispecific antibodies can be prepared.\",\n \"Tutt et al., J. Immunol.\",\n \"147:60 (1991).\",\n \"Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.\",\n \"Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.\",\n \"CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.\",\n \"Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.\",\n \"These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.\",\n \"Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (° F.).\",\n \"5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.\",\n \"Heteroconjugate antibodies are composed of two covalently joined antibodies.\",\n \"Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.\",\n \"No.\",\n \"4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).\",\n \"It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.\",\n \"For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.\",\n \"Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.\",\n \"No.\",\n \"4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.\",\n \"For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.\",\n \"The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).\",\n \"See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.\",\n \"Immunol., 148: 2918-2922 (1992).\",\n \"Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.\",\n \"Cancer Research, 53: 2560-2565 (1993).\",\n \"Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.\",\n \"See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).\",\n \"5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).\",\n \"Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.\",\n \"Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.\",\n \"A variety of radionuclides are available for the production of radioconjugated antibodies.\",\n \"Examples include 212Bi, 131I, 90Y, 90Y, and 186Re.\",\n \"Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).\",\n \"For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).\",\n \"Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.\",\n \"See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.\",\n \"4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.\",\n \"As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.\",\n \"Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.\",\n \"As used herein, “recorded” refers to a process for storing information on computer readable medium.\",\n \"A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.\",\n \"A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.\",\n \"The choice of the data storage structure will generally be based on the means chosen to access the stored information.\",\n \"In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.\",\n \"The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.\",\n \"A skilled artisan can readily adapt any number of data processor structuring formats (e.g.\",\n \"text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.\",\n \"By providing any of the nucleotide sequences SEQ ID NO: 1-30368 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-30368 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.\",\n \"Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.\",\n \"The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.\",\n \"Biol.\",\n \"215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.\",\n \"Chem.\",\n \"17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.\",\n \"Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.\",\n \"As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.\",\n \"The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.\",\n \"A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.\",\n \"As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.\",\n \"As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.\",\n \"As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.\",\n \"Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.\",\n \"A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.\",\n \"Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASIN and BLASTA (NPOLYPEPTIDEIA).\",\n \"A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.\",\n \"As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.\",\n \"A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.\",\n \"The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.\",\n \"However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.\",\n \"As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.\",\n \"There are a variety of target motifs known in the art.\",\n \"Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.\",\n \"Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).\",\n \"4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.\",\n \"Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.\",\n \"Acids Res.\",\n \"6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olnno, J. Neurochem.\",\n \"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).\",\n \"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.\",\n \"Both techniques have been demonstrated to be effective in model systems.\",\n \"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.\",\n \"4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.\",\n \"In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.\",\n \"Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.\",\n \"In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.\",\n \"In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.\",\n \"Conditions for incubating a nucleic acid probe or antibody with a test sample vary.\",\n \"Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.\",\n \"One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.\",\n \"Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.\",\n \"1 (1982), Vol.\",\n \"2 (1983), Vol.\",\n \"3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).\",\n \"The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.\",\n \"The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.\",\n \"Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.\",\n \"In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.\",\n \"Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.\",\n \"In detail, a compartment kit includes any kit in which reagents are contained in separate containers.\",\n \"Such containers include small glass containers, plastic containers or strips of plastic or paper.\",\n \"Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.\",\n \"Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.\",\n \"), and containers which contain the reagents used to detect the bound antibody or probe.\",\n \"Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.\",\n \"One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.\",\n \"4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).\",\n \"See, e.g., Kunkel et al., U.S. Pat.\",\n \"No.\",\n \"5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.\",\n \"4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-30368, or bind to a specific domain of the polypeptide encoded by the nucleic acid.\",\n \"In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.\",\n \"In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.\",\n \"Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.\",\n \"Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.\",\n \"Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).\",\n \"Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).\",\n \"Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.\",\n \"The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.\",\n \"The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.\",\n \"For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.\",\n \"Alternatively, agents may be rationally selected or designed.\",\n \"As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.\",\n \"For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.\",\n \"Freeman, N.Y. (1992), pp.\",\n \"289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.\",\n \"In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.\",\n \"As described above, such agents can be randomly screened or rationally designed/selected.\",\n \"Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.\",\n \"One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.\",\n \"Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.\",\n \"Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.\",\n \"Acids Res.\",\n \"6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.\",\n \"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).\",\n \"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.\",\n \"Both techniques have been demonstrated to be effective in model systems.\",\n \"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.\",\n \"Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.\",\n \"Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.\",\n \"4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.\",\n \"The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-30368.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-30368 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.\",\n \"Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.\",\n \"PCR as described in U.S. Pat.\",\n \"Nos.\",\n \"4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.\",\n \"Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.\",\n \"The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.\",\n \"Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.\",\n \"Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.\",\n \"The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.\",\n \"The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.\",\n \"These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.\",\n \"The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.\",\n \"Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f).\",\n \"Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.\",\n \"The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.\",\n \"4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.\",\n \"Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.\",\n \"One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.\",\n \"Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.\",\n \"Microbiol.\",\n \"28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey& Collins, (1989) Mol.\",\n \"Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.\",\n \"Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.\",\n \"For example, Broude et al.\",\n \"(1994) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.\",\n \"Streptavidin-coated beads may be purchased from Dynal, Oslo.\",\n \"Of course, this same linking chemistry is applicableto coating any surface with streptavidin Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).\",\n \"Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.\",\n \"Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.\",\n \"CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.\",\n \"CovaLink Modules may be purchased from Nunc Laboratories.\",\n \"DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.\",\n \"Biochem.\",\n \"198(1) 138-42).\",\n \"The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).\",\n \"In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.\",\n \"1 (8) 6513-29).\",\n \"This is beneficial as immobilization using only a single covalent bond is preferred.\",\n \"The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.\",\n \"To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.\",\n \"It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.\",\n \"More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.\",\n \"at 95° C. and cooling on ice for 10 min.\",\n \"Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.\",\n \"Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well.\",\n \"The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).\",\n \"It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.\",\n \"This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.\",\n \"The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.\",\n \"Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.\",\n \"An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.\",\n \"For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al.\",\n \"(1991) Science 251(4995) 767-73, incorporated herein by reference.\",\n \"Probes may also be immobilized on nylon supports as described by Van Ness et al.\",\n \"(1991) Nucleic Acids Res.\",\n \"19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.\",\n \"Biochem.\",\n \"169(1) 104-8; all references being specifically incorporated herein.\",\n \"To link an oligonucleotide to a nylon support, as described by Van Ness et at (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.\",\n \"One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference).\",\n \"These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).\",\n \"These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.\",\n \"A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.\",\n \"4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.\",\n \"For example, Sambrook et al.\",\n \"(1989) describes three protocols for the isolation ofhigh molecular weight DNA from mammalian cells (p. 9.14-9.23).\",\n \"DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.\",\n \"Samples may be prepared or dispensed in multiwell plates.\",\n \"About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.\",\n \"The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.\",\n \"(1989), shearing by ultrasound and NaOH treatment.\",\n \"Low pressure shearing is also appropriate, as described by Schriefer et at (1.990) Nucleic Acids Res.\",\n \"18(24) 7455-6, incorporated herein by reference).\",\n \"In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.\",\n \"A lever device allows controlled application of low to intermediate pressures to the cell.\",\n \"The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.\",\n \"One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.\",\n \"(1992) Nucleic Acids Res.\",\n \"20(14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.\",\n \"The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.\",\n \"A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).\",\n \"Fitzgerald et al.\",\n \"(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest ofpUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.\",\n \"Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.\",\n \"As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.\",\n \"Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.\",\n \"This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.\",\n \"Phosphate groups must also be removed from genomic DNA by methods known in the art.\",\n \"4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.\",\n \"Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.\",\n \"By offset printing, a density of dots higher than the density of the wells is achieved.\",\n \"One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.\",\n \"By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.\",\n \"Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.\",\n \"Each of the subarrays may represent replica spotting of the same samples.\",\n \"In one example, a selected gene segment may be amplified from 64 patients.\",\n \"For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).\",\n \"A plate for each of the 64 patients is prepared.\",\n \"By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.\",\n \"Subarrays may contain 64 samples, one from each patient.\",\n \"Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.\",\n \"Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.\",\n \"a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.\",\n \"A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.\",\n \"The present invention is illustrated in the following examples.\",\n \"Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.\",\n \"Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.\",\n \"The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.\",\n \"Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.\",\n \"Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.\",\n \"All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.\",\n \"5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.\",\n \"The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.\",\n \"Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.\",\n \"The clones were clustered into groups of similar or identical sequences.\",\n \"Representative clones were selected for sequencing.\",\n \"In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.\",\n \"PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.\",\n \"Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.\",\n \"In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.\",\n \"5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.\",\n \"The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-30368 and are provided in the attached Sequence Listing.\",\n \"The contigs were assembled using an EST sequence as a seed.\",\n \"Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.\",\n \"The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.\",\n \"Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.\",\n \"The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-30368) of the present invention are incorporated in the attached Sequence Listing.\",\n \"A subset the predicted polypeptide sequences contain an unknown amino acid, a stop codon, a possible nucleotide deletion or a possible nucleotide insertion.\",\n \"These sequences have been shown in their entirety with the special characters in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-30368.Table 2 also indicates the method by which the polypeptide was predicted.\",\n \"Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fastabioch.virginia.edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).\",\n \"Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.\",\n \"Biol., 268:78-94(1997), incorporated herein by reference).\",\n \"Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.\",\n \"The nearest neighbor results for SEQ ID NO: 1-30368 were obtained by a BLASTP version 2.0al 19 MP-WashU search against Genpept release 121 and Geneseq release 200103 (Derwent), using BLAST algorithm.\",\n \"The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-30368.The nearest neighbor results for SEQ ID NO: 1-30368 are incorporated in the attached Sequence Listing.\",\n \"Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.\",\n \"Biol., Vol.\",\n \"6 pp.\",\n \"219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions.\",\n \"The attached Sequence Listing provodes the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.\",\n \"Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.\",\n \"26(1) pp.\",\n \"320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.\",\n \"The attached Sequence Listing provides the results obtained by PFAM analysis for each peptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.\",\n \"Tables 1 and 2 follow.\",\n \"Table I shows the various tissue sources of SEQ ID NO: 1-30368.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.\",\n \"Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.\",\n \"No.\",\n \"09/540,217\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450763"}}},{"rowIdx":283,"cells":{"text":{"kind":"list like","value":[["Novel means for the diagnosis and therapy of ctcl","A means for the diagnosis and therapy of T lymphomas, in particular Cutaneous T Cell Lymphomas (“CTCL”) are provided.","Tumor markers which are universal for CTCL are described.","More particularly, a molecule, termed SC5 by the inventors, an allelic form of p140, and biological applications of SC5 and p140 molecules, notably in the diagnosis and therapy of CTCL are described."],["1 A monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","2 A hybridoma deposited as deposit number I-2575 at the C.N.C.M.","3 An isolated protein obtainable by: (i) collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating them with PHA at 1 microgram/ml, (ii) lysing the cells by incubation in a lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the compound onto which the mAb of claim 1 binds under conditions enabling this mAb to perform reactions of the antigen-antibody type.","4 An isolated protein obtainable by recovering the isolated protein compound of claim 3 under non reducing conditions.","5 An isolated cDNA obtainable by: collecting cells selected from the group consisting of total blood cells and PBL5 incubating the collected cell population with a CD3 activator at 1 microgram per ml, extracting and purifying the whole mRNA population from said cells, synthesizing every complementary cDNA, operably cloning each cDNA so that expression of this cDNA in this clone is under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody of claim 1 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or of which lysate would give an immuno-precipitation reaction with this mAb, optionally, amplifying those clones that have been thus selected, and recovering the inserted cDNA from the selected clones.","6 An isolated mRNA obtainable by selection among the mRNA population of cells selected from the group consisting of total blood cells and PBL, of a mRNA which is complementary to a cDNA of claim 5.7 An isolated genomic DNA encoding an isolated mRNA of claim 6.8 An engineered cell in which a cDNA according to claim 5, has been transfected.","9 An isolated protein encoded by an isolated cDNA according to claim 5.10 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 3, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.","11 An isolated polypeptide compound obtainable by: collecting PBL cells or total blood cells, incubating the collected cells with a CD3 activator at 1 microgram par ml, labeling the cells with a polypeptide-specific label, lysing the cells in a lysis buffer comprising Trition X-100 at 1%, and submitting the lysate to an immuno-precipitation reaction with the mAb of claim 1, and recovering from the digested immuno-precipitate any compound onto which bear a label.","12 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 3, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells maizy at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with We a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a protein according to claim 3, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","13 A fragment of a monoclonal antibody according to claim 1, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.14 An isolated compound, comprising a fragment according to claim 13.15 The isolated compound according to claim 14, wherein it is a humanized antibody.","16 The isolated compound according to claim 14, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.","17 An isolated protein of SEQ ID No.","2.18 The isolated DNA encoding an isolated protein according to claim 17.19 An isolated DNA of SEQ No.","1.20 A polypeptidic vector comprising a monoclonal antibody according to claim 1, or a fragment thereof selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2 and CDR3.21 A polypeptidic vector for use in the treatment of CTCL, wherein said vector comprises a p140 binding compound.","22 The polypeptidic vector according to claim 20, further comprising an element selected from the group consisting of tumoral toxins and radioelements.","23 The polypeptidic vector according to claim 20, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form.","24 A medical kit comprising a polypeptidic vector according to claim 23 and an antimitotic pro-drug.","25 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.26 A method for manufacturing an anti-CTCL medicament comprising including a p140 binding compound in said medicament.","27 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 3 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","28 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 3, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","29 A method for CTCL diagnosis, comprising measuring the percentage of CD4+ cells expressing an element selected from the group consisting of the proteins according to claim 3, in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","30 A method for CTCL diagnosis, comprising determining the presence of CD4+ cells expressing p140 in a biological sample which comprises potential CTCL cells, a CTCL-positive diagnosis being made when such a presence is significantly detected.","31 A method according to claim 27, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1 and the DNA encoding SEQ ID No.","3 or SEQ ID No.","4.32 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.33 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 3, or to a p140 molecule, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","34 The isolated protein of claim 4, wherein said non reducing conditions are of chromatography by affinity with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","35 The isolated cDNA of claim 5, wherein said CD3 activator is PHA at 1 microgram per ml.","36 An engineered cell in which a mRNA according to claim 6 has been transfected.","37 An engineered cell in which a DNA according to claim 7 has been transfected.","38 An isolated protein encoded by an isolated mRNA according to claim 6.39 An isolated protein encoded by an isolated cDNA according to claim 7.40 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 4, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.","41 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 9, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.","42 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 4, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a protein according to claim 4, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","43 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 9, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a protein according to claim 9, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","44 A monoclonal antibody obtainable by: (i) immunizing an animal against a polypeptide compound according to claim 11, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.","for binding to a polypeptide according to claim 11, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.","45 A fragment of a monoclonal antibody according to claim 12, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.46 The isolated compound according to claim 15, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.","47 The polypeptidic vector according to claim 20, wherein said fragment is the Fab or F(ab′)2 fragment.","48 A polypeptidic vector comprising an isolated compound according to claim 15, optionally including at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.","49 The polypeptidic vector according to claim 21, further comprising an element selected from the group consisting of tumoral toxins and radioelements.","50 The polypeptidic vector according to claim 21, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form 51 The polypeptidic vector according to claim 23, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.","52 The polypeptidic vector according to claim 50, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.","53 The medical kit according to claim 24, wherein said antimitotic pro-drug is phenol mustard pro-drug.","54 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","55 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","56 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","57 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.","58 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 4, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","59 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 9, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","60 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.","61 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","62 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","63 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing the polypeptide compounds of claim 11 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.","64 A method according to claim 28, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","65 A method according to claim 29, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","66 A method according to claim 30, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody, and the DNA encoding SEQ ID No.","3 or SEQ ID No.","4.67 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.","68 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises at least one polypeptide compound selected from the bank of polypeptide compounds according to claim 10.69 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting the cDNA according to claim 5, an mRNA complementary to said cDNA, a p140 binding compound, a p140 DNA, an isolated DNA encoding an isolated protein of SEQ ID NO: 2, an isolated DNA of SEQ ID NO: 1, the DNA encoding SEQ ID No.","3 and the DNA encoding SEQ ID No.","4.70 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 4, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","71 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 9, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","72 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a polypeptide compound according to claim 10, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.","73 The method according to claim 33, wherein said p140 molecule is of SEQ ID No.","2 or SEQ ID No.","4."],[" BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.","The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.","Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.","LP and CD30+ lymphomas also develop in the skin.","More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.","SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.","Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.","The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.","In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.","Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).","Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.","Such a diagnosis method does also not enable to stage the disease.","Technically speaking, it is also time-consuming.","Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.","There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL."],[" SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).","The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.","It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.","The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.","(C.N.C.M.","Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.","deposit number: I-2575).","When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.","The second marker of the invention, i.e.","p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.","The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.","They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.","Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.","lupus, lichen), or toxic epidermal necrolysis.","At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).","Allelic form KIR3D clone 24 (SEQ ID No.","1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.","1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.","3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.","2) when compared to p140 clone 1.1.mature protein (SEQ ID No.","4).","The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.","sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.","To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.","The invention thus provides with the first CTCL universal markers.","No prior art product was known to be such a CTCL universal marker.","The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.","CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.","CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.","In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.","cytokine profile) of non-tumoral T cells.","The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.","SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).","SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.","The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).","It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.","In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).","Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).","More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.","The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.","humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity)."],["FIELD OF THE INVENTION The present application relates to novel means for the diagnosis and therapy of T lymphomas, and more particularly of Cutaneous T Cell Lymphomas (abbreviated into CTCL).","The invention indeed provides novel tumor markers which are universal for CTCL, and describes biotechnological and medical uses thereof.","BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.","The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.","Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.","LP and CD30+ lymphomas also develop in the skin.","More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.","SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.","Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.","The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.","In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.","Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).","Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.","Such a diagnosis method does also not enable to stage the disease.","Technically speaking, it is also time-consuming.","Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.","There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.","SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).","The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.","It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.","The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.","(C.N.C.M.","Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.","deposit number: I-2575).","When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.","The second marker of the invention, i.e.","p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.","The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.","They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.","Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.","lupus, lichen), or toxic epidermal necrolysis.","At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).","Allelic form KIR3D clone 24 (SEQ ID No.","1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.","1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.","3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.","2) when compared to p140 clone 1.1.mature protein (SEQ ID No.","4).","The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.","sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.","To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.","The invention thus provides with the first CTCL universal markers.","No prior art product was known to be such a CTCL universal marker.","The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.","CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.","CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.","In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.","cytokine profile) of non-tumoral T cells.","The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.","SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).","SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.","The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).","It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.","In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).","Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).","More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.","The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.","humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).","DETAILED DESCRIPTION OF THE INVENTION The invention thus provides with an hybridoma deposited on Oct. 30, 2000 as deposit number I-2575 at the C.N.C.M.","in accordance with the Budapest Treaty (C.N.C.M.","Collection Nationale de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France), and with a method for preparing an anti-SC5 monoclonal antibody which comprises cultivating hybridoma I-2575 under conditions suitable for the metabolism of the hybridoma cells (e.g.","culture for three days at 37° C., with 5% CO2 in a humid atmosphere at 2.106 cells par ml of appropriate culture medium such as RPMI 1640 medium Gibco BRL Cat.","No21875-034, with 10% Fetal Calf Serum, penicillin 100 U/ml and streptomycin 100 microgram/ml).","The invention therefore encompasses the monoclonal antibody (mAb) which is produced by hybridoma I-2575, or which is obtainable from the culture supernatant of this deposited hybridoma.","This mAb is a pentameric anti-SC5 IgM.","It is a useful tool for the diagnosis and therapy of an inappropriate or undesired T cell proliferation and/or functional activity (cytokine profile), of an inappropriate or undesired IL-2 production.","Its inhibits the proliferation and/or functional activity of normal T cells, of virus-infected T cells (e.g.","HIV-infected CD4+ T cells), and of malignant T cells, such as notably malignant CTCL cells.","The monoclonal antibody produced by hybridoma I-2575 enables the isolation of a is protein, termed SC5 by the inventors.","SC5 protein appears as a biochemically and functionally new molecule, with an apparent molecular weight of 96 kD under reducing conditions.","It has been located in and on numerous PBL cells: normal T cells (both from CD4+ and CD8+ subsets), and in particular CD3+ T cells, CD45RO+ T cells, but also normal NK cells, B cells, granulocytes and macrophages.","In addition, SC5 has been located in the cytoplasm of cells from other lineages and other species such as NIH/3T3 (ATCC CRL-1658) and CHO-K1 (ATCC CRL-9618).","In resting normal PBL cells, SC5 is mainly intracellularly located, but CD3 stimulation induces the transfer of SC5 to the cell membrane.","Remarkably, SC5 has also been observed by the inventors on malignant T cells, such as malignant CTCL cells, and in particular malignant CD4+ CTCL cells, where they are mainly located at the cell surface (without any ex vivo CD3 stimulation).","As a further remarkable feature, SC5 surface expression has been observed for all the CTCL patients who were tested until now (see the examples below).","Another further remarkable feature of the invention is that the percentage of CD4+ T cells which co-express SC5 is statistically higher in patients developing tumoral CD4+ T cells than in healthy patients.","The percentage of SC5+ CD4+ cells in CD4+ CTCL patients is significantly higher than the percentage observed in healthy patients (average standard percentage of SC5+ CD4+ cells is in the 1-15% range, generally in the 5-10% range).","In the particular CD4+ CTCL case of Sézary Syndrome (SS), the percentage of CD4+ cells which co-express SC5 is furthermore statistically closely correlated to the percentage of malignant CTCL cells in the PBL of the CTCL patient: the percentage of SC5+ CD4+ T cells is substantially equal to the percentage of malignant CTCL cells (within ±10% of this percentage).","SC5 protein is therefore a new molecule whose transmembrane expression at the cell surface is linked to an activation and/or proliferation status for non-tumoral T cells (such as virus-infected cells, e.g.","HIV-infected cells, and such as normal CD4+ or CD8+ T cells), and to a malignant status for non-activated T cells.","According to a remarkable feature, it enables a CD4+ CTCL-positive diagnosis with a confidence of more than 90% (actually of 100% of those humans who have been tested up-to-date).","A further outstanding feature of the invention is that the SC5 new molecule is capable.","of acting as an inhibitory receptor for cell proliferation and/or functional activity when expressed at the cell surface: SC5 aggregation induces a decrease in the proliferation and activity of the cell expressing it.","For example, the anti-SC5 monoclonal antibody produced by hybridoma I-2575 (which is a pentameric Ig) inhibits the anti-CD3 induced proliferation of SC5+ cells such as normal CD3+ T cells collected from a human or an animal, or normal CD3+ T cell clones, and also inhibits the anti-CD3 induced in vitro proliferation of CTCL cells collected from a CTCL patient, and the in vivo natural proliferation of human CTCL cells in an animal model.","This mAb also binds HIV-infected CD4+ T cells, and is therefore useful as an active principle in an anti-HIV drug intended for killing infected cells, and therefore reducing HIV propagation.","Several procedures enabling the isolation of the SC5 protein are available to the skilled person to whom a monoclonal antibody of the invention has been made available.","Standard procedures comprise recovering the SC5 molecules from a cell lysate by affinity chromatography, or by electrophoresis under reducing conditions.","All human or animal cells which the monoclonal antibody produced by hybridoma I-2575 recognizes as an antigen, or of which lysate gives an immuno-precipitation reaction with this monoclonal antibody, are appropriate cell sources for isolating the SC5 molecule.","Such cells notably include normal cells (collected from a human or an animal, or cell clones) such as T cells, CD3+ T cells, CD45RO+ T cells, B cells, NK cells, macrophages, granulocytes.","Appropriate cells also include tumoral cells such as malignant T cells, e.g.","CD4+ CTCL cells collected from a CTCL patient, or tumoral clones such as the CTCL clone HUT78 (ATCC TIB-161).","Due to the wide range of appropriate cell sources, it will be understood that rough total blood cell or PBL may be directly used as a SC5 source without any further purification.","Normal cells enables the isolation of both the intracellular form and the transmembrane receptor form of SC5.When it is desired to isolate the SC5 receptor form, or a portion thereof, it may thus be preferable to use a source of SC5+ cells (i.e.","cells expressing SC5 at their surface).","Such a source include normal cells as above-mentioned but that have been CD3-stimulated so that SC5 expression increases at the cell surface (see examples below for CD3 stimulation procedures).","When total blood cells or PBL are used as a source of SC5+ cells, CD3 activation can be induced by incubation with a polyclonal activator such as PHA (obtainable from Wellcome) at a concentration of about 1 microgram per ml, because total blood cells or PBL also contain T cells and mononuclear cells.","When a more homogeneous normal cell population is used as a source of SC5+ cells (e.g.","only SC5+ NK cells), then CD3 activation is preferably performed by incubation with immobilized anti-CD3 antibodies, or alternatively with both soluble anti-CD3 antibodies and polyclonal activator such as PHA.","Optionally, a culture step may be performed before and/or after CD3 activation so as to increase cell amount (e.g.","culture for 3 days in RPMI 10% fetal calf serum at 37° C., 95% humidity, and 5% CO2).","When it is desired to isolate SC5 in its intracellular form, then CD3 stimulation may be omitted.","Tumoral cells such as CTCL cells, and notably malignant transformed MF or SS cells, enable the isolation of SC5 molecules in their receptor form, without any required ex vivo CD3 activation: CTCL cells already express SC5 in a transmembranar location.","The invention therefore encompasses any isolated protein obtainable by: (i) collecting cells selected from the group consisting of T cells, CD3+ T cells, CD45RO+ T cells, B cells, NIK cells, macrophages, granulocytes, and stimulating the collected cells with immobilized anti-CD3 antibodies, or with both soluble anti-CD3 antibodies and PHA at 1 micrograimn/ml, or collecting malignant CTCL cells such as malignant CD4+ CTCL cells, or collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating the collected cells with PHA at about 1 microgram per ml, (ii) lysing the cells under conditions enabling the dissociation of multimeric polypeptidic complexes, for example by incubation in a drastic lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the protein compound onto which the monoclonal antibody produced by hybridoma I-2575 binds under conditions enabling this monoclonal antibody to perform reactions of the antigen-antibody type (i.e.","the compound which is recognized as an antigen by this mAb).","The aminoacid sequence of the recovered protein compound can be obtained by any standard technique, such as mass spectrophotometry.","As above-indicated, the cells mentioned in step i. are activated cells, and therefore enables the isolation of SC5 in the form it has when it is a transmembrane receptor.","If the SC5 source used in step i. consists of resting normal T cells (without ex vivo stimulation), the the protein compound thus isolated will be SC5 in its cytoplamsic form.","When the cells selected in step i. are activated or malignant cells, lysis in step ii.","therefore may be performed either on whole cells, or on the membranar fraction of these cells.","The lysis step itself can be achieved by any technique enabling the dissociation of multimeric polypeptidic complexes from each others.","For example, cells (or their membrane fraction) may be incubated in a lysis buffer containing a drastic detergent such as Triton X-100 at about 1% for 1 hour at 4° C.; this enables the dissociation of the SC5 protein from the other cell components.","If a mild detergent such as digitonin 1% or Bridg58 1% is used, then SC5 won't dissociate from those compounds which are naturally associated with it; the use of a mild detergent in the above step ii.","therefore enables the isolation of SC5, and the further isolation of the SC5 transducers and effectors.","The invention therefore also encompasses any isolated compound obtainable by such processes.","The lysate obtained from step ii.","is then incubated with the monoclonal antibody produced by hybridoma I-2575 under conditions appropriate for this mAb to perform reactions of the antigen-antibody type, such as coating the antibodies into microwells containing the cell lysate (e.g.","2 microgrammes of the monoclonal antibody produced by hybridoma I-2575 for a lysate of 107 cells) and incubating the monoclonal antibodies with the cell lysate for two hours at 4° C. The step of recovering the compound onto which the monoclonal antibody produced by hybridoma I-2575 binds (step iii.)","can be achieved by any technique available the skilled person.","Recovering under non reducing conditions enables the isolation of SC5 in its native conformation, and will be preferred when it is desired to retain the native biological properties of SC5.Appropriate techniques for recovering SC5 protein under non reducing conditions namely include chromatography by affinity, and, for example: percolation of the lysate through a column in which the monoclonal antibody (mAb) produced by hybridoma I-2575 has been immobilized (e.g.","by coupling this mAb onto agar beads and placing these beads inside said column); preferably, the lysate is mixed with a buffer which has a.physiological pH, e.g.","at a pH comprised between 6 and 8, such as 7, before percolation so as to make the percolation easier without substantially altering the conformation of the compounds contained in the lysate, rinsing the column with a buffer at the same physiological pH, and recovering the compound which has bound to the immobilized mAb, e.g.","by elution with a buffer which disrupts the binding of the compound onto the mAb without substantially modifying the native conformation of the compound.","Such an elution buffer usually has a slightly acidic pH (usually comprised between 2 and 5, e.g.","a pH of 3).","For the preparation of a matrix which has an optimal orientation of antibody molecules, and which thereby enables antigen binding at a high efficiency, and for a one-step purification procedure for the isolation of membrane proteins, see e.g.","Schneider et al.","1982 (J. Biol.","Chem.","257: 10766-10769) of which content is herein incorporated by reference.","The protein compound thus recovered is the isolated SC5 protein in its native form.","Alternatively, step iii.","under non reducing conditions can be achieved by placing the lysate obtained from step ii.","for immuno-precipitation with the monoclonal produced by hybridoma I-2575, and the immuno-precipitate thus obtained can be percolated through a column comprising antibodies capable of binding mice IgM.","The protein compound which has bound onto the monoclonal antibody produced by hybridoma I-2575 is then recovered by elution similarly to what has been above described.","The protein compound thus recovered is also the isolated SC5 protein in its native form.","Another standard way for recovering the SC5 protein (step iii.)","is to proceed through electrophoresis under reducing conditions.","This notably includes incubating the monoclonal antibody produced by hybridoma I-2575 with the lysate obtained from step ii.","under conditions enabling this mAb to perform reactions of the antigen-antibody type, collecting the immuno-precipitate thus formed, separating the compounds contained in this immuno-precipitate under reducing conditions, and recovering the protein compound which has an apparent molecular weight of 96 kD.","An alternative way to localize on the electrophoresis gel or membrane the desired SC5 protein (and to subsequently isolate it from the gel or membrane) is to proceed with a labeling step prior to lysis so that surface polypeptidic components of the cells are labeled (e.g.","125 iode or fluorescent labeling with e.g.","sulfo-NHS-LC-biotin), and to recover from the immuno-precipitate those compounds which bear the label.","It has to be noted that if the labeling step is performed after lysis, then both the intra-cellular form and the trans-membranar receptor form of SC5 will be isolated (in this case, as above-indicated, non-activated T cells may be used as a SC5 source).","Because denaturing conditions are used, the protein compound thus recovered is in a non native form: it still has the native amino acid sequence, but its native biological properties have been substantially lost.","It is however the SC5 protein in its non-native conformation that is observed (and has to be searched for) when the biological sample to be tested has been treated under denaturing conditions (such as e.g.","paraffin sections of skin sample).","The SC5 protein in its non-native form may therefore be used for the production of monoclonal antibodies directed against it, such monoclonal antibodies being then useful for SC5 detection on denaturated samples.","If the SC5 protein in its native conformation is desired, portions of at least 10 amino acids of the denaturated protein may then be sequenced (e.g.","by mass spectrophotometry), oligonucleotides deduced from the sequenced portions may be synthetized, and then used as probes to screen a cDNA library obtained from SC5+ cells.","The cDNA thus selected may the be produced and cloned into a cell under conditions enabling the production, and preferably the excretion, of the cDNA-encoded product by culture of the clone (e.g.","via insertion of the SC5-encoding cDNA in a baculo virus vector and transformation of insect cells—e.g.","Sf9 cells—, or via the vaccina virus and EBV cells, see Rindis Bacher 1995, J. Biol.","Chem.","270(23): 14220-14228).","The invention therefore encompasses any isolated protein of which sequence is obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, labeling the stimulated cells with a polypeptide-specific label such as biotin, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which bears the label, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, recovering the protein produced by the transfected cells, e.g.","with the monoclonal antibody produced by hybridoma I-2575, or obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which has an apparent molecular weight of 96 kD under reducing conditions, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, the protein compound produced by the transfected insect cells, e.g.","with the monoclonal antibody produced by hybridoma I-2575.The invention thus encompasses any isolated protein compound obtainable by any of the above-mentioned processes, as well as any amino acid sequence of such an isolated protein compound, and any isolated protein of which sequence comprises such a sequence.","Falls within the scope of the invention any solid support onto which a protein compound or protein of the invention has been placed.","Such solid supports notably include sepharose beads.","The invention also encompasses any isolated DNA (or cDNA) of which sequence codes for an isolated protein of the invention.","It also relates to engineered cells transfected by such a DNA, to engineered cells which excrete an isolated protein of the invention, and to isolated protein obtainable by isolation from the culture medium of such engineered cells.","The invention notably encompasses any cDNA obtainable by: collecting a cell population which consists of, or comprises cells onto which the monoclonal antibody produced by hybridoma I-2575 binds (i.e.","onto which this mAb recognizes an antigen), or cells of which lysate would give an immuno-precipitation reaction with this monoclonal antibody, e.g.","collecting cells selected from.","the group consisting of total blood cells and PBL, if the sole transmembranar form of SC5 is desired: stimulating the CD3 pathway if said cell population mainly comprises or consists of non-activated cells (e.g.","resting non-tumoral cells) so as to increase SC5 expression at the cell surface, e.g.","by incubating the collected cell population with a CD3 activator (for example a polyclonal activator such as PHA at 1 microgram par ml when the collected cell population is PBL or total blood, or immobilized anti-CD3 antibodies, or both a polyclonal activator such as PHA and soluble anti-CD3 antibodies when the collected cell population is more homogeneous such as a cell population mainly comprising, or consisting of CD3+ T cells, or of CD45RO+ cells, or of NK cells, or of macrophages, or of granulocytes); if the collected cell population consists of, or mainly comprises activated cells (such as CD3-activated non-tumoral cells, or such as tumoral cells such as CD4+ CTCL cells), then CD3 stimulation is not necessary because SC5 location is already mainly transmembranar in these activated cells; if it is desired to isolate both the intracellular form and the trans-membranar form of SC5, then non-activated cells are preferred and CD3 activation is omitted, extracting and purifying the whole mRNA population from said cell population (commercial kits are available for mRNA extraction and purification, e.g.","a polydT columns), synthetising every complementary cDNA (with a reverse transcriptase, e.g.","such as described in Seed B. and Arrufo A.","1987, Proc.","Natl.","Acad.","Sci.","USA 84: 3365-3369, of which content is herewith incorporated by reference), operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody produced by hybridoma I-2575 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or those clones of which lysate would give an immuno-precipitation reaction with the monoclonal antibody produced by hybridoma I-2575 will be selected.","optionally amplifying those clones that have been thus selected, recovering the inserted cDNA from the selected clones.","The cDNA thus recovered is an SC5 cDNA of the invention, it can be sequenced using standard DNA sequencing techniques.","Operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions may be achieved by any conventional cloning procedure available to the skilled person.","An example of cloning procedure comprises inserting each cDNA in a transfection vector under the control of a promoter suitable for the expression of this cDNA in the host cell, transfecting the host cells with this transfection vector, and cultivating the transfected host cells under conditions appropriate to their metabolism.","Appropriate transfection vectors and host cells comprise virus such as Baculo virus and host cells such as insect cells (Sf9 cells), or vaccinia virus as a vector and EBV cells as host cells (see Rindis Bacher reference supra).","Such a cDNA can be used for cell transfection, and for SC5 production from the clones thus obtained.","It may also be used as probe for identifying or isolating the native SC5 mRNA.","The invention therefore also encompasses any isolated mRNA obtainable by selection among the above-mentioned mRNA population, of a mRNA which is complementary to a cDNA of the invention (e.g.","via selection of a mRNA which hybridizes to the above-mentioned cDNA under stringent conditions such as described in Freeman G. J. et al.","1992, J. Immunol.","149: 3745).","Are particularly encompassed any isolated mRNA obtainable by using an isolated cDNA of the invention as a probe so as to recover from the whole mRNA population of said cell population, the mRNA which is complementary to this extracted cDNA.","Alternatively, the one of ordinary skill in the art may directly sequence the cDNA that is recovered from the selected clones, and deduce from this sequence the sequence of the mRNA.","The invention also encompasses any DNA encoding an isolated mRNA according to the invention.","It particularly encompasses any genomic DNA obtainable by searching a genomic bank (such as www.ucsc.edu) for a genomic DNA matching with the above-mentioned SC5 cDNA.","The invention also encompasses any engineered cell in which a cDNA, a mRNA or a DNA of the invention has been transfected.","Are also encompassed SC5-specific probes and primers.","Such probes and primers can be obtained by any technique available to the skilled person (e.g.","selecting, as candidate sequences, portions of SC5 sequence that may be unique to SC5, comparing these candidate sequences on DNA/RNA banks with the sequences of cell receptors other than SC5, and selecting those candidate sequences which target a SC5 sequence that appears as unique after said comparison, and/or a SC5 sequence of which length appears as unique after said comparison).","Any solid support onto which is placed a cDNA, a mRNA or a genomic DNA of the invention falls within the scope of the present invention.","Such solid supports notably include DNA chips, or DNA microspheres.","The invention also encompasses any isolated protein encoded by an isolated cDNA, mRNA, or DNA according to the invention.","The invention further encompasses any isolated portion of a protein of the invention, provided that this portion is still characteristic of CTCL cells by comparison with normal resting cells.","It particularly encompasses any portion that is recognized by the monoclonal antibody produced by the hybridoma I-2575 at the C.N.C.M.","It also encompasses any isolated portion of a protein compound of the invention, provided that this portion is capable of inducing a modulation (inhibition or stimulation) of the CD3-induced proliferation of a normal T cell, and/or is capable of inducing a modulation of the in vivo proliferation of a malignant T cell such as a CTCL cell, and/or is capable of inducing a modulation of the IL-2 production of a normal T cell or of a malignant T cell, when said portion is expressed by said cell and contacted with the mAb produced by hybridoma I-2575.It particularly encompasses any isolated portion of a protein compound of the invention, provided that this portion is a part of a molecule that is expressed in a trans-membrane location by malignant CTCL cells and that is expressed in the cytoplasm compartment by non-tumoral resting T cells.","The invention notably provides with a bank of polypeptidic fragments which is obtainable by enzymatic cleavage of the SC5 protein of the invention.","Preferred enzymes comprises proteolytic enzymes such as those serine endopeptidases which cleave at the level of Tyr, Phe and Trp (e.g.","alpha-chymotrypsin), and also those enzymes which cleaves at the C-terminus of glutamic acid and aspartic acid (e.g.","V8 protease); see e.g.","Shesberadaran and Payne 1988, Proc.","Natl.","Acad.","USA 85:1-5, of which content is herein incorporated by reference.","Such a bank of SC5 polypeptidic fragments is of particular use in analysis such as mass spectrophotometry analysis: such analysis use the characteristic separation of such a polypeptidic bank, and give a (polypeptide) profile that is characteristic of SC5, and that can be used for SC5 detection.","Such a use and such a profile is encompassed by the present application.","The invention particularly encompasses the extra-cellular, trans-membranar and intra-cytoplasmic portions of any SC5 protein compound of the invention.","Determining the extra-cellular, trans-membranar and intra-cytoplasmic portions of a receptor molecule may be achieved by the skilled person according to any appropriate technique.","They may be identified on the whole molecule by identification of the region which contains glycosylation sites (extra-cellular portion), of the region that is hydrophobic (trans-membrane portion), and of the region that is hydrophylic (intra-cytoplasmic region), and they may then be isolated either by cleavage or synthesis.","An alternative way to isolate the extra-cellular portion of a protein compound of the invention is to proceed as above-described for the isolation of the whole protein compound, but with an additional step of enzymatic digestion so as to cleave from the whole molecule its extra-cellular portion.","Appropriate enzymes for such a cleavage includes enzymes such as V8 protease or alpha-chymotrypsin as above-mentioned.","The invention thus encompasses any isolated polypeptide compound obtainable by: collecting a SC5 cell source as above-described, such as e.g.","PBL or total blood cells, if it is desired to isolate a polypeptide compound in the form it has when it is naturally expressed at the cell surface, and if the collected SC5 cell source is, or mainly comprises normal resting cells, then stimulating the CD3 pathway, e.g.","via incubation with an appropriate CD3 activator (e.g.","incubation with PHA at 1 microgramme per ml when said SC5 source is total blood cells or PBL), labeling the cells with a polypeptide-specific label, such as biotin (surface labeling on whole cells), lysing the SC5 cell source (such as PBL or total blood cells) so as to dissociate multimeric polypeptidic complexes (and thereby recovering SC5 dissociated from the transducers and effectors naturally associated therewith) e.g.","by incubating the cells in a lysis buffer comprising a drastic detergent such as Triton X-100 at 1%, and submitting the lysate to an enzymatic digestion enabling the cleavage of SC5 into portions, e.g.","by incubating the lysate with a proteolytic enzyme such as an enzyme selected from the group consisting of V8 protease or alpha-chemotrypsin, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, and recovering from the immuno-precipitate a polypeptide compound which bears a label.","Such recovered compounds are SC5 portions which either are the complete SC5 extra-cellular portion, or are part of such a complete SC5 extra-cellular portion, or comprise such a SC5 extra-cellular portion.","The set of polypeptide compounds that are thus obtainable constitute a bank of SC5 “extra-cellular” portions.","Such a bank, and its medical uses, such as e.g.","its use for the diagnosis of a T-related disease (e.g.","via mass spectrophotometry analysis), falls within the scope of the present invention.","The person of ordinary skill in the art will note that lysis and enzymatic digestion may be performed in a single step.","Labeling on whole cells is performed prior to cell lysis so as to identify SC5 portions which are extra-cellular portions, or which are part of such extra-cellular portions, or which comprise such portions.","The invention also encompasses any isolated compound which comprises a portion of a protein of the invention.","The mAb of the invention also enables the identification of the SC5 epitope onto which this mAb binds.","Conventional techniques such as directed mutagenesis are appropriate (see e.g.","Chang H. C. et al.","1989, J. Exp.","Med.","169: 2073-2083, of which content is herein incorporated by reference).","The proteins of the invention and their portions according to the invention, and notably their extra-cellular portions, enable the production of monoclonal antibodies directed against them.","An example of such monoclonal antibodies is produced by hybridoma I-2575.The monoclonal antibodies of the invention are particularly useful for the detection of SC5, for the diagnosis of T lymphomas such as CTCL, and are also useful as modulators of an undesired or inappropriate proliferation and/or activity of T cells, such as normal CD4+ and CD8+ T cells, virus-infected T cells such as HIV-infected T cells, T lymphomas such as CTCL.","The invention therefore encompasses any monoclonal antibody obtainable by: (i) immunizing an animal against a protein of the invention or against a portion thereof as above-defined, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or protein portion that as been used as an immunogen in step i., and which has at least one property selected from the group consisting of the property of: binding resting non-tumoral T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of malignant T cells such as CD4+ CTCL cells, modulating the proliferation of malignant T cells such as CD4+ CTCL cells in an animal, and preferably in a human, competing with a monoclonal antibody of the invention, such as the monoclonal antibody produced by hybridoma I-2575, for binding to a protein of the invention, or to a portion thereof as above-defined, and in particular with the extra-cellular portion of such a protein, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.","Production of hybridomas may be achieved using any conventional technique.","This includes removing the spleen from said animal, making a suspension of spleen cells, fusing the spleen cells with myeloma cells in the presence of a fusion promoter, diluting and cultivating the fused cells in separate wells in a medium which will not support unfused cells.","The invention also encompasses any ascite isolated from said immunized animal.","For step iii., T and malignant T cells can be collected from a human using standard procedures.","Alternatively, clones can be used such as the CD4+ CTCL HUT 28 cell line (ATCC TIB-161).","In the case of CD4+ tumors, collection is made from the body part which will display the CD4+ tumoral cells (PBL for SS, cutaneous erythroderma for transformed MF, etc.).","The above-mentioned properties can be easily assessed by the skilled person using common knowledge in the art.","Examples of appropriate experimental conditions for assessing the properties mentioned in step iii.","can be found in the below examples.","The person of ordinary skill in the art will adjust them to the particular cells, or concentrations used.","Modulation of cell proliferation herein refers to stimulation as well as inhibition of this proliferation.","When CTCL cells are used, inhibition of proliferation is preferred in perspective of therapeutic application.","The mAbs of the invention are all anti-SC5 mAbs which have either diagnostic or therapeutic applications, or both.","For certain applications, it may be desired to immobilize one or several of these mAb onto a support, e.g.","for protein purification purposes, or for exerting the modulation property of which they are capable.","The present application also encompasses any support onto which a mAb of the invention has been immobilized.","Such supports notably include those appropriate for columns of chromatography by affinity (e.g.","agar beads).","The present application is also directed to any fragment of a mAb of the invention (anti-SC5 fragments) selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3.It is directed to any compound, and namely to any antibody, comprising such a fragment.","It notably relates to any humanized antibody comprising at least one of such fragments.","Techniques for the production of humanized antibodies have been widely described in the prior art (see e.g.","Farah et al.","1998, Crit.","Rev.","Eucaryote Gene Exp.","Vol.","8 pp 321-345, of which content is incorporated herein by reference).","In particular, it relates to any compound, and notably to any antibody and humanized antibody, comprising at least one anti-SC5 fragment as herein defined and also at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3 (see e.g.","WO 94/13804 “Multivalent and multispecific binding proteins, their manufacture and use”, Inventors: Holliger et al.","Applicants: Cambridge Antibody Technology Ltd and Medical Research Council; see also Merchant et al.","“An efficient route to human bi-specific IgG” Nat.","Biotechnol.","1998 vol.","16 pp 677-681).","As previously indicated, the invention provides with two molecules linked by a common general inventive concept: SC5 and p140.The SC5 is a novel molecule fully herein described.","The p140 molecule has been described in the prior art as an inhibitory receptor which is expressed by sub-groups of NK cells and of CD3+ CD8+ cells from healthy (non-cancerous) patients.","The present invention now demonstrates that p140 is expressed by malignant T cells, and notably by CTCL cells, and provides with a novel p140 allelic form (clone 24 in the below example 3; SEQ ID No.","1) encoding a novel protein (SEQ ID No.","2).","The cDNA and amino acid sequences of this novel allelic form are as follow: SEQ ID No.1: CATGTCGCTCACTGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGC AGGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCC CGGCGCAGCACTGTGGTGCCTCAAGGAGGACACGTGGCTCTTCAGTGTCA CTATCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCC ACGTTCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTGATCATGGGC CCTGTGACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCC ACACTCCCTCACTGGGTGGTCGGCACCCAGCAACCCCCTGGTGATCATGG TCACAGGAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGACCCTG CTGAAATCAGGAGAGACAGTCATCGTGCAATGTTGGTCAGATGTCATGTT TGAGCACTTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCC TCGTTGGACAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGT CCCTTGATGCCTGTCCTTGCAGGAACCTACAGATGTTATGGTTCTGTTCC TCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCCCTGGACATCGTGA TCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACG GTTCAGGCAGGAGAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTA TGACATCTACCATCTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCC GTGCAGTGCCGAAGGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGG CCTGCCACCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGCCCT GCCCTGCGTGTGGTCAAACTCAAGTGACCCACTGCTTGTTTCTGTCACAG GAAACCCTTCAAGTAGTTGGCCTTCACCCACAGAACCAAGCTCCAAATCT GGTATCTGCAGACAGGTGCATGTTCTGATTGGGACGTCAGTGGTCATCTT CCTCTTCATCCTCCTCCTCTTCTTTCTCCTTTATCGCTGGTGCTCCAACA AAAAGAATGGTGCTGTAATGGACCAAGAGCCTGCGGGGGACAGAACAGTG AATAGGCAGGACTCTGATGAACAAGACCGTCAGGAGGTGACGTACGCACA GTTGGATCACTGCGTTTTCATACAGAGAAAAATCAGTCGCCCTTCTCAGA GGGCCAAGAGACCCCCAACAGATACCAGCGTGTACACGGAACTTCCAAAT GCTGAGCCCAGATCCAAAGTTGTCTCCTGCCCACGAGCACCACAGTCAGG TCTTGAGGGGGTTTTCTAGGGAGACAACAGCCCTGTCTCAAAACC SEQ ID No.2: pLMGGQDKPF LSARPSTVVP QGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPLVIMVTGN HRKPSLLAHP GTLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS ˜DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PPTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF The DNA and amino acid sequences of the previously described p140 allelic form (clone 1.1) are as follow: SEQ ID No.3: CATGTCGCTCACGGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGCA GGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCCCG GCCCAGCACTGTGGTGCCTCGAGGAGGACACGTGGCTCTTCAGTGTCACTA TCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCGACGT TCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTCATCATGGGCCCTGT GACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCCACACT CCCTCACTGGGTGGTCGGGACCCAGCAACCCCGTGGTGATCATGGTCACAG GAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGCCCCTGCTGAAA TCACGAGAGACAGTCATGCTGCAATGTTGGTCAGATGTCATGTTTGAGCAC TTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCCTCGTTGGA CAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTGGCTTGATG CCTGTGCTTGCAGGAACGTACAGATGTTATGGTTCTGTTCCTCACTCCGGC TATCAGTTGTCAGCTCGCAGTGACCCCCTCGACATCGTGATCACAGGTCTA TATGAGAAACCTTCTCTCTCAGCGCAGCGGGGCCCCACGGTTCAGGCAGGA GAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTATGACATCTACCAT CTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCCGTGCAGTGCCGAA GGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGCCCTGCCACCCACGG AGGGACCTACAGATGCTTCGGCTCTTTGCGTGCCCTGCCCTGCGTGTGGTC AAACTCAAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAGTAG TTGGCCTTCACCCACAGAACCAAGCTCCAAATCTGGTATCTGCAGACACCT GCATGTTCTGATTGGGACCTCAGTGGTCATCTTCCTCTTCATCCTCCTCCT CTTCTTTCTCCTTTATCGCTGGTGCTCCAACAAAAAGAATGCTGCTGTAAT GGACCAAGAGCCTGCGGGGGACAGAACAGTGAATAGGCAGGACTCTGATG AACAAGACCCTCAGGAGGTGACGTACGCACAGTTGGATCACTGCGTTTTC ATACAGAGAAAAATCAGTCGCCCTTCTCAGAGGCCCAAGACACCCCTAAC AGATACCAGCGTGTACACGGAACTTCCAAATGCTGAGCCCAGATCCAAAG TTGTCTCCTGCCCACGAGCACCACAGTCAGGTCTTGAGGGGGTTTTCTAGG GAGACAACAGCCCTGTCTCAAAACC SEQ ID No.4: pLMGGQDKPF LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPVVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQBVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF SEQ ID No.","1 differs from SEQ ID No.","3 in five locations.","The resulting SEQ ID No.2 differs from the p140 disclosed in prior art (SEQ ID No.","4) by four amino acid substitutions (shown in bold and underlined characters): R instead of Q in position 20, V instead of L in position 92, P instead of T in position 102, and L instead of P in position 401 (see FIG.","11).","The present application encompasses any isolated compound of which sequence is, or comprises SEQ ID No.","1 and/or SEQ ID No.","2.It also encompasses any mRNA that is complementary to SEQ ID No.","1, and any genomic DNA coding for SEQ ID No.","2.Appropriate techniques for producing p140 proteins are available to the skilled persons.","They notably include the production of cells which have been engineered so as to produce p140 proteins in their culture medium.","An example of such techniques comprises inserting SEQ ID No.","1 or No.","3 in a baculo virus vector, operably transfecting an insect cell such as Sf9 cell line with this vector, recovering the p140 protein produced in the culture medium (e.g.","by percolation of the culture medium is through a column—e.g.","a Sephadex column—which protein compounds as a function of their molecular weight, and isolating the 140 kD eluate).","Compounds capable of binding to p140 under conditions of the physiological type (in vivo conditions, or in vitro conditions mimicking the in vivo ones) have been previously described, and may be obtained by the person of ordinary skill in the art by any appropriate available technique.","By “p140 binding compound”, it is herein meant any compound which is capable under said conditions of the physiological type to recognize a p140 molecule as an antigen (SEQ ID No.","2 and/or SEQ ID No.","4) when it is expressed by a cell in a transmembrane receptor location (i.e.","recognition of the SEQ ID No.","2 and/or No.","4 portion that is above the lipid bi-layer of said cell).","Such p140 binding compound notably comprise HLA-A11 and HLA-A3 molecules (natural p140 ligands), and notably fusion proteins such as Fc-HLA-A11 and Fc-HLA-A3 fusion proteins.","p140 binding compounds also comprise p140 antiserum (obtainable by immunizing an animal such as a rabbit against p140, and by recovering the antiserum thus produced—an additional step of immuno-purification may be performed so as to increase p140 concentration in the antiserum, e.g.","via percolation through a p140 column).","Other p140 binding compounds comprise monoclonal antibodies directed to p140 SEQ ID No.","4, such as e.g.","AZ158, Q66, Q241 described in Pende et al.","1996 (J. Exp.","Med.","184: 505-518).","Such anti-SEQ ID No.","4 mAbs may also recognize the new SEQ ID No.","2 as an antigen.","Any available technique for mAb production is appropriate for production of anti-p140 mAbs.","An example of such a technique comprise the steps of: (i) immunizing an animal against p140, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the p140 molecule that as been used as an immunogen in step i., and which is also capable of binding resting non-malignant T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.","Preferred p140 binding compounds do also comprise an anti-CD4 entity (e.g.","bi-specific mAb).","Such p140 binding compounds are useful for p140 detection on malignant T cells, such as CTCL cells, in particular CD4+ CTCL.","They are thus useful for CD4+ cancer detection.","They are also useful as polypeptidic vectors, as described below.","According to another aspect of the invention, there are provided polypeptidic vectors comprising at least one element selected from the group consisting of the anti-SC5 mAbs of the invention and the anti-SC5 mAb fragments of the invention.","Such polypeptidic vectors are useful for molecules, such as active principle molecules, to reach activated T cells, or their vicinity (e.g.","activated T cells observed in inflammatory diseases, or in virus-infected T cells, or malignant T cells such as malignant CTCL cells, and malignant CD4+ CTCL cells in particular).","It also relates to polypeptidic vectors comprising a p140 binding compound such as a mAb directed against SEQ ID No.","2 and/or SEQ ID No.","4 extra-cellular portion.","Such polypeptidic vectors are useful for delivering molecules to, or in the vicinity of malignant T cells, such as notably CTCL cells, and malignant CD4+ CTCL cells in particular.","Molecules which may be usefully delivered to malignant T cells, such as CTCL cells notably comprise molecules capable of inducing cell death or apoptosis, such as a radio-element or a toxin.","Such molecules also comprise any enzyme capable of transforming an antimititotic pro-drug into an active drug form, such as a carboxypeptidase.","A polypeptidic vector carrying such an enzyme is then advantageously used in combination with an anti-mitotic pro-drug (such as phenol mustard pro-drug).","The invention therefore also encompasses a medical kit comprising such a vector and such an anti-mitotic pro-drug.","Molecules which may be usefully delivered in the vicinity of malignant T cells such as CTCL cells notably comprise molecules capable of stimulating the immune functions of cells which are in the vicinity-of malignant T cells in an effort to induce an anti-tumor effect from these neighboring cells towards said malignant T cells.","Examples of such molecules notably comprise cytokines such as interferon gamma, and interleukine such as IL-2 (see e.g.","Xu et al.","2000, Cancer Research 60: 4475-4484).","When a polypeptide vector of the invention is intended for targeting CD4+ cells such as CD4+ CTCL cells, then this vector advantageously further comprises an anti-CD4 entity.","The invention also encompasses any pharmaceutical composition comprising such polypeptidic vectors, and in particular any medicament comprising such polypeptidic vectors.","Such medicaments are useful in the control of an inappropriate or undesired T cell proliferation and/or functional activity (including undesired IL-2 production).","They are particularly useful for the prevention, palliation, relieve or therapy of an inappropriate immune activity, and notably for the prevention or inhibition of a T lymphoma such as CTCL and in particular CD4+ CTCL.","The use of such vectors for the manufacture of a drug intended for the prevention, palliation, relieve or therapy of an inappropriate T cell proliferation and/or activity, and notably for the prevention or inhibition of a CTCL proliferation is thus also an object of the present application.","A compound which binds to SC5 under conditions of the physiological type may be used as a SC5 ligand agonist so as to stimulate the activation of SC5, the SC5 molecule being then regarded as a cell inhibitory receptor.","Such anti-SC5 compounds namely comprise anti-SC5 antisera, the anti-SC5 mAb of the invention, the Fab, F(ab′)2, fragments thereof, the humanized mAbs derived therefrom.","When CD4+ cells are more particularly concerned, said anti-SC5 compound advantageously further comprises an anti-CD4 entity.","Anti-SC5 compounds are particularly useful for modulating an inappropriate or undesired proliferation and/or functional activity of SC5+ cells.","They are thus useful for modulating the proliferation of T cells, CD45RO+ cells, CD3+ cells, CD4+ T cells, CD8+ T cells, or of virus-infected T cells (e.g.","HIV-infected CD4+ T cells), or of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.","They are also useful for modulating IL-2 production from T cells.","When the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M.","(which is a pentameric IgM) is used, this modulation goes in favor of an inhibition.","It is therefore particularly appropriate for inhibition of T lymphomas such as CTCL.","The application encompasses any drug comprising, as an active principle, at least one anti-SC5 compound.","Such a drug may be intended for the prevention, palliation, therapy of T lymphomas, of CTCL, of T infections such as viral (e.g.","HIV) infections, of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis—e.g.","spondyloarthropathis—or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).","Such a drug may also be intended for graft improvement (inhibition of graft rejection).","The invention also encompasses the use of such anti-SC5 compounds as complement recruiting agents, or as ADCC activators or stimulators.","The p140 binding compounds as defined above, may also be used as complement recruiting agents, or as ADCC stimulators or activators in the prevention, palliation, relieve or treatment of T lymphomas, such as CTCL and in particular CD4+ CTCL.","The invention therefore encompasses the use of such p140 binding compounds in the manufacture of an anti-T lymphoma drug, of an anti-CTCL drug, of an anti-CD4+ CTCL drugs, and also encompasses such drugs.","The invention also provides with a method for the assessment of the development level of a CTCL (staging): it enables to evaluate the proportion (e.g.","percentage) of malignant CD4+ CTCL cells present within a certain body compartment of a patient.","According to this method, the proportion of CD4+ cells expressing at their surface an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from said body compartment.","The proportion of CD4+ CTCL cells that are actually present in said body compartment can be considered as substantially equal to said measured proportion.","It usually falls within a ±10% range around this measured proportion.","The invention also provides with a method for CTCL diagnosis, wherein the percentage of T cells expressing an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from a patient, and is compared to the average percentage observed in non-CTCL humans (preferably in healthy humans), and in that a CTCL-positive diagnosis is decided when said measured percentage is significantly higher than said average percentage.","This CTCL diagnosis method particularly applies to those CTCL which are CD4+; in this case the measured T cell percentage is preferably a CD4+ percentage.","The average standard proportion of CD4+ T cells expressing SC5 is generally in between 1-15%, usually in between 5-10% among healthy humans.","When the measured percentage of SC5+ CD4+ T cells is higher than such an average percentage, then a CD4+ CTCL positive diagnosis can be concluded.","Of note is that, as far as SC5 protein or portions thereof are concerned, the methods of the invention more generally enables to evaluate the proportion of CD4+ activated T cells that are present in said body compartment.","This may e.g.","be useful for assessing whether a graft induces rejection reactions, and for the diagnosis and follow-up of inflammatory diseases such as those of the auto-immune type (e.g.","rhumatoid arthritis such as spondyloarthropathis, skin immune mediated disease such as psoriasis, eczema, atopic dermatitis).","Said biological sample may then be intra-synovial fluid.","This may be also useful for the diagnosis and monitoring of non-CTCL T lymphomas (e.g.","CD4+ ALL, or CD4+ T-LL).","The CTCL assessing/diagnostic methods of the invention are highly efficient for all kinds of CD4+ CTCL, and notably for transformed MF, for SS, for LP, as well as for CD30+ lymphomas.","Said biological sample may e.g.","then be a skin sample collected from an erythroderma (e.g; suspicion of transformed MF), and/or a PBL or total blood sample (in order to evaluate whether a transformed MF has evolved into a more aggressive form such as SS).","When the element detected at the surface of said CD4+ cells is a SC5 compound, then a (preliminary or ulterior) step of cytological observation (search for tumor-alike cytology) is the preferably performed to conclude with a confidence of more than 90% to an actual CD4+ CTCL.","When p140 is detected at the surface of said CD4+ cells, then no additional step is required: the simple fact of detecting p140 at the surface of CD4+ T cells directs to a positive diagnosis of CD4+ CTCL.","The methods of the invention can be implemented with any appropriate element, and notably with an element selected from the group consisting of the anti-SC5 monoclonal antibodies of the invention, the Fab, F(ab′)2 fragments thereof, the humanized mAb derived therefrom, the SC5 “extra-cellular” bank of the invention (as above-defined), the SC5 cDNA, mRNA, genomic DNA of the invention, the p140 binding compounds as herein defined, the p140 DNA, the DNA comprising SEQ ID No.","1 or SEQ ID No.","3 or encoding SEQ ID No.","2 or SEQ ID No.","4.Such an element is then used as a detection means for SC5 or p140 cell surface expression.","Preferably, said detection means also comprises an anti-CD4 entity so as to detect SC5+ /p140+ CD4+ cells in a one-step procedure.","When cDNA/mRNA material is used as a detection means, then CD4+ cells have to be lysed so as to detect those SC5 or p140 hybridizing mRNA which are actually present in said cells (detection of the transcripts).","Such cDNA/mRNA detection means may advantageously be placed onto or into a solid support such as a DNA chip or a DNA microsphere.","When mAb material or mAb-derived material is used as a detection means, then surface expression of SC5 or p140 is detected.","Any appropriate antibody-antigen technique is convenient.","Examples notably comprise flow cytometry analysis.","The invention also encompasses a method for the identification of a compound which is useful in the palliation, prevention, relieve, therapy of an undesired or inappropriate T cell activity, such as notably a proliferation of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.","This method is characterized in that it comprises the detection of a compound that is capable of binding to a SC5 protein as above-defined, or to a SC5 polypeptide compound as above-defined, or to a p140 molecule (such as SEQ ID No.","2 and/or SEQ ID No.","4).","Such compounds can be used as polypeptidic vectors as above-described.","Those compounds which, in addition to binding to SC5 molecules, are also capable of aggregating these SC5 molecules at the cell surface, are preferred for stimulating SC5 inhibitory functions.","An easy way to implement this identification method is to proceed with SC5 and/or p140 molecules immobilized onto a solid support such as protein A Sepharose CL-4B.","Candidates compounds for implementation of the method notably comprise sera immunized against SC5 or p140.The invention is illustrated by the examples, which are in no way intended to restrict the invention to the particular embodiments described below.","The person of ordinary skill in the art will contemplate alternative embodiments, and such alternative embodiments derived from common knowledge in the art are within the scope of the invention.","The skilled person will in particular appreciate that various standard immuno-techniques, and various standard molecular biology techniques are available to him (see e.g.","“Antibodies: a laboratory manual” Ed Harlow, David lane, Cold Spring Harbor Laboratory 1988; Maniatis 1982, “Molecular cloning: a laboratory manual”, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory; “Current protocols in Immunology” protocols on CD-ROM, John Wiley, West Sussex, England; “Immunological techniques made easy”, edited by Olivier Cochet et at.","; John Wiley, West Sussex, England; and the publication specifically referred to in the present application).","The content of such prior art documents is herein incorporated by reference.","It is also to be understood that experiments described herein in full details for one particular form of CD4+ CTCL can be directly transposed by the skilled person to any other form of CD4+ CTCL.","In these examples, reference is made to the following figures: FIGS.","1A, 1B and 1C illustrate cell membrane expression of SC5 molecule on PBLs from normal individual.","PBMC from a normal individual were stained with anti-SC5 mAb (ascites ({fraction (1/200)}) followed by FITC-conjugated isotype specific goat anti-mouse IgM second reagent and one of the following PE-conjugated mAbs: anti-CD3 (FIG.","1A), anti-CD56 (FIG.","1C) and anti-CD45RO (FIG.","1B).","The percentage of double-stained lymphocyte-gated cells is shown in the upper right quadrant (3.3 for FIG.","1A; 5.1 for FIG.","1B; 0.6 for FIG.","1C).","This experiment is representative for the 10 donors studied.","FIG.","2 illustrates cell membrane expression of SC5 molecule during activation of PBLs.","PBMC were stimulated with 1 μg/mL PHA and the kinetics of SC5 and CD69 expression were studied in parallel on the CD3+ cells.","FIG.","3 illustrates intracellular localization of SC5 molecule in PBL.","Anti-SC5, anti-CD3ζ chain mAb and anti-BY55 isotype-matched mAb were used for staining permeabilized and non-permeabilized gated lymphocytes from a normal donor.","The shaded histograms represent the labeling obtained with the indicated mAbs compared to an irrelevant control mAb.","FIG.","4 illustrates biochemical analysis of SC5 molecule.","DS6 T cell clone was surface labeled with biotin and 1% Triton X-100 lysates were immunoprecipitated, using anti SC5 mAb.","The samples were analyzed by SDS-10%-PAGE under reducing conditions.","Anti-SC5 mAb was used at two concentrations {fraction (1/200)} (lanes 2) and {fraction (1/500)} (lane 3).","The negative control samples were precipitated with goat anti-mouse Ig alone (lane 1) and isotype-matched irrelevant mAb (lane 4).","The molecular weight markers (kD), are indicated on the left.","FIGS.","5A, 5B, 5C, 5D illkustrate that anti-SC5 mAb modulates the anti-CD3-induced proliferation and cytokine secretion of normal T lymphocytes whithout affecting their cytotoxic activity: FIG.","5A: PBL or T cell clones LSO and GDS.3 were stimulated with immobilized anti-CD3 mAb (1 μg of mAb coated per well) or with IL-2 (50 IU/ml) in the presence of anti-SC5 mAb (1:200 final dilution of ascites) or an isotype-matched irrelevant mAb.","Results shown are representative of at least three separate experiments and are expressed as mean cpm±SD of triplicate wells.","FIG.","5B: GDS.3 CD4+ T cell clone was stimulated with the indicated concentrations of pre-coated anti-CD3 mAb in the presence of different concentrations of anti-SC5 mAb.","FIG.","5C: IL-2 secretion in the supernatant of anti-CD3-stimulated PBL, in the presence of anti-SC5 or control mAb, was assessed by measuring the proliferation of an IL-2 dependant T cell clone in the presence of: medium alone (a), supernatant from PBL/anti-CD3+ control mAb (b), supernatant from PBL/anti-CD3+ anti-SC5 mAb (c).","Data represent the mean values±SD of triplicate determinations of one out of three representative experiments.","FIG.","5D: JF1, a cytotoxic CD8+ cell clone was incubated with anti-SC5 mAb ({fraction (1/200)} final dilution of ascites) or with an isotype matched control mAb before the coculture with the FcγR+ murine tumor cells P815 at an E/T ratio of 5/1.Target cells were preincubated with the indicated final concentrations of anti-CD3 mAb.","Results are expressed as mean of triplicate wells.","FIGS.","6A, 6B illustrate cell membrane expression of SC5 molecule on PBL from CTCL patients.","Cells from peripheral blood of 8 patients with Sézary syndrome, 3 patients with Mycosis fungoides and 8 healthy donors were analysed by two-color fluorescence for the expression of SC5 molecule by CD4+ lymphocytes.","FIG.","6A: co-expression of SC5 and CD4 molecules in PBL from a patient with Sézary syndrome in comparison to a normal individual.","FIG.","6B: histograms showing the mean values of SC5 expression in the 3 indicated groups.","Statistically significant differences between SS patients and 2 other groups are marked, (Mann-Witney U test, p<0.001) FIGS.","7A, 7B, 7C illustrate surface membrane expression on malignant cell from Sézary syndrome patient.","FIG.","7A: two-color immunofluorescence analysis of PBMC from SS patient with a circulating TCRVβ22+ malignant T cell clone.","Cells were stained with anti-SC5 mAb followed by PE-conjugated isotype specific goat anti-mouse IgM and FITC-conjugated anti-TCRVP22 mAb.","FIG.","7B: anti-SC5-mAb labeled weakly (shaded histogram) the TCRVβ22-positive malignant cells.","FIG.","7C: cells from the same patient stained with PE-conjugated anti-CD69 mAb and FITC-conjugated anti-TCRVβ22 mAb.","FIG.","8 illustrates the modulation of anti-CD3-induced proliferation of CTCL malignant cell line by anti-SC5 mAb.","Pno cell line was stimulated with IL-7 (10 ng/mL), PMA alone (2 ng/mL), immobilized anti-CD3 mAb or a combination of PMA/anti-CD3 mAb.","SC5 mAb or an isotype-matched irrelevant mAb (1:200 final dilution of ascites) was added at the start of cultures.","Results shown are representative of four separate experiments and are expressed as mean cpm±SD of triplicate wells.","FIGS.","9A, 9B illustrate the detection of p140/KIR molecules on CTCL cell lines (FIG.","9A) and on fresh tumor lymphocytes (FIG.","9B).","Double immunostaining flow cytometric analysis was performed as described in Bagot et al.","1998 (Blood 91:4331-434 1) and in Poszepczynska et al.","2000 (Blood 96: 1056-1063).","FIG.","10 illustrates the biochemical analysis of p140 molecules: the NK clone AM61 (p140+/p70−), derived from a healthy donor, and the cell lines Pno and Cou-L were surface labeled with biotin and immunoprecipitated with anti-p140 and anti-CD8 mAbs.","Samples were treated (+) or not (−) with N-glycosidase F and analyzed in an 8% (left panel) or 11% (right panel) SDS-PAGE under reducing conditions.","Sepharose Protein-A alone represents the negative control.","Molecular weight markers (kD) are indicated on the right.","FIG.","11 shows the amino acid sequence alignment of KIR3D cl.","24 (SEQ ID No.","2) and cl.","1.1 (SEQ ID No.","4) encoded proteins.","Amino acids corresponding to the signal peptide are in small case letters, while transmembrane region is underlined in the consensus sequence.","Amino acids identical to the consensus sequence are indicated by dots.","EXAMPLE 1 SC5 Isolation, SC5 Biochemical and Functional Characterization, Production of Anti-SC5 mAbs T lymphocyte immune responses are regulated by functional cell surface molecules providing positive signals that lead to the expansion of antigen-specific clones, and negative signals which prevent excessive stimulation or responsiveness to self antigens.","The activation of resting T lymphocytes requires two independent signals.","The signal provided by the engagement of T-cell receptor (TCR) must be accompanied by a second positive signal in order to result in optimal T cell response.","CD28 is considered as the major co-stimulatory receptor inducing T lymphocyte activation and IL-2 synthesis and preventing cell-death.","Recent studies demonstrated that CD3/TCR stimulation leads to a redistribution of the detergent-insoluble glycolipid-enriched membrane fraction (DIG) or raft, which results in the aggregation of TCR/CD3 and DIG-associated signal-transducing molecules.","CD28 was reported to enhance raft redistribution to the site of TCR engagement.","Further, a series of other molecules may provide co-stimulatory signals to T cells, acting at different time points, affecting particular subsets or promoting distinct effector functions.","Negative regulation of T lymphocyte responses can be mediated by CD152-induced signals or by apoptosis through the members of TNF receptor superfamily.","Recently, a novel type of negative regulation has been reported for subsets of NK cells and CD8+ T lymphocytes (Lan998, Annu.","Rev.","Immunol.","16: 359-393).","The immunoglobulin-like (KIRs) or C-type lectin structures responsible for these signals prevent unwanted effector functions such as activation of CTL activity or cytokine production.","We now describe a novel 96 kD transmembrane receptor, SC5, which delineates a minor subset of peripheral blood lymphocytes in normal individuals.","Anti-SC5 mAb stained mainly CD45RO+ lymphocytes, from both CD8 and CD4 subsets, as well as NK lineage cells.","We found that SC5 was predominantly located in the intracellular compartment of resting T lymphocytes and its surface membrane expression increased rapidly after cell activation.","SC5 molecule engagement inhibited the anti-CD3 mAb-induced proliferation of resting T lymphocytes or T cell clones, whereas it had no effect on the cytokine-induced proliferation of these cells.","At the same time, the effector cytotoxic activity mediated by CD8+ T cell clones was not altered by anti-SC5 mAb.","Further on, we observed that the percentage of SC5+CD4+ circulating lymphocytes was significantly increased in Sézary syndrome (SS) patients as compared to peripheral blood lymphocytes of normal individuals.","Importantly, we were able to clearly demonstrate that Sézary cells express SC5 molecules.","In addition, we found that ligation of SC5 molecules in a cutaneous T cell lymphoma (CTCL) cell line resulted in a strong inhibition of the malignant cell proliferation induced by anti-CD3 mAbs.","Thus, SC5 molecule expression can serve to detect the presence of circulating malignant CD4+ cells in SS patients.","Moreover, the identification of a cell surface molecule providing negative signals upon ligation constitutes a new tool to investigate the mechanisms of pathological T cell growth and could be used to prevent it.","Material and Methods Production of Anti-CS mAb SC5 mAb was obtained by immunizing 6 wk old Balb/c mice with the NK cell line Ytindi, using an established protocol (David et al.","1990; J. Immunol.","144: 1-6).","Hybridoma supernatants were screened for selective reactivity with the immunizing cell line and a CTCL cell line termed Pno (Poszcepczynska et al.","2000; Blood 96: 1056-1063) by indirect immunofluorescence and flow cytometry.","Pno cell line derives from CTCL cells from a patient which had a CD3+ Vβ22+ CD4+ CD8αα+ CD25-phenotype, and which proliferates in response to IL-7 and to anti-CD3 mAb stimulation.","Any CTCL cell line is appropriate for this screening.","Examples of other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).","The reactive supernatants were further tested for their reactivity with peripheral blood lymphocytes (PBL).","A hybridoma supernatant was selected for its reactivity with both tumoral cell lines and with a minor PBL sub-population.","The hybridoma was cloned twice and cloned hybridomas were passaged into pristane-primed Balb/c mice to produce ascites.","The antibody isotype was determined as IgM and termed anti-SC5 mAb.","The ascites was dialysed against PBS, sterilized by ultrafiltration and further utilized at final dilution {fraction (1/200)}.","This hybridoma has been deposited on Oct. 30, 2000 at the C.N.C.M.","(Collection Nationale de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty; the C.N.C.M.","deposit number is I-2575.In this example, “anti-SC5 mAb” refers to the mAb produced by hybridoma I-2575.Patients After informed consent and approval by an ethic committee (CCPPRB, Hôpital Henri Mondor, Creteil, France), we obtained blood samples from eleven patients with CTCL.","Eight patients had a Sézary syndrome with 10 to 80% circulating CD3+, CD4+, CD8-Sézary cells, whereas three patients had a transformed mycosis fungoides (MF) and presented with disseminated skin tumors with a CD3+, CD4+, CD8-phenotype.","All patients were not previously treated with chemotherapy.","Cells and Cell Lines Peripheral blood mononuclear cells (PBMC) were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.) density gradient centrifuigation.","Human T cell clones GDS.3 (CD3+TCRαβ+CD4+CD8−), DS6 (CD3+TCRγδ+CD4−CD8−), LSO (CD3+TCRγδ+CD4−CD8−) and JF1 (CD3+TCRαβ+CD4−CD8+), described in Bensussan et al.","1984 (J. Exp.","Med.","159: 947-952) and Yssel et al 1984.(J.","Immunol.","Methods 72: 219), were fed each 8 days with irradiated allogenic PBMC in culture medium consisting of RPMI 1640 (GIBCO, Paisley, UK), 2 mmol/L L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), 10% heat-inactivated human serum, 50 IU/ml recombinant interleukin-2 (rIL-2, Sanofi Synthélabo, Labège, France) and 1 μg/ml phytohemagglutinin (PHA), (Wellcome, Beckenham, UK).","Functional studies were performed at day 7 after the feeding.","Standard human leukemic cell lines were mycoplasma-free and maintained in logarithmic growth in complete RPMI medium supplemented with 10% fetal calf serum (FCS) and antibiotics.","The human CTCL cell clone, Pno, was established from peripheral blood of a Sézary patient and maintained in culture as previously described (Poszcepczynska et al.","2000; see supra).","Pno cell line has a CD3+Vβ22+CD4+CD8αα+CD25-phenotype and proliferates in response to IL-7 and to anti-CD3 mAb stimulation.","Other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).","Monoclonal Antibodies and Flow Cytometry Studies Indirect immuno-fluorescent staining was performed with hybridoma supernatants or ascites fluid using FITC-conjugated goat anti-mouse Ig from Caltag laboratories (San Francisco, Calif.).","For two-color analysis the immuno-fluorescent staining was performed by incubating 3×105 cells with anti-SC5 mAb for 30 min at 4° C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgM (Caltag laboratories) followed by a second PE-, ECD- or TRI-conjugated specific mAb of IgG isotype.","Stained cells were analyzed using a single argon flow cytometer analyser (Epics XL, Bekman-Coulter, Miami, Fla.) as described in Schiavon V et al.","1999; Tissue Antigens 53: 23-32.For intracellular labeling, cells were fixed in PBS 4% p-formaldehyde for 20 min at 4° C., washed, and then permeabilized with staining buffer containing 0.1% saponin.","Conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD69, and anti-TCRVβ22 mAbs were purchased from Immunotech (Marseille, France).","Other mAbs are available from Beckman Coulter (see Cell Analysis 2000 catalogue): anti-CD3 (PE) Cell Analysis 2000 catalogue reference IM1282; purified anti-CD3 Cell Analysis 2000 catalogue reference IM0178; Cell Analysis 2000 catalogue reference anti-CD4 IM0449; anti-CD8 Cell Analysis 2000 catalogue reference IM0452; anti-CD45RO Cell Analysis 2000 catalogue reference IM1307; anti-CD69 Cell Analysis 2000 catalogue reference IM1943; anti-CD94 Cell Analysis 2000 catalogue reference IM2276; anti-CD158a Cell Analysis 2000 catalogue reference IM2277; anti-CD158b Cell Analysis 2000 catalogue reference IM2278; anti-NKG2a Cell Analysis 2000 catalogue reference IM3291; anti-BY55 Cell Analysis 2000 catalogue reference IM2745.Immunoprecipitation of Biotinylated Surface Molecules.","2×107 cells were washed twice in PBS and surface-labeled with Sulfo-NHS-LC biotin (Pierce Europe, Interchim, Montlucon, France) (2 mg/mL in PBS) for 20 min at 4° C. After quenching for 20 min with RPMI 1640, cells were washed twice in PBS and re-suspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 1 h at 4° C. Postnuclear supernatant was then incubated for 2 h at 4° C. in a 96-well plate (Maxisorp Nunc immuno-plate) pre-coated with goat anti-mouse IgG+IgM (Caltag laboratories) followed by anti-SC5 mAb or anti-TCRβ (anti-TCRβ C305 isotype matched mAb, Cell Analysis 2000 catalogue reference IM1466; anti-TCRVbeta22, Cell Analysis 2000 catalogue reference IM2051; anti-TCRzeta, Cell Analysis 2000 catalogue reference IM3169; all from Beckman Coulter).","Immuno-precipitates were washed 4 times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF) and precipitated proteins were subjected to SDS-PAGE.","Western blot analysis was performed using streptavidin-peroxidase (Immunotech) and the ECL detection system according to manufacturers' recommendations (Amersham Pharmacia, Orsay, France).","Proliferation Assays For lymphocyte activation, PBMC were cultured in medium containing 15% human serum in the presence of 1 μg/ml PHA (Wellcome, Beckenham, UK).","For proliferation assays, 5×104 cells were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium.","When needed, the plates were pre-coated with anti-CD3 mAb at the indicated concentrations as previously described (Poszcepczynska et al.","2000, see supra).","For stimulation of CTCL cell line Pno, pre-coated anti-CD3 mAb was used in combination with 2 ng/ml phorbol 12 β-myristate 13 α-acetate (PMA, Sigma Biochemicals).","Cells were cultured for 4 days and were pulsed with 1 μCi of 3H[TdR] during the last 8-16 h of culture.","3H[TdR] incorporation was measured in a liquid scintillation counter (Topcount; Packard Instrument Co, Meriden, Conn.).","For the determination of IL-2 production, 105 PBL were stimulated with immobilized anti-CD3 mAb in the presence of anti-SC5 or control mAb.","After 32 hours of culture, 100 μL of supernatant was added to 105 cells from an IL-2-dependent T cell clone.","CD3-Induced Redirected Cytotoxicity Assays The redirected cytotoxicity assay was carried out as described in Le Cleach et al.","2000, Clin.","Exp.","Immunol.","119: 225-230.The target murine mastocytoma P815 tumor cells were loaded with 100 μl 51Cr (2.5 mCi/mL) for 90 min at 37° C., washed and incubated with anti-CD3 mAb for 15 min at room temperature.","Effector cells were likewise pre-incubated with anti-SC5 mAb (1:200 final dilution of ascites) and added to target cells in ratio 5:1, in a final volume of 150 μl in 96 well V-bottomed microtiter plates for 4 h. After centrifugation 100 μl aliquots were counted in a gamma-counter to determine 51Cr release.","The spontaneous release was always less than 20% from the maximum release (target cells with 1% NP40).","Percentage of specific 51Cr release was calculated as described in David et al.","1987, J. Immunol.","138: 2831-2836.Statistical Analysis Statistical analysis of the results was performed with Statistica software vers.","5.0 (StatSoft, Los Angeles, Calif.).","Significant differences between SC5 expression in patients and control group were evaluated by the Mann-Whitney U test, and the correlation between the percentage of SC5+CD4+ cells and the percentage of malignant cells in patients peripheral blood was evaluated by the Spearman Rank Order Correlations test.","Results The results herein reported have been obtained with the CTCL cell line Pno, and with CTCL cells collected from patients.","Equivalent experiments and results can be performed and obtained with any CTCL cell line, such as e.g.","HUT 78 (ATCC TIB-161).","Expression of SC5 Molecules in Normal Resting and Activated Peripheral Blood Lymphocytes Monoclonal antibodies raised against the functional tumor cell line Ytindi were analyzed for their simultaneous reactivity with the CTCL cell line Pno (Poszcepczynska et al.","2000, see supra) and normal peripheral blood T cells.","The SC5-specific mAb, of IgM isotype, stained both the immunizing and CTCL cell lines.","Among peripheral blood lymphocytes, anti-SC5 mAb delineated a small sub-population (see FIGS.","1A, 1B, 1C).","The SC5 mAb reactive molecule was expressed by a variable percentage of CD3+ T cells, never exceeding 20% (mean=10.5%, SD±5.6).","Both CD4+ and CD8+ lymphocytes were stained by anti-SC5 mAb.","A sub-population of peripheral blood CD56+ NK cells was also reactive with anti-SC5 mAb, though with more important inter-individual variations (mean=19.4%, SD±11.4); see the below Table 1.TABLE 1 expression of SC5 in peripheral blood lymphocyte sub-populations Cell population N tested mean (%) SD Total Ly 15 14.1 6.8 CD3+ Ly 10 10.5 5.6 CD4+ Ly 8 9.4 5.1 CD8+ Ly 8 14.8 5.1 CD56+ Ly 10 19.4 11.4 A representative example of SC5 co-expression with CD3 or CD56 molecules on gated lymphocytes from PBL of normal donors is shown in FIG.","1A.","It should be noted that most SC5+ lymphocytes belonged to the activation/memory pool, as demonstrated in FIG.","1B.","Over 70% of the SC5-positive cells co-expressed CD45RO.","As SC5 molecule expression was detected in all IL-2 dependent T lymphocyte clones tested, we verified whether its cell surface expression was induced during T lymphocyte activation.","We found that stimulation of PBL with PHA promptly increased the expression of SC5 on CD3+ lymphocytes.","Both the percentage of SC5+ T cells and the level of SC5 expression rose very rapidly after stimulation (see FIG.","2).","As early as 4 hours after stimulation with PHA, the percentage of SC5+CD3+ cells increased two to threefold and reached a maximum after 24 to 48 hours.","In the course of 5 to 7 days of in vitro stimulation SC5 expression returned to the basal level.","In parallel we studied the expression of the very early activation antigen CD69 and we observed similar kinetic profiles of CD69 and SC5 on CD3+ lymphocytes.","It should be noted, that unlike CD69 molecules, low levels of SC5 were already present on circulating PBL before stimulation (FIG.","2) Activation-induced antigen expression results either from de novo protein synthesis or cell surface export of preexisting intracellular molecules.","Therefore, we studied the expression of SC5 in non-activated permeabilized PBL.","As shown in FIG.","3, anti-SC5 mAb stained over 90% of permeabilized lymphocytes vs. 16% of the same non-permeabilized cells.","The specificity of anti-SC5 mAb staining was confirmed by using the isotype-matched mAb BY55 which detects a GPI-anchored cell surface structure expressed by a subset of circulating lymphocytes (see Agrawal et al.","1999, J. Immunol.","162: 1223-1226).","A comparable percentage of BY55+ cells was found in permeabilized and non-permeabilized lymphocytes.","As a positive control for the detection of an intracellular epitope, we used an anti-CD3ζ chain mAb which only labeled permeabilized T lymphocytes.","Thus, the surface expression of SC5 is dynamically regulated by the activation status of cells and its induction during T cell activation is due to the enhanced transport of the molecule from an intracellular pool to the surface membrane.","Biochemical Characterization of SC5 Antigen In order to characterize the molecular weight of the structure identified by anti-SC5 mAb, an IL-2 dependent TCRγδ+ clone, termed DS6, was surface labeled with biotin and the cell lysates were immun-precipitated with either anti-SC5 mAb or an isotype-matched anti-TCRβ mAb (as negative control).","Anti-SC5 mAb recognized a single molecule with apparent molecular weight of 96 kD under reducing conditions (see FIG.","4).","As tumor cell lines (the immunizing Ytindi cells) are often characterized by aberrant expression of carbohydrate epitopes (20, 22), we studied the reactivity of anti-SC5 mAb with the SC5+ NK cell line NK3.3 and with the CTCL cell line HUT78 (ATCC TIB-161) after treatment with neuraminidase or sodium periodate in concentrations destroying known sialylated epitopes (digestion of CD75 epitope on Raji cells was used as a positive control).","Expression of SC5 was not affected by neuraminidase or sodium periodate treatment.","Thus, we excluded a possible carbohydrate nature of the epitope.","Inhibition of Tile Anti-CD3 mAb-Induced T Lymphocyte Proliferation by Anti-SC5 mAb In order to examine the possible function of SC5 molecule in normal T cells, we studied the effect of anti-SC5 mAb alone or during the proliferative responses induced by immobilized anti-CD3 mAb.","We found that anti-SC5 mAb alone or in combination with PMA did not induce the proliferation of peripheral blood T lymphocytes.","Interestingly, when soluble anti-SC5 mAb was present together with immobilized anti-CD3 mAb, a significant inhibition of lymphocyte proliferation was observed.","The inhibition varied between 33 and 66% at day 4 depending on the donor as compared to the effect of an isotype-matched irrelevant control mAb (see FIG.","5A, upper panel).","As SC5 molecule is expressed on monocytes, it was important to determine whether the inhibitory effect was monocyte-independent.","We studied the proliferation of two T cell clones, GDS.3 and LSO, stimulated by immobilized anti-CD3 mAb in the presence of anti-SC5 mAb or an isotype control mAb.","The results obtained with both T cell clones indicated that engagement of SC5 molecules resulted in an inhibition of the proliferation to anti-CD3 mAb (see FIG.","5A, lower panel).","It should be noted that the inhibitory effect of anti-SC5 mAb on T cell proliferation depended on its concentration, as well as on the concentration of the agonistic anti-CD3 mAb.","The inhibition obtained with anti-SC5 mAb was significant only when T cells were stimulated with optimally diluted anti-CD3 mAb (see FIG.","5B).","Such inhibitory behaviour was also reported for anti-KIRs mAbs (Cambiaggi et al.","1999, Blood 94: 2396-2402).","The decrease of T cell proliferation following SC5 engagement could be due to a direct induction of cell death, a perturbation of the IL-2R expression, or the inhibition of cytokine synthesis.","We found that anti-SC5 mAb did not inhibit the IL-2-dependent proliferation of PBL or T cell clones (FIG.","5A).","Furthermore, the addition of rIL-2 to T cells pre-incubated with anti-SC5 mAb (ascites diluted to {fraction (1/200)}) in combination with anti-CD3 for 48 h restored their proliferation.","This would not be the case if the specific mAb inhibited IL-2R expression or caused cell death.","Finally, in order to prove that SC5 triggering may inhibit cytokine production, we compared the amount of IL-2 secreted by anti CD3-activated PBL in the presence or absence of anti-SC5 mAb, using an IL-2 dependant T cell line.","The quantity of IL-2 produced in the presence of anti-SC5 mAb represented 40 to 65% of the control values obtained with an isotype-matched antibody (see FIG.","5C).","Thus, the simultaneous engagement of SC5 and CD3 molecules in T cells results in the decrease of cytokine production.","We next studied the effect of anti-SC5 mnAb on T cell effector cytotoxic activity using an anti-CD3 mAb redirected killing assay against the FcgR+ murine cell line P815.FIG.","5D shows a representative experiment performed with a cytotoxic T cell clone at an E/T ratio of 5/1.The CD8+ T cell clone JF1, expressing high amount of SC5 molecules, was induced to kill murine target cells following anti-CD3 mAb is stimulation.","The simultaneous targeting of SC5 by its specific mAb did not influence JF1 cytotoxic activity, regardless of the E/T ratio or the concentration of stimulating anti-CD3 mAb.","Expression of SC5 Molecules on Malignant Lymphocytes Obtainedfrom Patients with Sézary Syndrome As we initially screened anti-SC5 mAb for its reactivity with a CTCL cell line, we further studied the expression of SC5 molecule in the periphcral blood lymphocytes isolated from 8 patients with Sézary syndrome.","Two-color flow cytometry analysis showed that expression of SC5 was significantly increased in the CD4+ subset (see representative results obtained with one patient in FIG.","6A).","On the average, 35.5% of the CD4+ lymphocytes in SS patient blood expressed SC5 as compared to 9.4% in healthy controls (p<0.001), (FIG.","6B).","In the 8 SS patients studied, the expression of SC5 on CD4+ cells correlated with the percentage of circulating malignant cells detected by cytomorphology (r=0.91, p=0.0012).","It should be noted that in these patients the percentage of total CD4+ cells (mean=88%, SD±8.8) was higher than that of Sézary cells (mean=26%, SD±12.8).","We also studied SC5 molecule expression in peripheral blood samples from 3 MF patients without blood involvement.","No significant difference in the levels of SC5 expression on peripheral blood CD4+ cells was observed between MF patients and healthy donors (FIG.","6B).","Still, an increased percentage of SC5+CD4+ was detected in the skin samples from two of these patients, that we were able to to test (26% and 19% respectively).","In one SS patient we had previously demonstrated that the circulating malignant cells were clonal lymphocytes with a CD4+TCRVβ22+ phenotype (Poszcepczynska et al.","2000, see supra).","Here we demonstrated that SC5 was co-expressed on the TCRVβ22+ cells, i.e.—by the malignant cell population (FIG.","7A).","It should be noted that the profile of SC5-mAb binding to TCRVβ22+ cells corresponded to a weak homogeneous expression on the whole malignant clonal cell population (FIG.","7B).","Interestingly, the malignant TCRVβ22+ cells did not co-express common activation markers as CD69 (FIG.","7C), CD25 or CD30 as previously described (Poszcepczynska et al.","2000, see supra).","Thus, the increased levels of SC5 in SS PBL were mostly due to the expression of the antigen on circulating malignant T cells.","Inhibition of the Anti-CD3mAb-Induced CTCL Cell Line Proliferation by Anti-SC5 mAb We next investigated whether SC5 molecules were able to provide a negative signal in malignant CTCL cells.","We used the long-term cultured Pno CTCL tumor T cell line which strongly proliferates in the presence of IL-7.As previously reported that Pno cell line expresses functional T cell receptors, since immobilized anti-CD3 mAbs or the combination of soluble anti-CD3 mAbs and PMA induced significantly their proliferation (Poszcepczynska et al.","2000, see supra).","We examined the effect of SC5 molecule engagement on the proliferation of Pno cells.","FIG.","8 shows that anti-SC5 mAb strongly inhibited the anti-CD3 mAb-induced proliferation of the CTCL cell line.","In contrast, PMA alone (2 ng/mL) produced a low level of proliferation which was not significantly affected by anti-SC5.Notably, anti-SC5 mAb had no effect on the IL-7-dependent proliferation, since Pno cells incubated with IL-7 in the presence of anti-SC5 mAb proliferated as vigorously as in the presence of the isotype control mAb.","Thus, we concluded that SC5 antigen is functional in CTCL malignant cells and may specifically and profoundly inhibit their proliferation triggered through the TCR/CD3 signaling pathway.","A CTCL tumor was then successfully grafted onto transgenic mice having a substantial deficiency in functionally active NK cells and T lymphocytes (SCID mice) as described in Charley et al.","(1990) “Establishment of a human cutaneous T cell lymphoma in CB-17 SCID mice” J.","Invest.","Dermatol.","94: 381-384 (see also U.S. Pat.","No.","5,530,179 Cornelius P. Terhorst and Baoping Wang for obtention of SCID mice).","Anti-SC5 mAb was intraveneously or intracutaneously administered at different times to half of the mice at various concentrations of purified mAb per gram of mouse, whereas the other half received the same amount of purified CD56 (isotype IgM) as a control.","PBL and cutaneous cells were collected for standard cytological observations of infiltrating large T cells, and the percentage of CTCL cells was assessed for each mouse of the experiment.","This procedure is an example of procedure that enables the is skilled person to check that anti-SC5 compounds, such as the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M., are capable of inhibiting the in vivo proliferation of human CTCL.","Discussion A number of receptors with dynamic surface expression mediate T cell interactions with other immune cells or cytokines and, consequently modulate T cell immune responses.","In this study, we identified a novel 96 kD functional molecule expressed by a minor subset of PBL whose surface expression is induced rapidly during in vitro T-cell activation.","SC5 is distinct from the established molecules with similar molecular weight and/or T cell modulatory functions.","First, SC5 was induced very early upon T cell activation, with important increase of its surface expression detected after 4 h. A similar kinetics has been observed for the very early activation antigen CD69, which is detected on the cell surface between 2 and 4 h after stimulation.","Most established T-cell specific inducible receptors such as 4-1BB, OX-40 LIGHT or CD152 (CTLA-4) appear on T-cell surface between 10 to 24 h following stimulation.","Further, SC5 expression is not T cell restricted.","The antigen was detected on sub-populations of NK and B lymphocytes, monocytes and granulocytes.","Such a wide distribution pattern is typical of certain killer cell Ig-like receptors (KIRs), as the recently described AIRM1 and IRp60, which are found on NK cells, T and B lymphocytes, myeloid and other antigen-presenting cells.","The family of immunoglobulin—like transcript (ILT) inhibitory receptors has also a pan-leukocyte type of expression, the individual receptors being detected on different NK, T, B and myeloid sub-populations.","Moreover, ILT4 has a molecular weight very close to SC5.However, neither the expression of AIRM1 and IRp60, nor of ILTRs depends on the activation status of cells.","The prompt induction of SC5 molecule at the surface of activated T cells followed by its down-regulation may result from trafficking of intracellular molecules to the cell membrane and vice versa, as we detected SC5 in most non-activated T lymphocytes after cell permeabilization.","Such a regulatory mechanism has been already reported for another T cell receptor, CD152.Further on, a heterogeneous family of lysosome associated membrane proteins (LAMP) exists, including molecules involved in endocytosis, lysosomal trafficking and secretion from intracellular granules and that may be induced on the surface of monocytes, neutrophiles, NK, B or T cells upon activation.","Additional studies on the intracellular localization and transport of SC5 would precise its proper role in T cell activation and functions.","Our studies rcvealed that triggering of SC5 by its specific mAb inhibited the anti-CD3 mAb-induced T cell proliferation.","This inhibitory effect required an optimal anti-CD3 mAb stimulation, which is necessary to induce maximal SC5 surface expression in PBL (see FIG.","5B).","A 1:200 final dilution of anti-SC5 ascites significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram par well and this effect of inhibition increases with lower anti-CD3 mAb concentrations −1 microgram par well; 0.5 microgram per well—A 1:400 final dilution of anti-SC5 ascites do not significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram per well, but inhibition is significant when a lower concentration of anti-CD3 is used −1 microgram per well; 0.5 microgram per well—The person of ordinary skill in the art can proceed with such adjustments by standard interpolation from the particular results described herein, or from his own trials.","At the same time, the inhibitory.","threshold posed by SC5 engagement could be bypassed by excessive anti-CD3 mAb stimulation.","It should be noted that the inhibitory functional effects obtained by ligation of SC5 molecules were observed in an APC-independent system.","Further experiments are needed to determine whether SC5 stimulation may have different effects on the proliferation of APC or allo-stimulated T cells.","Recently, the negative regulation of immune responses has been extensively investigated, in particular in T lymphocytes and NK cells.","The best studied T cell specific inhibitory receptor, CD152 (CTLA-4), is a typical activation-induced antigen, functionally coupled to CD28 co-stimulatory receptor.","Indeed, CD152 may overcome CD28 positive signals at low antigenic concentrations and prevent immune response.","Alternatively, it may downregulate and terminate T-cell proliferation induced through CD28 by affecting IL-2R expression and IL-2 synthesis.","Similarly to CD152, SC5 is an intracellular molecule whose surface expression is regulated by the activation status of cells.","Both receptors mediate their effects by affecting IL-2 synthesis.","The identification of SC5 specific ligands and signaling pathways will precise the temporal and spatial correlation between these negative T-cell regulators.","In consideration to its wide cell distribution, SC5 functional effects are probably not restricted to T cells.","In several aspects, SC5 is comparable to NK cell inhibitory receptors, equally expressed by T cells and other lymphocytes sub-populations.","Both KIRs and CD94/NKG2 receptors were shown to inhibit antigen-specific and superantigen-driven activation of TCRαβ and TCRγδ T cells, including cytotoxicity, proliferation and cytokine secretion.","The KIR-induced inhibition depends on the activation status of T cells and the potency of T cell stimulation and may be bypassed under specific in vivo conditions.","However, SC5 is definitely distinct from KIRs, since we demonstrated that it selectively inhibited the proliferation of anti-CD3 mAb stimulated T cells without affecting their effector cytotoxic functions.","An important characteristic of SC5 molecule is its significantly increased expression in peripheral blood lymphocytes of patients with SS in comparison to healthy subjects.","SS is characterized by the generalization of a primary epidermotropic malignant T cell clone with mature phenotype.","Actually, none of the established T cell functional receptors conclusively identifies peripheral blood SS tumor cells.","MF/SS has been considered as the expansion of a T cell subset with a particular phenotype, CD4+CD7−, corresponding to a normally existing T memory subset.","However, a recent study demonstrated that the CD4+CD7− population does not always represent the dominant T cell clone in SS patients (Dummer et al.","1999, Arch.","Dermatol.","Res.","291: 307-311).","SS is regarded as a proliferation of specifically activated T cells although the nature of the stimulus is not known (Fargnoli et al.","1997, Leukemia 11: 1338-1346).","For this reason the distinct expression of activation induced antigens on SS and normal T cells is of special interest.","Established activation antigens such as CD69, CD25, CD30 are not consistently detected on Sézary cells, as confirmed by our own investigations.","Expression of CD25 rather correlates with the progression of the disease and the transformation of MF/SS cells into a large cell variant in a minority of the cases.","An increased rate of expression of T cell activation molecules as HLA-DR, CD25 or CD71 in SS has been primarily associated with reactive tumor-infiltrating T cells.","A 78 kD very late activation antigen, BE2, was reported to be specifically increased in SS peripheral blood, while not detected on normal T cells.","In fact, BE2 was not consistently detected in all SS patients studied, a comparatively low percentage of peripheral blood cells in relation to the reported percentage of malignant cells were BE2-positive, and BE2 was equally detected in patients without clinical evidence of blood involvement.","In contrast, SC5 was detected in 8/8 SS cases studied.","Although the percentage of malignant cells determined by cytomorphology varied considerably from patient to patient (10-80%), the expression of SC5 on CD4+ cells correlated with this percentage.","Finally, no elevated expression of SC5 on CD4+ cells was detected in MF patients without blood involvement.","Further, we demonstrated that Sézary cells identified by the expression of the clonotypic TCRVβ chain co-expressed SC5 molecule, confirming that increased levels of SC5 in the peripheral blood of SS patients were due to its expression on the malignant T cell clone.","A very important observation was that SC5 triggering on Sézary cells induced down-modulation of the CD3-mediated proliferation of tumor cells, analogous to the inhibition observed in normal T cells.","It has been already demonstrated that malignant CTCL lymphocytes preserve certain functional properties as they may be activated through basic signaling pathways although producing aberrant profiles of cytokines.","We now demonstrate that an inhibitory pathway is preserved in CTCL tumor cells, that may permit to counterbalance their proliferation.","Notably, the inhibitory effect observed after anti-SC5 mAb binding on anti-CD3 mAb-stimulated CTCL Pno cells was much more important than in normal T cells.","Anti-SC5 mAb does not seem to directly mediate cell death of neither normal nor Sézary T cells, since it does not modulate the rIL-induced proliferation.","Moreover, T-cells that had been cultivated in the presence of anti-SC5 mAb can be re-stimulated by addition of rIL-2.At the same time, we observed a diminished IL-2 production by anti-SC5 mAb-treated T cells.","which should account at least partially for the inhibition of their proliferation.","Inhibitory effects of functional receptors on the proliferation of malignant cells have been already observed.","It has been recently reported the inhibition of the proliferation of normal and leukemic myeloid cells mediated by the homologous NK cell sialoadhesin receptor AIRMI and CD33 myeloid-specific antigen (Vitale et al.","1999, Proc.","Natl.","Acad.","Sci.","USA 96: 15091-15096).","Unlike SC5, these receptors inhibited proliferation in the presence of growvth factors and most probably by induction of apoptosis.","Interestingly, another T cell activation antigen, CD30, expressed by a subset of normal CD45RO+ T cells, is likely to mediate the regression of primary CTCL other than MF/SS.","This effect is related to the natural ligand of CD30 (CD30L) which is co-located within the cytoplasm of malignant cells and may induce cytolytic cell death independently of Fas/FasL system (Mori et al.","1999, Blood 94: 3077-3083).","In this aspect, the identification of SC5 ligand will enlighten the mechanism of its inhibitory action.","In conclusion, we have identified an early activation antigen which is distinct, by phenotypic, biochemical and functional criteria, from the established T lymphocyte receptors.","As it was underlined, SC5 molecule is expressed by a sub-population of the post-activation/memory peripheral blood T cell subset.","Likewise, continuous expression of KIRs is detected on minor T cell clones, and this expression seems to depend on TCR-occupancy by specific antigens.","It was recently proposed that such clones might be auto-reactive and maintained tolerant by KIRs signaling.","We may speculate that SC5 surface expression is also preserved on auto-reactive T cells, suggesting that Sézary syndrome tumor cells possibly originate from such clones.","Importantly, the expression of SC5 on CTCL malignant cells will serve to better define their origin and the nature of the signal driving mature T cells to uncontrolled proliferation.","In addition, the inhibitory effect of anti-SC5 mAb on tumor T cells constitutes a novel therapeutic approach in patients with advanced CTCL.","Furthermore, expression of SC5 has also been observed at the surface of CD8+ CTCL, such as CD8+ transformed MF.","EXAMPLE 2 P140 is Expressed by CD4+ CTCL Cells, and Identification of a Novel P140 Allelic Form Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin.","Mycosis fungoides (MF) is characterized by skin invasion of clonally-derived malignant CD4+ T lymphocytes that phenotypically resemble mature T helper cells.","A more aggressive form of CTCL develops when the is malignant cells become non-epidermotropic and is associated with extra-cutaneous involvement.","Sézary syndrome (SS) is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.","The biology of the disease remains poorly understood, as it is difficult to identify the malignant cell, due to the lack of specific cell surface markers.","Thus, in cutaneous lesions it is difficult to distinguish CD4+ CTCL cells from reactive infiltrating CD4+ T lymphocytes.","Bagot et al.","previously described a unique CD4+ T cell line derived from CTCL lesions (Bagot et al.","1998, Blood 91: 4331-4341); this cell line and the in vivo tumor cells expressed an identical size of the complementarily-determining region 3 of TCR-Vβ transcripts.","More recently, Poszepczynska et al.","(Blood 2000, 96: 1056-1063) functionally characterized an IL-7-dependent CD4+CD8αα+ tumor T cell line isolated from the blood of another patient with a cutaneous erythrodermic CTCL.","This tumor T-cell line was identical to the major circulating T cell populations, as demonstrated by the expression of TCR-Vβ22 and the identity of the TCRβ-VDJ sequences.","Here, we show that these two different CTCL lines express the p140/KIR inhibitory receptor for HLA-A alleles.","Importantly, this receptor is detected on freshly isolated tumor cells derived from the same patients.","Moreover, the p140/KIR was also co-expressed by a major subset of CD4+ lymphocytes in seven other patients with SS, and by tumor skin CD4+ cells in two additional patients with advanced MF.","The p140/KIR is detected in normal individuals on a minor NK cell-subset and on rare peripheral blood CD3+CD8+ cells.","Moreover, T cells obtained from skin in other dermatological diseases such as inflammatory skin diseases and toxic epidermal necrolysis did not express this receptor (Le Cleach et al.","2000, Clin.","Exp.","Immunol.","119: 225-230).","Thus, our present findings demonstrate that the p140/KIR represents a suitable marker on CD4+ cells for the identification of CTCL.","Materials and Methods Patients After informed consent and approval by an ethic committee, we obtained skin and blood samples from eleven patients with a CTCL.","Eight patients had a Sézary syndrome with 10 to 45% circulating Sézary cells in the blood.","In seven cases, the phenotype of tumor cells was CD3+,CD4+,CD8−.","In one case, the phenotype was CD3+,CD4+,CD8αα+ (patient Pno).","Three patients had a transformed mycosis fungoides (Lez, Cou, Bic) and presented with disseminated skin tumors with a CD3+,CD4+,CD8− phenotype.","All patients were not previously treated with chemotherapy.","Isolation of Tumoral Lymphocytes Fresh CTCL tumor cells were obtained from tumor fragments mechanically dispersed into single-cell suspensions (Bagot et al.","1998, Blood 91: 4331-4341).","The mononuclear cells were then washed and frozen in human serum plus 10% dimethyl sulfoxide for later use.","For Sézary syndrome patients, the mononuclear blood cells were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.).","Long Term Culture of Tumor Cell Lines We established the long term culture of Pno cell line (TCRVβ22+,CD3+, CD4+,CD8αα+,MHC class I+,MHC class II−) in vitro from the peripheral blood of the patient as previously described (Bagot et al.","1998, Blood 91: 4331-4341).","We demonstrated that both the malignant clone circulating in the patient blood and the derived cultured T-cell line were identical for their cell surface phenotype and for their size and sequence of the TCRβ VDJ region (Poszepczynska et al.","2000, cf.","supra).","Other sources of SS cell lines may be found at the ATCC (e.g.","HUT 78 ATCC TIB-161).","The Cou-L cell line (TCRVβ13+) was cultured in vitro with rIL-2 for more than three years.","It corresponds to a subelone of the CD4+ Cou-LS CTCL line previously described (Bagot et al.","1998, cf supra).","The Cou-L cell line, the original TCRVβ13+, CD4+ Cou-LS CTCL, and the tumor cells freshly isolated from the skin shared the same size and sequence of the TCRβ VDJ region (Bagot et al.","1998, cf.","supra).","Monoclonal Antibodies (mAbs) and Flow Cytometry Studies One- and two-color immunofluorescence analysis was performed as previously described (Bagot et al.","1998, cf.","supra).","Anti-CD3 (Cell Analysis 2000 catalogue reference 6604629), anti-CD4 (Cell Analysis 2000 catalogue reference 6602138), anti-MHC Class I (Cell Analysis 2000 catalogue reference IM0107) and II (Cell Analysis 2000 catalogue reference IM0108) are obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue).","The anti-TCRVβ13+ mAb was purchased from Bioadvance (Emerainville, France, catalogue reference TCR 1738), and the anti-TCRVβ22+ mAb was obtained from Beckman-Coulter (Marseille, France, Cell Analysis 2000 catalogue reference IM 1484/IM1365).","The anti-CD8 αβ 2ST8.5H7 mAb is obtainable from Beckman Coulter (Cell Analysis 2000 catalogue reference IM 2014.As anti-p140 mAbs, Q66 (IgM, anti-p140) and AZ158 (IgG2a, recognizing both p70/NKB1 and p140) which have been described in Pende et al.","1996 (J. Exp.","Med.","184: 505-518) have been used; however any anti-140 binding compound is appropriate (e.g.","anti-p140 antiserum, Fc-HLA-A11, Fc-HLA-A3 fuision proteins).","Z27 (IgG1, anti-p70/NKB1), EB6 (IgG1, anti-p58.1/S50.1), GL183 (IgG1, anti-p58.2/p50.2), XA185 (IgG1, anti-CD94), Z199 and Z270 (IgG2b and IgG2a respectively, anti-NKG2A), and B9.4 (IgG2b, anti-CD8) mAbs are all obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue references IM2748, IM2277, IM2278, IM1610, IM2750, IM0102).","Goat anti-mouse IgG FITC and goat anti-mouse IgM PE are also obtainable from Beckman Coulter (see Cell Analysis Catalogue references IM08 and IM0555).","Biochemical Characterization 20×106 cells were incubated (15′ at 20° C.) in 1 ml PBS pH8 containing 250 μg of EZ-LINK SULFO-NHS-LC-LC-BIOTIN (Pierce, Rockford, Ill.) and washed three times in Washing Buffer (10 mM TRIS pH 8, 0.14 M NaCl).","Cells were then lysed in 1% NP-40 and immuno-precipitated with Sepharose-PA (Pharmacia Biotech Inc. Piscataway, N.J.)-coupled AZ158 (IgG2a, antip70/140) or B9.4 ([gG2b, anti-CD8) mAbs.","Samples were analyzed by discontinuous SDS-PAGE either undigested or digested with N-glycosidase F (Boehringer Mannheim, GmbH, Germany) and transferred to Immobilon P (Millipore Corp, Bedform, Mass.).","After staining with Neutravidin (Pierce) the Renaissance Chemiluminescence Kit (NEN, Boston, Mass.)","was used for detection.","NK cell clones were obtained by limiting dilution as described in Pende et al.","1996 (J. Exp.","Med.","184: 505-518).","RT-PCR Analysis Total RNA was extracted from CTCL cell lines Pno and Cou-LCD8 αα using RNA-Clean System (AGS GmbH, Heidelberg, Germany).","cDNA synthesis was performed using oligodT priming.","Primers used for cDNA amplification of the complete ORF (open reading frame) of KIR displaying three Ig-like domains (1395 bp) were the following: 5′CATGT(CT)GCTCA(CT)GGTCGTC (Ig3 UP; SEQ ID No.","5) and 5′ GGTTTTGAGACAGGGCTG (Ig3 DOWN; SEQ ID No.","6).","Amplification was performed for 30 cycles (30 sec.","at 94° C., 30 sec.","at 55° C., 30 sec.","at 72° C.), followed by a 7 min.","incubation at 72° C., utilizing AmpliTAQ (Perkin Elmer-Applied Biosystems, Foster City, Calif.).","PCR products were sub-cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, Calif.).","DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer (Perkin Elmer-Applied Biosystems).","Transient Transfections COS-7 cells were transfected with pcDNA3.1 TOPO-KIR3D cl.24 or with pCR3-cl.1.1 (Pende et al.","1996, J. Exp.","Med.","184: 505-518) utilizing Fugene 6 (Roche, Monza Italy).","Briefly, cells were seeded at 5×105/plate; 24 hr later they were incubated with 6 μg plasmid and 10 μl of Fugene-6 reagent in DMEM/10% FCS.","After 48 or 72 hrs, transfected cells were used for cytofluorimetric analysis.","Cell transfectants were stained with Q66 and AZ158 mAbs, followed by a phycoerythrin-conjugated goat antibody to mouse IgG2a or IgM and analyzed by flow cytometry using a FACSort (Becton Dickinson, San Jose, Calif.).","Results CTCL Cell Lines are Stained by mAbs to the p140/KIR Two long-term CTCL tumor lines Pno (labeled with anti-TCR-Vβ22 mAb) (Poszepczynska et al.","2000, cf supra) and Cou-L (labeled with anti-CD3 mAb) (Bagot et al.","1998, cf.","supra) were analyzed for reactivity with different anti-KIR mAbs.","We found that both cell lines were reactive with mAbs Q66 (see FIG.","9A) and AZ158, both recognizing the p140/KIR.","In contrast, these cell lines did not express other inhibitory receptors specific for HLA class I molecules, including p58.1, p58.2, p70 KIRs and the CD94/NKG2A lectin-like receptor (Poszepczynska et al.","2000, cf supra).","Tumor T Lymph Ocytes Freshly Isolated from CTCL Patients are Stained by Anti-p140/KIR mAbs To determine whether p140/KIR was expressed by freshly isolated tumor cells, we tested the reactivity of Q66 mAb with uncultured tumor cells isolated from the blood in SS patient Pno and from tumoral skin fragments of MF patient Cou.","We found that the majority of tumor cells were stained by this mAb (FIG.","9B).","In particular, we observed that most TCRVβ22+ tumor lymphocytes isolated from the blood of patient Pno were reactive with Q66 antibody, and that most of the TCRVβ13+ tumor lymphocytes isolated from the skin of patient Cou were stained by the same antibody (FIG.","9B).","Next, we studied the phenotype of tumor T lymphocytes from the blood of seven additional patients with a Sézary syndrome, with malignant cells representing 10 to 45% of circulating CD4+ lymphocytes, and tumor T lymphocytes isolated from skin tumors of two other patients with a MF.","Remarkably, all patients tested exhibited a significant population co-expressing CD4 and p140/KIR (see Table 2 below).","TABLE 2 Anti-Q66 mAb stained CD4+ lymphocytes isolated from the skin of patients with transformed mycosis fungoides and from blood of patients with Sezary syndrome.","Percentage of positive cells with Patient anti- anti-CD4 mAb + samples CD4 mAb anti-Q66 mAb MF Lez 65 29 Bic 53 44 SS Bri 85 35 Bar 90 18 Att 95 35 Ros 98 15 Can 78 36 Pet 45 9 Riv 98 19 It should be noted that all Q66+ cells were included in the CD4+ cell population.","Thus, the expression of p140/KIR, which in normal individuals is restricted to subsets of lymphocytes from the NK and CD8+ populations, appears to be a characteristic of CTCL tumor CD4+ T lymphocytes, both in the skin and in the blood.","As control, skin T lymphocytes derived from another dermatological disease, toxic epidermal necrolysis, which were shown to contain small percentages of various KIR expressing T lymphocytes (Le Cleach et al.","2000, Clin.","Exp.","Immunol.","119: 225-230), failed to express p140/KIR.","Molecular Characterization of the p140 Receptor Expressed by CTCL.","The Pno and Cou-L cell lines were surface labeled with biotin and cell lysates were immuno-precipitated with an anti-p140 (AZ158 mAb).","As shown in FIG.","10, this mAb immuno-precipitated from a NK clone and from the Pno and Cou-L cell lines a molecule with a molecular mass of approximately 70 kD under reducing conditions.","Treatment with N-glycosidase revealed a protein backbone of approximately 50 kD with a slightly higher mobility for Pno and Cou-L compared to NK clone.","These data suggested that the p140 inhibitory receptor expressed by these CTCL tumor cell lines could be almost similar to that previously detected on normal NK cells (Pende et al.","1996, J. Exp.","Med.","184: 505-518).","Next, we determined whether the cDNA encoding the molecule recognized by Q66 and AZ158 mAbs on Pno and Cou-L CTCL cell lines corresponded to one of the already described cDNA encoding p140/KIR (SEQ ID No.","3).","To this end, RT-PCR was performed on RNA derived from these cell lines using a set of primers able to amplify all cDNA encoding KIR with three Ig-like domains.","From the Pno cell line, we isolated a full-length cDNA, termed KIR3D cl.24 (SEQ ID No.","1).","Comparison of its nucleotide sequence with DNA sequences coding for all KIR characterized by three extra-cellular Ig-like domains revealed that KIR3D cl.24 represents a novel allelic form of p140 receptor.","In particular, its nucleotide sequence displays five differences compared to the previously described cl.","1.1 cDNA (Pende et al.","1996, J. Exp.","Med.","184: 505-518), resulting in four amino acid substitutions in the mature protein (see FIG.","11; SEQ ID No.","2: clone 24 protein; SEQ ID No.","4: clone 1.1 protein).","Three of the four substitutions are found in the extra-cellular Ig-like domain (pos.","20, 92 and 111 of the mature protein), whereas the other is located in the cytoplasmic region (pos.401).","RT-PCR performed on Cou-L CTCL cell line revealed two different allelic fonns of p140/KIR one corresponding to cl.","1.1 cDNA and the other identical to KIR3D cl.24 (isolated from Pno cell line).","The cDNA derived from the CTCL cell lines were then transiently transfected in COS-7 cells.","As expected, all cell transfectants were brightly stained by Q66 and AZ158 mAb, while they were unreactive with p70/KIR-specific Z27 mAb used as a negative control.","Finally, RT-PCR was performed on RNA extracted from PBL derived from three SS patients (including patient Pno) and from skin-derived T cells of one additional MF patients.","Also in these samples one or another allelic isoform of p140/KIR described above could be identified.","The same results were obtained on malignant CTCL cells collected from patients.","Discussion Skin lesions in CTCL contain a heterogeneous lymphocytic infiltrate composed of malignant T cells, which are most often CD4+, and non-neoplastic tumor infiltrating T lymphocytes.","We previously reported CD4+ cytotoxic tumor infiltrating lymphocytes specifically directed against autologous malignant CTCL CD4+ cell line.","However, as no tumor restricted cell surface structure, besides the clonotypic TCR expressed by tumor cells, has been identified on CTCL, it is difficult using standard methods to distinguish malignant from non-malignant reactive CD4+ lymphocytes.","In the present study, we report for the first time that tumor cells from MF and SS patients express the p140/KIR.","This receptor has been identified in both skin and blood tumor cells from CTCL patients as well as in two long-term culture CTCL lines.","Two color fluorescence analysis indicated that p140/KIR expression is restricted to T cells characterized by a given TCRβ-VDJ previously identified on tumor cells.","Thus, p140/KIR allows rapid identification of tumor cells from tumor reactive cells among the T lymphocyte CD4+ population.","This could be particularly useful in SS patients, in which the tumor T cell clone is not always easily identified within the peripheral blood CD4+ lymphocyte population.","Moreover, during the course of the disease or after treatment, it is crucial to assess whether an increase of the CD4+ population is due to an expansion of the tumor cell population or of reactive T lymphocytes.","Since the anti-p140/KIR mAbs appear to recognize tumor cells in all CTCL patients analyzed, they may be considered as a suitable tool for the direct identification of CTCLs.","In addition, the use of p140/KIR co-expression on CD4+ CTCL cells provides a unique tool to distinguish malignant cells from normal cells in every patient.","This is in contrast to TCR determinations which is unique to an individual patient.","Moreover, the p140/KIR represents a possible target for the development of novel specific immuno-therapy of CTCL.","Previous studies indicated that this receptor was able to generate inhibitory signals upon recognition of HLA.A3 and HLA.A11 alleles.","It is of note, however, that p140/KIR expression in the various patients analyzed appears apparently independent from their own HLA Class I haplotype.","Nevertheless, the actual role of p140/KIR in the patho-physiology of CTCL is an important feature that remains to be studied by taking into account the potential role of this receptor in the tolerance to self.","In conclusion, the present study demonstrates for the first time the expression of the p140/KIR in CD4+ CTCL cells and the isolation of a novel allelic form of this receptor in tumor cells.","This finding is an important new issue, both for the patho-physiology and for the clinical management of CTCL patients.","Furthermore, p140 expression has also been observed at the surface of CD8+ CTCL such as CD8+ transformed MF.","EXAMPLE 3 Production of Anti-CTCL Drugs The person of ordinary skill in the art is enabled to produce anti-CTCL drugs pursuant to the following outlines, given for illustration purposes: use of anti-SC5 or anti-p140 mAb with a cytokine such as Interferon gamma or IL-2 for stimulating the immune system of cells in the vicinity of CTCL cells, use of these anti-SC5 and anti-p140 mAb as vectors for delivering anti-CTCL ingredients onto CTCL cells; examples of anti-CTCL ingredients comprise standard compounds used in chimiotherapy such as radioelements, toxines.","mAb vectors for radioelements can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.","Press O W et al.","“Phase two trial of iode131-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphoma”, Lancet 1995 vol.","346 pp336-340, of which content is herein incorporated by reference).","mAb vectors for toxine can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.","Ghetie U et al.","“Large scale preparation of an immunoconjuguate constructed with human recombinant CD4 and deglycosylated ricin A chain”, J. Immunol.","Methods 1990, vol.","126 pp 135-141, of which content is herein incorporated by reference).","combined use of an antimitotic pro-drug and of anti-SC5 mAb and/or anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form (mitotic agent in a functional form).","Anti-SC5 mAb and anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form can be produced following standard techniques applied to an anti-SC5 mAb of the invention and to an anti-p140 mAb (see e.g.","the ADEPT technique, for Antibody Directed Enzymre Prodrug Therapy, described in Plakey T C et al.","“Anti-tumor effects of an antibody-carboxypeptidase G2 conjuguate in combination with phenol mustard prodrugs”, British J.","Cancer 1995 vol.","72 pp 1083-1088, of which content is herein incorporated by reference).","use of anti-SC5 or anti-p140 mAb as ligand agonists (SC5 stimulation or p140 stimulation) use of anti-SC5 or anti-p140 mAb as complement recruiting agent For the above-mentioned uses, mAbs with a double anti-SC5/anti-CD4 or anti-p140/anti-CD4 specificity are preferred.","use of anti-SC5 or anti-p140 (no CD4+ specificity needed) as activators of ADCC (Antibody Dependent Cell Cytotoxicity): polynuclear cells, macrophages, NK cells bear Fc receptors which are activated by the Fc portion of the anti-SC5 and anti-p140 mAb, these mAb therefore enable the ADCC activation in the vicinity of CTCL cells.","Abbreviations: APC: Antigen Presenting Cell, CTCL: cutaneous T cell lymphoma, DIG: detergent-insoluble glycolipid-enriched fraction, ILT: immunoglobulin-like transcript, KIR: killer cell immunoglobulin receptor, mAb: monoclonal antibody, MF: mycosis fungoides, PBL: peripheral blood lymphocytes, PBMC: peripheral blood mononuclear cells, PHA: phytohemagglutinin, PMA: phorbo112 beta-myristate13 alpha-acetate, SS: Sézary syndrome, TCR: T cell receptor"]],"string":"[\n [\n \"Novel means for the diagnosis and therapy of ctcl\",\n \"A means for the diagnosis and therapy of T lymphomas, in particular Cutaneous T Cell Lymphomas (“CTCL”) are provided.\",\n \"Tumor markers which are universal for CTCL are described.\",\n \"More particularly, a molecule, termed SC5 by the inventors, an allelic form of p140, and biological applications of SC5 and p140 molecules, notably in the diagnosis and therapy of CTCL are described.\"\n ],\n [\n \"1 A monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"2 A hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"3 An isolated protein obtainable by: (i) collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating them with PHA at 1 microgram/ml, (ii) lysing the cells by incubation in a lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the compound onto which the mAb of claim 1 binds under conditions enabling this mAb to perform reactions of the antigen-antibody type.\",\n \"4 An isolated protein obtainable by recovering the isolated protein compound of claim 3 under non reducing conditions.\",\n \"5 An isolated cDNA obtainable by: collecting cells selected from the group consisting of total blood cells and PBL5 incubating the collected cell population with a CD3 activator at 1 microgram per ml, extracting and purifying the whole mRNA population from said cells, synthesizing every complementary cDNA, operably cloning each cDNA so that expression of this cDNA in this clone is under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody of claim 1 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or of which lysate would give an immuno-precipitation reaction with this mAb, optionally, amplifying those clones that have been thus selected, and recovering the inserted cDNA from the selected clones.\",\n \"6 An isolated mRNA obtainable by selection among the mRNA population of cells selected from the group consisting of total blood cells and PBL, of a mRNA which is complementary to a cDNA of claim 5.7 An isolated genomic DNA encoding an isolated mRNA of claim 6.8 An engineered cell in which a cDNA according to claim 5, has been transfected.\",\n \"9 An isolated protein encoded by an isolated cDNA according to claim 5.10 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 3, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.\",\n \"11 An isolated polypeptide compound obtainable by: collecting PBL cells or total blood cells, incubating the collected cells with a CD3 activator at 1 microgram par ml, labeling the cells with a polypeptide-specific label, lysing the cells in a lysis buffer comprising Trition X-100 at 1%, and submitting the lysate to an immuno-precipitation reaction with the mAb of claim 1, and recovering from the digested immuno-precipitate any compound onto which bear a label.\",\n \"12 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 3, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells maizy at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with We a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a protein according to claim 3, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"13 A fragment of a monoclonal antibody according to claim 1, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.14 An isolated compound, comprising a fragment according to claim 13.15 The isolated compound according to claim 14, wherein it is a humanized antibody.\",\n \"16 The isolated compound according to claim 14, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.\",\n \"17 An isolated protein of SEQ ID No.\",\n \"2.18 The isolated DNA encoding an isolated protein according to claim 17.19 An isolated DNA of SEQ No.\",\n \"1.20 A polypeptidic vector comprising a monoclonal antibody according to claim 1, or a fragment thereof selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2 and CDR3.21 A polypeptidic vector for use in the treatment of CTCL, wherein said vector comprises a p140 binding compound.\",\n \"22 The polypeptidic vector according to claim 20, further comprising an element selected from the group consisting of tumoral toxins and radioelements.\",\n \"23 The polypeptidic vector according to claim 20, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form.\",\n \"24 A medical kit comprising a polypeptidic vector according to claim 23 and an antimitotic pro-drug.\",\n \"25 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.26 A method for manufacturing an anti-CTCL medicament comprising including a p140 binding compound in said medicament.\",\n \"27 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 3 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"28 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 3, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"29 A method for CTCL diagnosis, comprising measuring the percentage of CD4+ cells expressing an element selected from the group consisting of the proteins according to claim 3, in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"30 A method for CTCL diagnosis, comprising determining the presence of CD4+ cells expressing p140 in a biological sample which comprises potential CTCL cells, a CTCL-positive diagnosis being made when such a presence is significantly detected.\",\n \"31 A method according to claim 27, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1 and the DNA encoding SEQ ID No.\",\n \"3 or SEQ ID No.\",\n \"4.32 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.33 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 3, or to a p140 molecule, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"34 The isolated protein of claim 4, wherein said non reducing conditions are of chromatography by affinity with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"35 The isolated cDNA of claim 5, wherein said CD3 activator is PHA at 1 microgram per ml.\",\n \"36 An engineered cell in which a mRNA according to claim 6 has been transfected.\",\n \"37 An engineered cell in which a DNA according to claim 7 has been transfected.\",\n \"38 An isolated protein encoded by an isolated mRNA according to claim 6.39 An isolated protein encoded by an isolated cDNA according to claim 7.40 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 4, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.\",\n \"41 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 9, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.\",\n \"42 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 4, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a protein according to claim 4, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"43 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 9, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a protein according to claim 9, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"44 A monoclonal antibody obtainable by: (i) immunizing an animal against a polypeptide compound according to claim 11, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"for binding to a polypeptide according to claim 11, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.\",\n \"45 A fragment of a monoclonal antibody according to claim 12, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.46 The isolated compound according to claim 15, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.\",\n \"47 The polypeptidic vector according to claim 20, wherein said fragment is the Fab or F(ab′)2 fragment.\",\n \"48 A polypeptidic vector comprising an isolated compound according to claim 15, optionally including at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.\",\n \"49 The polypeptidic vector according to claim 21, further comprising an element selected from the group consisting of tumoral toxins and radioelements.\",\n \"50 The polypeptidic vector according to claim 21, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form 51 The polypeptidic vector according to claim 23, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.\",\n \"52 The polypeptidic vector according to claim 50, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.\",\n \"53 The medical kit according to claim 24, wherein said antimitotic pro-drug is phenol mustard pro-drug.\",\n \"54 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"55 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"56 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"57 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.\",\n \"58 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 4, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"59 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 9, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"60 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.\",\n \"61 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"62 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"63 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing the polypeptide compounds of claim 11 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.\",\n \"64 A method according to claim 28, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"65 A method according to claim 29, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"66 A method according to claim 30, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody, and the DNA encoding SEQ ID No.\",\n \"3 or SEQ ID No.\",\n \"4.67 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.\",\n \"68 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises at least one polypeptide compound selected from the bank of polypeptide compounds according to claim 10.69 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting the cDNA according to claim 5, an mRNA complementary to said cDNA, a p140 binding compound, a p140 DNA, an isolated DNA encoding an isolated protein of SEQ ID NO: 2, an isolated DNA of SEQ ID NO: 1, the DNA encoding SEQ ID No.\",\n \"3 and the DNA encoding SEQ ID No.\",\n \"4.70 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 4, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"71 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 9, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"72 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a polypeptide compound according to claim 10, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.\",\n \"73 The method according to claim 33, wherein said p140 molecule is of SEQ ID No.\",\n \"2 or SEQ ID No.\",\n \"4.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.\",\n \"The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.\",\n \"Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.\",\n \"LP and CD30+ lymphomas also develop in the skin.\",\n \"More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.\",\n \"SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.\",\n \"Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.\",\n \"The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.\",\n \"In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.\",\n \"Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).\",\n \"Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.\",\n \"Such a diagnosis method does also not enable to stage the disease.\",\n \"Technically speaking, it is also time-consuming.\",\n \"Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.\",\n \"There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.\"\n ],\n [\n \" SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).\",\n \"The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.\",\n \"It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.\",\n \"The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.\",\n \"(C.N.C.M.\",\n \"Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.\",\n \"deposit number: I-2575).\",\n \"When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.\",\n \"The second marker of the invention, i.e.\",\n \"p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.\",\n \"The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.\",\n \"They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.\",\n \"Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.\",\n \"lupus, lichen), or toxic epidermal necrolysis.\",\n \"At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).\",\n \"Allelic form KIR3D clone 24 (SEQ ID No.\",\n \"1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.\",\n \"1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.\",\n \"3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.\",\n \"2) when compared to p140 clone 1.1.mature protein (SEQ ID No.\",\n \"4).\",\n \"The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.\",\n \"sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.\",\n \"To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.\",\n \"The invention thus provides with the first CTCL universal markers.\",\n \"No prior art product was known to be such a CTCL universal marker.\",\n \"The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.\",\n \"CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.\",\n \"CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.\",\n \"In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.\",\n \"cytokine profile) of non-tumoral T cells.\",\n \"The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.\",\n \"SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).\",\n \"SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.\",\n \"The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).\",\n \"It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.\",\n \"In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).\",\n \"Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).\",\n \"More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.\",\n \"The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.\",\n \"humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).\"\n ],\n [\n \"FIELD OF THE INVENTION The present application relates to novel means for the diagnosis and therapy of T lymphomas, and more particularly of Cutaneous T Cell Lymphomas (abbreviated into CTCL).\",\n \"The invention indeed provides novel tumor markers which are universal for CTCL, and describes biotechnological and medical uses thereof.\",\n \"BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.\",\n \"The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.\",\n \"Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.\",\n \"LP and CD30+ lymphomas also develop in the skin.\",\n \"More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.\",\n \"SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.\",\n \"Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.\",\n \"The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.\",\n \"In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.\",\n \"Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).\",\n \"Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.\",\n \"Such a diagnosis method does also not enable to stage the disease.\",\n \"Technically speaking, it is also time-consuming.\",\n \"Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.\",\n \"There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.\",\n \"SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).\",\n \"The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.\",\n \"It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.\",\n \"The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.\",\n \"(C.N.C.M.\",\n \"Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.\",\n \"deposit number: I-2575).\",\n \"When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.\",\n \"The second marker of the invention, i.e.\",\n \"p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.\",\n \"The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.\",\n \"They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.\",\n \"Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.\",\n \"lupus, lichen), or toxic epidermal necrolysis.\",\n \"At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).\",\n \"Allelic form KIR3D clone 24 (SEQ ID No.\",\n \"1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.\",\n \"1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.\",\n \"3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.\",\n \"2) when compared to p140 clone 1.1.mature protein (SEQ ID No.\",\n \"4).\",\n \"The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.\",\n \"sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.\",\n \"To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.\",\n \"The invention thus provides with the first CTCL universal markers.\",\n \"No prior art product was known to be such a CTCL universal marker.\",\n \"The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.\",\n \"CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.\",\n \"CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.\",\n \"In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.\",\n \"cytokine profile) of non-tumoral T cells.\",\n \"The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.\",\n \"SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).\",\n \"SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.\",\n \"The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).\",\n \"It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.\",\n \"In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).\",\n \"Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).\",\n \"More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.\",\n \"The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.\",\n \"humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).\",\n \"DETAILED DESCRIPTION OF THE INVENTION The invention thus provides with an hybridoma deposited on Oct. 30, 2000 as deposit number I-2575 at the C.N.C.M.\",\n \"in accordance with the Budapest Treaty (C.N.C.M.\",\n \"Collection Nationale de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France), and with a method for preparing an anti-SC5 monoclonal antibody which comprises cultivating hybridoma I-2575 under conditions suitable for the metabolism of the hybridoma cells (e.g.\",\n \"culture for three days at 37° C., with 5% CO2 in a humid atmosphere at 2.106 cells par ml of appropriate culture medium such as RPMI 1640 medium Gibco BRL Cat.\",\n \"No21875-034, with 10% Fetal Calf Serum, penicillin 100 U/ml and streptomycin 100 microgram/ml).\",\n \"The invention therefore encompasses the monoclonal antibody (mAb) which is produced by hybridoma I-2575, or which is obtainable from the culture supernatant of this deposited hybridoma.\",\n \"This mAb is a pentameric anti-SC5 IgM.\",\n \"It is a useful tool for the diagnosis and therapy of an inappropriate or undesired T cell proliferation and/or functional activity (cytokine profile), of an inappropriate or undesired IL-2 production.\",\n \"Its inhibits the proliferation and/or functional activity of normal T cells, of virus-infected T cells (e.g.\",\n \"HIV-infected CD4+ T cells), and of malignant T cells, such as notably malignant CTCL cells.\",\n \"The monoclonal antibody produced by hybridoma I-2575 enables the isolation of a is protein, termed SC5 by the inventors.\",\n \"SC5 protein appears as a biochemically and functionally new molecule, with an apparent molecular weight of 96 kD under reducing conditions.\",\n \"It has been located in and on numerous PBL cells: normal T cells (both from CD4+ and CD8+ subsets), and in particular CD3+ T cells, CD45RO+ T cells, but also normal NK cells, B cells, granulocytes and macrophages.\",\n \"In addition, SC5 has been located in the cytoplasm of cells from other lineages and other species such as NIH/3T3 (ATCC CRL-1658) and CHO-K1 (ATCC CRL-9618).\",\n \"In resting normal PBL cells, SC5 is mainly intracellularly located, but CD3 stimulation induces the transfer of SC5 to the cell membrane.\",\n \"Remarkably, SC5 has also been observed by the inventors on malignant T cells, such as malignant CTCL cells, and in particular malignant CD4+ CTCL cells, where they are mainly located at the cell surface (without any ex vivo CD3 stimulation).\",\n \"As a further remarkable feature, SC5 surface expression has been observed for all the CTCL patients who were tested until now (see the examples below).\",\n \"Another further remarkable feature of the invention is that the percentage of CD4+ T cells which co-express SC5 is statistically higher in patients developing tumoral CD4+ T cells than in healthy patients.\",\n \"The percentage of SC5+ CD4+ cells in CD4+ CTCL patients is significantly higher than the percentage observed in healthy patients (average standard percentage of SC5+ CD4+ cells is in the 1-15% range, generally in the 5-10% range).\",\n \"In the particular CD4+ CTCL case of Sézary Syndrome (SS), the percentage of CD4+ cells which co-express SC5 is furthermore statistically closely correlated to the percentage of malignant CTCL cells in the PBL of the CTCL patient: the percentage of SC5+ CD4+ T cells is substantially equal to the percentage of malignant CTCL cells (within ±10% of this percentage).\",\n \"SC5 protein is therefore a new molecule whose transmembrane expression at the cell surface is linked to an activation and/or proliferation status for non-tumoral T cells (such as virus-infected cells, e.g.\",\n \"HIV-infected cells, and such as normal CD4+ or CD8+ T cells), and to a malignant status for non-activated T cells.\",\n \"According to a remarkable feature, it enables a CD4+ CTCL-positive diagnosis with a confidence of more than 90% (actually of 100% of those humans who have been tested up-to-date).\",\n \"A further outstanding feature of the invention is that the SC5 new molecule is capable.\",\n \"of acting as an inhibitory receptor for cell proliferation and/or functional activity when expressed at the cell surface: SC5 aggregation induces a decrease in the proliferation and activity of the cell expressing it.\",\n \"For example, the anti-SC5 monoclonal antibody produced by hybridoma I-2575 (which is a pentameric Ig) inhibits the anti-CD3 induced proliferation of SC5+ cells such as normal CD3+ T cells collected from a human or an animal, or normal CD3+ T cell clones, and also inhibits the anti-CD3 induced in vitro proliferation of CTCL cells collected from a CTCL patient, and the in vivo natural proliferation of human CTCL cells in an animal model.\",\n \"This mAb also binds HIV-infected CD4+ T cells, and is therefore useful as an active principle in an anti-HIV drug intended for killing infected cells, and therefore reducing HIV propagation.\",\n \"Several procedures enabling the isolation of the SC5 protein are available to the skilled person to whom a monoclonal antibody of the invention has been made available.\",\n \"Standard procedures comprise recovering the SC5 molecules from a cell lysate by affinity chromatography, or by electrophoresis under reducing conditions.\",\n \"All human or animal cells which the monoclonal antibody produced by hybridoma I-2575 recognizes as an antigen, or of which lysate gives an immuno-precipitation reaction with this monoclonal antibody, are appropriate cell sources for isolating the SC5 molecule.\",\n \"Such cells notably include normal cells (collected from a human or an animal, or cell clones) such as T cells, CD3+ T cells, CD45RO+ T cells, B cells, NK cells, macrophages, granulocytes.\",\n \"Appropriate cells also include tumoral cells such as malignant T cells, e.g.\",\n \"CD4+ CTCL cells collected from a CTCL patient, or tumoral clones such as the CTCL clone HUT78 (ATCC TIB-161).\",\n \"Due to the wide range of appropriate cell sources, it will be understood that rough total blood cell or PBL may be directly used as a SC5 source without any further purification.\",\n \"Normal cells enables the isolation of both the intracellular form and the transmembrane receptor form of SC5.When it is desired to isolate the SC5 receptor form, or a portion thereof, it may thus be preferable to use a source of SC5+ cells (i.e.\",\n \"cells expressing SC5 at their surface).\",\n \"Such a source include normal cells as above-mentioned but that have been CD3-stimulated so that SC5 expression increases at the cell surface (see examples below for CD3 stimulation procedures).\",\n \"When total blood cells or PBL are used as a source of SC5+ cells, CD3 activation can be induced by incubation with a polyclonal activator such as PHA (obtainable from Wellcome) at a concentration of about 1 microgram per ml, because total blood cells or PBL also contain T cells and mononuclear cells.\",\n \"When a more homogeneous normal cell population is used as a source of SC5+ cells (e.g.\",\n \"only SC5+ NK cells), then CD3 activation is preferably performed by incubation with immobilized anti-CD3 antibodies, or alternatively with both soluble anti-CD3 antibodies and polyclonal activator such as PHA.\",\n \"Optionally, a culture step may be performed before and/or after CD3 activation so as to increase cell amount (e.g.\",\n \"culture for 3 days in RPMI 10% fetal calf serum at 37° C., 95% humidity, and 5% CO2).\",\n \"When it is desired to isolate SC5 in its intracellular form, then CD3 stimulation may be omitted.\",\n \"Tumoral cells such as CTCL cells, and notably malignant transformed MF or SS cells, enable the isolation of SC5 molecules in their receptor form, without any required ex vivo CD3 activation: CTCL cells already express SC5 in a transmembranar location.\",\n \"The invention therefore encompasses any isolated protein obtainable by: (i) collecting cells selected from the group consisting of T cells, CD3+ T cells, CD45RO+ T cells, B cells, NIK cells, macrophages, granulocytes, and stimulating the collected cells with immobilized anti-CD3 antibodies, or with both soluble anti-CD3 antibodies and PHA at 1 micrograimn/ml, or collecting malignant CTCL cells such as malignant CD4+ CTCL cells, or collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating the collected cells with PHA at about 1 microgram per ml, (ii) lysing the cells under conditions enabling the dissociation of multimeric polypeptidic complexes, for example by incubation in a drastic lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the protein compound onto which the monoclonal antibody produced by hybridoma I-2575 binds under conditions enabling this monoclonal antibody to perform reactions of the antigen-antibody type (i.e.\",\n \"the compound which is recognized as an antigen by this mAb).\",\n \"The aminoacid sequence of the recovered protein compound can be obtained by any standard technique, such as mass spectrophotometry.\",\n \"As above-indicated, the cells mentioned in step i. are activated cells, and therefore enables the isolation of SC5 in the form it has when it is a transmembrane receptor.\",\n \"If the SC5 source used in step i. consists of resting normal T cells (without ex vivo stimulation), the the protein compound thus isolated will be SC5 in its cytoplamsic form.\",\n \"When the cells selected in step i. are activated or malignant cells, lysis in step ii.\",\n \"therefore may be performed either on whole cells, or on the membranar fraction of these cells.\",\n \"The lysis step itself can be achieved by any technique enabling the dissociation of multimeric polypeptidic complexes from each others.\",\n \"For example, cells (or their membrane fraction) may be incubated in a lysis buffer containing a drastic detergent such as Triton X-100 at about 1% for 1 hour at 4° C.; this enables the dissociation of the SC5 protein from the other cell components.\",\n \"If a mild detergent such as digitonin 1% or Bridg58 1% is used, then SC5 won't dissociate from those compounds which are naturally associated with it; the use of a mild detergent in the above step ii.\",\n \"therefore enables the isolation of SC5, and the further isolation of the SC5 transducers and effectors.\",\n \"The invention therefore also encompasses any isolated compound obtainable by such processes.\",\n \"The lysate obtained from step ii.\",\n \"is then incubated with the monoclonal antibody produced by hybridoma I-2575 under conditions appropriate for this mAb to perform reactions of the antigen-antibody type, such as coating the antibodies into microwells containing the cell lysate (e.g.\",\n \"2 microgrammes of the monoclonal antibody produced by hybridoma I-2575 for a lysate of 107 cells) and incubating the monoclonal antibodies with the cell lysate for two hours at 4° C. The step of recovering the compound onto which the monoclonal antibody produced by hybridoma I-2575 binds (step iii.)\",\n \"can be achieved by any technique available the skilled person.\",\n \"Recovering under non reducing conditions enables the isolation of SC5 in its native conformation, and will be preferred when it is desired to retain the native biological properties of SC5.Appropriate techniques for recovering SC5 protein under non reducing conditions namely include chromatography by affinity, and, for example: percolation of the lysate through a column in which the monoclonal antibody (mAb) produced by hybridoma I-2575 has been immobilized (e.g.\",\n \"by coupling this mAb onto agar beads and placing these beads inside said column); preferably, the lysate is mixed with a buffer which has a.physiological pH, e.g.\",\n \"at a pH comprised between 6 and 8, such as 7, before percolation so as to make the percolation easier without substantially altering the conformation of the compounds contained in the lysate, rinsing the column with a buffer at the same physiological pH, and recovering the compound which has bound to the immobilized mAb, e.g.\",\n \"by elution with a buffer which disrupts the binding of the compound onto the mAb without substantially modifying the native conformation of the compound.\",\n \"Such an elution buffer usually has a slightly acidic pH (usually comprised between 2 and 5, e.g.\",\n \"a pH of 3).\",\n \"For the preparation of a matrix which has an optimal orientation of antibody molecules, and which thereby enables antigen binding at a high efficiency, and for a one-step purification procedure for the isolation of membrane proteins, see e.g.\",\n \"Schneider et al.\",\n \"1982 (J. Biol.\",\n \"Chem.\",\n \"257: 10766-10769) of which content is herein incorporated by reference.\",\n \"The protein compound thus recovered is the isolated SC5 protein in its native form.\",\n \"Alternatively, step iii.\",\n \"under non reducing conditions can be achieved by placing the lysate obtained from step ii.\",\n \"for immuno-precipitation with the monoclonal produced by hybridoma I-2575, and the immuno-precipitate thus obtained can be percolated through a column comprising antibodies capable of binding mice IgM.\",\n \"The protein compound which has bound onto the monoclonal antibody produced by hybridoma I-2575 is then recovered by elution similarly to what has been above described.\",\n \"The protein compound thus recovered is also the isolated SC5 protein in its native form.\",\n \"Another standard way for recovering the SC5 protein (step iii.)\",\n \"is to proceed through electrophoresis under reducing conditions.\",\n \"This notably includes incubating the monoclonal antibody produced by hybridoma I-2575 with the lysate obtained from step ii.\",\n \"under conditions enabling this mAb to perform reactions of the antigen-antibody type, collecting the immuno-precipitate thus formed, separating the compounds contained in this immuno-precipitate under reducing conditions, and recovering the protein compound which has an apparent molecular weight of 96 kD.\",\n \"An alternative way to localize on the electrophoresis gel or membrane the desired SC5 protein (and to subsequently isolate it from the gel or membrane) is to proceed with a labeling step prior to lysis so that surface polypeptidic components of the cells are labeled (e.g.\",\n \"125 iode or fluorescent labeling with e.g.\",\n \"sulfo-NHS-LC-biotin), and to recover from the immuno-precipitate those compounds which bear the label.\",\n \"It has to be noted that if the labeling step is performed after lysis, then both the intra-cellular form and the trans-membranar receptor form of SC5 will be isolated (in this case, as above-indicated, non-activated T cells may be used as a SC5 source).\",\n \"Because denaturing conditions are used, the protein compound thus recovered is in a non native form: it still has the native amino acid sequence, but its native biological properties have been substantially lost.\",\n \"It is however the SC5 protein in its non-native conformation that is observed (and has to be searched for) when the biological sample to be tested has been treated under denaturing conditions (such as e.g.\",\n \"paraffin sections of skin sample).\",\n \"The SC5 protein in its non-native form may therefore be used for the production of monoclonal antibodies directed against it, such monoclonal antibodies being then useful for SC5 detection on denaturated samples.\",\n \"If the SC5 protein in its native conformation is desired, portions of at least 10 amino acids of the denaturated protein may then be sequenced (e.g.\",\n \"by mass spectrophotometry), oligonucleotides deduced from the sequenced portions may be synthetized, and then used as probes to screen a cDNA library obtained from SC5+ cells.\",\n \"The cDNA thus selected may the be produced and cloned into a cell under conditions enabling the production, and preferably the excretion, of the cDNA-encoded product by culture of the clone (e.g.\",\n \"via insertion of the SC5-encoding cDNA in a baculo virus vector and transformation of insect cells—e.g.\",\n \"Sf9 cells—, or via the vaccina virus and EBV cells, see Rindis Bacher 1995, J. Biol.\",\n \"Chem.\",\n \"270(23): 14220-14228).\",\n \"The invention therefore encompasses any isolated protein of which sequence is obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, labeling the stimulated cells with a polypeptide-specific label such as biotin, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which bears the label, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, recovering the protein produced by the transfected cells, e.g.\",\n \"with the monoclonal antibody produced by hybridoma I-2575, or obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which has an apparent molecular weight of 96 kD under reducing conditions, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, the protein compound produced by the transfected insect cells, e.g.\",\n \"with the monoclonal antibody produced by hybridoma I-2575.The invention thus encompasses any isolated protein compound obtainable by any of the above-mentioned processes, as well as any amino acid sequence of such an isolated protein compound, and any isolated protein of which sequence comprises such a sequence.\",\n \"Falls within the scope of the invention any solid support onto which a protein compound or protein of the invention has been placed.\",\n \"Such solid supports notably include sepharose beads.\",\n \"The invention also encompasses any isolated DNA (or cDNA) of which sequence codes for an isolated protein of the invention.\",\n \"It also relates to engineered cells transfected by such a DNA, to engineered cells which excrete an isolated protein of the invention, and to isolated protein obtainable by isolation from the culture medium of such engineered cells.\",\n \"The invention notably encompasses any cDNA obtainable by: collecting a cell population which consists of, or comprises cells onto which the monoclonal antibody produced by hybridoma I-2575 binds (i.e.\",\n \"onto which this mAb recognizes an antigen), or cells of which lysate would give an immuno-precipitation reaction with this monoclonal antibody, e.g.\",\n \"collecting cells selected from.\",\n \"the group consisting of total blood cells and PBL, if the sole transmembranar form of SC5 is desired: stimulating the CD3 pathway if said cell population mainly comprises or consists of non-activated cells (e.g.\",\n \"resting non-tumoral cells) so as to increase SC5 expression at the cell surface, e.g.\",\n \"by incubating the collected cell population with a CD3 activator (for example a polyclonal activator such as PHA at 1 microgram par ml when the collected cell population is PBL or total blood, or immobilized anti-CD3 antibodies, or both a polyclonal activator such as PHA and soluble anti-CD3 antibodies when the collected cell population is more homogeneous such as a cell population mainly comprising, or consisting of CD3+ T cells, or of CD45RO+ cells, or of NK cells, or of macrophages, or of granulocytes); if the collected cell population consists of, or mainly comprises activated cells (such as CD3-activated non-tumoral cells, or such as tumoral cells such as CD4+ CTCL cells), then CD3 stimulation is not necessary because SC5 location is already mainly transmembranar in these activated cells; if it is desired to isolate both the intracellular form and the trans-membranar form of SC5, then non-activated cells are preferred and CD3 activation is omitted, extracting and purifying the whole mRNA population from said cell population (commercial kits are available for mRNA extraction and purification, e.g.\",\n \"a polydT columns), synthetising every complementary cDNA (with a reverse transcriptase, e.g.\",\n \"such as described in Seed B. and Arrufo A.\",\n \"1987, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84: 3365-3369, of which content is herewith incorporated by reference), operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody produced by hybridoma I-2575 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or those clones of which lysate would give an immuno-precipitation reaction with the monoclonal antibody produced by hybridoma I-2575 will be selected.\",\n \"optionally amplifying those clones that have been thus selected, recovering the inserted cDNA from the selected clones.\",\n \"The cDNA thus recovered is an SC5 cDNA of the invention, it can be sequenced using standard DNA sequencing techniques.\",\n \"Operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions may be achieved by any conventional cloning procedure available to the skilled person.\",\n \"An example of cloning procedure comprises inserting each cDNA in a transfection vector under the control of a promoter suitable for the expression of this cDNA in the host cell, transfecting the host cells with this transfection vector, and cultivating the transfected host cells under conditions appropriate to their metabolism.\",\n \"Appropriate transfection vectors and host cells comprise virus such as Baculo virus and host cells such as insect cells (Sf9 cells), or vaccinia virus as a vector and EBV cells as host cells (see Rindis Bacher reference supra).\",\n \"Such a cDNA can be used for cell transfection, and for SC5 production from the clones thus obtained.\",\n \"It may also be used as probe for identifying or isolating the native SC5 mRNA.\",\n \"The invention therefore also encompasses any isolated mRNA obtainable by selection among the above-mentioned mRNA population, of a mRNA which is complementary to a cDNA of the invention (e.g.\",\n \"via selection of a mRNA which hybridizes to the above-mentioned cDNA under stringent conditions such as described in Freeman G. J. et al.\",\n \"1992, J. Immunol.\",\n \"149: 3745).\",\n \"Are particularly encompassed any isolated mRNA obtainable by using an isolated cDNA of the invention as a probe so as to recover from the whole mRNA population of said cell population, the mRNA which is complementary to this extracted cDNA.\",\n \"Alternatively, the one of ordinary skill in the art may directly sequence the cDNA that is recovered from the selected clones, and deduce from this sequence the sequence of the mRNA.\",\n \"The invention also encompasses any DNA encoding an isolated mRNA according to the invention.\",\n \"It particularly encompasses any genomic DNA obtainable by searching a genomic bank (such as www.ucsc.edu) for a genomic DNA matching with the above-mentioned SC5 cDNA.\",\n \"The invention also encompasses any engineered cell in which a cDNA, a mRNA or a DNA of the invention has been transfected.\",\n \"Are also encompassed SC5-specific probes and primers.\",\n \"Such probes and primers can be obtained by any technique available to the skilled person (e.g.\",\n \"selecting, as candidate sequences, portions of SC5 sequence that may be unique to SC5, comparing these candidate sequences on DNA/RNA banks with the sequences of cell receptors other than SC5, and selecting those candidate sequences which target a SC5 sequence that appears as unique after said comparison, and/or a SC5 sequence of which length appears as unique after said comparison).\",\n \"Any solid support onto which is placed a cDNA, a mRNA or a genomic DNA of the invention falls within the scope of the present invention.\",\n \"Such solid supports notably include DNA chips, or DNA microspheres.\",\n \"The invention also encompasses any isolated protein encoded by an isolated cDNA, mRNA, or DNA according to the invention.\",\n \"The invention further encompasses any isolated portion of a protein of the invention, provided that this portion is still characteristic of CTCL cells by comparison with normal resting cells.\",\n \"It particularly encompasses any portion that is recognized by the monoclonal antibody produced by the hybridoma I-2575 at the C.N.C.M.\",\n \"It also encompasses any isolated portion of a protein compound of the invention, provided that this portion is capable of inducing a modulation (inhibition or stimulation) of the CD3-induced proliferation of a normal T cell, and/or is capable of inducing a modulation of the in vivo proliferation of a malignant T cell such as a CTCL cell, and/or is capable of inducing a modulation of the IL-2 production of a normal T cell or of a malignant T cell, when said portion is expressed by said cell and contacted with the mAb produced by hybridoma I-2575.It particularly encompasses any isolated portion of a protein compound of the invention, provided that this portion is a part of a molecule that is expressed in a trans-membrane location by malignant CTCL cells and that is expressed in the cytoplasm compartment by non-tumoral resting T cells.\",\n \"The invention notably provides with a bank of polypeptidic fragments which is obtainable by enzymatic cleavage of the SC5 protein of the invention.\",\n \"Preferred enzymes comprises proteolytic enzymes such as those serine endopeptidases which cleave at the level of Tyr, Phe and Trp (e.g.\",\n \"alpha-chymotrypsin), and also those enzymes which cleaves at the C-terminus of glutamic acid and aspartic acid (e.g.\",\n \"V8 protease); see e.g.\",\n \"Shesberadaran and Payne 1988, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"USA 85:1-5, of which content is herein incorporated by reference.\",\n \"Such a bank of SC5 polypeptidic fragments is of particular use in analysis such as mass spectrophotometry analysis: such analysis use the characteristic separation of such a polypeptidic bank, and give a (polypeptide) profile that is characteristic of SC5, and that can be used for SC5 detection.\",\n \"Such a use and such a profile is encompassed by the present application.\",\n \"The invention particularly encompasses the extra-cellular, trans-membranar and intra-cytoplasmic portions of any SC5 protein compound of the invention.\",\n \"Determining the extra-cellular, trans-membranar and intra-cytoplasmic portions of a receptor molecule may be achieved by the skilled person according to any appropriate technique.\",\n \"They may be identified on the whole molecule by identification of the region which contains glycosylation sites (extra-cellular portion), of the region that is hydrophobic (trans-membrane portion), and of the region that is hydrophylic (intra-cytoplasmic region), and they may then be isolated either by cleavage or synthesis.\",\n \"An alternative way to isolate the extra-cellular portion of a protein compound of the invention is to proceed as above-described for the isolation of the whole protein compound, but with an additional step of enzymatic digestion so as to cleave from the whole molecule its extra-cellular portion.\",\n \"Appropriate enzymes for such a cleavage includes enzymes such as V8 protease or alpha-chymotrypsin as above-mentioned.\",\n \"The invention thus encompasses any isolated polypeptide compound obtainable by: collecting a SC5 cell source as above-described, such as e.g.\",\n \"PBL or total blood cells, if it is desired to isolate a polypeptide compound in the form it has when it is naturally expressed at the cell surface, and if the collected SC5 cell source is, or mainly comprises normal resting cells, then stimulating the CD3 pathway, e.g.\",\n \"via incubation with an appropriate CD3 activator (e.g.\",\n \"incubation with PHA at 1 microgramme per ml when said SC5 source is total blood cells or PBL), labeling the cells with a polypeptide-specific label, such as biotin (surface labeling on whole cells), lysing the SC5 cell source (such as PBL or total blood cells) so as to dissociate multimeric polypeptidic complexes (and thereby recovering SC5 dissociated from the transducers and effectors naturally associated therewith) e.g.\",\n \"by incubating the cells in a lysis buffer comprising a drastic detergent such as Triton X-100 at 1%, and submitting the lysate to an enzymatic digestion enabling the cleavage of SC5 into portions, e.g.\",\n \"by incubating the lysate with a proteolytic enzyme such as an enzyme selected from the group consisting of V8 protease or alpha-chemotrypsin, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, and recovering from the immuno-precipitate a polypeptide compound which bears a label.\",\n \"Such recovered compounds are SC5 portions which either are the complete SC5 extra-cellular portion, or are part of such a complete SC5 extra-cellular portion, or comprise such a SC5 extra-cellular portion.\",\n \"The set of polypeptide compounds that are thus obtainable constitute a bank of SC5 “extra-cellular” portions.\",\n \"Such a bank, and its medical uses, such as e.g.\",\n \"its use for the diagnosis of a T-related disease (e.g.\",\n \"via mass spectrophotometry analysis), falls within the scope of the present invention.\",\n \"The person of ordinary skill in the art will note that lysis and enzymatic digestion may be performed in a single step.\",\n \"Labeling on whole cells is performed prior to cell lysis so as to identify SC5 portions which are extra-cellular portions, or which are part of such extra-cellular portions, or which comprise such portions.\",\n \"The invention also encompasses any isolated compound which comprises a portion of a protein of the invention.\",\n \"The mAb of the invention also enables the identification of the SC5 epitope onto which this mAb binds.\",\n \"Conventional techniques such as directed mutagenesis are appropriate (see e.g.\",\n \"Chang H. C. et al.\",\n \"1989, J. Exp.\",\n \"Med.\",\n \"169: 2073-2083, of which content is herein incorporated by reference).\",\n \"The proteins of the invention and their portions according to the invention, and notably their extra-cellular portions, enable the production of monoclonal antibodies directed against them.\",\n \"An example of such monoclonal antibodies is produced by hybridoma I-2575.The monoclonal antibodies of the invention are particularly useful for the detection of SC5, for the diagnosis of T lymphomas such as CTCL, and are also useful as modulators of an undesired or inappropriate proliferation and/or activity of T cells, such as normal CD4+ and CD8+ T cells, virus-infected T cells such as HIV-infected T cells, T lymphomas such as CTCL.\",\n \"The invention therefore encompasses any monoclonal antibody obtainable by: (i) immunizing an animal against a protein of the invention or against a portion thereof as above-defined, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or protein portion that as been used as an immunogen in step i., and which has at least one property selected from the group consisting of the property of: binding resting non-tumoral T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of malignant T cells such as CD4+ CTCL cells, modulating the proliferation of malignant T cells such as CD4+ CTCL cells in an animal, and preferably in a human, competing with a monoclonal antibody of the invention, such as the monoclonal antibody produced by hybridoma I-2575, for binding to a protein of the invention, or to a portion thereof as above-defined, and in particular with the extra-cellular portion of such a protein, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.\",\n \"Production of hybridomas may be achieved using any conventional technique.\",\n \"This includes removing the spleen from said animal, making a suspension of spleen cells, fusing the spleen cells with myeloma cells in the presence of a fusion promoter, diluting and cultivating the fused cells in separate wells in a medium which will not support unfused cells.\",\n \"The invention also encompasses any ascite isolated from said immunized animal.\",\n \"For step iii., T and malignant T cells can be collected from a human using standard procedures.\",\n \"Alternatively, clones can be used such as the CD4+ CTCL HUT 28 cell line (ATCC TIB-161).\",\n \"In the case of CD4+ tumors, collection is made from the body part which will display the CD4+ tumoral cells (PBL for SS, cutaneous erythroderma for transformed MF, etc.).\",\n \"The above-mentioned properties can be easily assessed by the skilled person using common knowledge in the art.\",\n \"Examples of appropriate experimental conditions for assessing the properties mentioned in step iii.\",\n \"can be found in the below examples.\",\n \"The person of ordinary skill in the art will adjust them to the particular cells, or concentrations used.\",\n \"Modulation of cell proliferation herein refers to stimulation as well as inhibition of this proliferation.\",\n \"When CTCL cells are used, inhibition of proliferation is preferred in perspective of therapeutic application.\",\n \"The mAbs of the invention are all anti-SC5 mAbs which have either diagnostic or therapeutic applications, or both.\",\n \"For certain applications, it may be desired to immobilize one or several of these mAb onto a support, e.g.\",\n \"for protein purification purposes, or for exerting the modulation property of which they are capable.\",\n \"The present application also encompasses any support onto which a mAb of the invention has been immobilized.\",\n \"Such supports notably include those appropriate for columns of chromatography by affinity (e.g.\",\n \"agar beads).\",\n \"The present application is also directed to any fragment of a mAb of the invention (anti-SC5 fragments) selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3.It is directed to any compound, and namely to any antibody, comprising such a fragment.\",\n \"It notably relates to any humanized antibody comprising at least one of such fragments.\",\n \"Techniques for the production of humanized antibodies have been widely described in the prior art (see e.g.\",\n \"Farah et al.\",\n \"1998, Crit.\",\n \"Rev.\",\n \"Eucaryote Gene Exp.\",\n \"Vol.\",\n \"8 pp 321-345, of which content is incorporated herein by reference).\",\n \"In particular, it relates to any compound, and notably to any antibody and humanized antibody, comprising at least one anti-SC5 fragment as herein defined and also at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3 (see e.g.\",\n \"WO 94/13804 “Multivalent and multispecific binding proteins, their manufacture and use”, Inventors: Holliger et al.\",\n \"Applicants: Cambridge Antibody Technology Ltd and Medical Research Council; see also Merchant et al.\",\n \"“An efficient route to human bi-specific IgG” Nat.\",\n \"Biotechnol.\",\n \"1998 vol.\",\n \"16 pp 677-681).\",\n \"As previously indicated, the invention provides with two molecules linked by a common general inventive concept: SC5 and p140.The SC5 is a novel molecule fully herein described.\",\n \"The p140 molecule has been described in the prior art as an inhibitory receptor which is expressed by sub-groups of NK cells and of CD3+ CD8+ cells from healthy (non-cancerous) patients.\",\n \"The present invention now demonstrates that p140 is expressed by malignant T cells, and notably by CTCL cells, and provides with a novel p140 allelic form (clone 24 in the below example 3; SEQ ID No.\",\n \"1) encoding a novel protein (SEQ ID No.\",\n \"2).\",\n \"The cDNA and amino acid sequences of this novel allelic form are as follow: SEQ ID No.1: CATGTCGCTCACTGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGC AGGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCC CGGCGCAGCACTGTGGTGCCTCAAGGAGGACACGTGGCTCTTCAGTGTCA CTATCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCC ACGTTCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTGATCATGGGC CCTGTGACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCC ACACTCCCTCACTGGGTGGTCGGCACCCAGCAACCCCCTGGTGATCATGG TCACAGGAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGACCCTG CTGAAATCAGGAGAGACAGTCATCGTGCAATGTTGGTCAGATGTCATGTT TGAGCACTTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCC TCGTTGGACAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGT CCCTTGATGCCTGTCCTTGCAGGAACCTACAGATGTTATGGTTCTGTTCC TCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCCCTGGACATCGTGA TCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACG GTTCAGGCAGGAGAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTA TGACATCTACCATCTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCC GTGCAGTGCCGAAGGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGG CCTGCCACCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGCCCT GCCCTGCGTGTGGTCAAACTCAAGTGACCCACTGCTTGTTTCTGTCACAG GAAACCCTTCAAGTAGTTGGCCTTCACCCACAGAACCAAGCTCCAAATCT GGTATCTGCAGACAGGTGCATGTTCTGATTGGGACGTCAGTGGTCATCTT CCTCTTCATCCTCCTCCTCTTCTTTCTCCTTTATCGCTGGTGCTCCAACA AAAAGAATGGTGCTGTAATGGACCAAGAGCCTGCGGGGGACAGAACAGTG AATAGGCAGGACTCTGATGAACAAGACCGTCAGGAGGTGACGTACGCACA GTTGGATCACTGCGTTTTCATACAGAGAAAAATCAGTCGCCCTTCTCAGA GGGCCAAGAGACCCCCAACAGATACCAGCGTGTACACGGAACTTCCAAAT GCTGAGCCCAGATCCAAAGTTGTCTCCTGCCCACGAGCACCACAGTCAGG TCTTGAGGGGGTTTTCTAGGGAGACAACAGCCCTGTCTCAAAACC SEQ ID No.2: pLMGGQDKPF LSARPSTVVP QGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPLVIMVTGN HRKPSLLAHP GTLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS ˜DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PPTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF The DNA and amino acid sequences of the previously described p140 allelic form (clone 1.1) are as follow: SEQ ID No.3: CATGTCGCTCACGGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGCA GGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCCCG GCCCAGCACTGTGGTGCCTCGAGGAGGACACGTGGCTCTTCAGTGTCACTA TCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCGACGT TCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTCATCATGGGCCCTGT GACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCCACACT CCCTCACTGGGTGGTCGGGACCCAGCAACCCCGTGGTGATCATGGTCACAG GAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGCCCCTGCTGAAA TCACGAGAGACAGTCATGCTGCAATGTTGGTCAGATGTCATGTTTGAGCAC TTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCCTCGTTGGA CAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTGGCTTGATG CCTGTGCTTGCAGGAACGTACAGATGTTATGGTTCTGTTCCTCACTCCGGC TATCAGTTGTCAGCTCGCAGTGACCCCCTCGACATCGTGATCACAGGTCTA TATGAGAAACCTTCTCTCTCAGCGCAGCGGGGCCCCACGGTTCAGGCAGGA GAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTATGACATCTACCAT CTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCCGTGCAGTGCCGAA GGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGCCCTGCCACCCACGG AGGGACCTACAGATGCTTCGGCTCTTTGCGTGCCCTGCCCTGCGTGTGGTC AAACTCAAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAGTAG TTGGCCTTCACCCACAGAACCAAGCTCCAAATCTGGTATCTGCAGACACCT GCATGTTCTGATTGGGACCTCAGTGGTCATCTTCCTCTTCATCCTCCTCCT CTTCTTTCTCCTTTATCGCTGGTGCTCCAACAAAAAGAATGCTGCTGTAAT GGACCAAGAGCCTGCGGGGGACAGAACAGTGAATAGGCAGGACTCTGATG AACAAGACCCTCAGGAGGTGACGTACGCACAGTTGGATCACTGCGTTTTC ATACAGAGAAAAATCAGTCGCCCTTCTCAGAGGCCCAAGACACCCCTAAC AGATACCAGCGTGTACACGGAACTTCCAAATGCTGAGCCCAGATCCAAAG TTGTCTCCTGCCCACGAGCACCACAGTCAGGTCTTGAGGGGGTTTTCTAGG GAGACAACAGCCCTGTCTCAAAACC SEQ ID No.4: pLMGGQDKPF LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPVVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQBVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF SEQ ID No.\",\n \"1 differs from SEQ ID No.\",\n \"3 in five locations.\",\n \"The resulting SEQ ID No.2 differs from the p140 disclosed in prior art (SEQ ID No.\",\n \"4) by four amino acid substitutions (shown in bold and underlined characters): R instead of Q in position 20, V instead of L in position 92, P instead of T in position 102, and L instead of P in position 401 (see FIG.\",\n \"11).\",\n \"The present application encompasses any isolated compound of which sequence is, or comprises SEQ ID No.\",\n \"1 and/or SEQ ID No.\",\n \"2.It also encompasses any mRNA that is complementary to SEQ ID No.\",\n \"1, and any genomic DNA coding for SEQ ID No.\",\n \"2.Appropriate techniques for producing p140 proteins are available to the skilled persons.\",\n \"They notably include the production of cells which have been engineered so as to produce p140 proteins in their culture medium.\",\n \"An example of such techniques comprises inserting SEQ ID No.\",\n \"1 or No.\",\n \"3 in a baculo virus vector, operably transfecting an insect cell such as Sf9 cell line with this vector, recovering the p140 protein produced in the culture medium (e.g.\",\n \"by percolation of the culture medium is through a column—e.g.\",\n \"a Sephadex column—which protein compounds as a function of their molecular weight, and isolating the 140 kD eluate).\",\n \"Compounds capable of binding to p140 under conditions of the physiological type (in vivo conditions, or in vitro conditions mimicking the in vivo ones) have been previously described, and may be obtained by the person of ordinary skill in the art by any appropriate available technique.\",\n \"By “p140 binding compound”, it is herein meant any compound which is capable under said conditions of the physiological type to recognize a p140 molecule as an antigen (SEQ ID No.\",\n \"2 and/or SEQ ID No.\",\n \"4) when it is expressed by a cell in a transmembrane receptor location (i.e.\",\n \"recognition of the SEQ ID No.\",\n \"2 and/or No.\",\n \"4 portion that is above the lipid bi-layer of said cell).\",\n \"Such p140 binding compound notably comprise HLA-A11 and HLA-A3 molecules (natural p140 ligands), and notably fusion proteins such as Fc-HLA-A11 and Fc-HLA-A3 fusion proteins.\",\n \"p140 binding compounds also comprise p140 antiserum (obtainable by immunizing an animal such as a rabbit against p140, and by recovering the antiserum thus produced—an additional step of immuno-purification may be performed so as to increase p140 concentration in the antiserum, e.g.\",\n \"via percolation through a p140 column).\",\n \"Other p140 binding compounds comprise monoclonal antibodies directed to p140 SEQ ID No.\",\n \"4, such as e.g.\",\n \"AZ158, Q66, Q241 described in Pende et al.\",\n \"1996 (J. Exp.\",\n \"Med.\",\n \"184: 505-518).\",\n \"Such anti-SEQ ID No.\",\n \"4 mAbs may also recognize the new SEQ ID No.\",\n \"2 as an antigen.\",\n \"Any available technique for mAb production is appropriate for production of anti-p140 mAbs.\",\n \"An example of such a technique comprise the steps of: (i) immunizing an animal against p140, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the p140 molecule that as been used as an immunogen in step i., and which is also capable of binding resting non-malignant T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.\",\n \"Preferred p140 binding compounds do also comprise an anti-CD4 entity (e.g.\",\n \"bi-specific mAb).\",\n \"Such p140 binding compounds are useful for p140 detection on malignant T cells, such as CTCL cells, in particular CD4+ CTCL.\",\n \"They are thus useful for CD4+ cancer detection.\",\n \"They are also useful as polypeptidic vectors, as described below.\",\n \"According to another aspect of the invention, there are provided polypeptidic vectors comprising at least one element selected from the group consisting of the anti-SC5 mAbs of the invention and the anti-SC5 mAb fragments of the invention.\",\n \"Such polypeptidic vectors are useful for molecules, such as active principle molecules, to reach activated T cells, or their vicinity (e.g.\",\n \"activated T cells observed in inflammatory diseases, or in virus-infected T cells, or malignant T cells such as malignant CTCL cells, and malignant CD4+ CTCL cells in particular).\",\n \"It also relates to polypeptidic vectors comprising a p140 binding compound such as a mAb directed against SEQ ID No.\",\n \"2 and/or SEQ ID No.\",\n \"4 extra-cellular portion.\",\n \"Such polypeptidic vectors are useful for delivering molecules to, or in the vicinity of malignant T cells, such as notably CTCL cells, and malignant CD4+ CTCL cells in particular.\",\n \"Molecules which may be usefully delivered to malignant T cells, such as CTCL cells notably comprise molecules capable of inducing cell death or apoptosis, such as a radio-element or a toxin.\",\n \"Such molecules also comprise any enzyme capable of transforming an antimititotic pro-drug into an active drug form, such as a carboxypeptidase.\",\n \"A polypeptidic vector carrying such an enzyme is then advantageously used in combination with an anti-mitotic pro-drug (such as phenol mustard pro-drug).\",\n \"The invention therefore also encompasses a medical kit comprising such a vector and such an anti-mitotic pro-drug.\",\n \"Molecules which may be usefully delivered in the vicinity of malignant T cells such as CTCL cells notably comprise molecules capable of stimulating the immune functions of cells which are in the vicinity-of malignant T cells in an effort to induce an anti-tumor effect from these neighboring cells towards said malignant T cells.\",\n \"Examples of such molecules notably comprise cytokines such as interferon gamma, and interleukine such as IL-2 (see e.g.\",\n \"Xu et al.\",\n \"2000, Cancer Research 60: 4475-4484).\",\n \"When a polypeptide vector of the invention is intended for targeting CD4+ cells such as CD4+ CTCL cells, then this vector advantageously further comprises an anti-CD4 entity.\",\n \"The invention also encompasses any pharmaceutical composition comprising such polypeptidic vectors, and in particular any medicament comprising such polypeptidic vectors.\",\n \"Such medicaments are useful in the control of an inappropriate or undesired T cell proliferation and/or functional activity (including undesired IL-2 production).\",\n \"They are particularly useful for the prevention, palliation, relieve or therapy of an inappropriate immune activity, and notably for the prevention or inhibition of a T lymphoma such as CTCL and in particular CD4+ CTCL.\",\n \"The use of such vectors for the manufacture of a drug intended for the prevention, palliation, relieve or therapy of an inappropriate T cell proliferation and/or activity, and notably for the prevention or inhibition of a CTCL proliferation is thus also an object of the present application.\",\n \"A compound which binds to SC5 under conditions of the physiological type may be used as a SC5 ligand agonist so as to stimulate the activation of SC5, the SC5 molecule being then regarded as a cell inhibitory receptor.\",\n \"Such anti-SC5 compounds namely comprise anti-SC5 antisera, the anti-SC5 mAb of the invention, the Fab, F(ab′)2, fragments thereof, the humanized mAbs derived therefrom.\",\n \"When CD4+ cells are more particularly concerned, said anti-SC5 compound advantageously further comprises an anti-CD4 entity.\",\n \"Anti-SC5 compounds are particularly useful for modulating an inappropriate or undesired proliferation and/or functional activity of SC5+ cells.\",\n \"They are thus useful for modulating the proliferation of T cells, CD45RO+ cells, CD3+ cells, CD4+ T cells, CD8+ T cells, or of virus-infected T cells (e.g.\",\n \"HIV-infected CD4+ T cells), or of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.\",\n \"They are also useful for modulating IL-2 production from T cells.\",\n \"When the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M.\",\n \"(which is a pentameric IgM) is used, this modulation goes in favor of an inhibition.\",\n \"It is therefore particularly appropriate for inhibition of T lymphomas such as CTCL.\",\n \"The application encompasses any drug comprising, as an active principle, at least one anti-SC5 compound.\",\n \"Such a drug may be intended for the prevention, palliation, therapy of T lymphomas, of CTCL, of T infections such as viral (e.g.\",\n \"HIV) infections, of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis—e.g.\",\n \"spondyloarthropathis—or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).\",\n \"Such a drug may also be intended for graft improvement (inhibition of graft rejection).\",\n \"The invention also encompasses the use of such anti-SC5 compounds as complement recruiting agents, or as ADCC activators or stimulators.\",\n \"The p140 binding compounds as defined above, may also be used as complement recruiting agents, or as ADCC stimulators or activators in the prevention, palliation, relieve or treatment of T lymphomas, such as CTCL and in particular CD4+ CTCL.\",\n \"The invention therefore encompasses the use of such p140 binding compounds in the manufacture of an anti-T lymphoma drug, of an anti-CTCL drug, of an anti-CD4+ CTCL drugs, and also encompasses such drugs.\",\n \"The invention also provides with a method for the assessment of the development level of a CTCL (staging): it enables to evaluate the proportion (e.g.\",\n \"percentage) of malignant CD4+ CTCL cells present within a certain body compartment of a patient.\",\n \"According to this method, the proportion of CD4+ cells expressing at their surface an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from said body compartment.\",\n \"The proportion of CD4+ CTCL cells that are actually present in said body compartment can be considered as substantially equal to said measured proportion.\",\n \"It usually falls within a ±10% range around this measured proportion.\",\n \"The invention also provides with a method for CTCL diagnosis, wherein the percentage of T cells expressing an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from a patient, and is compared to the average percentage observed in non-CTCL humans (preferably in healthy humans), and in that a CTCL-positive diagnosis is decided when said measured percentage is significantly higher than said average percentage.\",\n \"This CTCL diagnosis method particularly applies to those CTCL which are CD4+; in this case the measured T cell percentage is preferably a CD4+ percentage.\",\n \"The average standard proportion of CD4+ T cells expressing SC5 is generally in between 1-15%, usually in between 5-10% among healthy humans.\",\n \"When the measured percentage of SC5+ CD4+ T cells is higher than such an average percentage, then a CD4+ CTCL positive diagnosis can be concluded.\",\n \"Of note is that, as far as SC5 protein or portions thereof are concerned, the methods of the invention more generally enables to evaluate the proportion of CD4+ activated T cells that are present in said body compartment.\",\n \"This may e.g.\",\n \"be useful for assessing whether a graft induces rejection reactions, and for the diagnosis and follow-up of inflammatory diseases such as those of the auto-immune type (e.g.\",\n \"rhumatoid arthritis such as spondyloarthropathis, skin immune mediated disease such as psoriasis, eczema, atopic dermatitis).\",\n \"Said biological sample may then be intra-synovial fluid.\",\n \"This may be also useful for the diagnosis and monitoring of non-CTCL T lymphomas (e.g.\",\n \"CD4+ ALL, or CD4+ T-LL).\",\n \"The CTCL assessing/diagnostic methods of the invention are highly efficient for all kinds of CD4+ CTCL, and notably for transformed MF, for SS, for LP, as well as for CD30+ lymphomas.\",\n \"Said biological sample may e.g.\",\n \"then be a skin sample collected from an erythroderma (e.g; suspicion of transformed MF), and/or a PBL or total blood sample (in order to evaluate whether a transformed MF has evolved into a more aggressive form such as SS).\",\n \"When the element detected at the surface of said CD4+ cells is a SC5 compound, then a (preliminary or ulterior) step of cytological observation (search for tumor-alike cytology) is the preferably performed to conclude with a confidence of more than 90% to an actual CD4+ CTCL.\",\n \"When p140 is detected at the surface of said CD4+ cells, then no additional step is required: the simple fact of detecting p140 at the surface of CD4+ T cells directs to a positive diagnosis of CD4+ CTCL.\",\n \"The methods of the invention can be implemented with any appropriate element, and notably with an element selected from the group consisting of the anti-SC5 monoclonal antibodies of the invention, the Fab, F(ab′)2 fragments thereof, the humanized mAb derived therefrom, the SC5 “extra-cellular” bank of the invention (as above-defined), the SC5 cDNA, mRNA, genomic DNA of the invention, the p140 binding compounds as herein defined, the p140 DNA, the DNA comprising SEQ ID No.\",\n \"1 or SEQ ID No.\",\n \"3 or encoding SEQ ID No.\",\n \"2 or SEQ ID No.\",\n \"4.Such an element is then used as a detection means for SC5 or p140 cell surface expression.\",\n \"Preferably, said detection means also comprises an anti-CD4 entity so as to detect SC5+ /p140+ CD4+ cells in a one-step procedure.\",\n \"When cDNA/mRNA material is used as a detection means, then CD4+ cells have to be lysed so as to detect those SC5 or p140 hybridizing mRNA which are actually present in said cells (detection of the transcripts).\",\n \"Such cDNA/mRNA detection means may advantageously be placed onto or into a solid support such as a DNA chip or a DNA microsphere.\",\n \"When mAb material or mAb-derived material is used as a detection means, then surface expression of SC5 or p140 is detected.\",\n \"Any appropriate antibody-antigen technique is convenient.\",\n \"Examples notably comprise flow cytometry analysis.\",\n \"The invention also encompasses a method for the identification of a compound which is useful in the palliation, prevention, relieve, therapy of an undesired or inappropriate T cell activity, such as notably a proliferation of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.\",\n \"This method is characterized in that it comprises the detection of a compound that is capable of binding to a SC5 protein as above-defined, or to a SC5 polypeptide compound as above-defined, or to a p140 molecule (such as SEQ ID No.\",\n \"2 and/or SEQ ID No.\",\n \"4).\",\n \"Such compounds can be used as polypeptidic vectors as above-described.\",\n \"Those compounds which, in addition to binding to SC5 molecules, are also capable of aggregating these SC5 molecules at the cell surface, are preferred for stimulating SC5 inhibitory functions.\",\n \"An easy way to implement this identification method is to proceed with SC5 and/or p140 molecules immobilized onto a solid support such as protein A Sepharose CL-4B.\",\n \"Candidates compounds for implementation of the method notably comprise sera immunized against SC5 or p140.The invention is illustrated by the examples, which are in no way intended to restrict the invention to the particular embodiments described below.\",\n \"The person of ordinary skill in the art will contemplate alternative embodiments, and such alternative embodiments derived from common knowledge in the art are within the scope of the invention.\",\n \"The skilled person will in particular appreciate that various standard immuno-techniques, and various standard molecular biology techniques are available to him (see e.g.\",\n \"“Antibodies: a laboratory manual” Ed Harlow, David lane, Cold Spring Harbor Laboratory 1988; Maniatis 1982, “Molecular cloning: a laboratory manual”, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory; “Current protocols in Immunology” protocols on CD-ROM, John Wiley, West Sussex, England; “Immunological techniques made easy”, edited by Olivier Cochet et at.\",\n \"; John Wiley, West Sussex, England; and the publication specifically referred to in the present application).\",\n \"The content of such prior art documents is herein incorporated by reference.\",\n \"It is also to be understood that experiments described herein in full details for one particular form of CD4+ CTCL can be directly transposed by the skilled person to any other form of CD4+ CTCL.\",\n \"In these examples, reference is made to the following figures: FIGS.\",\n \"1A, 1B and 1C illustrate cell membrane expression of SC5 molecule on PBLs from normal individual.\",\n \"PBMC from a normal individual were stained with anti-SC5 mAb (ascites ({fraction (1/200)}) followed by FITC-conjugated isotype specific goat anti-mouse IgM second reagent and one of the following PE-conjugated mAbs: anti-CD3 (FIG.\",\n \"1A), anti-CD56 (FIG.\",\n \"1C) and anti-CD45RO (FIG.\",\n \"1B).\",\n \"The percentage of double-stained lymphocyte-gated cells is shown in the upper right quadrant (3.3 for FIG.\",\n \"1A; 5.1 for FIG.\",\n \"1B; 0.6 for FIG.\",\n \"1C).\",\n \"This experiment is representative for the 10 donors studied.\",\n \"FIG.\",\n \"2 illustrates cell membrane expression of SC5 molecule during activation of PBLs.\",\n \"PBMC were stimulated with 1 μg/mL PHA and the kinetics of SC5 and CD69 expression were studied in parallel on the CD3+ cells.\",\n \"FIG.\",\n \"3 illustrates intracellular localization of SC5 molecule in PBL.\",\n \"Anti-SC5, anti-CD3ζ chain mAb and anti-BY55 isotype-matched mAb were used for staining permeabilized and non-permeabilized gated lymphocytes from a normal donor.\",\n \"The shaded histograms represent the labeling obtained with the indicated mAbs compared to an irrelevant control mAb.\",\n \"FIG.\",\n \"4 illustrates biochemical analysis of SC5 molecule.\",\n \"DS6 T cell clone was surface labeled with biotin and 1% Triton X-100 lysates were immunoprecipitated, using anti SC5 mAb.\",\n \"The samples were analyzed by SDS-10%-PAGE under reducing conditions.\",\n \"Anti-SC5 mAb was used at two concentrations {fraction (1/200)} (lanes 2) and {fraction (1/500)} (lane 3).\",\n \"The negative control samples were precipitated with goat anti-mouse Ig alone (lane 1) and isotype-matched irrelevant mAb (lane 4).\",\n \"The molecular weight markers (kD), are indicated on the left.\",\n \"FIGS.\",\n \"5A, 5B, 5C, 5D illkustrate that anti-SC5 mAb modulates the anti-CD3-induced proliferation and cytokine secretion of normal T lymphocytes whithout affecting their cytotoxic activity: FIG.\",\n \"5A: PBL or T cell clones LSO and GDS.3 were stimulated with immobilized anti-CD3 mAb (1 μg of mAb coated per well) or with IL-2 (50 IU/ml) in the presence of anti-SC5 mAb (1:200 final dilution of ascites) or an isotype-matched irrelevant mAb.\",\n \"Results shown are representative of at least three separate experiments and are expressed as mean cpm±SD of triplicate wells.\",\n \"FIG.\",\n \"5B: GDS.3 CD4+ T cell clone was stimulated with the indicated concentrations of pre-coated anti-CD3 mAb in the presence of different concentrations of anti-SC5 mAb.\",\n \"FIG.\",\n \"5C: IL-2 secretion in the supernatant of anti-CD3-stimulated PBL, in the presence of anti-SC5 or control mAb, was assessed by measuring the proliferation of an IL-2 dependant T cell clone in the presence of: medium alone (a), supernatant from PBL/anti-CD3+ control mAb (b), supernatant from PBL/anti-CD3+ anti-SC5 mAb (c).\",\n \"Data represent the mean values±SD of triplicate determinations of one out of three representative experiments.\",\n \"FIG.\",\n \"5D: JF1, a cytotoxic CD8+ cell clone was incubated with anti-SC5 mAb ({fraction (1/200)} final dilution of ascites) or with an isotype matched control mAb before the coculture with the FcγR+ murine tumor cells P815 at an E/T ratio of 5/1.Target cells were preincubated with the indicated final concentrations of anti-CD3 mAb.\",\n \"Results are expressed as mean of triplicate wells.\",\n \"FIGS.\",\n \"6A, 6B illustrate cell membrane expression of SC5 molecule on PBL from CTCL patients.\",\n \"Cells from peripheral blood of 8 patients with Sézary syndrome, 3 patients with Mycosis fungoides and 8 healthy donors were analysed by two-color fluorescence for the expression of SC5 molecule by CD4+ lymphocytes.\",\n \"FIG.\",\n \"6A: co-expression of SC5 and CD4 molecules in PBL from a patient with Sézary syndrome in comparison to a normal individual.\",\n \"FIG.\",\n \"6B: histograms showing the mean values of SC5 expression in the 3 indicated groups.\",\n \"Statistically significant differences between SS patients and 2 other groups are marked, (Mann-Witney U test, p<0.001) FIGS.\",\n \"7A, 7B, 7C illustrate surface membrane expression on malignant cell from Sézary syndrome patient.\",\n \"FIG.\",\n \"7A: two-color immunofluorescence analysis of PBMC from SS patient with a circulating TCRVβ22+ malignant T cell clone.\",\n \"Cells were stained with anti-SC5 mAb followed by PE-conjugated isotype specific goat anti-mouse IgM and FITC-conjugated anti-TCRVP22 mAb.\",\n \"FIG.\",\n \"7B: anti-SC5-mAb labeled weakly (shaded histogram) the TCRVβ22-positive malignant cells.\",\n \"FIG.\",\n \"7C: cells from the same patient stained with PE-conjugated anti-CD69 mAb and FITC-conjugated anti-TCRVβ22 mAb.\",\n \"FIG.\",\n \"8 illustrates the modulation of anti-CD3-induced proliferation of CTCL malignant cell line by anti-SC5 mAb.\",\n \"Pno cell line was stimulated with IL-7 (10 ng/mL), PMA alone (2 ng/mL), immobilized anti-CD3 mAb or a combination of PMA/anti-CD3 mAb.\",\n \"SC5 mAb or an isotype-matched irrelevant mAb (1:200 final dilution of ascites) was added at the start of cultures.\",\n \"Results shown are representative of four separate experiments and are expressed as mean cpm±SD of triplicate wells.\",\n \"FIGS.\",\n \"9A, 9B illustrate the detection of p140/KIR molecules on CTCL cell lines (FIG.\",\n \"9A) and on fresh tumor lymphocytes (FIG.\",\n \"9B).\",\n \"Double immunostaining flow cytometric analysis was performed as described in Bagot et al.\",\n \"1998 (Blood 91:4331-434 1) and in Poszepczynska et al.\",\n \"2000 (Blood 96: 1056-1063).\",\n \"FIG.\",\n \"10 illustrates the biochemical analysis of p140 molecules: the NK clone AM61 (p140+/p70−), derived from a healthy donor, and the cell lines Pno and Cou-L were surface labeled with biotin and immunoprecipitated with anti-p140 and anti-CD8 mAbs.\",\n \"Samples were treated (+) or not (−) with N-glycosidase F and analyzed in an 8% (left panel) or 11% (right panel) SDS-PAGE under reducing conditions.\",\n \"Sepharose Protein-A alone represents the negative control.\",\n \"Molecular weight markers (kD) are indicated on the right.\",\n \"FIG.\",\n \"11 shows the amino acid sequence alignment of KIR3D cl.\",\n \"24 (SEQ ID No.\",\n \"2) and cl.\",\n \"1.1 (SEQ ID No.\",\n \"4) encoded proteins.\",\n \"Amino acids corresponding to the signal peptide are in small case letters, while transmembrane region is underlined in the consensus sequence.\",\n \"Amino acids identical to the consensus sequence are indicated by dots.\",\n \"EXAMPLE 1 SC5 Isolation, SC5 Biochemical and Functional Characterization, Production of Anti-SC5 mAbs T lymphocyte immune responses are regulated by functional cell surface molecules providing positive signals that lead to the expansion of antigen-specific clones, and negative signals which prevent excessive stimulation or responsiveness to self antigens.\",\n \"The activation of resting T lymphocytes requires two independent signals.\",\n \"The signal provided by the engagement of T-cell receptor (TCR) must be accompanied by a second positive signal in order to result in optimal T cell response.\",\n \"CD28 is considered as the major co-stimulatory receptor inducing T lymphocyte activation and IL-2 synthesis and preventing cell-death.\",\n \"Recent studies demonstrated that CD3/TCR stimulation leads to a redistribution of the detergent-insoluble glycolipid-enriched membrane fraction (DIG) or raft, which results in the aggregation of TCR/CD3 and DIG-associated signal-transducing molecules.\",\n \"CD28 was reported to enhance raft redistribution to the site of TCR engagement.\",\n \"Further, a series of other molecules may provide co-stimulatory signals to T cells, acting at different time points, affecting particular subsets or promoting distinct effector functions.\",\n \"Negative regulation of T lymphocyte responses can be mediated by CD152-induced signals or by apoptosis through the members of TNF receptor superfamily.\",\n \"Recently, a novel type of negative regulation has been reported for subsets of NK cells and CD8+ T lymphocytes (Lan998, Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"16: 359-393).\",\n \"The immunoglobulin-like (KIRs) or C-type lectin structures responsible for these signals prevent unwanted effector functions such as activation of CTL activity or cytokine production.\",\n \"We now describe a novel 96 kD transmembrane receptor, SC5, which delineates a minor subset of peripheral blood lymphocytes in normal individuals.\",\n \"Anti-SC5 mAb stained mainly CD45RO+ lymphocytes, from both CD8 and CD4 subsets, as well as NK lineage cells.\",\n \"We found that SC5 was predominantly located in the intracellular compartment of resting T lymphocytes and its surface membrane expression increased rapidly after cell activation.\",\n \"SC5 molecule engagement inhibited the anti-CD3 mAb-induced proliferation of resting T lymphocytes or T cell clones, whereas it had no effect on the cytokine-induced proliferation of these cells.\",\n \"At the same time, the effector cytotoxic activity mediated by CD8+ T cell clones was not altered by anti-SC5 mAb.\",\n \"Further on, we observed that the percentage of SC5+CD4+ circulating lymphocytes was significantly increased in Sézary syndrome (SS) patients as compared to peripheral blood lymphocytes of normal individuals.\",\n \"Importantly, we were able to clearly demonstrate that Sézary cells express SC5 molecules.\",\n \"In addition, we found that ligation of SC5 molecules in a cutaneous T cell lymphoma (CTCL) cell line resulted in a strong inhibition of the malignant cell proliferation induced by anti-CD3 mAbs.\",\n \"Thus, SC5 molecule expression can serve to detect the presence of circulating malignant CD4+ cells in SS patients.\",\n \"Moreover, the identification of a cell surface molecule providing negative signals upon ligation constitutes a new tool to investigate the mechanisms of pathological T cell growth and could be used to prevent it.\",\n \"Material and Methods Production of Anti-CS mAb SC5 mAb was obtained by immunizing 6 wk old Balb/c mice with the NK cell line Ytindi, using an established protocol (David et al.\",\n \"1990; J. Immunol.\",\n \"144: 1-6).\",\n \"Hybridoma supernatants were screened for selective reactivity with the immunizing cell line and a CTCL cell line termed Pno (Poszcepczynska et al.\",\n \"2000; Blood 96: 1056-1063) by indirect immunofluorescence and flow cytometry.\",\n \"Pno cell line derives from CTCL cells from a patient which had a CD3+ Vβ22+ CD4+ CD8αα+ CD25-phenotype, and which proliferates in response to IL-7 and to anti-CD3 mAb stimulation.\",\n \"Any CTCL cell line is appropriate for this screening.\",\n \"Examples of other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).\",\n \"The reactive supernatants were further tested for their reactivity with peripheral blood lymphocytes (PBL).\",\n \"A hybridoma supernatant was selected for its reactivity with both tumoral cell lines and with a minor PBL sub-population.\",\n \"The hybridoma was cloned twice and cloned hybridomas were passaged into pristane-primed Balb/c mice to produce ascites.\",\n \"The antibody isotype was determined as IgM and termed anti-SC5 mAb.\",\n \"The ascites was dialysed against PBS, sterilized by ultrafiltration and further utilized at final dilution {fraction (1/200)}.\",\n \"This hybridoma has been deposited on Oct. 30, 2000 at the C.N.C.M.\",\n \"(Collection Nationale de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty; the C.N.C.M.\",\n \"deposit number is I-2575.In this example, “anti-SC5 mAb” refers to the mAb produced by hybridoma I-2575.Patients After informed consent and approval by an ethic committee (CCPPRB, Hôpital Henri Mondor, Creteil, France), we obtained blood samples from eleven patients with CTCL.\",\n \"Eight patients had a Sézary syndrome with 10 to 80% circulating CD3+, CD4+, CD8-Sézary cells, whereas three patients had a transformed mycosis fungoides (MF) and presented with disseminated skin tumors with a CD3+, CD4+, CD8-phenotype.\",\n \"All patients were not previously treated with chemotherapy.\",\n \"Cells and Cell Lines Peripheral blood mononuclear cells (PBMC) were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.) density gradient centrifuigation.\",\n \"Human T cell clones GDS.3 (CD3+TCRαβ+CD4+CD8−), DS6 (CD3+TCRγδ+CD4−CD8−), LSO (CD3+TCRγδ+CD4−CD8−) and JF1 (CD3+TCRαβ+CD4−CD8+), described in Bensussan et al.\",\n \"1984 (J. Exp.\",\n \"Med.\",\n \"159: 947-952) and Yssel et al 1984.(J.\",\n \"Immunol.\",\n \"Methods 72: 219), were fed each 8 days with irradiated allogenic PBMC in culture medium consisting of RPMI 1640 (GIBCO, Paisley, UK), 2 mmol/L L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), 10% heat-inactivated human serum, 50 IU/ml recombinant interleukin-2 (rIL-2, Sanofi Synthélabo, Labège, France) and 1 μg/ml phytohemagglutinin (PHA), (Wellcome, Beckenham, UK).\",\n \"Functional studies were performed at day 7 after the feeding.\",\n \"Standard human leukemic cell lines were mycoplasma-free and maintained in logarithmic growth in complete RPMI medium supplemented with 10% fetal calf serum (FCS) and antibiotics.\",\n \"The human CTCL cell clone, Pno, was established from peripheral blood of a Sézary patient and maintained in culture as previously described (Poszcepczynska et al.\",\n \"2000; see supra).\",\n \"Pno cell line has a CD3+Vβ22+CD4+CD8αα+CD25-phenotype and proliferates in response to IL-7 and to anti-CD3 mAb stimulation.\",\n \"Other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).\",\n \"Monoclonal Antibodies and Flow Cytometry Studies Indirect immuno-fluorescent staining was performed with hybridoma supernatants or ascites fluid using FITC-conjugated goat anti-mouse Ig from Caltag laboratories (San Francisco, Calif.).\",\n \"For two-color analysis the immuno-fluorescent staining was performed by incubating 3×105 cells with anti-SC5 mAb for 30 min at 4° C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgM (Caltag laboratories) followed by a second PE-, ECD- or TRI-conjugated specific mAb of IgG isotype.\",\n \"Stained cells were analyzed using a single argon flow cytometer analyser (Epics XL, Bekman-Coulter, Miami, Fla.) as described in Schiavon V et al.\",\n \"1999; Tissue Antigens 53: 23-32.For intracellular labeling, cells were fixed in PBS 4% p-formaldehyde for 20 min at 4° C., washed, and then permeabilized with staining buffer containing 0.1% saponin.\",\n \"Conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD69, and anti-TCRVβ22 mAbs were purchased from Immunotech (Marseille, France).\",\n \"Other mAbs are available from Beckman Coulter (see Cell Analysis 2000 catalogue): anti-CD3 (PE) Cell Analysis 2000 catalogue reference IM1282; purified anti-CD3 Cell Analysis 2000 catalogue reference IM0178; Cell Analysis 2000 catalogue reference anti-CD4 IM0449; anti-CD8 Cell Analysis 2000 catalogue reference IM0452; anti-CD45RO Cell Analysis 2000 catalogue reference IM1307; anti-CD69 Cell Analysis 2000 catalogue reference IM1943; anti-CD94 Cell Analysis 2000 catalogue reference IM2276; anti-CD158a Cell Analysis 2000 catalogue reference IM2277; anti-CD158b Cell Analysis 2000 catalogue reference IM2278; anti-NKG2a Cell Analysis 2000 catalogue reference IM3291; anti-BY55 Cell Analysis 2000 catalogue reference IM2745.Immunoprecipitation of Biotinylated Surface Molecules.\",\n \"2×107 cells were washed twice in PBS and surface-labeled with Sulfo-NHS-LC biotin (Pierce Europe, Interchim, Montlucon, France) (2 mg/mL in PBS) for 20 min at 4° C. After quenching for 20 min with RPMI 1640, cells were washed twice in PBS and re-suspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 1 h at 4° C. Postnuclear supernatant was then incubated for 2 h at 4° C. in a 96-well plate (Maxisorp Nunc immuno-plate) pre-coated with goat anti-mouse IgG+IgM (Caltag laboratories) followed by anti-SC5 mAb or anti-TCRβ (anti-TCRβ C305 isotype matched mAb, Cell Analysis 2000 catalogue reference IM1466; anti-TCRVbeta22, Cell Analysis 2000 catalogue reference IM2051; anti-TCRzeta, Cell Analysis 2000 catalogue reference IM3169; all from Beckman Coulter).\",\n \"Immuno-precipitates were washed 4 times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF) and precipitated proteins were subjected to SDS-PAGE.\",\n \"Western blot analysis was performed using streptavidin-peroxidase (Immunotech) and the ECL detection system according to manufacturers' recommendations (Amersham Pharmacia, Orsay, France).\",\n \"Proliferation Assays For lymphocyte activation, PBMC were cultured in medium containing 15% human serum in the presence of 1 μg/ml PHA (Wellcome, Beckenham, UK).\",\n \"For proliferation assays, 5×104 cells were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium.\",\n \"When needed, the plates were pre-coated with anti-CD3 mAb at the indicated concentrations as previously described (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"For stimulation of CTCL cell line Pno, pre-coated anti-CD3 mAb was used in combination with 2 ng/ml phorbol 12 β-myristate 13 α-acetate (PMA, Sigma Biochemicals).\",\n \"Cells were cultured for 4 days and were pulsed with 1 μCi of 3H[TdR] during the last 8-16 h of culture.\",\n \"3H[TdR] incorporation was measured in a liquid scintillation counter (Topcount; Packard Instrument Co, Meriden, Conn.).\",\n \"For the determination of IL-2 production, 105 PBL were stimulated with immobilized anti-CD3 mAb in the presence of anti-SC5 or control mAb.\",\n \"After 32 hours of culture, 100 μL of supernatant was added to 105 cells from an IL-2-dependent T cell clone.\",\n \"CD3-Induced Redirected Cytotoxicity Assays The redirected cytotoxicity assay was carried out as described in Le Cleach et al.\",\n \"2000, Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"119: 225-230.The target murine mastocytoma P815 tumor cells were loaded with 100 μl 51Cr (2.5 mCi/mL) for 90 min at 37° C., washed and incubated with anti-CD3 mAb for 15 min at room temperature.\",\n \"Effector cells were likewise pre-incubated with anti-SC5 mAb (1:200 final dilution of ascites) and added to target cells in ratio 5:1, in a final volume of 150 μl in 96 well V-bottomed microtiter plates for 4 h. After centrifugation 100 μl aliquots were counted in a gamma-counter to determine 51Cr release.\",\n \"The spontaneous release was always less than 20% from the maximum release (target cells with 1% NP40).\",\n \"Percentage of specific 51Cr release was calculated as described in David et al.\",\n \"1987, J. Immunol.\",\n \"138: 2831-2836.Statistical Analysis Statistical analysis of the results was performed with Statistica software vers.\",\n \"5.0 (StatSoft, Los Angeles, Calif.).\",\n \"Significant differences between SC5 expression in patients and control group were evaluated by the Mann-Whitney U test, and the correlation between the percentage of SC5+CD4+ cells and the percentage of malignant cells in patients peripheral blood was evaluated by the Spearman Rank Order Correlations test.\",\n \"Results The results herein reported have been obtained with the CTCL cell line Pno, and with CTCL cells collected from patients.\",\n \"Equivalent experiments and results can be performed and obtained with any CTCL cell line, such as e.g.\",\n \"HUT 78 (ATCC TIB-161).\",\n \"Expression of SC5 Molecules in Normal Resting and Activated Peripheral Blood Lymphocytes Monoclonal antibodies raised against the functional tumor cell line Ytindi were analyzed for their simultaneous reactivity with the CTCL cell line Pno (Poszcepczynska et al.\",\n \"2000, see supra) and normal peripheral blood T cells.\",\n \"The SC5-specific mAb, of IgM isotype, stained both the immunizing and CTCL cell lines.\",\n \"Among peripheral blood lymphocytes, anti-SC5 mAb delineated a small sub-population (see FIGS.\",\n \"1A, 1B, 1C).\",\n \"The SC5 mAb reactive molecule was expressed by a variable percentage of CD3+ T cells, never exceeding 20% (mean=10.5%, SD±5.6).\",\n \"Both CD4+ and CD8+ lymphocytes were stained by anti-SC5 mAb.\",\n \"A sub-population of peripheral blood CD56+ NK cells was also reactive with anti-SC5 mAb, though with more important inter-individual variations (mean=19.4%, SD±11.4); see the below Table 1.TABLE 1 expression of SC5 in peripheral blood lymphocyte sub-populations Cell population N tested mean (%) SD Total Ly 15 14.1 6.8 CD3+ Ly 10 10.5 5.6 CD4+ Ly 8 9.4 5.1 CD8+ Ly 8 14.8 5.1 CD56+ Ly 10 19.4 11.4 A representative example of SC5 co-expression with CD3 or CD56 molecules on gated lymphocytes from PBL of normal donors is shown in FIG.\",\n \"1A.\",\n \"It should be noted that most SC5+ lymphocytes belonged to the activation/memory pool, as demonstrated in FIG.\",\n \"1B.\",\n \"Over 70% of the SC5-positive cells co-expressed CD45RO.\",\n \"As SC5 molecule expression was detected in all IL-2 dependent T lymphocyte clones tested, we verified whether its cell surface expression was induced during T lymphocyte activation.\",\n \"We found that stimulation of PBL with PHA promptly increased the expression of SC5 on CD3+ lymphocytes.\",\n \"Both the percentage of SC5+ T cells and the level of SC5 expression rose very rapidly after stimulation (see FIG.\",\n \"2).\",\n \"As early as 4 hours after stimulation with PHA, the percentage of SC5+CD3+ cells increased two to threefold and reached a maximum after 24 to 48 hours.\",\n \"In the course of 5 to 7 days of in vitro stimulation SC5 expression returned to the basal level.\",\n \"In parallel we studied the expression of the very early activation antigen CD69 and we observed similar kinetic profiles of CD69 and SC5 on CD3+ lymphocytes.\",\n \"It should be noted, that unlike CD69 molecules, low levels of SC5 were already present on circulating PBL before stimulation (FIG.\",\n \"2) Activation-induced antigen expression results either from de novo protein synthesis or cell surface export of preexisting intracellular molecules.\",\n \"Therefore, we studied the expression of SC5 in non-activated permeabilized PBL.\",\n \"As shown in FIG.\",\n \"3, anti-SC5 mAb stained over 90% of permeabilized lymphocytes vs. 16% of the same non-permeabilized cells.\",\n \"The specificity of anti-SC5 mAb staining was confirmed by using the isotype-matched mAb BY55 which detects a GPI-anchored cell surface structure expressed by a subset of circulating lymphocytes (see Agrawal et al.\",\n \"1999, J. Immunol.\",\n \"162: 1223-1226).\",\n \"A comparable percentage of BY55+ cells was found in permeabilized and non-permeabilized lymphocytes.\",\n \"As a positive control for the detection of an intracellular epitope, we used an anti-CD3ζ chain mAb which only labeled permeabilized T lymphocytes.\",\n \"Thus, the surface expression of SC5 is dynamically regulated by the activation status of cells and its induction during T cell activation is due to the enhanced transport of the molecule from an intracellular pool to the surface membrane.\",\n \"Biochemical Characterization of SC5 Antigen In order to characterize the molecular weight of the structure identified by anti-SC5 mAb, an IL-2 dependent TCRγδ+ clone, termed DS6, was surface labeled with biotin and the cell lysates were immun-precipitated with either anti-SC5 mAb or an isotype-matched anti-TCRβ mAb (as negative control).\",\n \"Anti-SC5 mAb recognized a single molecule with apparent molecular weight of 96 kD under reducing conditions (see FIG.\",\n \"4).\",\n \"As tumor cell lines (the immunizing Ytindi cells) are often characterized by aberrant expression of carbohydrate epitopes (20, 22), we studied the reactivity of anti-SC5 mAb with the SC5+ NK cell line NK3.3 and with the CTCL cell line HUT78 (ATCC TIB-161) after treatment with neuraminidase or sodium periodate in concentrations destroying known sialylated epitopes (digestion of CD75 epitope on Raji cells was used as a positive control).\",\n \"Expression of SC5 was not affected by neuraminidase or sodium periodate treatment.\",\n \"Thus, we excluded a possible carbohydrate nature of the epitope.\",\n \"Inhibition of Tile Anti-CD3 mAb-Induced T Lymphocyte Proliferation by Anti-SC5 mAb In order to examine the possible function of SC5 molecule in normal T cells, we studied the effect of anti-SC5 mAb alone or during the proliferative responses induced by immobilized anti-CD3 mAb.\",\n \"We found that anti-SC5 mAb alone or in combination with PMA did not induce the proliferation of peripheral blood T lymphocytes.\",\n \"Interestingly, when soluble anti-SC5 mAb was present together with immobilized anti-CD3 mAb, a significant inhibition of lymphocyte proliferation was observed.\",\n \"The inhibition varied between 33 and 66% at day 4 depending on the donor as compared to the effect of an isotype-matched irrelevant control mAb (see FIG.\",\n \"5A, upper panel).\",\n \"As SC5 molecule is expressed on monocytes, it was important to determine whether the inhibitory effect was monocyte-independent.\",\n \"We studied the proliferation of two T cell clones, GDS.3 and LSO, stimulated by immobilized anti-CD3 mAb in the presence of anti-SC5 mAb or an isotype control mAb.\",\n \"The results obtained with both T cell clones indicated that engagement of SC5 molecules resulted in an inhibition of the proliferation to anti-CD3 mAb (see FIG.\",\n \"5A, lower panel).\",\n \"It should be noted that the inhibitory effect of anti-SC5 mAb on T cell proliferation depended on its concentration, as well as on the concentration of the agonistic anti-CD3 mAb.\",\n \"The inhibition obtained with anti-SC5 mAb was significant only when T cells were stimulated with optimally diluted anti-CD3 mAb (see FIG.\",\n \"5B).\",\n \"Such inhibitory behaviour was also reported for anti-KIRs mAbs (Cambiaggi et al.\",\n \"1999, Blood 94: 2396-2402).\",\n \"The decrease of T cell proliferation following SC5 engagement could be due to a direct induction of cell death, a perturbation of the IL-2R expression, or the inhibition of cytokine synthesis.\",\n \"We found that anti-SC5 mAb did not inhibit the IL-2-dependent proliferation of PBL or T cell clones (FIG.\",\n \"5A).\",\n \"Furthermore, the addition of rIL-2 to T cells pre-incubated with anti-SC5 mAb (ascites diluted to {fraction (1/200)}) in combination with anti-CD3 for 48 h restored their proliferation.\",\n \"This would not be the case if the specific mAb inhibited IL-2R expression or caused cell death.\",\n \"Finally, in order to prove that SC5 triggering may inhibit cytokine production, we compared the amount of IL-2 secreted by anti CD3-activated PBL in the presence or absence of anti-SC5 mAb, using an IL-2 dependant T cell line.\",\n \"The quantity of IL-2 produced in the presence of anti-SC5 mAb represented 40 to 65% of the control values obtained with an isotype-matched antibody (see FIG.\",\n \"5C).\",\n \"Thus, the simultaneous engagement of SC5 and CD3 molecules in T cells results in the decrease of cytokine production.\",\n \"We next studied the effect of anti-SC5 mnAb on T cell effector cytotoxic activity using an anti-CD3 mAb redirected killing assay against the FcgR+ murine cell line P815.FIG.\",\n \"5D shows a representative experiment performed with a cytotoxic T cell clone at an E/T ratio of 5/1.The CD8+ T cell clone JF1, expressing high amount of SC5 molecules, was induced to kill murine target cells following anti-CD3 mAb is stimulation.\",\n \"The simultaneous targeting of SC5 by its specific mAb did not influence JF1 cytotoxic activity, regardless of the E/T ratio or the concentration of stimulating anti-CD3 mAb.\",\n \"Expression of SC5 Molecules on Malignant Lymphocytes Obtainedfrom Patients with Sézary Syndrome As we initially screened anti-SC5 mAb for its reactivity with a CTCL cell line, we further studied the expression of SC5 molecule in the periphcral blood lymphocytes isolated from 8 patients with Sézary syndrome.\",\n \"Two-color flow cytometry analysis showed that expression of SC5 was significantly increased in the CD4+ subset (see representative results obtained with one patient in FIG.\",\n \"6A).\",\n \"On the average, 35.5% of the CD4+ lymphocytes in SS patient blood expressed SC5 as compared to 9.4% in healthy controls (p<0.001), (FIG.\",\n \"6B).\",\n \"In the 8 SS patients studied, the expression of SC5 on CD4+ cells correlated with the percentage of circulating malignant cells detected by cytomorphology (r=0.91, p=0.0012).\",\n \"It should be noted that in these patients the percentage of total CD4+ cells (mean=88%, SD±8.8) was higher than that of Sézary cells (mean=26%, SD±12.8).\",\n \"We also studied SC5 molecule expression in peripheral blood samples from 3 MF patients without blood involvement.\",\n \"No significant difference in the levels of SC5 expression on peripheral blood CD4+ cells was observed between MF patients and healthy donors (FIG.\",\n \"6B).\",\n \"Still, an increased percentage of SC5+CD4+ was detected in the skin samples from two of these patients, that we were able to to test (26% and 19% respectively).\",\n \"In one SS patient we had previously demonstrated that the circulating malignant cells were clonal lymphocytes with a CD4+TCRVβ22+ phenotype (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"Here we demonstrated that SC5 was co-expressed on the TCRVβ22+ cells, i.e.—by the malignant cell population (FIG.\",\n \"7A).\",\n \"It should be noted that the profile of SC5-mAb binding to TCRVβ22+ cells corresponded to a weak homogeneous expression on the whole malignant clonal cell population (FIG.\",\n \"7B).\",\n \"Interestingly, the malignant TCRVβ22+ cells did not co-express common activation markers as CD69 (FIG.\",\n \"7C), CD25 or CD30 as previously described (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"Thus, the increased levels of SC5 in SS PBL were mostly due to the expression of the antigen on circulating malignant T cells.\",\n \"Inhibition of the Anti-CD3mAb-Induced CTCL Cell Line Proliferation by Anti-SC5 mAb We next investigated whether SC5 molecules were able to provide a negative signal in malignant CTCL cells.\",\n \"We used the long-term cultured Pno CTCL tumor T cell line which strongly proliferates in the presence of IL-7.As previously reported that Pno cell line expresses functional T cell receptors, since immobilized anti-CD3 mAbs or the combination of soluble anti-CD3 mAbs and PMA induced significantly their proliferation (Poszcepczynska et al.\",\n \"2000, see supra).\",\n \"We examined the effect of SC5 molecule engagement on the proliferation of Pno cells.\",\n \"FIG.\",\n \"8 shows that anti-SC5 mAb strongly inhibited the anti-CD3 mAb-induced proliferation of the CTCL cell line.\",\n \"In contrast, PMA alone (2 ng/mL) produced a low level of proliferation which was not significantly affected by anti-SC5.Notably, anti-SC5 mAb had no effect on the IL-7-dependent proliferation, since Pno cells incubated with IL-7 in the presence of anti-SC5 mAb proliferated as vigorously as in the presence of the isotype control mAb.\",\n \"Thus, we concluded that SC5 antigen is functional in CTCL malignant cells and may specifically and profoundly inhibit their proliferation triggered through the TCR/CD3 signaling pathway.\",\n \"A CTCL tumor was then successfully grafted onto transgenic mice having a substantial deficiency in functionally active NK cells and T lymphocytes (SCID mice) as described in Charley et al.\",\n \"(1990) “Establishment of a human cutaneous T cell lymphoma in CB-17 SCID mice” J.\",\n \"Invest.\",\n \"Dermatol.\",\n \"94: 381-384 (see also U.S. Pat.\",\n \"No.\",\n \"5,530,179 Cornelius P. Terhorst and Baoping Wang for obtention of SCID mice).\",\n \"Anti-SC5 mAb was intraveneously or intracutaneously administered at different times to half of the mice at various concentrations of purified mAb per gram of mouse, whereas the other half received the same amount of purified CD56 (isotype IgM) as a control.\",\n \"PBL and cutaneous cells were collected for standard cytological observations of infiltrating large T cells, and the percentage of CTCL cells was assessed for each mouse of the experiment.\",\n \"This procedure is an example of procedure that enables the is skilled person to check that anti-SC5 compounds, such as the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M., are capable of inhibiting the in vivo proliferation of human CTCL.\",\n \"Discussion A number of receptors with dynamic surface expression mediate T cell interactions with other immune cells or cytokines and, consequently modulate T cell immune responses.\",\n \"In this study, we identified a novel 96 kD functional molecule expressed by a minor subset of PBL whose surface expression is induced rapidly during in vitro T-cell activation.\",\n \"SC5 is distinct from the established molecules with similar molecular weight and/or T cell modulatory functions.\",\n \"First, SC5 was induced very early upon T cell activation, with important increase of its surface expression detected after 4 h. A similar kinetics has been observed for the very early activation antigen CD69, which is detected on the cell surface between 2 and 4 h after stimulation.\",\n \"Most established T-cell specific inducible receptors such as 4-1BB, OX-40 LIGHT or CD152 (CTLA-4) appear on T-cell surface between 10 to 24 h following stimulation.\",\n \"Further, SC5 expression is not T cell restricted.\",\n \"The antigen was detected on sub-populations of NK and B lymphocytes, monocytes and granulocytes.\",\n \"Such a wide distribution pattern is typical of certain killer cell Ig-like receptors (KIRs), as the recently described AIRM1 and IRp60, which are found on NK cells, T and B lymphocytes, myeloid and other antigen-presenting cells.\",\n \"The family of immunoglobulin—like transcript (ILT) inhibitory receptors has also a pan-leukocyte type of expression, the individual receptors being detected on different NK, T, B and myeloid sub-populations.\",\n \"Moreover, ILT4 has a molecular weight very close to SC5.However, neither the expression of AIRM1 and IRp60, nor of ILTRs depends on the activation status of cells.\",\n \"The prompt induction of SC5 molecule at the surface of activated T cells followed by its down-regulation may result from trafficking of intracellular molecules to the cell membrane and vice versa, as we detected SC5 in most non-activated T lymphocytes after cell permeabilization.\",\n \"Such a regulatory mechanism has been already reported for another T cell receptor, CD152.Further on, a heterogeneous family of lysosome associated membrane proteins (LAMP) exists, including molecules involved in endocytosis, lysosomal trafficking and secretion from intracellular granules and that may be induced on the surface of monocytes, neutrophiles, NK, B or T cells upon activation.\",\n \"Additional studies on the intracellular localization and transport of SC5 would precise its proper role in T cell activation and functions.\",\n \"Our studies rcvealed that triggering of SC5 by its specific mAb inhibited the anti-CD3 mAb-induced T cell proliferation.\",\n \"This inhibitory effect required an optimal anti-CD3 mAb stimulation, which is necessary to induce maximal SC5 surface expression in PBL (see FIG.\",\n \"5B).\",\n \"A 1:200 final dilution of anti-SC5 ascites significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram par well and this effect of inhibition increases with lower anti-CD3 mAb concentrations −1 microgram par well; 0.5 microgram per well—A 1:400 final dilution of anti-SC5 ascites do not significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram per well, but inhibition is significant when a lower concentration of anti-CD3 is used −1 microgram per well; 0.5 microgram per well—The person of ordinary skill in the art can proceed with such adjustments by standard interpolation from the particular results described herein, or from his own trials.\",\n \"At the same time, the inhibitory.\",\n \"threshold posed by SC5 engagement could be bypassed by excessive anti-CD3 mAb stimulation.\",\n \"It should be noted that the inhibitory functional effects obtained by ligation of SC5 molecules were observed in an APC-independent system.\",\n \"Further experiments are needed to determine whether SC5 stimulation may have different effects on the proliferation of APC or allo-stimulated T cells.\",\n \"Recently, the negative regulation of immune responses has been extensively investigated, in particular in T lymphocytes and NK cells.\",\n \"The best studied T cell specific inhibitory receptor, CD152 (CTLA-4), is a typical activation-induced antigen, functionally coupled to CD28 co-stimulatory receptor.\",\n \"Indeed, CD152 may overcome CD28 positive signals at low antigenic concentrations and prevent immune response.\",\n \"Alternatively, it may downregulate and terminate T-cell proliferation induced through CD28 by affecting IL-2R expression and IL-2 synthesis.\",\n \"Similarly to CD152, SC5 is an intracellular molecule whose surface expression is regulated by the activation status of cells.\",\n \"Both receptors mediate their effects by affecting IL-2 synthesis.\",\n \"The identification of SC5 specific ligands and signaling pathways will precise the temporal and spatial correlation between these negative T-cell regulators.\",\n \"In consideration to its wide cell distribution, SC5 functional effects are probably not restricted to T cells.\",\n \"In several aspects, SC5 is comparable to NK cell inhibitory receptors, equally expressed by T cells and other lymphocytes sub-populations.\",\n \"Both KIRs and CD94/NKG2 receptors were shown to inhibit antigen-specific and superantigen-driven activation of TCRαβ and TCRγδ T cells, including cytotoxicity, proliferation and cytokine secretion.\",\n \"The KIR-induced inhibition depends on the activation status of T cells and the potency of T cell stimulation and may be bypassed under specific in vivo conditions.\",\n \"However, SC5 is definitely distinct from KIRs, since we demonstrated that it selectively inhibited the proliferation of anti-CD3 mAb stimulated T cells without affecting their effector cytotoxic functions.\",\n \"An important characteristic of SC5 molecule is its significantly increased expression in peripheral blood lymphocytes of patients with SS in comparison to healthy subjects.\",\n \"SS is characterized by the generalization of a primary epidermotropic malignant T cell clone with mature phenotype.\",\n \"Actually, none of the established T cell functional receptors conclusively identifies peripheral blood SS tumor cells.\",\n \"MF/SS has been considered as the expansion of a T cell subset with a particular phenotype, CD4+CD7−, corresponding to a normally existing T memory subset.\",\n \"However, a recent study demonstrated that the CD4+CD7− population does not always represent the dominant T cell clone in SS patients (Dummer et al.\",\n \"1999, Arch.\",\n \"Dermatol.\",\n \"Res.\",\n \"291: 307-311).\",\n \"SS is regarded as a proliferation of specifically activated T cells although the nature of the stimulus is not known (Fargnoli et al.\",\n \"1997, Leukemia 11: 1338-1346).\",\n \"For this reason the distinct expression of activation induced antigens on SS and normal T cells is of special interest.\",\n \"Established activation antigens such as CD69, CD25, CD30 are not consistently detected on Sézary cells, as confirmed by our own investigations.\",\n \"Expression of CD25 rather correlates with the progression of the disease and the transformation of MF/SS cells into a large cell variant in a minority of the cases.\",\n \"An increased rate of expression of T cell activation molecules as HLA-DR, CD25 or CD71 in SS has been primarily associated with reactive tumor-infiltrating T cells.\",\n \"A 78 kD very late activation antigen, BE2, was reported to be specifically increased in SS peripheral blood, while not detected on normal T cells.\",\n \"In fact, BE2 was not consistently detected in all SS patients studied, a comparatively low percentage of peripheral blood cells in relation to the reported percentage of malignant cells were BE2-positive, and BE2 was equally detected in patients without clinical evidence of blood involvement.\",\n \"In contrast, SC5 was detected in 8/8 SS cases studied.\",\n \"Although the percentage of malignant cells determined by cytomorphology varied considerably from patient to patient (10-80%), the expression of SC5 on CD4+ cells correlated with this percentage.\",\n \"Finally, no elevated expression of SC5 on CD4+ cells was detected in MF patients without blood involvement.\",\n \"Further, we demonstrated that Sézary cells identified by the expression of the clonotypic TCRVβ chain co-expressed SC5 molecule, confirming that increased levels of SC5 in the peripheral blood of SS patients were due to its expression on the malignant T cell clone.\",\n \"A very important observation was that SC5 triggering on Sézary cells induced down-modulation of the CD3-mediated proliferation of tumor cells, analogous to the inhibition observed in normal T cells.\",\n \"It has been already demonstrated that malignant CTCL lymphocytes preserve certain functional properties as they may be activated through basic signaling pathways although producing aberrant profiles of cytokines.\",\n \"We now demonstrate that an inhibitory pathway is preserved in CTCL tumor cells, that may permit to counterbalance their proliferation.\",\n \"Notably, the inhibitory effect observed after anti-SC5 mAb binding on anti-CD3 mAb-stimulated CTCL Pno cells was much more important than in normal T cells.\",\n \"Anti-SC5 mAb does not seem to directly mediate cell death of neither normal nor Sézary T cells, since it does not modulate the rIL-induced proliferation.\",\n \"Moreover, T-cells that had been cultivated in the presence of anti-SC5 mAb can be re-stimulated by addition of rIL-2.At the same time, we observed a diminished IL-2 production by anti-SC5 mAb-treated T cells.\",\n \"which should account at least partially for the inhibition of their proliferation.\",\n \"Inhibitory effects of functional receptors on the proliferation of malignant cells have been already observed.\",\n \"It has been recently reported the inhibition of the proliferation of normal and leukemic myeloid cells mediated by the homologous NK cell sialoadhesin receptor AIRMI and CD33 myeloid-specific antigen (Vitale et al.\",\n \"1999, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 96: 15091-15096).\",\n \"Unlike SC5, these receptors inhibited proliferation in the presence of growvth factors and most probably by induction of apoptosis.\",\n \"Interestingly, another T cell activation antigen, CD30, expressed by a subset of normal CD45RO+ T cells, is likely to mediate the regression of primary CTCL other than MF/SS.\",\n \"This effect is related to the natural ligand of CD30 (CD30L) which is co-located within the cytoplasm of malignant cells and may induce cytolytic cell death independently of Fas/FasL system (Mori et al.\",\n \"1999, Blood 94: 3077-3083).\",\n \"In this aspect, the identification of SC5 ligand will enlighten the mechanism of its inhibitory action.\",\n \"In conclusion, we have identified an early activation antigen which is distinct, by phenotypic, biochemical and functional criteria, from the established T lymphocyte receptors.\",\n \"As it was underlined, SC5 molecule is expressed by a sub-population of the post-activation/memory peripheral blood T cell subset.\",\n \"Likewise, continuous expression of KIRs is detected on minor T cell clones, and this expression seems to depend on TCR-occupancy by specific antigens.\",\n \"It was recently proposed that such clones might be auto-reactive and maintained tolerant by KIRs signaling.\",\n \"We may speculate that SC5 surface expression is also preserved on auto-reactive T cells, suggesting that Sézary syndrome tumor cells possibly originate from such clones.\",\n \"Importantly, the expression of SC5 on CTCL malignant cells will serve to better define their origin and the nature of the signal driving mature T cells to uncontrolled proliferation.\",\n \"In addition, the inhibitory effect of anti-SC5 mAb on tumor T cells constitutes a novel therapeutic approach in patients with advanced CTCL.\",\n \"Furthermore, expression of SC5 has also been observed at the surface of CD8+ CTCL, such as CD8+ transformed MF.\",\n \"EXAMPLE 2 P140 is Expressed by CD4+ CTCL Cells, and Identification of a Novel P140 Allelic Form Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin.\",\n \"Mycosis fungoides (MF) is characterized by skin invasion of clonally-derived malignant CD4+ T lymphocytes that phenotypically resemble mature T helper cells.\",\n \"A more aggressive form of CTCL develops when the is malignant cells become non-epidermotropic and is associated with extra-cutaneous involvement.\",\n \"Sézary syndrome (SS) is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.\",\n \"The biology of the disease remains poorly understood, as it is difficult to identify the malignant cell, due to the lack of specific cell surface markers.\",\n \"Thus, in cutaneous lesions it is difficult to distinguish CD4+ CTCL cells from reactive infiltrating CD4+ T lymphocytes.\",\n \"Bagot et al.\",\n \"previously described a unique CD4+ T cell line derived from CTCL lesions (Bagot et al.\",\n \"1998, Blood 91: 4331-4341); this cell line and the in vivo tumor cells expressed an identical size of the complementarily-determining region 3 of TCR-Vβ transcripts.\",\n \"More recently, Poszepczynska et al.\",\n \"(Blood 2000, 96: 1056-1063) functionally characterized an IL-7-dependent CD4+CD8αα+ tumor T cell line isolated from the blood of another patient with a cutaneous erythrodermic CTCL.\",\n \"This tumor T-cell line was identical to the major circulating T cell populations, as demonstrated by the expression of TCR-Vβ22 and the identity of the TCRβ-VDJ sequences.\",\n \"Here, we show that these two different CTCL lines express the p140/KIR inhibitory receptor for HLA-A alleles.\",\n \"Importantly, this receptor is detected on freshly isolated tumor cells derived from the same patients.\",\n \"Moreover, the p140/KIR was also co-expressed by a major subset of CD4+ lymphocytes in seven other patients with SS, and by tumor skin CD4+ cells in two additional patients with advanced MF.\",\n \"The p140/KIR is detected in normal individuals on a minor NK cell-subset and on rare peripheral blood CD3+CD8+ cells.\",\n \"Moreover, T cells obtained from skin in other dermatological diseases such as inflammatory skin diseases and toxic epidermal necrolysis did not express this receptor (Le Cleach et al.\",\n \"2000, Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"119: 225-230).\",\n \"Thus, our present findings demonstrate that the p140/KIR represents a suitable marker on CD4+ cells for the identification of CTCL.\",\n \"Materials and Methods Patients After informed consent and approval by an ethic committee, we obtained skin and blood samples from eleven patients with a CTCL.\",\n \"Eight patients had a Sézary syndrome with 10 to 45% circulating Sézary cells in the blood.\",\n \"In seven cases, the phenotype of tumor cells was CD3+,CD4+,CD8−.\",\n \"In one case, the phenotype was CD3+,CD4+,CD8αα+ (patient Pno).\",\n \"Three patients had a transformed mycosis fungoides (Lez, Cou, Bic) and presented with disseminated skin tumors with a CD3+,CD4+,CD8− phenotype.\",\n \"All patients were not previously treated with chemotherapy.\",\n \"Isolation of Tumoral Lymphocytes Fresh CTCL tumor cells were obtained from tumor fragments mechanically dispersed into single-cell suspensions (Bagot et al.\",\n \"1998, Blood 91: 4331-4341).\",\n \"The mononuclear cells were then washed and frozen in human serum plus 10% dimethyl sulfoxide for later use.\",\n \"For Sézary syndrome patients, the mononuclear blood cells were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.).\",\n \"Long Term Culture of Tumor Cell Lines We established the long term culture of Pno cell line (TCRVβ22+,CD3+, CD4+,CD8αα+,MHC class I+,MHC class II−) in vitro from the peripheral blood of the patient as previously described (Bagot et al.\",\n \"1998, Blood 91: 4331-4341).\",\n \"We demonstrated that both the malignant clone circulating in the patient blood and the derived cultured T-cell line were identical for their cell surface phenotype and for their size and sequence of the TCRβ VDJ region (Poszepczynska et al.\",\n \"2000, cf.\",\n \"supra).\",\n \"Other sources of SS cell lines may be found at the ATCC (e.g.\",\n \"HUT 78 ATCC TIB-161).\",\n \"The Cou-L cell line (TCRVβ13+) was cultured in vitro with rIL-2 for more than three years.\",\n \"It corresponds to a subelone of the CD4+ Cou-LS CTCL line previously described (Bagot et al.\",\n \"1998, cf supra).\",\n \"The Cou-L cell line, the original TCRVβ13+, CD4+ Cou-LS CTCL, and the tumor cells freshly isolated from the skin shared the same size and sequence of the TCRβ VDJ region (Bagot et al.\",\n \"1998, cf.\",\n \"supra).\",\n \"Monoclonal Antibodies (mAbs) and Flow Cytometry Studies One- and two-color immunofluorescence analysis was performed as previously described (Bagot et al.\",\n \"1998, cf.\",\n \"supra).\",\n \"Anti-CD3 (Cell Analysis 2000 catalogue reference 6604629), anti-CD4 (Cell Analysis 2000 catalogue reference 6602138), anti-MHC Class I (Cell Analysis 2000 catalogue reference IM0107) and II (Cell Analysis 2000 catalogue reference IM0108) are obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue).\",\n \"The anti-TCRVβ13+ mAb was purchased from Bioadvance (Emerainville, France, catalogue reference TCR 1738), and the anti-TCRVβ22+ mAb was obtained from Beckman-Coulter (Marseille, France, Cell Analysis 2000 catalogue reference IM 1484/IM1365).\",\n \"The anti-CD8 αβ 2ST8.5H7 mAb is obtainable from Beckman Coulter (Cell Analysis 2000 catalogue reference IM 2014.As anti-p140 mAbs, Q66 (IgM, anti-p140) and AZ158 (IgG2a, recognizing both p70/NKB1 and p140) which have been described in Pende et al.\",\n \"1996 (J. Exp.\",\n \"Med.\",\n \"184: 505-518) have been used; however any anti-140 binding compound is appropriate (e.g.\",\n \"anti-p140 antiserum, Fc-HLA-A11, Fc-HLA-A3 fuision proteins).\",\n \"Z27 (IgG1, anti-p70/NKB1), EB6 (IgG1, anti-p58.1/S50.1), GL183 (IgG1, anti-p58.2/p50.2), XA185 (IgG1, anti-CD94), Z199 and Z270 (IgG2b and IgG2a respectively, anti-NKG2A), and B9.4 (IgG2b, anti-CD8) mAbs are all obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue references IM2748, IM2277, IM2278, IM1610, IM2750, IM0102).\",\n \"Goat anti-mouse IgG FITC and goat anti-mouse IgM PE are also obtainable from Beckman Coulter (see Cell Analysis Catalogue references IM08 and IM0555).\",\n \"Biochemical Characterization 20×106 cells were incubated (15′ at 20° C.) in 1 ml PBS pH8 containing 250 μg of EZ-LINK SULFO-NHS-LC-LC-BIOTIN (Pierce, Rockford, Ill.) and washed three times in Washing Buffer (10 mM TRIS pH 8, 0.14 M NaCl).\",\n \"Cells were then lysed in 1% NP-40 and immuno-precipitated with Sepharose-PA (Pharmacia Biotech Inc. Piscataway, N.J.)-coupled AZ158 (IgG2a, antip70/140) or B9.4 ([gG2b, anti-CD8) mAbs.\",\n \"Samples were analyzed by discontinuous SDS-PAGE either undigested or digested with N-glycosidase F (Boehringer Mannheim, GmbH, Germany) and transferred to Immobilon P (Millipore Corp, Bedform, Mass.).\",\n \"After staining with Neutravidin (Pierce) the Renaissance Chemiluminescence Kit (NEN, Boston, Mass.)\",\n \"was used for detection.\",\n \"NK cell clones were obtained by limiting dilution as described in Pende et al.\",\n \"1996 (J. Exp.\",\n \"Med.\",\n \"184: 505-518).\",\n \"RT-PCR Analysis Total RNA was extracted from CTCL cell lines Pno and Cou-LCD8 αα using RNA-Clean System (AGS GmbH, Heidelberg, Germany).\",\n \"cDNA synthesis was performed using oligodT priming.\",\n \"Primers used for cDNA amplification of the complete ORF (open reading frame) of KIR displaying three Ig-like domains (1395 bp) were the following: 5′CATGT(CT)GCTCA(CT)GGTCGTC (Ig3 UP; SEQ ID No.\",\n \"5) and 5′ GGTTTTGAGACAGGGCTG (Ig3 DOWN; SEQ ID No.\",\n \"6).\",\n \"Amplification was performed for 30 cycles (30 sec.\",\n \"at 94° C., 30 sec.\",\n \"at 55° C., 30 sec.\",\n \"at 72° C.), followed by a 7 min.\",\n \"incubation at 72° C., utilizing AmpliTAQ (Perkin Elmer-Applied Biosystems, Foster City, Calif.).\",\n \"PCR products were sub-cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, Calif.).\",\n \"DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer (Perkin Elmer-Applied Biosystems).\",\n \"Transient Transfections COS-7 cells were transfected with pcDNA3.1 TOPO-KIR3D cl.24 or with pCR3-cl.1.1 (Pende et al.\",\n \"1996, J. Exp.\",\n \"Med.\",\n \"184: 505-518) utilizing Fugene 6 (Roche, Monza Italy).\",\n \"Briefly, cells were seeded at 5×105/plate; 24 hr later they were incubated with 6 μg plasmid and 10 μl of Fugene-6 reagent in DMEM/10% FCS.\",\n \"After 48 or 72 hrs, transfected cells were used for cytofluorimetric analysis.\",\n \"Cell transfectants were stained with Q66 and AZ158 mAbs, followed by a phycoerythrin-conjugated goat antibody to mouse IgG2a or IgM and analyzed by flow cytometry using a FACSort (Becton Dickinson, San Jose, Calif.).\",\n \"Results CTCL Cell Lines are Stained by mAbs to the p140/KIR Two long-term CTCL tumor lines Pno (labeled with anti-TCR-Vβ22 mAb) (Poszepczynska et al.\",\n \"2000, cf supra) and Cou-L (labeled with anti-CD3 mAb) (Bagot et al.\",\n \"1998, cf.\",\n \"supra) were analyzed for reactivity with different anti-KIR mAbs.\",\n \"We found that both cell lines were reactive with mAbs Q66 (see FIG.\",\n \"9A) and AZ158, both recognizing the p140/KIR.\",\n \"In contrast, these cell lines did not express other inhibitory receptors specific for HLA class I molecules, including p58.1, p58.2, p70 KIRs and the CD94/NKG2A lectin-like receptor (Poszepczynska et al.\",\n \"2000, cf supra).\",\n \"Tumor T Lymph Ocytes Freshly Isolated from CTCL Patients are Stained by Anti-p140/KIR mAbs To determine whether p140/KIR was expressed by freshly isolated tumor cells, we tested the reactivity of Q66 mAb with uncultured tumor cells isolated from the blood in SS patient Pno and from tumoral skin fragments of MF patient Cou.\",\n \"We found that the majority of tumor cells were stained by this mAb (FIG.\",\n \"9B).\",\n \"In particular, we observed that most TCRVβ22+ tumor lymphocytes isolated from the blood of patient Pno were reactive with Q66 antibody, and that most of the TCRVβ13+ tumor lymphocytes isolated from the skin of patient Cou were stained by the same antibody (FIG.\",\n \"9B).\",\n \"Next, we studied the phenotype of tumor T lymphocytes from the blood of seven additional patients with a Sézary syndrome, with malignant cells representing 10 to 45% of circulating CD4+ lymphocytes, and tumor T lymphocytes isolated from skin tumors of two other patients with a MF.\",\n \"Remarkably, all patients tested exhibited a significant population co-expressing CD4 and p140/KIR (see Table 2 below).\",\n \"TABLE 2 Anti-Q66 mAb stained CD4+ lymphocytes isolated from the skin of patients with transformed mycosis fungoides and from blood of patients with Sezary syndrome.\",\n \"Percentage of positive cells with Patient anti- anti-CD4 mAb + samples CD4 mAb anti-Q66 mAb MF Lez 65 29 Bic 53 44 SS Bri 85 35 Bar 90 18 Att 95 35 Ros 98 15 Can 78 36 Pet 45 9 Riv 98 19 It should be noted that all Q66+ cells were included in the CD4+ cell population.\",\n \"Thus, the expression of p140/KIR, which in normal individuals is restricted to subsets of lymphocytes from the NK and CD8+ populations, appears to be a characteristic of CTCL tumor CD4+ T lymphocytes, both in the skin and in the blood.\",\n \"As control, skin T lymphocytes derived from another dermatological disease, toxic epidermal necrolysis, which were shown to contain small percentages of various KIR expressing T lymphocytes (Le Cleach et al.\",\n \"2000, Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"119: 225-230), failed to express p140/KIR.\",\n \"Molecular Characterization of the p140 Receptor Expressed by CTCL.\",\n \"The Pno and Cou-L cell lines were surface labeled with biotin and cell lysates were immuno-precipitated with an anti-p140 (AZ158 mAb).\",\n \"As shown in FIG.\",\n \"10, this mAb immuno-precipitated from a NK clone and from the Pno and Cou-L cell lines a molecule with a molecular mass of approximately 70 kD under reducing conditions.\",\n \"Treatment with N-glycosidase revealed a protein backbone of approximately 50 kD with a slightly higher mobility for Pno and Cou-L compared to NK clone.\",\n \"These data suggested that the p140 inhibitory receptor expressed by these CTCL tumor cell lines could be almost similar to that previously detected on normal NK cells (Pende et al.\",\n \"1996, J. Exp.\",\n \"Med.\",\n \"184: 505-518).\",\n \"Next, we determined whether the cDNA encoding the molecule recognized by Q66 and AZ158 mAbs on Pno and Cou-L CTCL cell lines corresponded to one of the already described cDNA encoding p140/KIR (SEQ ID No.\",\n \"3).\",\n \"To this end, RT-PCR was performed on RNA derived from these cell lines using a set of primers able to amplify all cDNA encoding KIR with three Ig-like domains.\",\n \"From the Pno cell line, we isolated a full-length cDNA, termed KIR3D cl.24 (SEQ ID No.\",\n \"1).\",\n \"Comparison of its nucleotide sequence with DNA sequences coding for all KIR characterized by three extra-cellular Ig-like domains revealed that KIR3D cl.24 represents a novel allelic form of p140 receptor.\",\n \"In particular, its nucleotide sequence displays five differences compared to the previously described cl.\",\n \"1.1 cDNA (Pende et al.\",\n \"1996, J. Exp.\",\n \"Med.\",\n \"184: 505-518), resulting in four amino acid substitutions in the mature protein (see FIG.\",\n \"11; SEQ ID No.\",\n \"2: clone 24 protein; SEQ ID No.\",\n \"4: clone 1.1 protein).\",\n \"Three of the four substitutions are found in the extra-cellular Ig-like domain (pos.\",\n \"20, 92 and 111 of the mature protein), whereas the other is located in the cytoplasmic region (pos.401).\",\n \"RT-PCR performed on Cou-L CTCL cell line revealed two different allelic fonns of p140/KIR one corresponding to cl.\",\n \"1.1 cDNA and the other identical to KIR3D cl.24 (isolated from Pno cell line).\",\n \"The cDNA derived from the CTCL cell lines were then transiently transfected in COS-7 cells.\",\n \"As expected, all cell transfectants were brightly stained by Q66 and AZ158 mAb, while they were unreactive with p70/KIR-specific Z27 mAb used as a negative control.\",\n \"Finally, RT-PCR was performed on RNA extracted from PBL derived from three SS patients (including patient Pno) and from skin-derived T cells of one additional MF patients.\",\n \"Also in these samples one or another allelic isoform of p140/KIR described above could be identified.\",\n \"The same results were obtained on malignant CTCL cells collected from patients.\",\n \"Discussion Skin lesions in CTCL contain a heterogeneous lymphocytic infiltrate composed of malignant T cells, which are most often CD4+, and non-neoplastic tumor infiltrating T lymphocytes.\",\n \"We previously reported CD4+ cytotoxic tumor infiltrating lymphocytes specifically directed against autologous malignant CTCL CD4+ cell line.\",\n \"However, as no tumor restricted cell surface structure, besides the clonotypic TCR expressed by tumor cells, has been identified on CTCL, it is difficult using standard methods to distinguish malignant from non-malignant reactive CD4+ lymphocytes.\",\n \"In the present study, we report for the first time that tumor cells from MF and SS patients express the p140/KIR.\",\n \"This receptor has been identified in both skin and blood tumor cells from CTCL patients as well as in two long-term culture CTCL lines.\",\n \"Two color fluorescence analysis indicated that p140/KIR expression is restricted to T cells characterized by a given TCRβ-VDJ previously identified on tumor cells.\",\n \"Thus, p140/KIR allows rapid identification of tumor cells from tumor reactive cells among the T lymphocyte CD4+ population.\",\n \"This could be particularly useful in SS patients, in which the tumor T cell clone is not always easily identified within the peripheral blood CD4+ lymphocyte population.\",\n \"Moreover, during the course of the disease or after treatment, it is crucial to assess whether an increase of the CD4+ population is due to an expansion of the tumor cell population or of reactive T lymphocytes.\",\n \"Since the anti-p140/KIR mAbs appear to recognize tumor cells in all CTCL patients analyzed, they may be considered as a suitable tool for the direct identification of CTCLs.\",\n \"In addition, the use of p140/KIR co-expression on CD4+ CTCL cells provides a unique tool to distinguish malignant cells from normal cells in every patient.\",\n \"This is in contrast to TCR determinations which is unique to an individual patient.\",\n \"Moreover, the p140/KIR represents a possible target for the development of novel specific immuno-therapy of CTCL.\",\n \"Previous studies indicated that this receptor was able to generate inhibitory signals upon recognition of HLA.A3 and HLA.A11 alleles.\",\n \"It is of note, however, that p140/KIR expression in the various patients analyzed appears apparently independent from their own HLA Class I haplotype.\",\n \"Nevertheless, the actual role of p140/KIR in the patho-physiology of CTCL is an important feature that remains to be studied by taking into account the potential role of this receptor in the tolerance to self.\",\n \"In conclusion, the present study demonstrates for the first time the expression of the p140/KIR in CD4+ CTCL cells and the isolation of a novel allelic form of this receptor in tumor cells.\",\n \"This finding is an important new issue, both for the patho-physiology and for the clinical management of CTCL patients.\",\n \"Furthermore, p140 expression has also been observed at the surface of CD8+ CTCL such as CD8+ transformed MF.\",\n \"EXAMPLE 3 Production of Anti-CTCL Drugs The person of ordinary skill in the art is enabled to produce anti-CTCL drugs pursuant to the following outlines, given for illustration purposes: use of anti-SC5 or anti-p140 mAb with a cytokine such as Interferon gamma or IL-2 for stimulating the immune system of cells in the vicinity of CTCL cells, use of these anti-SC5 and anti-p140 mAb as vectors for delivering anti-CTCL ingredients onto CTCL cells; examples of anti-CTCL ingredients comprise standard compounds used in chimiotherapy such as radioelements, toxines.\",\n \"mAb vectors for radioelements can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.\",\n \"Press O W et al.\",\n \"“Phase two trial of iode131-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphoma”, Lancet 1995 vol.\",\n \"346 pp336-340, of which content is herein incorporated by reference).\",\n \"mAb vectors for toxine can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.\",\n \"Ghetie U et al.\",\n \"“Large scale preparation of an immunoconjuguate constructed with human recombinant CD4 and deglycosylated ricin A chain”, J. Immunol.\",\n \"Methods 1990, vol.\",\n \"126 pp 135-141, of which content is herein incorporated by reference).\",\n \"combined use of an antimitotic pro-drug and of anti-SC5 mAb and/or anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form (mitotic agent in a functional form).\",\n \"Anti-SC5 mAb and anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form can be produced following standard techniques applied to an anti-SC5 mAb of the invention and to an anti-p140 mAb (see e.g.\",\n \"the ADEPT technique, for Antibody Directed Enzymre Prodrug Therapy, described in Plakey T C et al.\",\n \"“Anti-tumor effects of an antibody-carboxypeptidase G2 conjuguate in combination with phenol mustard prodrugs”, British J.\",\n \"Cancer 1995 vol.\",\n \"72 pp 1083-1088, of which content is herein incorporated by reference).\",\n \"use of anti-SC5 or anti-p140 mAb as ligand agonists (SC5 stimulation or p140 stimulation) use of anti-SC5 or anti-p140 mAb as complement recruiting agent For the above-mentioned uses, mAbs with a double anti-SC5/anti-CD4 or anti-p140/anti-CD4 specificity are preferred.\",\n \"use of anti-SC5 or anti-p140 (no CD4+ specificity needed) as activators of ADCC (Antibody Dependent Cell Cytotoxicity): polynuclear cells, macrophages, NK cells bear Fc receptors which are activated by the Fc portion of the anti-SC5 and anti-p140 mAb, these mAb therefore enable the ADCC activation in the vicinity of CTCL cells.\",\n \"Abbreviations: APC: Antigen Presenting Cell, CTCL: cutaneous T cell lymphoma, DIG: detergent-insoluble glycolipid-enriched fraction, ILT: immunoglobulin-like transcript, KIR: killer cell immunoglobulin receptor, mAb: monoclonal antibody, MF: mycosis fungoides, PBL: peripheral blood lymphocytes, PBMC: peripheral blood mononuclear cells, PHA: phytohemagglutinin, PMA: phorbo112 beta-myristate13 alpha-acetate, SS: Sézary syndrome, TCR: T cell receptor\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450818"}}},{"rowIdx":284,"cells":{"text":{"kind":"list like","value":[["Anti-Cd28 Antibody","The invention concerns an antibody directed against the CD28 receptor and capable of blocking CD28/B7 interaction, and proteins derived from said antibody, for use in particular to block CD28-dependent activation of lymphocytes."],["1.A protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.2.The protein as claimed in claim 1, selected from the group consisting of: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) Fv, Fab, Fab′2 or scFv fragments of the antibody CD28.3; c) chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) Fv, Fab, Fab′2 or scFv fragments of an antibody b); and e) recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.","3.A nucleic acid molecule encoding a protein as claimed claim 1.4.An expression vector comprising a nucleic acid molecule as claimed in claim 3.5.The expression vector as claimed in claim 4, wherein the expression vector is the plasmid CNCM I-2762.6.A cell expressing a protein as claimed in claim 1.7.The cell as claimed in claim 6, wherein the cell is the hybridoma CNCM I-2582.8.A cell transformed with a nucleic acid molecule as claimed in claim 3, and expressing a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CO28/B7 interaction and comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.9.A method for preparing a protein as claimed in claim 1, comprising culturing at least one cell as claimed in claim 6, and recovering said protein from said culture.","10.A medicinal product comprising the protein as claimed in claim 1.11.The medicinal product as claimed in claim 10, wherein in that said protein has a single CD28 receptor-binding site, and said medicinal product is an immunosuppressor which selectively blocks T cell activation via the CD28 receptor.","12.A method of treating a pathological condition selected from the group consisting of transplant rejection, graft-versus-host disease, T-lymphocyte-mediated autoimmune diseases, allergic phenomena and chronic inflammatory diseases wherein the method comprises administering to a patient in need thereof an effective amount of the medicinal product of claim 11.13.A nucleic acid molecule encoding a protein as claimed in claim 2.14.A cell expressing a protein as claimed in claim 2."],[" Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment "],["The invention relates to antibodies directed against the CD28 lymphocyte receptor and to their fragments, and to their therapeutic uses, in particular in the context of regulating T cell activation.","Abnormal activation of T cells is involved in the pathogenesis of many autoimmune diseases, and also in transplant rejection phenomena, where they cause an immune response directed against the transplanted organ to develop.","T lymphocyte activation requires an activating signal, induced by the recognition, by T receptors (TCRs) of the antigen associated with the class II major histocompatibility complex (MHC) and presented by antigen-presenting cells (APCs).","However, this activation only causes proliferation of T cells and secretion of specific immunomodulatory cytokines (such as interleukin 2, gamma interferon or interleukin 4) if other T co-stimulation systems are also activated.","One of the most important systems for regulating T lymphocyte activation is the molecular system B7/CD28/CTLA4.This system plays, for example, an essential role in the mechanisms of transplant rejection [Woodward et al., Transplantation, 66, 14-20, (1998)].","The molecules B7.1 (CD80) and B7.2 (CD86) borne by the APCs can activate the CD28 receptor and also the CTLA4 receptor of T lymphocytes.","The activation of CD28 sends the T lymphocyte a positive signal which stimulates the cell; on the other hand, the activation of CTLA4 sends a negative signal which leads to a non-response (anergy) [FALLARINO et al., J. Exp.","Med., 188, 205-210, (1998)].","Dormant T lymphocytes express a large amount of CD28 and very little CTLA4.When there is a first cognitive contact between an APC and a T lymphocyte, the CD28/B7 interaction is favored, which activates the cell.","It is only several hours after the initiation of activation that, due to the increase in membrane expression of CTLA4, the affinity of which for B7 is 5 to 10 times greater than that of CD28, the B7/CD28 interaction shifts in favor of a B7/CTLA4 interaction.","Currently, cyclosporin is mainly used to block T lymphocyte activation, in particular in the context of organ transplants.","Despite the effectiveness of this medicinal product, it does not, however, confer absolute protection.","In addition, it acts by blocking all the calcium-dependent cell activation pathways, and therefore has a biological activity which is not strictly T lyphocyte specific and leads to a considerable number of side effects.","It is therefore desirable to develop new immunosuppressants which have a defined method of action and greater specificity.","It has been postulated that selective inhibition of the agonist signal given to the T cell by CD28, leaving the antagonist system consisting of the pair CTLA4/B7 intact, via specific blocking of the CD28/B7 interaction, would make it possible to prevent T lymphocyte activation.","Such specific blocking of the CD28/B7 interaction can be obtained using an antibody directed against CD28.Anti-CD28 antibodies capable of preventing CD28 binding to B7 are known.","However, they have the drawback, when they are used in their divalent native form, of bringing about the dimerization and the activation of CD28 via their binding with this receptor.","However, monovalent fragments derived from these antibodies are capable of blocking the CD28 receptor without activating it [DAMLE et al., J. Immunol.","140, 1753-1761, (1988); NUNES et al., Int.","Immunol., 5, 311-315 (1993); PAGES et al., J. Biol.","Chem., 271, 9403, (1996)].","It has thus been reported [PERRIN et al., J. Immunol.","163, 1704-1710, (1999)] that Fab fragments derived from an anti-CD28 antibody can curb the clinical symptoms of experimental autoimmune encephalitis induced in mice by the administration of myelin or the transfer of T cells from an affected animal.","Monovalent Fab or scFv fragments derived from an anti-CD28 antibody can potentially be used to prevent T lymphocyte activation via specific blocking of the CD28/B7 interaction.","Fab fragments result from the action of papain on an immunoglobulin molecule, and each contain a light chain and the first half of a heavy chain; scfv fragments consist of the variable portions of the heavy and light chains of an antibody, connected to one another via a flexible linker [CLACKSON et al., Nature, 352, 624-628, (1991)], thus forming a single-chain protein.","These monovalent fragments frequently exhibit less affinity for the antigen than the native antibodies, which can limit their possibilities for use in diagnostic or therapeutic applications.","The inventors have succeeded in selecting, among various antibodies which recognize the CD28 antigen, an antibody capable of blocking the CD28/B7 interaction, the monovalent fragments of which exhibit sufficient affinity for the antigen so that they can be used, in vitro or in vivo, to block the CD28 receptor without activation of this receptor.","This antibody, called CD28.3, is produced by the hybridoma deposited, according to the terms of the Treaty of Budapest, on Nov. 28, 2000, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2582.A subject of the present invention is a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, characterized in that it comprises at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3.CDRs (complementarity determining regions) are the portions of the variable regions of an immunoglobulin which are involved in antigen recognition specificity.","Proteins in accordance with the invention thus encompass in particular: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) the Fv, Fab, Fab′2 or scfv fragments of the antibody CD28.3; c) the chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) the fragments of the antibodies b) above comprising the CDRs of the antibody CD28.3, with a monovalent Fv, Fab or scfv fragments, or divalent Fab′2 fragments; e) the recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.","They may, for example, be: divalent or plurivalent derivatives of scfv fragments, such as “diabodies” or “triabodies”, resulting from the association of 2 or 3 scFv fragments; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with at least one antibody fragment comprising the CDRs of an antibody of different specificity; by way of examples, mention will be made of bispecific immunoglobulins, conjugates of an Fv or Fab fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity, and “bispecific diabodies” resulting from the association of an scFv fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule having pharmacological activity (for example a toxin) or effector properties (for example an Fc fragment); proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule which makes it possible to prolong its plasma halflife when it is administered in vivo; it is, for example, possible to combine said antibody fragment with a water-soluble polypeptide of sufficient molecular mass for the molecular mass of the fusion polypeptide thus obtained to be greater than the renal filtration threshold.","In this case, a polypeptide will be chosen which, unlike Fc fragments, cannot associate as dimers, and which does not have its own effector activity liable to cause unfortunate side effects.","Polypeptides which have these properties can advantageously be obtained from water-soluble serum proteins, namely, in particular, serum albumin, haptoglobulin, ITIH2 (inter-alpha (globulin) inhibitor, H2 polypeptide), transferrin, CBG (corticosteroid binding globulin), α1-antitrypsin, ITIH4 (inter-alpha (globulin) inhibitor, H4 polypeptide), AACT (alpha-1-antichymotrypsin), TBG (thyroxine binding globulin), fibrinogen and prothrombin, in order to prepare fusion proteins with scFv fragments derived from anti-CD28 antibodies.","It is also possible to conjugate a protein in accordance with the invention with a polyol, for example polyethylene glycol, as described, for example, in U.S. Pat.","No.","4,179,337.An example of a protein in accordance with the invention is illustrated in FIG.","1, which represents an scFv fragment derived from the antibody CD28.3.The sequences of the CDRs of the antibody CD28.3 are boxed in the sequence represented in FIG.","1.The nucleotide sequence encoding this scFv fragment is represented in the attached sequence listing under the number SEQ ID No.","1, and the corresponding peptide sequence is represented under the number SEQ ID No.","2.Fv, Fab or Fab′2 fragments in accordance with the invention can be obtained by the conventional techniques of enzyme digestion, from the antibody CD28.3.A plasmid containing a polynucleotide encoding an scFv fragment of CD28.3, fused to a polynucleotide encoding amino acids 53 to 425 of α1-antitrypsin was deposited, according to the terms of the Treaty of Budapest, on Dec. 11, 2001, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2762.Proteins in accordance with the invention such as chimeric or recombinant antibodies, scFv fragments and their derivatives, etc., can be obtained by conventional genetic engineering techniques, such as those described by SAMBROOK et al.","[MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989)].","Polynucleotides encoding the variable regions of the anti-CD28.3 antibody can, for example, be obtained by cloning said variable regions from a cDNA library of the hybridoma CD28.3, or from the plasmid CNCM I-2762.They can also be prepared, completely or partially, by nucleic acid synthesis, based on the nucleotide sequences of said variable regions.","It is, for example, possible to synthesize polynucleotides encoding the CDRs of CD28.3, and to incorporate them into the framework regions (FRs) of another antibody, in particular of an antibody of human origin, using techniques, known in themselves, of CDR grafting, such as those described by ROUTLEDGE et al.","[“Reshaping antibodies for therapy”, in Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, 13-44, Academic Titles, Nottingham, England (1993)] or by ROGUSKA et al.","Protein Engineering, 9(10), 895-904, (1996)].","A subject of the present invention is also any nucleic acid molecule encoding a protein in accordance with the invention comprising the CDRs of the antibody CD28.3, and also any recombinant vector, in particular any expression vector, comprising said nucleic acid molecule.","A subject of the present invention is also any cell expressing a protein in accordance with the invention comprising the CDRs of the antibody CD28.3.This encompasses in particular the hybridoma CNCM I-2582, and also the host cells transformed with a nucleic acid molecule in accordance with the invention.","Nucleic acid molecules in accordance with the invention may advantageously comprise, besides a sequence encoding a protein in accordance with the invention, a sequence encoding a signal peptide allowing secretion of said protein; they may also comprise one or more sequence(s) encoding one or more marker peptide(s) for detecting, and/or facilitating the purification of, said protein.","Expression vectors in accordance with the invention comprise at least one nucleic acid sequence encoding a protein in accordance with the invention, associated with transcription- and translation-controlling elements which are active in the host cell chosen.","Vectors which can be used to construct expression vectors in accordance with the invention are known in themselves, and will be chosen in particular as a function of the host cell intended to be used.","Host cells which can be used in the context of the present invention can be prokaryotic or eukaryotic cells.","Among the eukaryotic cells which can be used, mention will in particular be made of plant cells, cells from yeast, such as Saccharomyces, insect cells, such as Drosophila or Spodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc., cells.","The construction of expression vectors in accordance with the invention and the transformation of the host cells can be carried out by the conventional techniques of molecular biology.","A subject of the invention is also a method for producing a protein in accordance with the invention, characterized in that it comprises culturing at least one cell in accordance with the invention, and recovering said protein from said culture.","If the protein is secreted, it can be recovered directly from the culture medium; if not, cell lysis will be carried out beforehand.","The protein can then be purified from the culture medium or from the cell lysate, by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular precipitation with ammonium sulfate, electrophoresis, gel filtration, affinity chromatography, etc.","The proteins in accordance with the invention can be used, in vitro, to study the proliferative response or the differentiation of T lymphocytes responding to an antigenic, viral, allogenic or xenogenic stimulation.","They can also be used, in vitro, to induce the differentiation of T lymphocytes taken from a patient, for example the induction of tolerance with respect to an antigen or to an alloantigen, intended to be subsequently re-administered in vivo.","They may also be used to obtain medicinal products, or diagnostic reagents.","Proteins in accordance with the invention which are divalent, i.e.","which have 2 CD28 receptor-binding sites, and thus capable of inducing dimerization of this receptor, can be used in all situations where it is desired to activate this CD28 receptor, i.e.","to increase the response of a T lymphocyte with respect to an antigen.","Proteins in accordance with the invention which are monovalent, i.e.","which have a single CD28 receptor-binding site, can be used in all situations where it is desired to selectively block this receptor without activating it, in order to induce immunosuppression.","A protein in accordance with the invention, comprising a monovalent fragment derived from an anti-CD28 antibody, can in particular be used to obtain an immunosuppressant medicinal product which selectively blocks T cell activation phenomena involving the CD28 receptor, and which does not have the drawbacks of known immunosuppressants such as cyclosporin.","The T immunosuppression by selective blocking of CD28 with protein in accordance with the invention has applications in all T lymphocyte-dependent pathological conditions.","These are essentially transplant rejection, graft-versus-host disease, T lymphocyte-mediated autoimmune diseases, such as type I diabetes or multiple sclerosis, and type IV hypersensitivity, which is involved in allergic phenomena and also in the pathogenesis of chronic inflammatory diseases following infection with a pathogenic agent (in particular leprosy, tuberculosis, leishmaniasis, listeriosis, etc.).","The present invention will be understood more clearly from the further description which follows, which refers to nonlimiting examples of preparation and of use of antibodies in accordance with the invention.","EXAMPLE 1 Choice of an Antibody Producing Monovalent Fragments, Properties of Monovalent Fab Fragments Derived from CD28.3 Some of the properties of several anti-CD28 antibodies (CD28.1, CD28.2, CD28.3, CD28.4, CD28.5 and CD28.6) are described in the publication by NUNES et al.","[Int.","Immunol., 5, 311, (1993)].","These various antibodies, which are not accessible to the public, were provided by the laboratory of Daniel OLIVE (INSERM).","The antigen-binding properties of the monovalent Fab fragments of these various antibodies were compared.","5 mg of Fab fragments of each of these antibodies were prepared by digestion with papain (papain/antibody molar ratio= 1/100) for 24 hours at 37° C., followed by inactivation of the enzyme with 0.03M iodoacetamide, and dialysis against PBS to remove the iodoacetamide.","1) Binding of the Fab Fragments to CD28+ Jurkat T Cells: 100,000 CD28+ Jurkat cells in 100 μl are incubated in PBS-1% BSA-0.1% NaN3 at 4° C. for 30 minutes with increasing concentrations of anti-CD28 antibodies or of their Fab fragments.","After washing, the cells are incubated in a similar way with an FITC-conjugated anti-mouse IgG goat antibody, washed, and analyzed by cytofluorometry.","The results are given in FIG.","2: Legend of FIG.","2: X-axis: antibody or Fab fragments concentration Y-axis: mean fluorescence intensity (MFI) —⋄—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —Δ—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —◯—: F6=Fab fragments of the antibody CD28.6 ····: W132 whole antibody CD28.1 ··-··: W2=whole antibody CD28.2 : W3=whole antibody CD28.3 ——: W5=whole antibody CD28.5 —*—: W6=whole antibody CD28.6 —▴—: Mara-1=negative control (mouse IgG1).","These results show that, among the Fab fragments, only those derived from CD28.3 are capable of significantly binding to the CD28+ Jurkat cells at concentrations of less than 10 μg/ml.","2) Effect of the Fab Fragments on the Adhesion of CD28+ Jurkat T Cells to Transfected Murine L Cells Expressing the B7-1 Molecule: 4×105 human T cells (Jurkat, CD28-positive) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.","Chem., 271, 9403, (1996)] have been seeded 24 hours beforehand.","These incubations are carried out in the presence of the Fab fragments derived from the antibodies CD28.1 to CD28.6, or of the antibody CD28.3, diluted to various dilutions in PBS buffer without Ca2+ or Mg2+.","The adherent cells after washing are quantified by reading the residual radioactivity in a beta counter (PACKARD TOPCOUNT).","The results are given in FIG.","3: Legend of FIG.","3: X-axis: percentage of adherent cells Y-axis: antibody concentration —♦—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —▴—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —*—: F6=Fab fragments of the antibody CD28.6 ····: whole antibody CD28.3 ◯: no antibody.","These results show that the Fab fragments derived from CD28.3 are the most effective for inhibiting CD28/B7 interactions.","They give 90% inhibition of adhesion at a concentration of 3 μg/ml, and with an effectiveness comparable to that of the whole antibody CD28.3, whereas, at this concentration, the Fab fragments derived from the other antibodies give no more than 50% inhibition of adhesion.","3) Effect of the Fab Fragments on Proliferation in a Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells (PBMCs) are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, in the presence of varying concentrations of the antibodies CD28.1 to CD28.6 or of the Fab fragments derived from these antibodies.","The proliferative response in these cultures is evaluated after 3 days, by incorporation of (3H) thymidine for a period of 16 hours.","The results are given in FIG.","4: Legend of FIG.","4: X-axis: antibody concentration Y-axis: proliferative response (cpm) Basal level of proliferation=6 500 cpm.","—♦—: 28.1=antibody CD28.1 —▪—: 28.2=antibody CD28.2 —▴—: 28.3=antibody CD28.3 —x—: 28.5=antibody CD28.5 ···*···: 28.6=antibody CD28.6 ——: Fab.","1=Fab fragments of the antibody CD28.1 ··+··: Fab.","2=Fab fragments of the antibody CD28.2 ——: Fab.","3=Fab fragments of the antibody CD28.3 —+—: Fab.","5=Fab fragment of the antibody CD28.5 —⋄—: Fab.","6=Fab fragment of the antibody CD28.6.These results show that the Fab fragments derived from CD28.3 or from CD28.6 are the most effective for inhibiting mononuclear cell proliferation.","The whole antibodies CD28.1 to CD28.6, tested in parallel, have no inhibitory effect or indeed stimulate the proliferation by virtue of their stimulator action on CD28.Effect of the Fab Fragments Derived from CD28.3 on Proliferation Induced by a Superantigen For this experiment, responder CD4+ T cells were mixed with irradiated isogenic PBMCs, in the presence of 50 ng/ml of toxic shock syndrome toxin-1 (TSST-1), which specifically stimulates the vβ2+ T cell, either in the absence of antibody or in the presence of anti-B7-1 (1 μg/ml), of anti-B7-2 (0.5 μg/ml), of CTLA4Ig (10 μg/ml), or of Fab fragments derived from CD28.3 (10 μg/ml).","The proliferative response in these cultures is evaluated after 1, 3, 6 and 8 days, by incorporation of (3H) thymidine for a period of 16 hours.","The results are given in FIG.","5: Legend of FIG.","5: X-axis: culturing time Y-axis: proliferation index=PI P I = cpm mixed lumphocyte reaction - cpm irradiated stimulating cells only cpm unstimulated responder cells —♦—: anti-CD28.3 Fab —X—: anti-B7-2 —▪—: CTLA-4 Ig ··*··: anti-B7-1+2 —▴—: anti-B7-1 —◯—: no antibody TSST-1 induces considerable proliferation of CD4+ T cells.","In the presence of anti-B7, of CTLA4Ig or of the Fab fragments of CD28.3, 70% inhibition of this proliferation is observed after 6 days.","Effect of the Fab Fragments Derived from CD28.3 on Cytokine Production In order to determine whether the Fab fragments derived from CD28.3 could induce an immune deviation in vitro, a mixed lymphocyte reaction (PBMCs derived from a donor A/irradiated PBMCs derived from a donor B) was carried out, in the presence of Fab fragments derived from CD28.3.105 peripheral blood mononuclear cells from a donor are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, and cultured for 5 days in the presence or absence of 10 μg/ml of Fab derived from the antibody CD28.3.The RNA of the responder cells was extracted, and the amount of cytokine mRNA was evaluated by quantitative measurement of the number of transcripts, related to the amount of HPRT, using a TaqMan (Perkin Elmer).","In the presence of Fab fragments derived from CD28.3, a decrease in the production of γIFN and of IL2, and an increase in the production of IL10 are observed.","This deviation in the immune response suggests an orientation toward a Th2-type response.","This result is unexpected insofar as it has been reported that the involvement of CTLA4 (which is supposed to intervene in the blocking of CD28 alone) leads to a Th1-type response.","In Vitro Processing of the Antibody CD28.3 and of the Fab Fragments Derived Therefrom, by Human T Cells A possible internalization of the Fab fragments of the antibody CD28.3 in human T cells was investigated, in comparison with the whole antibody CD28.3.Jurkat T cells were incubated in culture medium with 100 μg/ml of antibody CD28.3, at 37° C. or at 0° C. At various times, the cells were washed with cold PBS buffer containing 0.1% of bovine serum albumin, and NaN3, in order to block membrane motility.","The bound antibodies were revealed with a fluorescein-labeled goat anti-mouse secondary antibody.","The cells were mounted in MOVIOL and analyzed by confocal microscopy.","It is thus observed that the whole CD28.3 antibodies which bind to the Jurkat T cells are captured and disappear from the cell surface at 37° C., but not at 0° C. On the other hand, the Fab fragments remain attached at the surface of the cell.","This indicates that the attachment of the divalent antibodies CD28.3 leads to dimerization of CD28, which brings about their entry into the cell, whereas the monovalent Fab fragments, which do not induce this dimerization, remain at the surface.","EXAMPLE 2 Properties of an scFv Fragment Derived from the Antibody CD28.3 FIG.","1 represents the nucleotide sequence and the deduced polypeptide sequence of an scFv fragment derived from the antibody CD28.3.The portions of this sequence corresponding to the variable fragment of the heavy chain and of the light chain are represented in capital letters.","The sequence corresponding to the variable fragment of the light chain is also underlined.","The sequence of the linker is represented in lower case letters.","The sequences of the CDRs of the heavy chain and of the light chain are boxed in.","The nucleotide sequence encoding this scFv fragment is also represented in the attached sequence listing, under the number SEQ ID No.1.The cDNA encoding this scFv fragment was inserted into the vector pIG6 (Biochemisches Institut, University of Zurich).","This vector comprises in particular an ampicillin resistance marker and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.","The cDNA encoding the scFv fragment described above was introduced between the EcoRI and EcoRV sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.","The construct obtained is called pIg6-28.3.Production in Prokaryotic Cells The vector pIg6-28.3 was used to transform E.coli JM83 cells.","The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the scFv fragment is produced in soluble form in the periplasma.","After electrophoresis and Western blotting, it appears in the form of a band at approximately 30 kDa.","It is purified from the periplasmic extracts of the bacteria, obtained after osmotic shock in 50 mM Tris-Cl, and ultracentrifugation of the insoluble material, by chromatography on an Ni-NTA matrix and ion exchange on DEAE-Sepharose.","The binding of the scFv fragments present in the eluate of the NiNTA column, to CD28+ Jurkat cells, is comparable to that obtained with Fab fragments obtained from the antibody CD28.3 by digestion with papain.","Production in Eukaryotic Cells The vector pSec-28.3 was used to transfect Cos cells.","The cells are cultured at 37° C. for 3 days.","The scFv fragment is produced in soluble form in the supernatant.","This supernatant inhibits the mixed lymphocyte reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, healthy allogenic donor.","The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.","Considerable inhibition of the incorporation, dependent on the supernatant dilution used, is observed.","A control supernatant exhibits no proliferation-inhibiting activity.","EXAMPLE 3 Production of a Fusion Protein Comprising an scFv Fragment of CD28.3 The nucleotide sequence encoding the scfv fragment described in example 2 was linked to the 5′ end of a portion of the cDNA of human α1-antitrypsin (GENBANK accession number K01396) corresponding to amino acids 53 to 425, via a hinged peptide of sequence VAAPS.","The resulting sequence is represented in the attached sequence listing under the number SEQ ID No.","3, and the corresponding polypeptide under the number SEQ ID No.","4.EXAMPLE 4 Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment Prokaryotic Expression Vector: The vector pIG6 was used (Biochemisches Institut, University of Zurich).","This vector comprises in particular an ampicillin resistance marker, and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.","The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the EcoRI and EcoRV sites of pIg6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.","FIG.","1 illustrates diagrammatically the construct obtained, called pIg6-Haat.","Eukaryotic Expression Vector: The vector pSECTagB (Invitrogen, De Schelp, The Netherlands) was used.","This vector comprises in particular an ampicillin resistance marker, a zeocin resistance marker, and an expression cassette which comprises a CMV promoter under the control of which are placed: a sequence encoding a signal peptide of the IgG kappa light chain, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-6 marker.","The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the BamHI and EcoRI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.","FIG.","2 illustrates diagrammatically the construct obtained, called pSecHaat.","EXAMPLE 5 Construction of Expression Vectors Integrating the Sequence Encoding the CD28.3 scFv/α1-Antitrypsin Fusion Protein Prokaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the EcoRI and XhoI sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.","FIG.","3 illustrates diagrammatically the construct obtained, called pIg6-28.3Haat.","Eukaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the BamHI and XhoI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.","FIG.","4 illustrates diagrammatically the construct obtained, called pSec-28.3Haat.","This vector, harbored by E.coli DH5α, was deposited with the CNCM on Dec. 11, 2001, under the number I-2762.EXAMPLE 6 Expression and Purification of the Fusion Proteins In Prokaryotic Cells: The vector pIg6-28.3Haat was used to transform E. coli JM83 cells.","The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the protein is produced in soluble form in the periplasm.","After electrophoresis and Western blotting, it appears in the form of a band at approximately 74 kDa.","It can be purified from the periplasmic extracts using an NI-NTA affinity chromatography matrix and/or an anti-c-myc affinity chromatography matrix.","It can also be purified using an anti-α1-antitrypsin affinity column.","In Eukaryotic Cells: The vector pSec-28.3Haat was used to transfect CHO cells by lipofection.","The cells are cultured in the presence of 200 μg/ml on zeocin in MEM medium containing 10% of fetal calf serum.","The protein is secreted into the culture medium.","After separation by electrophoresis, Western blotting, and revelation with an anti-c-myc antibody, it appears in the form of a band at approximately 80 kDa.","EXAMPLE 7 Assays for Activity of an scFv/α1-Antitrypsin Fusion Protein The anti-CD28 activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above was evaluated by its binding to the CD28 molecule, or to cells expressing CD28 on their membrane, and its lack of binding to cells which do not express CD28.The immunosuppressor activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above is evaluated by the inhibition of adhesion to B7, and the inhibition of the induced activation of the T lymphocyte.","These anti-CD28 and immunosuppresor activities were measured using the following assays: Anti-CD28 Activity Biosensor Measurement of the CD28-Binding Parameters: Recombinant human CD28 was immobilized on the biosensor (BIACORE) detector.","A CD28.3 scFv/α1-antitrypsin fusion protein obtained as described in example 6 above was brought into contact with the detector.","The binding parameters are: KA (1/M)2.86e9; KD (M): 3.49e-10.In comparison, these parameters measured for the Fab fragment of the antibody CD28.3 are: KA (1/M): 9.69e8; KD (M): 1.03e-9.The affinity for CD28, of the Fab fragment of the antibody CD28.3 and of the fusion protein, are therefore comparable.","Cytofluorometry Assay for Specific Recognition of CD28: 105 Jurkat (Cd28+) and U937 (CD28−) cells are incubated in PBS-1% BSA-0.1% NaN3, at 4° C., for 1 hour with increasing concentrations of the CD28.3 scFv/α1-antitrypsin fusion protein.","After washing, the cells are incubated with an anti-alpha-1-antitrypsin rabbit antibody and then with an FITC-conjugated goat anti-rabbit antibody, washed, and analyzed by cytofluorometry.","Binding dependent on the dose of the Jurkat cells (CD28+), and no binding to the U937 (CD28−) cells, were observed.","This shows the specificity of the fusion protein for the CD28 molecule and its lack of reactivity toward other molecules expressed by human hematopoietic cells.","Immunosuppressor Activity CD28/B7-Dependent Adhesion Assay: 4×105 human T cells (CD28-positive Jurkat cells) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.","Chem., 271, 9403 (1996)] had been seeded 24 hours beforehand.","These incubations are carried out in the absence or in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, diluted to various concentrations in PBS buffer without Ca2+ or Mg2+.","The adherent cells after washing are quantified by reading the residual radioactivity using a beta counter (PACKARD TOPCOUNT).","Inhibition of the adhesion in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein is observed, which inhibition is directly dependent on the dose of fusion protein used.","Inhibition of the Activation: 5×104 T cells (human polyclonal cells depleted of CD11b cells) are stimulated with 1×104 irradiated OKT3 hybridoma cells (anti-CD3), or with allogenic CD28− B cells (depleted of CD28+ cells), in the absence or in the presence of varying amounts of the CD28.3 scFv/α1-antitrypsin fusion protein.","The proliferative response in these cultures is evaluated after 3 days when the stimulation is performed with anti-CD3s, or after 7 days when the stimulation is performed with allogenic cells, by incorporation of (3H) thymidine for a period of 16 hours.","Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.","Inhibition of the Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, allogenic, healthy donor.","The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.","Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used."]],"string":"[\n [\n \"Anti-Cd28 Antibody\",\n \"The invention concerns an antibody directed against the CD28 receptor and capable of blocking CD28/B7 interaction, and proteins derived from said antibody, for use in particular to block CD28-dependent activation of lymphocytes.\"\n ],\n [\n \"1.A protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.2.The protein as claimed in claim 1, selected from the group consisting of: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) Fv, Fab, Fab′2 or scFv fragments of the antibody CD28.3; c) chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) Fv, Fab, Fab′2 or scFv fragments of an antibody b); and e) recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.\",\n \"3.A nucleic acid molecule encoding a protein as claimed claim 1.4.An expression vector comprising a nucleic acid molecule as claimed in claim 3.5.The expression vector as claimed in claim 4, wherein the expression vector is the plasmid CNCM I-2762.6.A cell expressing a protein as claimed in claim 1.7.The cell as claimed in claim 6, wherein the cell is the hybridoma CNCM I-2582.8.A cell transformed with a nucleic acid molecule as claimed in claim 3, and expressing a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CO28/B7 interaction and comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.9.A method for preparing a protein as claimed in claim 1, comprising culturing at least one cell as claimed in claim 6, and recovering said protein from said culture.\",\n \"10.A medicinal product comprising the protein as claimed in claim 1.11.The medicinal product as claimed in claim 10, wherein in that said protein has a single CD28 receptor-binding site, and said medicinal product is an immunosuppressor which selectively blocks T cell activation via the CD28 receptor.\",\n \"12.A method of treating a pathological condition selected from the group consisting of transplant rejection, graft-versus-host disease, T-lymphocyte-mediated autoimmune diseases, allergic phenomena and chronic inflammatory diseases wherein the method comprises administering to a patient in need thereof an effective amount of the medicinal product of claim 11.13.A nucleic acid molecule encoding a protein as claimed in claim 2.14.A cell expressing a protein as claimed in claim 2.\"\n ],\n [\n \" Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment \"\n ],\n [\n \"The invention relates to antibodies directed against the CD28 lymphocyte receptor and to their fragments, and to their therapeutic uses, in particular in the context of regulating T cell activation.\",\n \"Abnormal activation of T cells is involved in the pathogenesis of many autoimmune diseases, and also in transplant rejection phenomena, where they cause an immune response directed against the transplanted organ to develop.\",\n \"T lymphocyte activation requires an activating signal, induced by the recognition, by T receptors (TCRs) of the antigen associated with the class II major histocompatibility complex (MHC) and presented by antigen-presenting cells (APCs).\",\n \"However, this activation only causes proliferation of T cells and secretion of specific immunomodulatory cytokines (such as interleukin 2, gamma interferon or interleukin 4) if other T co-stimulation systems are also activated.\",\n \"One of the most important systems for regulating T lymphocyte activation is the molecular system B7/CD28/CTLA4.This system plays, for example, an essential role in the mechanisms of transplant rejection [Woodward et al., Transplantation, 66, 14-20, (1998)].\",\n \"The molecules B7.1 (CD80) and B7.2 (CD86) borne by the APCs can activate the CD28 receptor and also the CTLA4 receptor of T lymphocytes.\",\n \"The activation of CD28 sends the T lymphocyte a positive signal which stimulates the cell; on the other hand, the activation of CTLA4 sends a negative signal which leads to a non-response (anergy) [FALLARINO et al., J. Exp.\",\n \"Med., 188, 205-210, (1998)].\",\n \"Dormant T lymphocytes express a large amount of CD28 and very little CTLA4.When there is a first cognitive contact between an APC and a T lymphocyte, the CD28/B7 interaction is favored, which activates the cell.\",\n \"It is only several hours after the initiation of activation that, due to the increase in membrane expression of CTLA4, the affinity of which for B7 is 5 to 10 times greater than that of CD28, the B7/CD28 interaction shifts in favor of a B7/CTLA4 interaction.\",\n \"Currently, cyclosporin is mainly used to block T lymphocyte activation, in particular in the context of organ transplants.\",\n \"Despite the effectiveness of this medicinal product, it does not, however, confer absolute protection.\",\n \"In addition, it acts by blocking all the calcium-dependent cell activation pathways, and therefore has a biological activity which is not strictly T lyphocyte specific and leads to a considerable number of side effects.\",\n \"It is therefore desirable to develop new immunosuppressants which have a defined method of action and greater specificity.\",\n \"It has been postulated that selective inhibition of the agonist signal given to the T cell by CD28, leaving the antagonist system consisting of the pair CTLA4/B7 intact, via specific blocking of the CD28/B7 interaction, would make it possible to prevent T lymphocyte activation.\",\n \"Such specific blocking of the CD28/B7 interaction can be obtained using an antibody directed against CD28.Anti-CD28 antibodies capable of preventing CD28 binding to B7 are known.\",\n \"However, they have the drawback, when they are used in their divalent native form, of bringing about the dimerization and the activation of CD28 via their binding with this receptor.\",\n \"However, monovalent fragments derived from these antibodies are capable of blocking the CD28 receptor without activating it [DAMLE et al., J. Immunol.\",\n \"140, 1753-1761, (1988); NUNES et al., Int.\",\n \"Immunol., 5, 311-315 (1993); PAGES et al., J. Biol.\",\n \"Chem., 271, 9403, (1996)].\",\n \"It has thus been reported [PERRIN et al., J. Immunol.\",\n \"163, 1704-1710, (1999)] that Fab fragments derived from an anti-CD28 antibody can curb the clinical symptoms of experimental autoimmune encephalitis induced in mice by the administration of myelin or the transfer of T cells from an affected animal.\",\n \"Monovalent Fab or scFv fragments derived from an anti-CD28 antibody can potentially be used to prevent T lymphocyte activation via specific blocking of the CD28/B7 interaction.\",\n \"Fab fragments result from the action of papain on an immunoglobulin molecule, and each contain a light chain and the first half of a heavy chain; scfv fragments consist of the variable portions of the heavy and light chains of an antibody, connected to one another via a flexible linker [CLACKSON et al., Nature, 352, 624-628, (1991)], thus forming a single-chain protein.\",\n \"These monovalent fragments frequently exhibit less affinity for the antigen than the native antibodies, which can limit their possibilities for use in diagnostic or therapeutic applications.\",\n \"The inventors have succeeded in selecting, among various antibodies which recognize the CD28 antigen, an antibody capable of blocking the CD28/B7 interaction, the monovalent fragments of which exhibit sufficient affinity for the antigen so that they can be used, in vitro or in vivo, to block the CD28 receptor without activation of this receptor.\",\n \"This antibody, called CD28.3, is produced by the hybridoma deposited, according to the terms of the Treaty of Budapest, on Nov. 28, 2000, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2582.A subject of the present invention is a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, characterized in that it comprises at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3.CDRs (complementarity determining regions) are the portions of the variable regions of an immunoglobulin which are involved in antigen recognition specificity.\",\n \"Proteins in accordance with the invention thus encompass in particular: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) the Fv, Fab, Fab′2 or scfv fragments of the antibody CD28.3; c) the chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) the fragments of the antibodies b) above comprising the CDRs of the antibody CD28.3, with a monovalent Fv, Fab or scfv fragments, or divalent Fab′2 fragments; e) the recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.\",\n \"They may, for example, be: divalent or plurivalent derivatives of scfv fragments, such as “diabodies” or “triabodies”, resulting from the association of 2 or 3 scFv fragments; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with at least one antibody fragment comprising the CDRs of an antibody of different specificity; by way of examples, mention will be made of bispecific immunoglobulins, conjugates of an Fv or Fab fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity, and “bispecific diabodies” resulting from the association of an scFv fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule having pharmacological activity (for example a toxin) or effector properties (for example an Fc fragment); proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule which makes it possible to prolong its plasma halflife when it is administered in vivo; it is, for example, possible to combine said antibody fragment with a water-soluble polypeptide of sufficient molecular mass for the molecular mass of the fusion polypeptide thus obtained to be greater than the renal filtration threshold.\",\n \"In this case, a polypeptide will be chosen which, unlike Fc fragments, cannot associate as dimers, and which does not have its own effector activity liable to cause unfortunate side effects.\",\n \"Polypeptides which have these properties can advantageously be obtained from water-soluble serum proteins, namely, in particular, serum albumin, haptoglobulin, ITIH2 (inter-alpha (globulin) inhibitor, H2 polypeptide), transferrin, CBG (corticosteroid binding globulin), α1-antitrypsin, ITIH4 (inter-alpha (globulin) inhibitor, H4 polypeptide), AACT (alpha-1-antichymotrypsin), TBG (thyroxine binding globulin), fibrinogen and prothrombin, in order to prepare fusion proteins with scFv fragments derived from anti-CD28 antibodies.\",\n \"It is also possible to conjugate a protein in accordance with the invention with a polyol, for example polyethylene glycol, as described, for example, in U.S. Pat.\",\n \"No.\",\n \"4,179,337.An example of a protein in accordance with the invention is illustrated in FIG.\",\n \"1, which represents an scFv fragment derived from the antibody CD28.3.The sequences of the CDRs of the antibody CD28.3 are boxed in the sequence represented in FIG.\",\n \"1.The nucleotide sequence encoding this scFv fragment is represented in the attached sequence listing under the number SEQ ID No.\",\n \"1, and the corresponding peptide sequence is represented under the number SEQ ID No.\",\n \"2.Fv, Fab or Fab′2 fragments in accordance with the invention can be obtained by the conventional techniques of enzyme digestion, from the antibody CD28.3.A plasmid containing a polynucleotide encoding an scFv fragment of CD28.3, fused to a polynucleotide encoding amino acids 53 to 425 of α1-antitrypsin was deposited, according to the terms of the Treaty of Budapest, on Dec. 11, 2001, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2762.Proteins in accordance with the invention such as chimeric or recombinant antibodies, scFv fragments and their derivatives, etc., can be obtained by conventional genetic engineering techniques, such as those described by SAMBROOK et al.\",\n \"[MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989)].\",\n \"Polynucleotides encoding the variable regions of the anti-CD28.3 antibody can, for example, be obtained by cloning said variable regions from a cDNA library of the hybridoma CD28.3, or from the plasmid CNCM I-2762.They can also be prepared, completely or partially, by nucleic acid synthesis, based on the nucleotide sequences of said variable regions.\",\n \"It is, for example, possible to synthesize polynucleotides encoding the CDRs of CD28.3, and to incorporate them into the framework regions (FRs) of another antibody, in particular of an antibody of human origin, using techniques, known in themselves, of CDR grafting, such as those described by ROUTLEDGE et al.\",\n \"[“Reshaping antibodies for therapy”, in Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, 13-44, Academic Titles, Nottingham, England (1993)] or by ROGUSKA et al.\",\n \"Protein Engineering, 9(10), 895-904, (1996)].\",\n \"A subject of the present invention is also any nucleic acid molecule encoding a protein in accordance with the invention comprising the CDRs of the antibody CD28.3, and also any recombinant vector, in particular any expression vector, comprising said nucleic acid molecule.\",\n \"A subject of the present invention is also any cell expressing a protein in accordance with the invention comprising the CDRs of the antibody CD28.3.This encompasses in particular the hybridoma CNCM I-2582, and also the host cells transformed with a nucleic acid molecule in accordance with the invention.\",\n \"Nucleic acid molecules in accordance with the invention may advantageously comprise, besides a sequence encoding a protein in accordance with the invention, a sequence encoding a signal peptide allowing secretion of said protein; they may also comprise one or more sequence(s) encoding one or more marker peptide(s) for detecting, and/or facilitating the purification of, said protein.\",\n \"Expression vectors in accordance with the invention comprise at least one nucleic acid sequence encoding a protein in accordance with the invention, associated with transcription- and translation-controlling elements which are active in the host cell chosen.\",\n \"Vectors which can be used to construct expression vectors in accordance with the invention are known in themselves, and will be chosen in particular as a function of the host cell intended to be used.\",\n \"Host cells which can be used in the context of the present invention can be prokaryotic or eukaryotic cells.\",\n \"Among the eukaryotic cells which can be used, mention will in particular be made of plant cells, cells from yeast, such as Saccharomyces, insect cells, such as Drosophila or Spodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc., cells.\",\n \"The construction of expression vectors in accordance with the invention and the transformation of the host cells can be carried out by the conventional techniques of molecular biology.\",\n \"A subject of the invention is also a method for producing a protein in accordance with the invention, characterized in that it comprises culturing at least one cell in accordance with the invention, and recovering said protein from said culture.\",\n \"If the protein is secreted, it can be recovered directly from the culture medium; if not, cell lysis will be carried out beforehand.\",\n \"The protein can then be purified from the culture medium or from the cell lysate, by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular precipitation with ammonium sulfate, electrophoresis, gel filtration, affinity chromatography, etc.\",\n \"The proteins in accordance with the invention can be used, in vitro, to study the proliferative response or the differentiation of T lymphocytes responding to an antigenic, viral, allogenic or xenogenic stimulation.\",\n \"They can also be used, in vitro, to induce the differentiation of T lymphocytes taken from a patient, for example the induction of tolerance with respect to an antigen or to an alloantigen, intended to be subsequently re-administered in vivo.\",\n \"They may also be used to obtain medicinal products, or diagnostic reagents.\",\n \"Proteins in accordance with the invention which are divalent, i.e.\",\n \"which have 2 CD28 receptor-binding sites, and thus capable of inducing dimerization of this receptor, can be used in all situations where it is desired to activate this CD28 receptor, i.e.\",\n \"to increase the response of a T lymphocyte with respect to an antigen.\",\n \"Proteins in accordance with the invention which are monovalent, i.e.\",\n \"which have a single CD28 receptor-binding site, can be used in all situations where it is desired to selectively block this receptor without activating it, in order to induce immunosuppression.\",\n \"A protein in accordance with the invention, comprising a monovalent fragment derived from an anti-CD28 antibody, can in particular be used to obtain an immunosuppressant medicinal product which selectively blocks T cell activation phenomena involving the CD28 receptor, and which does not have the drawbacks of known immunosuppressants such as cyclosporin.\",\n \"The T immunosuppression by selective blocking of CD28 with protein in accordance with the invention has applications in all T lymphocyte-dependent pathological conditions.\",\n \"These are essentially transplant rejection, graft-versus-host disease, T lymphocyte-mediated autoimmune diseases, such as type I diabetes or multiple sclerosis, and type IV hypersensitivity, which is involved in allergic phenomena and also in the pathogenesis of chronic inflammatory diseases following infection with a pathogenic agent (in particular leprosy, tuberculosis, leishmaniasis, listeriosis, etc.).\",\n \"The present invention will be understood more clearly from the further description which follows, which refers to nonlimiting examples of preparation and of use of antibodies in accordance with the invention.\",\n \"EXAMPLE 1 Choice of an Antibody Producing Monovalent Fragments, Properties of Monovalent Fab Fragments Derived from CD28.3 Some of the properties of several anti-CD28 antibodies (CD28.1, CD28.2, CD28.3, CD28.4, CD28.5 and CD28.6) are described in the publication by NUNES et al.\",\n \"[Int.\",\n \"Immunol., 5, 311, (1993)].\",\n \"These various antibodies, which are not accessible to the public, were provided by the laboratory of Daniel OLIVE (INSERM).\",\n \"The antigen-binding properties of the monovalent Fab fragments of these various antibodies were compared.\",\n \"5 mg of Fab fragments of each of these antibodies were prepared by digestion with papain (papain/antibody molar ratio= 1/100) for 24 hours at 37° C., followed by inactivation of the enzyme with 0.03M iodoacetamide, and dialysis against PBS to remove the iodoacetamide.\",\n \"1) Binding of the Fab Fragments to CD28+ Jurkat T Cells: 100,000 CD28+ Jurkat cells in 100 μl are incubated in PBS-1% BSA-0.1% NaN3 at 4° C. for 30 minutes with increasing concentrations of anti-CD28 antibodies or of their Fab fragments.\",\n \"After washing, the cells are incubated in a similar way with an FITC-conjugated anti-mouse IgG goat antibody, washed, and analyzed by cytofluorometry.\",\n \"The results are given in FIG.\",\n \"2: Legend of FIG.\",\n \"2: X-axis: antibody or Fab fragments concentration Y-axis: mean fluorescence intensity (MFI) —⋄—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —Δ—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —◯—: F6=Fab fragments of the antibody CD28.6 ····: W132 whole antibody CD28.1 ··-··: W2=whole antibody CD28.2 : W3=whole antibody CD28.3 ——: W5=whole antibody CD28.5 —*—: W6=whole antibody CD28.6 —▴—: Mara-1=negative control (mouse IgG1).\",\n \"These results show that, among the Fab fragments, only those derived from CD28.3 are capable of significantly binding to the CD28+ Jurkat cells at concentrations of less than 10 μg/ml.\",\n \"2) Effect of the Fab Fragments on the Adhesion of CD28+ Jurkat T Cells to Transfected Murine L Cells Expressing the B7-1 Molecule: 4×105 human T cells (Jurkat, CD28-positive) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.\",\n \"Chem., 271, 9403, (1996)] have been seeded 24 hours beforehand.\",\n \"These incubations are carried out in the presence of the Fab fragments derived from the antibodies CD28.1 to CD28.6, or of the antibody CD28.3, diluted to various dilutions in PBS buffer without Ca2+ or Mg2+.\",\n \"The adherent cells after washing are quantified by reading the residual radioactivity in a beta counter (PACKARD TOPCOUNT).\",\n \"The results are given in FIG.\",\n \"3: Legend of FIG.\",\n \"3: X-axis: percentage of adherent cells Y-axis: antibody concentration —♦—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —▴—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —*—: F6=Fab fragments of the antibody CD28.6 ····: whole antibody CD28.3 ◯: no antibody.\",\n \"These results show that the Fab fragments derived from CD28.3 are the most effective for inhibiting CD28/B7 interactions.\",\n \"They give 90% inhibition of adhesion at a concentration of 3 μg/ml, and with an effectiveness comparable to that of the whole antibody CD28.3, whereas, at this concentration, the Fab fragments derived from the other antibodies give no more than 50% inhibition of adhesion.\",\n \"3) Effect of the Fab Fragments on Proliferation in a Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells (PBMCs) are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, in the presence of varying concentrations of the antibodies CD28.1 to CD28.6 or of the Fab fragments derived from these antibodies.\",\n \"The proliferative response in these cultures is evaluated after 3 days, by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"The results are given in FIG.\",\n \"4: Legend of FIG.\",\n \"4: X-axis: antibody concentration Y-axis: proliferative response (cpm) Basal level of proliferation=6 500 cpm.\",\n \"—♦—: 28.1=antibody CD28.1 —▪—: 28.2=antibody CD28.2 —▴—: 28.3=antibody CD28.3 —x—: 28.5=antibody CD28.5 ···*···: 28.6=antibody CD28.6 ——: Fab.\",\n \"1=Fab fragments of the antibody CD28.1 ··+··: Fab.\",\n \"2=Fab fragments of the antibody CD28.2 ——: Fab.\",\n \"3=Fab fragments of the antibody CD28.3 —+—: Fab.\",\n \"5=Fab fragment of the antibody CD28.5 —⋄—: Fab.\",\n \"6=Fab fragment of the antibody CD28.6.These results show that the Fab fragments derived from CD28.3 or from CD28.6 are the most effective for inhibiting mononuclear cell proliferation.\",\n \"The whole antibodies CD28.1 to CD28.6, tested in parallel, have no inhibitory effect or indeed stimulate the proliferation by virtue of their stimulator action on CD28.Effect of the Fab Fragments Derived from CD28.3 on Proliferation Induced by a Superantigen For this experiment, responder CD4+ T cells were mixed with irradiated isogenic PBMCs, in the presence of 50 ng/ml of toxic shock syndrome toxin-1 (TSST-1), which specifically stimulates the vβ2+ T cell, either in the absence of antibody or in the presence of anti-B7-1 (1 μg/ml), of anti-B7-2 (0.5 μg/ml), of CTLA4Ig (10 μg/ml), or of Fab fragments derived from CD28.3 (10 μg/ml).\",\n \"The proliferative response in these cultures is evaluated after 1, 3, 6 and 8 days, by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"The results are given in FIG.\",\n \"5: Legend of FIG.\",\n \"5: X-axis: culturing time Y-axis: proliferation index=PI P I = cpm mixed lumphocyte reaction - cpm irradiated stimulating cells only cpm unstimulated responder cells —♦—: anti-CD28.3 Fab —X—: anti-B7-2 —▪—: CTLA-4 Ig ··*··: anti-B7-1+2 —▴—: anti-B7-1 —◯—: no antibody TSST-1 induces considerable proliferation of CD4+ T cells.\",\n \"In the presence of anti-B7, of CTLA4Ig or of the Fab fragments of CD28.3, 70% inhibition of this proliferation is observed after 6 days.\",\n \"Effect of the Fab Fragments Derived from CD28.3 on Cytokine Production In order to determine whether the Fab fragments derived from CD28.3 could induce an immune deviation in vitro, a mixed lymphocyte reaction (PBMCs derived from a donor A/irradiated PBMCs derived from a donor B) was carried out, in the presence of Fab fragments derived from CD28.3.105 peripheral blood mononuclear cells from a donor are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, and cultured for 5 days in the presence or absence of 10 μg/ml of Fab derived from the antibody CD28.3.The RNA of the responder cells was extracted, and the amount of cytokine mRNA was evaluated by quantitative measurement of the number of transcripts, related to the amount of HPRT, using a TaqMan (Perkin Elmer).\",\n \"In the presence of Fab fragments derived from CD28.3, a decrease in the production of γIFN and of IL2, and an increase in the production of IL10 are observed.\",\n \"This deviation in the immune response suggests an orientation toward a Th2-type response.\",\n \"This result is unexpected insofar as it has been reported that the involvement of CTLA4 (which is supposed to intervene in the blocking of CD28 alone) leads to a Th1-type response.\",\n \"In Vitro Processing of the Antibody CD28.3 and of the Fab Fragments Derived Therefrom, by Human T Cells A possible internalization of the Fab fragments of the antibody CD28.3 in human T cells was investigated, in comparison with the whole antibody CD28.3.Jurkat T cells were incubated in culture medium with 100 μg/ml of antibody CD28.3, at 37° C. or at 0° C. At various times, the cells were washed with cold PBS buffer containing 0.1% of bovine serum albumin, and NaN3, in order to block membrane motility.\",\n \"The bound antibodies were revealed with a fluorescein-labeled goat anti-mouse secondary antibody.\",\n \"The cells were mounted in MOVIOL and analyzed by confocal microscopy.\",\n \"It is thus observed that the whole CD28.3 antibodies which bind to the Jurkat T cells are captured and disappear from the cell surface at 37° C., but not at 0° C. On the other hand, the Fab fragments remain attached at the surface of the cell.\",\n \"This indicates that the attachment of the divalent antibodies CD28.3 leads to dimerization of CD28, which brings about their entry into the cell, whereas the monovalent Fab fragments, which do not induce this dimerization, remain at the surface.\",\n \"EXAMPLE 2 Properties of an scFv Fragment Derived from the Antibody CD28.3 FIG.\",\n \"1 represents the nucleotide sequence and the deduced polypeptide sequence of an scFv fragment derived from the antibody CD28.3.The portions of this sequence corresponding to the variable fragment of the heavy chain and of the light chain are represented in capital letters.\",\n \"The sequence corresponding to the variable fragment of the light chain is also underlined.\",\n \"The sequence of the linker is represented in lower case letters.\",\n \"The sequences of the CDRs of the heavy chain and of the light chain are boxed in.\",\n \"The nucleotide sequence encoding this scFv fragment is also represented in the attached sequence listing, under the number SEQ ID No.1.The cDNA encoding this scFv fragment was inserted into the vector pIG6 (Biochemisches Institut, University of Zurich).\",\n \"This vector comprises in particular an ampicillin resistance marker and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.\",\n \"The cDNA encoding the scFv fragment described above was introduced between the EcoRI and EcoRV sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.\",\n \"The construct obtained is called pIg6-28.3.Production in Prokaryotic Cells The vector pIg6-28.3 was used to transform E.coli JM83 cells.\",\n \"The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the scFv fragment is produced in soluble form in the periplasma.\",\n \"After electrophoresis and Western blotting, it appears in the form of a band at approximately 30 kDa.\",\n \"It is purified from the periplasmic extracts of the bacteria, obtained after osmotic shock in 50 mM Tris-Cl, and ultracentrifugation of the insoluble material, by chromatography on an Ni-NTA matrix and ion exchange on DEAE-Sepharose.\",\n \"The binding of the scFv fragments present in the eluate of the NiNTA column, to CD28+ Jurkat cells, is comparable to that obtained with Fab fragments obtained from the antibody CD28.3 by digestion with papain.\",\n \"Production in Eukaryotic Cells The vector pSec-28.3 was used to transfect Cos cells.\",\n \"The cells are cultured at 37° C. for 3 days.\",\n \"The scFv fragment is produced in soluble form in the supernatant.\",\n \"This supernatant inhibits the mixed lymphocyte reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, healthy allogenic donor.\",\n \"The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"Considerable inhibition of the incorporation, dependent on the supernatant dilution used, is observed.\",\n \"A control supernatant exhibits no proliferation-inhibiting activity.\",\n \"EXAMPLE 3 Production of a Fusion Protein Comprising an scFv Fragment of CD28.3 The nucleotide sequence encoding the scfv fragment described in example 2 was linked to the 5′ end of a portion of the cDNA of human α1-antitrypsin (GENBANK accession number K01396) corresponding to amino acids 53 to 425, via a hinged peptide of sequence VAAPS.\",\n \"The resulting sequence is represented in the attached sequence listing under the number SEQ ID No.\",\n \"3, and the corresponding polypeptide under the number SEQ ID No.\",\n \"4.EXAMPLE 4 Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment Prokaryotic Expression Vector: The vector pIG6 was used (Biochemisches Institut, University of Zurich).\",\n \"This vector comprises in particular an ampicillin resistance marker, and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.\",\n \"The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the EcoRI and EcoRV sites of pIg6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"1 illustrates diagrammatically the construct obtained, called pIg6-Haat.\",\n \"Eukaryotic Expression Vector: The vector pSECTagB (Invitrogen, De Schelp, The Netherlands) was used.\",\n \"This vector comprises in particular an ampicillin resistance marker, a zeocin resistance marker, and an expression cassette which comprises a CMV promoter under the control of which are placed: a sequence encoding a signal peptide of the IgG kappa light chain, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-6 marker.\",\n \"The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the BamHI and EcoRI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"2 illustrates diagrammatically the construct obtained, called pSecHaat.\",\n \"EXAMPLE 5 Construction of Expression Vectors Integrating the Sequence Encoding the CD28.3 scFv/α1-Antitrypsin Fusion Protein Prokaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the EcoRI and XhoI sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"3 illustrates diagrammatically the construct obtained, called pIg6-28.3Haat.\",\n \"Eukaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the BamHI and XhoI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.\",\n \"FIG.\",\n \"4 illustrates diagrammatically the construct obtained, called pSec-28.3Haat.\",\n \"This vector, harbored by E.coli DH5α, was deposited with the CNCM on Dec. 11, 2001, under the number I-2762.EXAMPLE 6 Expression and Purification of the Fusion Proteins In Prokaryotic Cells: The vector pIg6-28.3Haat was used to transform E. coli JM83 cells.\",\n \"The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the protein is produced in soluble form in the periplasm.\",\n \"After electrophoresis and Western blotting, it appears in the form of a band at approximately 74 kDa.\",\n \"It can be purified from the periplasmic extracts using an NI-NTA affinity chromatography matrix and/or an anti-c-myc affinity chromatography matrix.\",\n \"It can also be purified using an anti-α1-antitrypsin affinity column.\",\n \"In Eukaryotic Cells: The vector pSec-28.3Haat was used to transfect CHO cells by lipofection.\",\n \"The cells are cultured in the presence of 200 μg/ml on zeocin in MEM medium containing 10% of fetal calf serum.\",\n \"The protein is secreted into the culture medium.\",\n \"After separation by electrophoresis, Western blotting, and revelation with an anti-c-myc antibody, it appears in the form of a band at approximately 80 kDa.\",\n \"EXAMPLE 7 Assays for Activity of an scFv/α1-Antitrypsin Fusion Protein The anti-CD28 activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above was evaluated by its binding to the CD28 molecule, or to cells expressing CD28 on their membrane, and its lack of binding to cells which do not express CD28.The immunosuppressor activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above is evaluated by the inhibition of adhesion to B7, and the inhibition of the induced activation of the T lymphocyte.\",\n \"These anti-CD28 and immunosuppresor activities were measured using the following assays: Anti-CD28 Activity Biosensor Measurement of the CD28-Binding Parameters: Recombinant human CD28 was immobilized on the biosensor (BIACORE) detector.\",\n \"A CD28.3 scFv/α1-antitrypsin fusion protein obtained as described in example 6 above was brought into contact with the detector.\",\n \"The binding parameters are: KA (1/M)2.86e9; KD (M): 3.49e-10.In comparison, these parameters measured for the Fab fragment of the antibody CD28.3 are: KA (1/M): 9.69e8; KD (M): 1.03e-9.The affinity for CD28, of the Fab fragment of the antibody CD28.3 and of the fusion protein, are therefore comparable.\",\n \"Cytofluorometry Assay for Specific Recognition of CD28: 105 Jurkat (Cd28+) and U937 (CD28−) cells are incubated in PBS-1% BSA-0.1% NaN3, at 4° C., for 1 hour with increasing concentrations of the CD28.3 scFv/α1-antitrypsin fusion protein.\",\n \"After washing, the cells are incubated with an anti-alpha-1-antitrypsin rabbit antibody and then with an FITC-conjugated goat anti-rabbit antibody, washed, and analyzed by cytofluorometry.\",\n \"Binding dependent on the dose of the Jurkat cells (CD28+), and no binding to the U937 (CD28−) cells, were observed.\",\n \"This shows the specificity of the fusion protein for the CD28 molecule and its lack of reactivity toward other molecules expressed by human hematopoietic cells.\",\n \"Immunosuppressor Activity CD28/B7-Dependent Adhesion Assay: 4×105 human T cells (CD28-positive Jurkat cells) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.\",\n \"Chem., 271, 9403 (1996)] had been seeded 24 hours beforehand.\",\n \"These incubations are carried out in the absence or in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, diluted to various concentrations in PBS buffer without Ca2+ or Mg2+.\",\n \"The adherent cells after washing are quantified by reading the residual radioactivity using a beta counter (PACKARD TOPCOUNT).\",\n \"Inhibition of the adhesion in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein is observed, which inhibition is directly dependent on the dose of fusion protein used.\",\n \"Inhibition of the Activation: 5×104 T cells (human polyclonal cells depleted of CD11b cells) are stimulated with 1×104 irradiated OKT3 hybridoma cells (anti-CD3), or with allogenic CD28− B cells (depleted of CD28+ cells), in the absence or in the presence of varying amounts of the CD28.3 scFv/α1-antitrypsin fusion protein.\",\n \"The proliferative response in these cultures is evaluated after 3 days when the stimulation is performed with anti-CD3s, or after 7 days when the stimulation is performed with allogenic cells, by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.\",\n \"Inhibition of the Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, allogenic, healthy donor.\",\n \"The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.\",\n \"Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450832"}}},{"rowIdx":285,"cells":{"text":{"kind":"list like","value":[["Deflector Devices","A deflector device (22) for use with a tow line between a seismic survey vessel and a tow, in particular a seismic streamer or streamer array, in the water behind the vessel comprises a vertically oriented wing-shaped body (28) shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel.","The wing-shaped body (28) includes one or more buoyancy elements, and a rearwardly extending boom (32).","A pivotable control surface (54) extends sideways from the boom (32), and is shaped to produce in use a force having a substantial vertical component.","The angle of the control surface is remotely controllable, in order to control the depth of the deflector device."],["1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.","2.A deflector device as claimed in claim 1, wherein the one or more buoyancy elements have a buoyancy selected to give the complete device a small positive buoyancy.","3.A deflector device as claimed in claim 1, wherein the remotely-operable means comprises a telescopic member connected to pivot the control surface.","4.A deflector device as claimed in claim 3, wherein the telescopic member is hydraulically operated.","5.A deflector device as claimed in claim 1, further comprising an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.","6.A deflector device as claimed in claim 5, further comprising additional remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.","7.A deflector device as claimed in claim 6, wherein the additional remotely-operable adjusting means comprises a further telescopic member connected to the auxiliary wing-shaped body.","8.A deflector device as claimed in claim 7, wherein the further telescopic member is hydraulically operated.","9.A deflector device as claimed in claim 5, wherein the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.","10.A method of performing a marine seismic survey, comprising: towing a plurality of laterally-spaced seismic streamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.","11.The method of claim 10, further comprising selecting a buoyancy of the one or more buoyancy elements to give the complete device a small positive buoyancy.","12.The method of claim 10, wherein the remotely-operable means comprises a telescopic member connected to the control surface, and further comprising pivoting the control surface using the telescopic member.","13.The method of claim 10, wherein the deflector device includes an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in: use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and further comprising varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body."],["This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.","A deflector device of this kind is described in detail in our U.S. Pat.","No.","5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.","In use, the wing-shaped body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.","As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.","This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.","The deflector device of U.S. Pat.","No.","5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.","In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.","The MONOWING deflector devices in current use are very large, typically 7.5 m high by 2.5 m wide, and weigh several tonnes.","They are usually suspended around 2 m to 8 m below the float by means such as a fibre rope, and are also provided with a safety chain intended to prevent separation of the float and wing-shaped body in the event that the rope breaks.","In rough weather, the upper part of the wing-shaped body may rise up out of the water, allowing the rope connecting the wing-shaped body and the float to go slack.","If the wing-shaped body then drops abruptly, the rope, and possibly even the safety chain, may break, and/or their attachment points on the wing-shaped body may be badly damaged.","Additionally, the depth at which the current deflector device operates is effectively determined by the length of the rope connecting it to the float.","As a result of this, the operating depth of the deflector device cannot readily be varied while the device is deployed in the water.","And since the normal operating depth of the current deflector device is typically a few meters, in the event of the onset of bad weather during a survey, the device and all the streamers and other equipment directly or indirectly attached to it have to be recovered onto the towing vessel, and then re-deployed when the bad weather has passed, both of which operations are very time consuming.","It is an object of the present invention to alleviate the drawbacks arising from the connection of the deflector device to the float.","According to the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, one or more buoyancy elements disposed within and/or secured to the upper end of the wing-shaped body, a boom extending rearwardly from the wing-shaped body, and a remotely-operable pivotable control surface extending sideways from the boom and shaped to produce in use a force having a vertical component, whereby to control the depth of the deflector device.","It will be appreciated that since the deflector device of the invention can generate a controllable vertical force, this force, together with the buoyancy of the one or more buoyancy elements, can be selected and adjusted so that the separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.","In particular, at the onset of bad weather, the deflector device and its tow can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the weather improves.","Advantageously, the one or more buoyancy elements has or have a buoyancy selected to give the complete device a small positive buoyancy.","In a preferred embodiment of the invention, the deflector device further comprises an auxiliary wing-shaped body, smaller than the firstmentioned (or principal) wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.","Advantageously, this embodiment further includes remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.","The pivotable control surface and the remotely-operable means are preferably both hydraulically operated.","Advantageously, the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.","The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic steamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device in accordance with any one of the preceding statements of invention.","The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.","1 is a somewhat schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.","2 is a somewhat schematic part-sectional view of a first embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.","1; and FIGS.","3A and 3B are respective perspective view of the deflector device of FIG.","2.The seismic survey vessel shown in FIG.","1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.","PCT/GB98/01832 (WO 99/00295).","The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.","No.","4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.","PCT/IB98/01435 (WO 99/15913).","The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.","The deflector devices 22 act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.","One of the deflector devices 22 is shown in more detail in FIGS.","2, 3A and 3B.","The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.","No.","5,357,892, but is a much improved version of it.","In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.","No.","5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 29, both of which features enhance lift.","Also, the main wing-shaped body 28 is provided with vortex controlling end plates 30 (see FIGS.","3A and 3B) of the kind described in our PCT Patent Application No.","PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight, while the towing bridle 27 comprises a pair of titanium chains 52 (see FIGS.","3A and 3B).","Additionally, the angle lever 10 of U.S. Pat.","No.","5,357,892 is replaced by a rearwardly extending fixed angle boom 32, which is detachably connected at one end 34 to the low pressure side 36 of the body 28 near the trailing edge flap 29, at a mounting bracket 38.The boom 32 is of sandwich construction, and is made from two similarly shaped plates 39 which are bolted together at intervals along their length and which sandwich between them the mounting bracket 38.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 22 is on the vessel 10, for ease of stowage.","The other end 40 of the boom 32 has a towing eye 42, coupled in use to the forward end 24 of a respective one of the two outermost streamers 18.An auxiliary wing-shaped body 44, which is much smaller than the body 28 in length, thickness and chord, is pivotally secured as will be explained hereinafter to the end 40 of the boom 32, with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.","2) extending parallel to the longitudinal axis of the body 28.The shape of the body 44 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because as will become apparent, the direction of the force varies in use).","This sideways force is increased by providing the body 44 with a fixed trailing edge flap 46, angled away from the boom 32 at an angle of about 35°.","As best seen in FIGS.","3A and 3B, the auxiliary wing-shaped body 44 is implemented in two symmetrical halves 44a and 44b, which each have vortex-reducing end plates 45 and which are disposed on opposite sides of the boom 32.These two halves 44a, 44b are rotatable in unison about a common axis perpendicular to the plane of the boom 32, so as to vary the angle of the chord of the auxiliary wing-shaped body 44 with respect to the boom.","Rotation of the auxiliary wing-shaped body 44 is effected by a telescopic actuator 48 pivotally mounted between the plates 39 of the boom 32, the actuator being pivotally connected to a lever arm or eccentric 50 secured to each of the two halves 44a, 44b of the auxiliary wing-shaped body.","The telescopic actuator 48 is hydraulically operated by a remotely controllable electro-hydraulic control pack 52 also mounted between the plates 39 of the boom 32.It will be appreciated that varying the angle of the auxiliary wing-shaped body 44 of the deflector device 22 changes the angle of the main wing-shaped body 28 with respect to the direction of tow, and so changes the lift produced by the main wing-shaped body.","This in turn changes the lateral offset produced by the deflector device 22.In accordance with the present invention, the deflector device 22 is made approximately neutrally buoyant, by including gas-filled pipe-like buoyancy elements 58 extending longitudinally within it from top to bottom, and/or by providing an integral buoyancy element at its upper end similar to but smaller than that described in our co-pending United Kingdom Patent Applications Nos.","0023775.0, 0025719.6 and 0029451.2.In practice, the deflector device 22 is preferably designed to be slightly positively buoyant, so that in the event of a malfunction, it tends to float rather than to sink.","Additionally, the deflector device 22 is provided with a pivotable control surface (or flap) 54, which is secured to the boom 32 in the region of the auxiliary wing-shaped body 44 by a generally triangular bracket 56, and which is pivotable about an axis perpendicular to both the pivot axis of the body 44 and the direction of tow (indicated by the arrow 58 in FIG.","2).","The flap 54 and the bracket 56 are both made from titanium.","The angular position of the flap 54 is controlled by a further telescopic actuator 60, which is connected to a lever arm 62 provided on the flap, and which is hydraulically operated by the electro-hydraulic control pack 52.It will be appreciated that rotation of the flap 54 about its pivot axis produces in use an upward or downward force at the end 40 of the boom 32, and thus enables the depth of the deflector device 22 to be controlled.","It will be appreciated that as a result of making the deflector device 22 approximately neutrally buoyant and capable of generating a remotely-controllable vertical force, a separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.","In particular, in the event of the onset of bad weather, the deflector device 22 and the streamers 18 attached to it can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the bad weather passes.","Many modifications can be made to the described embodiment of the invention.","In particular, the flap 54 and the auxiliary wing-shaped body 44 can be made from a plastics material reinforced with high strength fibres, eg Kevlar fibres, and can be electrically actuated rather than hydraulically actuated, Additionally, the devices 22 and 60 can be used with tows other than streamers, for example seismic sources, and the tow need not be connected to the end 40 of the boom 32 (it could instead be connected to the lead-in 20, at a point near where the bridle 24 is connected to the lead-in).","Also, the invention can if desired be used with a deflector device in which the auxiliary wing-shaped body 44 is fixed, and the boom 32 is pivotable towards and away from the main deflector body 28, as described in our United Kingdom Patent Applications Nos.","0023755.2, 0025711.3 and 0029452.0.Indeed, the invention can even be used with a deflector device like that described in our U.S. Pat.","No.","5,357,892, ie a deflector device without the auxiliary wing-shaped body 44, by mounting a pivotable flap analogous to the flap 54 on a pivotable boom analogous to the angle lever 10 of the deflector device of the US patent.","Finally, although the invention has been described in relation to deflector devices whose lift can be varied by varying the angle of the device with respect to the direction of tow, it is also applicable in its broadest aspect to a fixed angle deflector device, eg of the kind referred to as a “door”."]],"string":"[\n [\n \"Deflector Devices\",\n \"A deflector device (22) for use with a tow line between a seismic survey vessel and a tow, in particular a seismic streamer or streamer array, in the water behind the vessel comprises a vertically oriented wing-shaped body (28) shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel.\",\n \"The wing-shaped body (28) includes one or more buoyancy elements, and a rearwardly extending boom (32).\",\n \"A pivotable control surface (54) extends sideways from the boom (32), and is shaped to produce in use a force having a substantial vertical component.\",\n \"The angle of the control surface is remotely controllable, in order to control the depth of the deflector device.\"\n ],\n [\n \"1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.\",\n \"2.A deflector device as claimed in claim 1, wherein the one or more buoyancy elements have a buoyancy selected to give the complete device a small positive buoyancy.\",\n \"3.A deflector device as claimed in claim 1, wherein the remotely-operable means comprises a telescopic member connected to pivot the control surface.\",\n \"4.A deflector device as claimed in claim 3, wherein the telescopic member is hydraulically operated.\",\n \"5.A deflector device as claimed in claim 1, further comprising an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.\",\n \"6.A deflector device as claimed in claim 5, further comprising additional remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.\",\n \"7.A deflector device as claimed in claim 6, wherein the additional remotely-operable adjusting means comprises a further telescopic member connected to the auxiliary wing-shaped body.\",\n \"8.A deflector device as claimed in claim 7, wherein the further telescopic member is hydraulically operated.\",\n \"9.A deflector device as claimed in claim 5, wherein the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.\",\n \"10.A method of performing a marine seismic survey, comprising: towing a plurality of laterally-spaced seismic streamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.\",\n \"11.The method of claim 10, further comprising selecting a buoyancy of the one or more buoyancy elements to give the complete device a small positive buoyancy.\",\n \"12.The method of claim 10, wherein the remotely-operable means comprises a telescopic member connected to the control surface, and further comprising pivoting the control surface using the telescopic member.\",\n \"13.The method of claim 10, wherein the deflector device includes an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in: use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and further comprising varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.\"\n ],\n [\n \"This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.\",\n \"A deflector device of this kind is described in detail in our U.S. Pat.\",\n \"No.\",\n \"5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.\",\n \"In use, the wing-shaped body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.\",\n \"As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.\",\n \"This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.\",\n \"The deflector device of U.S. Pat.\",\n \"No.\",\n \"5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.\",\n \"In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.\",\n \"The MONOWING deflector devices in current use are very large, typically 7.5 m high by 2.5 m wide, and weigh several tonnes.\",\n \"They are usually suspended around 2 m to 8 m below the float by means such as a fibre rope, and are also provided with a safety chain intended to prevent separation of the float and wing-shaped body in the event that the rope breaks.\",\n \"In rough weather, the upper part of the wing-shaped body may rise up out of the water, allowing the rope connecting the wing-shaped body and the float to go slack.\",\n \"If the wing-shaped body then drops abruptly, the rope, and possibly even the safety chain, may break, and/or their attachment points on the wing-shaped body may be badly damaged.\",\n \"Additionally, the depth at which the current deflector device operates is effectively determined by the length of the rope connecting it to the float.\",\n \"As a result of this, the operating depth of the deflector device cannot readily be varied while the device is deployed in the water.\",\n \"And since the normal operating depth of the current deflector device is typically a few meters, in the event of the onset of bad weather during a survey, the device and all the streamers and other equipment directly or indirectly attached to it have to be recovered onto the towing vessel, and then re-deployed when the bad weather has passed, both of which operations are very time consuming.\",\n \"It is an object of the present invention to alleviate the drawbacks arising from the connection of the deflector device to the float.\",\n \"According to the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, one or more buoyancy elements disposed within and/or secured to the upper end of the wing-shaped body, a boom extending rearwardly from the wing-shaped body, and a remotely-operable pivotable control surface extending sideways from the boom and shaped to produce in use a force having a vertical component, whereby to control the depth of the deflector device.\",\n \"It will be appreciated that since the deflector device of the invention can generate a controllable vertical force, this force, together with the buoyancy of the one or more buoyancy elements, can be selected and adjusted so that the separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.\",\n \"In particular, at the onset of bad weather, the deflector device and its tow can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the weather improves.\",\n \"Advantageously, the one or more buoyancy elements has or have a buoyancy selected to give the complete device a small positive buoyancy.\",\n \"In a preferred embodiment of the invention, the deflector device further comprises an auxiliary wing-shaped body, smaller than the firstmentioned (or principal) wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.\",\n \"Advantageously, this embodiment further includes remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.\",\n \"The pivotable control surface and the remotely-operable means are preferably both hydraulically operated.\",\n \"Advantageously, the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.\",\n \"The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic steamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device in accordance with any one of the preceding statements of invention.\",\n \"The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.\",\n \"1 is a somewhat schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.\",\n \"2 is a somewhat schematic part-sectional view of a first embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.\",\n \"1; and FIGS.\",\n \"3A and 3B are respective perspective view of the deflector device of FIG.\",\n \"2.The seismic survey vessel shown in FIG.\",\n \"1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.\",\n \"PCT/GB98/01832 (WO 99/00295).\",\n \"The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.\",\n \"No.\",\n \"4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.\",\n \"PCT/IB98/01435 (WO 99/15913).\",\n \"The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.\",\n \"The deflector devices 22 act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.\",\n \"One of the deflector devices 22 is shown in more detail in FIGS.\",\n \"2, 3A and 3B.\",\n \"The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.\",\n \"No.\",\n \"5,357,892, but is a much improved version of it.\",\n \"In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.\",\n \"No.\",\n \"5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 29, both of which features enhance lift.\",\n \"Also, the main wing-shaped body 28 is provided with vortex controlling end plates 30 (see FIGS.\",\n \"3A and 3B) of the kind described in our PCT Patent Application No.\",\n \"PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight, while the towing bridle 27 comprises a pair of titanium chains 52 (see FIGS.\",\n \"3A and 3B).\",\n \"Additionally, the angle lever 10 of U.S. Pat.\",\n \"No.\",\n \"5,357,892 is replaced by a rearwardly extending fixed angle boom 32, which is detachably connected at one end 34 to the low pressure side 36 of the body 28 near the trailing edge flap 29, at a mounting bracket 38.The boom 32 is of sandwich construction, and is made from two similarly shaped plates 39 which are bolted together at intervals along their length and which sandwich between them the mounting bracket 38.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 22 is on the vessel 10, for ease of stowage.\",\n \"The other end 40 of the boom 32 has a towing eye 42, coupled in use to the forward end 24 of a respective one of the two outermost streamers 18.An auxiliary wing-shaped body 44, which is much smaller than the body 28 in length, thickness and chord, is pivotally secured as will be explained hereinafter to the end 40 of the boom 32, with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.\",\n \"2) extending parallel to the longitudinal axis of the body 28.The shape of the body 44 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because as will become apparent, the direction of the force varies in use).\",\n \"This sideways force is increased by providing the body 44 with a fixed trailing edge flap 46, angled away from the boom 32 at an angle of about 35°.\",\n \"As best seen in FIGS.\",\n \"3A and 3B, the auxiliary wing-shaped body 44 is implemented in two symmetrical halves 44a and 44b, which each have vortex-reducing end plates 45 and which are disposed on opposite sides of the boom 32.These two halves 44a, 44b are rotatable in unison about a common axis perpendicular to the plane of the boom 32, so as to vary the angle of the chord of the auxiliary wing-shaped body 44 with respect to the boom.\",\n \"Rotation of the auxiliary wing-shaped body 44 is effected by a telescopic actuator 48 pivotally mounted between the plates 39 of the boom 32, the actuator being pivotally connected to a lever arm or eccentric 50 secured to each of the two halves 44a, 44b of the auxiliary wing-shaped body.\",\n \"The telescopic actuator 48 is hydraulically operated by a remotely controllable electro-hydraulic control pack 52 also mounted between the plates 39 of the boom 32.It will be appreciated that varying the angle of the auxiliary wing-shaped body 44 of the deflector device 22 changes the angle of the main wing-shaped body 28 with respect to the direction of tow, and so changes the lift produced by the main wing-shaped body.\",\n \"This in turn changes the lateral offset produced by the deflector device 22.In accordance with the present invention, the deflector device 22 is made approximately neutrally buoyant, by including gas-filled pipe-like buoyancy elements 58 extending longitudinally within it from top to bottom, and/or by providing an integral buoyancy element at its upper end similar to but smaller than that described in our co-pending United Kingdom Patent Applications Nos.\",\n \"0023775.0, 0025719.6 and 0029451.2.In practice, the deflector device 22 is preferably designed to be slightly positively buoyant, so that in the event of a malfunction, it tends to float rather than to sink.\",\n \"Additionally, the deflector device 22 is provided with a pivotable control surface (or flap) 54, which is secured to the boom 32 in the region of the auxiliary wing-shaped body 44 by a generally triangular bracket 56, and which is pivotable about an axis perpendicular to both the pivot axis of the body 44 and the direction of tow (indicated by the arrow 58 in FIG.\",\n \"2).\",\n \"The flap 54 and the bracket 56 are both made from titanium.\",\n \"The angular position of the flap 54 is controlled by a further telescopic actuator 60, which is connected to a lever arm 62 provided on the flap, and which is hydraulically operated by the electro-hydraulic control pack 52.It will be appreciated that rotation of the flap 54 about its pivot axis produces in use an upward or downward force at the end 40 of the boom 32, and thus enables the depth of the deflector device 22 to be controlled.\",\n \"It will be appreciated that as a result of making the deflector device 22 approximately neutrally buoyant and capable of generating a remotely-controllable vertical force, a separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.\",\n \"In particular, in the event of the onset of bad weather, the deflector device 22 and the streamers 18 attached to it can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the bad weather passes.\",\n \"Many modifications can be made to the described embodiment of the invention.\",\n \"In particular, the flap 54 and the auxiliary wing-shaped body 44 can be made from a plastics material reinforced with high strength fibres, eg Kevlar fibres, and can be electrically actuated rather than hydraulically actuated, Additionally, the devices 22 and 60 can be used with tows other than streamers, for example seismic sources, and the tow need not be connected to the end 40 of the boom 32 (it could instead be connected to the lead-in 20, at a point near where the bridle 24 is connected to the lead-in).\",\n \"Also, the invention can if desired be used with a deflector device in which the auxiliary wing-shaped body 44 is fixed, and the boom 32 is pivotable towards and away from the main deflector body 28, as described in our United Kingdom Patent Applications Nos.\",\n \"0023755.2, 0025711.3 and 0029452.0.Indeed, the invention can even be used with a deflector device like that described in our U.S. Pat.\",\n \"No.\",\n \"5,357,892, ie a deflector device without the auxiliary wing-shaped body 44, by mounting a pivotable flap analogous to the flap 54 on a pivotable boom analogous to the angle lever 10 of the deflector device of the US patent.\",\n \"Finally, although the invention has been described in relation to deflector devices whose lift can be varied by varying the angle of the device with respect to the direction of tow, it is also applicable in its broadest aspect to a fixed angle deflector device, eg of the kind referred to as a “door”.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450862"}}},{"rowIdx":286,"cells":{"text":{"kind":"list like","value":[["Methods for the production of multimeric proteins and related compositions","Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobins, in association with oil bodies are described.","The redox protein is enzymatically active when prepared in association with the oil bodies.","Also provided are related nucleic acids, proteins, cells, plants, and compositions."],["1-266.","(canceled) 267.A method of producing an oil body associated with a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.","268.A method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.","269.A method of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","270.A chimeric nucleic acid sequence encoding a multimeric-fusion-protein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","271.A recombinant multimeric-fusion-protein comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.","272.Isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","273.Isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","274.A cell comprising oil bodies and (i) an oil-body-targeting-protein, (ii) a first recombinant polypeptide and (iii) a second recombinant polypeptide wherein (1) said first recombinant polypeptide is capable of associating with said oil-body-targeting-protein; and (2) said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form a multimeric-protein-complex.","275.A composition comprising isolated oil bodies, thioredoxin and thioredoxin-reductase.","276.A food product, personal care product or pharmaceutical composition comprising the composition of claim 275.277.A method of reducing allergenicity of a food comprising the steps of: providing the isolated oil bodies of claim 273; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.","278.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing the recombinant fusion polypeptide of claim 269; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","279.A method for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","280.A method for preparing a redox protein associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide.","281.A chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","282.A transgenic plant comprising the chimeric nucleic acid sequence of claim 281.283.A safflower plant comprising the chimeric nucleic acid of anyone of claim 281.284.A plant seed comprising the chimeric nucleic acid of claim 281.285.A safflower seed comprising the chimeric nucleic acid of claim 281.286.An oil body preparation obtained by the method of claim 279.287.A food product comprising an oil body preparation of claim 286.288.A composition comprising an oil body preparation of claim 286 (New).","289.A personal care product comprising an oil body preparation of claim 286.290.A product capable of treating oxidative stress in a target comprising an oil body preparation of claim 286.291.A product capable of chemically reducing a target comprising an oil body preparation of claim 286.292.A detergent composition comprising the product of claim 286.293.A method of cleansing an item, comprising administering the product of claim 290 to said item under conditions that promote cleansing.","294.An emulsion formulation prepared by the method of claim 279.295.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.","296.The seed of the plant of claim 281.297.An extract of the seed of claim 296, wherein the extract comprises an activity of a thioredoxin-related protein.","298.An oil body from the seed of claim 296.299.Oil produced from the seed of claim 296.300.A method of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; obtaining seeds from the plant; and recovering the fusion protein by isolating oil bodies from the seeds.","301.Oil bodies in association with a fusion protein, obtained by the method of claim 300.302.A method of reducing allergenicity of a food comprising the steps of: providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.","303.A composition comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","304.A cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in an acceptable carrier.","305.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","306.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one recombinant polypeptide and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides."],[" BACKGROUND Multimeric proteins (i.e.","proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.","Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.","However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.","Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.","For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.","(1996) J. Cardiov.","Pharmacol.","(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.","(1999) J. Allerg.","Vlin.","Immunol.","103: 690-697) and wheat (Buchanan et al.","(1997) Proc.","Natl.","Acad.","Sci.","USA 94: 5372-5377).","However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.","The present invention satisfies this need and provides related advantages as well."],[" SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.","Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.","The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.","The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.","Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.","The oil-body-protein can be an oleosin or caleosin.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.","Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.","Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.","In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.","These formulations can further comprise the addition of NADP or NADPH.","The food product can be a milk or wheat based food product.","The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.","The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).","Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.","In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.","5 .","Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.","The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.","Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.","Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.","For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.","The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis .","In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.","Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.","Accordingly, an emulsion formulation composition is provided.","Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.","The chimeric nucleic acids can be contained within a plastid.","Accordingly, isolated plastids are provided having chimeric nucleic acids therein.","Also provided are plant seeds comprising the chimeric nucleic acids provided herein.","Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.","The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.","Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.","Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.","In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.","In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.","The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.","The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.","The second region can encode a thioredoxin and thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae .","In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.","The first region can precede, in a 5′ to 3′ direction, the second region.","Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.","In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.","In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.","In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.","Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.","The thioredoxin-related protein can be thioredoxin.","The nucleic acid construct can be contained within a plastid.","In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.","Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.","In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.","Also provided herein is an extract comprising an activity of a thioredoxin-related protein.","Also provided are oil bodies and/or oil obtained from various seeds.","Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.","In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.","The oil bodies can be in association with a fusion protein.","The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.","The cleaving step can make use of a protease or chemical proteolysis.","Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.","The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.","Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","The oil bodies can be associated with the fusion protein.","Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.","Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.","These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.","The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.","For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.","Other features and advantages of the present invention will become readily apparent from the following detailed description.","It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only."],["RELATED APPLICATIONS Benefit of priority under 35 U.S.C.","§119(e) is claimed to U.S. provisional application Ser.","No.","60/302,885, filed Jul.","5, 2001, to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”.","This application is also a continuation-in-part of U.S. utility application Ser.","No.","10/006,038, filed Dec. 4, 2001 to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”; which is a continuation-in-part of U.S. utility application Ser.","No.","09/742,900, filed Dec. 19, 2000 to Heifetz, et al., entitled “METHOD OF PRODUCTION AND DELIVERY OF THIOREDOXIN”.","This application is also a continuation-in-part of U.S. utility application Ser.","No.","09/742,900.The subject matter of each of the provisional and utility applications is incorporated herein by reference in its entirety.","FIELD OF THE INVENTION The present invention relates to multimeric-protein-complexes, redox proteins, and recombinant polypeptides; and improved methods for their production.","BACKGROUND Multimeric proteins (i.e.","proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.","Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.","However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.","Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.","For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.","(1996) J. Cardiov.","Pharmacol.","(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.","(1999) J. Allerg.","Vlin.","Immunol.","103: 690-697) and wheat (Buchanan et al.","(1997) Proc.","Natl.","Acad.","Sci.","USA 94: 5372-5377).","However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.","The present invention satisfies this need and provides related advantages as well.","SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.","Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.","This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.","The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.","Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.","The oil-body-targeting-protein can be an oleosin or caleosin.","When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.","Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.","In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.","In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.","Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.","The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.","The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.","The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.","Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.","The oil-body-protein can be an oleosin or caleosin.","The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.","Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.","Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.","The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.","The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.","In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.","In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.","These formulations can further comprise the addition of NADP or NADPH.","The food product can be a milk or wheat based food product.","The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.","The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).","Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.","In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.","For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.","5.Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.","The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.","Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.","Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.","For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.","In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.","Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.","The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis.","In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.","Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.","Accordingly, an emulsion formulation composition is provided.","Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.","In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.","Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.","The chimeric nucleic acids can be contained within a plastid.","Accordingly, isolated plastids are provided having chimeric nucleic acids therein.","Also provided are plant seeds comprising the chimeric nucleic acids provided herein.","Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.","The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.","Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.","Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.","In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.","The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.","In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.","The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.","The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.","The second region can encode a thioredoxin and thioredoxin-reductase.","In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae.","In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.","The first region can precede, in a 5′ to 3′ direction, the second region.","Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.","In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.","In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.","In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.","Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.","The thioredoxin-related protein can be thioredoxin.","The nucleic acid construct can be contained within a plastid.","In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.","The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.","The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.","The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.","The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.","Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.","In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.","Also provided herein is an extract comprising an activity of a thioredoxin-related protein.","Also provided are oil bodies and/or oil obtained from various seeds.","Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.","In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.","The oil bodies can be in association with a fusion protein.","The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.","The cleaving step can make use of a protease or chemical proteolysis.","Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.","The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.","In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.","Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","The oil bodies can be associated with the fusion protein.","Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.","Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.","The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.","Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.","Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.","These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.","The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.","For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.","Other features and advantages of the present invention will become readily apparent from the following detailed description.","It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows a ClustalW Formatted Alignment comparison of the published NADPH thioredoxin-reductase nucleic acid sequence (SEQ ID NO:9) (ATTHIREDB-Jacquot et al.","J. Mol.","Biol.","(1994) 235 (4):1357-63.)","with the sequence isolated herein in Example 1 (TR; SEQ ID NO:8).","FIG.","2 shows a ClustalW Formatted Alignment comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence (SEQ ID NO:12) (ATTHIREDB Jacquot et al.","J. Mol.","Biol.","(1994) 235 (4):1357-63.)","with the sequence isolated herein in Example 1 (TR; SEQ ID NO:13).","FIG.","3 shows a clustal alignment comparing the amino acid sequence of the Arabidopsis thaliana thioredoxin-reductase-linker-thioredoxin synthetic fusion (Arab TR-link-Trxh; SEQ ID NO:37) to the Mycobacterium leprae thioredoxin-reductase-thioredoxin natural fusion (M. lep TR/Trxh; SEQ ID NO:36) natural fusion.","Overall, the proteins are approximately 50% identical at the amino acid level.","FIG.","4 is a bar graph showing the thioredoxin/thioredoxin-reductase activity measurements for the various transgenic Arabidopsis seed fractions.","Relative specific activity is expressed as a percentage of the E. coli thioredoxin and thioredoxin-reductase activities.","The numbered bars in the graph correspond to the following: 1.W.T.+oleosin-thioredoxin 2.W.T.+thioredoxin-oleosin 3.W.T.+thioredoxin 4.W.T.+oleosin-thioredoxin-reductase 5.W.T.+thioredoxin-reductase-oleosin 6.W.T.+thioredoxin-reductase 7.thioredoxin+oleosin-thioredoxin-reductase 8.thioredoxin+thioredoxin-reductase-oleosin 9.thioredoxin+thioredoxin-reductase 10.thioredoxin-reductase+oleosin-thioredoxin 11.thioredoxin-reductase+thioredoxin-oleosin 12.oleosin-M. lep TR/Trxh 13.E.","coli thioredoxin-reductase+thioredoxin FIG.","5 provides a listing of exemplary proteins for use in the heteromultimeric-fusion-proteins and heteromultimeric-protein-complexes provided herein.","DETAILED DESCRIPTION As hereinbefore mentioned, the present invention relates to novel and improved methods for the production of multimeric proteins, including a first and second recombinant polypeptide, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and a first and second thioredoxin-related protein; and related products.","These methods permit the production of active multimeric-protein-complexes in association with oil bodies.","The oil bodies in association with the multimeric-protein-complex may be used to prepare various useful emulsions.","Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex associated with an oil body, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.","DEFINITIONS AND TERMS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.","Where permitted, all patents, applications, published applications and other publications and sequences from GenBank, SwissPro and other data bases referred to throughout in the disclosure herein are incorporated by reference in their entirety.","As used herein, the phrase “multimeric-protein-complex”, refers to two or more polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.","It should be noted that the polypeptides may be independently biologically active without interaction or coordination to form the complex.","The multimeric-protein-complex may provide a biological structure, or it may be capable of facilitating a chemical or biological reaction.","For example, one of the protein regions within the multimeric-protein-complex can repeatedly activate or repeatedly inactivate the biological or metabolic activity of one or more of the other proteins contained within the multimeric-protein-complex.","In one embodiment, the first and second recombinant polypeptide contained in a multimeric-protein-complex may either associate or interact as independent non-contiguous polypeptide chains or the multimeric-protein-complex may be prepared as a fusion polypeptide (multimeric-fusion-protein) between the first and second recombinant polypeptide.","One example of a repeated (e.g., reoccurring) interaction or association between the two or more polypeptides of a multimeric-protein-complex provided herein is the interaction between two or more non-identical redox proteins to form a heteromultimeric-protein-complex.","Exemplary redox proteins for use in this regard are thioredoxin and the thioredoxin-reductase.","A further example is the interaction between two or more immunoglobulin-polypeptide-chains to form an immunoglobulin.","As used herein, the phrase “heteromultimeric-protein-complex”, refers to two or more non-identical polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.","Other examples of multimeric-protein-complexes provided herein include a first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, first and second immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and second thioredoxin-related protein.","The recombinant polypeptide or multimeric-protein-complex is associated with an oil body.","As used herein, the phrase “oil body” or “oil bodies” refers to any oil or fat storage organelle in any cell type.","Accordingly, the oil bodies may be obtained from any cell comprising oil bodies, including plant cells (described in for example: Huang (1992) Ann.","Rev.","Plant Mol.","Biol.","43: 177-200), animal cells (described in for example: Murphy (1990) Prog Lipid Res 29(4): 299-324), including adipocytes, hepatocytes, steroidogenic cells, mammary epithelial cells, macrophages, algae cells (described in for example: Rossler (1988) J. Physiol.","London, 24: 394-400) fungal cells, including yeast cells (described in for example Leber et al.","(1994) Yeast 10: 1421-1428) and bacterial cells (described in for example: Pieper-Furst et al.","(1994) J. Bacteriol.","176: 4328-4337).","Preferably the oil bodies used herein are oil bodies obtainable from plant cells and more preferably the oil bodies obtainable from plant seed cells.","As used herein, the phrase “is capable of associating with”, “associate” or grammatical variations thereof, refers to any interaction between two or more polypeptides, including any covalent interactions (e.g.","multimeric-fusion-proteins) as well as non-covalent interactions.","Exemplary non-covalent interactions can be between the oil-body-targeting-protein and a redox protein or immunoglobulin-polypeptide-chain, as well as between two or more different proteins contained within two or more separate oil-body-protein fusion proteins (e.g., the redox proteins in oleosin-thioredoxin and oleosin-thioredoxin-reductase).","As used herein, the term “recombinant” (also referred to as heterologous) in the context of recombinant proteins and amino acids, means “of different natural origin” or represents a non-natural state.","For example, if a host cell is transformed with a nucleotide sequence derived from another organism, particularly from another species, that nucleotide sequence and amino acid sequence encoded thereby, is recombinant (heterologous) with respect to that host cell and also with respect to descendants of the host cell which carry that gene.","Similarly, recombinant (or heterologous) refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.","A transforming nucleotide sequence may include a recombinant coding sequence, or recombinant regulatory elements.","Alternatively, the transforming nucleotide sequence may be completely heterologous or may include any possible combination of heterologous and endogenous nucleic acid sequences.","In various embodiments of the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, are produced in a cell comprising oil bodies.","As used herein the phrase “in a cell”, “in the cell”, or grammatical variations thereof, mean that the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, may be produced in any cellular compartment of that cell, so long as that cell comprises oil bodies therein.","In embodiments of the invention in which plant cells are used, the phrase is intended to include the plant apoplast.","In various embodiments provided herein, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, associate with an oil body through an oil-body-targeting-protein.","As used herein, the phrase “oil-body-targeting-protein” refers to any protein, protein fragment or peptide capable of associating with an oil body.","Exemplary oil-body-targeting-proteins for use herein include oil-body-proteins, such as oleosin and caleosin; immunoglobulins, such as bi-specific antibodies; and the like.","In embodiments described herein in which an oil-body-protein is used, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, are preferably fused to the oil-body-protein.","The term “oil-body-protein” refers to any protein naturally present in cells and having the capability of association with oil bodies, including any oleosin or caleosin.","Accordingly, provided herein a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide, such as a redox protein, an immunoglobulin-polypeptide-chain or an thioredoxin-related protein, fused to an oil-body-protein; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide, such as a second redox protein, a second immunoglobulin-polypeptide-chain or a second thioredoxin-related protein; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex, preferably in said progeny cell; and (d) associating said first recombinant polypeptide with an oil body through said oil-body-protein.","The term “nucleic acid” as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.","The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.","The nucleic acid sequences may be ribonucleic acids (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.","The sequences also may contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo-uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol-adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8 thiol guanine, 8-thioalkyl guanines, 8 hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.","Multimeric-Protein-Complexes In accordance with the methods and compositions provided herein, any two recombinant polypeptides capable of forming a multimeric-protein-complex may be used.","The nucleic acid sequences encoding the two recombinant polypeptides may be obtained from any biological source or may be prepared synthetically.","In general nucleic acid sequence encoding multimeric proteins are known to the art and readily available.","Known nucleic acid sequences encoding multimeric-protein-complexes may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences encoding multimeric-protein-complexes, for example, by screening cDNA or genomic libraries or using 2- or multi-hybrid systems.","Thus, additional nucleic acid sequences encoding multimeric-protein-complexes may be discovered and used as described herein.","The first and/or second recombinant polypeptides that are comprised within a multimeric-protein-complex provided herein, can themselves be in the form of heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein.","The nucleic acid sequence encoding the first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein may be obtained from separate sources or may be obtained from the same source.","In general however, such nucleic acid sequence is obtained from the same or a similar biological source.","In certain embodiments wherein the nucleic acid sequence encoding the first and second recombinant polypeptide protein are obtained from the same source, the nucleic acid sequence encoding the first recombinant polypeptide and second recombinant polypeptide may be naturally fused.","In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second recombinant polypeptide are obtained from a plant source.","Oil-Body-Surface-Avoiding Linkers Polypeptide spacers or linkers of variable length and/or negative charge can be used herein to separate the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins from the in-frame oil-body-targeting-protein, to improve activity of and/or the accessibility of the polypeptide or complex.","For example, in one embodiment set forth herein, positioned between a nucleic acid sequence encoding a sufficient portion of an oil-body-protein and a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins; is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.","Oil-body-surface-avoiding linkers are positioned between the oil-body targeting sequence and an in-frame recombinant polypeptide of interest, e.g., the multimeric-protein-complexes provided herein, serve to increase the distance and or decrease the interaction between the negatively charged oil body surface and the recombinant polypeptide of interest.","A negatively charged linker is repelled by the negatively charged oil body surface, in turn increasing the distance or decreasing the interaction of its attached recombinant polypeptide with the oil body surface.","As a consequence of the increased distance from the oil body surface, the recombinant polypeptide will be more accessible, e.g.","to its target(s) substrate, protein substrate, protein partner, and less affected by the charged oil body surface.","Exemplary linker sequences for use herein can be either a negatively charged linker, or a linker having a molecular weight of at least about 35 kd or more.","As used herein, a “negatively charged linker” sequence, refers to any amino acid segment, or nucleic acid encoding such, that has a pI less than or equal to the pI of an oil body.","In certain embodiments, the pI of the negatively charged linker is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, down to about 25% or more, below that of the pI of an oil body in the particular plant or cell system being used.","Exemplary negatively charged linkers can be prepared comprising any combination of the negatively charged amino acid residues.","For example, in one embodiment, a negatively charged linker comprises either a poly-glutamate or poly-aspartate sequence, or any combination of both amino acid residues.","The negatively charged linker is typically at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.","The negatively charged linkers are preferably non-proteolytic (e.g., non-proteolytic linkers), having no site for efficient proteolysis.","When linker size rather than charge is used to minimize interaction of the recombinant polypeptide of interest with the oil body surface, then the linker is non-proteolytic and ranges in molecular weight from about 35 kd up to about 100 kd.","The upper size limit is chosen such that the expression of, the activity of, the conformation of, and/or the access to target of, the recombinant polypeptide of interest is not significantly affected by the linker.","In certain embodiments, described herein where a non-proteolytic linker amino acid sequence is employed, the gene-fusion or protein fusion (multimeric-fusion-protein) can optionally further comprise a linker nucleic or amino acid sequence encoding a sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the non-proteolytic linker sequence and sequence encoding the desired recombinant protein region, e.g., the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins set forth herein.","When a cleavable linker sequence is used herein, in a particular embodiment, it is further downstream than the non-proteolytic linker sequence from the oil-body-targeting-protein region of the fusion protein.","By virtue of cleavable linker, the recombinant fusion polypeptides provided herein, such as the multimeric-fusion-proteins and redox fusion polypeptides, can be isolated and purified by introducing an enzyme or chemical that cleaves said multimeric-fusion-protein and/or redox fusion polypeptide from said oil body, thereby obtaining and/or isolating the desired protein.","It is contemplated herein that the use of cleavable linker sequence downstream of the non-proteolytic linker/spacer sequence will improve the yield of protein recovery when isolating or purifying proteins using the methods provided herein.","The nucleic acid sequences encoding the first or second recombinant polypeptide may be altered to improve expression levels for example, by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the first and second recombinant polypeptide, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).","Comparison of the codon usage of the first and second recombinant polypeptide with codon usage of the host will enable the identification of codons that may be changed.","For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.","By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.","Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The first and second recombinant polypeptide can be altered using for example targeted mutagenesis, random mutagenesis (Shiraishi et al.","(1998) Arch.","Biochem.","Biophys.","358: 104-115; Galkin et al.","(1997) Protein Eng.","10: 687-690; Carugo et al.","(1997) Proteins 28: 10-28; Hurley et al.","(1996) Biochemistry 35: 5670-5678), gene shuffling, and/or by the addition of organic solvent (Holmberg et al.","(1999) Protein Eng.","12: 851-856).","Any polypeptide spacers that are used in accordance with the methods and products provided herein may be altered in similar ways.","In particular embodiments provided herein, the recombinant polypeptides or thioredoxin-related proteins capable of forming a multimeric-protein-complex are capable of forming a heteromultimeric-protein-complex.","Examples of heteromultimeric-protein-complexes that contain polypeptide chains that repeatedly interact, either to activate, inactivate, oxidize, reduce, stabilize, etc., with one another, that can be produced in association with oil bodies using the methods provided herein include those set forth in FIG.","5.Accordingly, exemplary proteins for use in the heteromultimeric-protein-complexes and nucleic acid constructs encoding such, provided herein include, among others described herein, those set forth in FIG.","5.Other polypeptide regions that can be used in the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, provided herein include, among other, those immunoglobulin regions set forth in Table 1.TABLE 1 ANTIBODY HETERODIMERS Class or molecule Subunits Fab Variable region and first constant region of heavy chain and complete light chain Fv Variable regions of heavy and light antibody chains IgA heavy chains, light chains and J (joining) chain IgG, IgD, IgE heavy and light chains IgM heavy chains, light chains and J (joining) chain Antibody chain(s) and a toxin Antibody chain(s) and a toxin Autoantigens, allergens and Autoantigens, allergens and transplant transplant antigens with an antigens with an adjuvant or tolerogen adjuvant or tolerogen Chimeras using antibody Fc Receptor subunits fused to the constant domain region of antibody heavy chains As set forth above, in one embodiment, exemplary heteromultimeric-protein-complexes and exemplary heteromultimeric-fusion-proteins provided herein comprise redox proteins, such as the thioredoxins and thioredoxin-reductases and immunoglobulins.","Oil-Body-Targeting-Proteins The nucleic acid sequence encoding the oil-body-targeting-protein that may be used in the methods and compositions provided herein may be any nucleic acid sequence encoding an oil-body-targeting-protein, protein fragment or peptide capable of association with first recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein and the oil bodies.","The nucleic acid sequence encoding the oil body targeting peptide may be synthesized or obtained from any biological source.","For example, in one embodiment the oil-body-targeting-protein is an immunoglobulin or an immunoglobulin derived molecule, for example, a bispecific single chain antibody.","The generation of single chain antibodies and bi-specific single chain antibodies is known to the art (see, e.g., U.S. Pat.","No.","5,763,733, U.S. Pat.","No.","5,767,260 and U.S. Pat.","No.","5,260,203).","Nucleic acid sequences encoding single chain antibodies functioning as oil-body-targeting-proteins may be prepared from hybridoma cell lines expressing monoclonal antibodies raised against an oleosin as described by Alting-Mees et al (2000) IBC's Annual International Conference on Antibody Engineering, Poster #1.In order to attain specificity for the first recombinant polypeptide a nucleic acid sequence encoding a second single chain antibody prepared from a monoclonal raised against the first recombinant polypeptide may be prepared and linked to the anti-oleosin single chain antibody.","In this embodiment the oil body associates with the first recombinant polypeptide through non-covalent interactions of the oil-body-targeting-protein with the first recombinant polypeptide and the oil body.","Alternatively the first recombinant polypeptide may be prepared as a fusion protein with an oil-body-targeting-protein.","For example, a nucleic acid sequence encoding a single chain antibody raised against an oleosin may be fused to a nucleic acid sequence encoding the first recombinant polypeptide Non-immunoglobulin-based oil-body-targeting-proteins capable of association with the first recombinant polypeptide may be discovered and prepared using for example phage display techniques (Pharmacia Biotech Catalogue Number 27-9401-011 Recombinant Phage Antibody System Expression Kit).","Oil-body-targeting-proteins may also be chemically modified.","For example, oleosins may be modified by changing chemical modification of the lysine residues using chemical agents such as biotinyl-N-hydroxysuccinimide ester resulting in a process referred to as biotinylation.","Conveniently this is accomplished by in vitro biotinylation of the oil bodies.","In vivo biotinylation may be accomplished using the biotinylation domain peptide from the biotin carboxy carrier protein of E. coli acetyl-CoA carboxylase (Smith et al.","(1998) Nucl.","Acids.","Res.","26: 1414-1420).","Avidin or streptavidin may subsequently be used to accomplish association of the redox protein with the oil body.","In a particular embodiment the oil-body-targeting-protein is an oil-body-protein such as for example an oleosin or a caleosin or a sufficient portion derived thereof capable of targeting to an oil body.","Nucleic acid sequences encoding oleosins are known to the art.","These include for example the Arabidopsis oleosin (van Rooijen et al (1991) Plant Mol.","Bio.","18:1177-1179); the maize oleosin (Qu and Huang (1990) J. Biol.","Chem.","Vol.","265 4:2238-2243); rapeseed oleosin (Lee and Huang (1991) Plant Physiol.","96:1395-1397); and the carrot oleosin (Hatzopoulos et al (1990) Plant Cell Vol.","2, 457-467.).","Caleosin nucleic acid sequences are also known to the art (Naested et al (2000) Plant Mol.","Biol.","44(4):463-476; Chen et al (1999) Plant Cell Physiol.","40(10):1079-1086).","Animal cell derived oil body proteins that may be used herein include adopihilin (Brasaemle et al, (1997) J. Lipid Res., 38: 2249-2263; Heid et al.","(1998) Cell Tissue Research 294: 309-321), perilipin (Blanchette-Mackie et al.","(1995), J. Lipid Res.","36: 1211-1226; Servetnick et al.","(1995) J. Biol.","Chem.","270: 16970-16973), apolipoproteins such as apo A-I, A-II, A-IV, C-I, C-II, CIII (Segrest et al.","(1990), Proteins 8:103-117) and apoB (Chatterton et al.","(1995) J. Lipid Res.","36: 2027-2037; Davis, R A in: Vance D E, Vance J. editors.","Lipoprotein structure and secretion.","The Netherlands, Elsevier, 191: 403-426.In one embodiment, the first recombinant polypeptide is fused to an oil-body-protein.","The methodology is further described in U.S. Pat.","No.","5,650,554, which is incorporated herein by reference in its entirety.","The first recombinant polypeptide may be fused to the N-terminus as well as to the C-terminus of the oil-body-protein (as described in: Moloney and van Rooijen (1996) INFORM 7:107-113) and fragments of the oil-body-protein such as for example the central domain of an oleosin molecule, or modified versions of the oil-body-protein may be used.","In this embodiment, the second recombinant polypeptide is expressed intracellularly and then intracellularly associates with the first recombinant polypeptide to form the multimeric-protein-complex in the cell.","Oil bodies comprising the multimeric-protein-complex are then conveniently isolated from the cells.","In a further embodiment both the first and second recombinant polypeptide are separately fused to an oil-body-protein.","In this embodiment nucleic acid sequences encoding the first and second polypeptides may be prepared separately and introduced in separate cell lines or they may be introduced in the same cell lines.","Where the nucleic acid sequences are introduced in the same cell line, these nucleic acid sequence may be prepared using two separate expression vectors, or they may be prepared using a single vector comprising nucleic acid sequences encoding both the first polypeptide fused to an oil body protein and the second polypeptide fused to an oil-body-protein.","Where separate cell lines are used subsequent mating of the offspring (e.g.","mating of plants) is used to prepare a generation of cells comprising oil bodies which comprise both the first and second recombinant polypeptide fused to an oil-body-protein.","In further alternate embodiment, the first and second recombinant polypeptide are fused to form a multimeric-fusion-protein comprising the multimeric-protein-complex.","In such an embodiment, the first and second polypeptide is associated with the oil body through an oil-body-targeting-protein capable of associating with both the fusion protein and with the oil body.","In a particular embodiment, the fusion protein comprising the multimeric-protein-complex is fused to an oil-body-protein, for example, an oleosin or caleosin.","In embodiments provided herein in which the multimeric-protein-complex is an immunoglobulin (e.g., a multimeric-immunoglobulin-complex), a particularly preferred oil body targeting protein is an oleosin or caleosin associated with an immunoglobulin binding protein, such as for example protein A (U.S. Pat.","No.","5,151,350), protein L (U.S. Pat.","No.","5,965,390) and protein G (U.S. Pat.","No.","4,954,618), or active fragments of such immunoglobulin binding proteins.","New oil-body-proteins may be discovered for example by preparing oil bodies (described in further detail below) and identifying proteins in these preparations using for example SDS gel electrophoresis.","Polyclonal antibodies may be raised against these proteins and used to screen cDNA libraries in order to identify nucleic acid sequences encoding oil-body-proteins.","The methodologies are familiar to the skilled artisan (Huynh et al.","(1985) in DNA Cloning Vol.","1.a Practical Approach ed.","D M Glover, IRL Press, pp 49-78).","New oil-body-proteins may further be discovered using known nucleic acid sequences encoding oil-body-proteins (e.g.","the Arabidopsis, rapeseed, carrot and corn nucleic acid sequences) to probe for example cDNA and genomic libraries for the presence of nucleic acid sequences encoding oil-body-proteins.","In one embodiment, the first and second polypeptide are a first and second redox protein.","Accordingly, one embodiment provided herein relates to novel and improved methods for the production of redox proteins.","It has unexpectedly been found that a redox protein when prepared as a fusion protein with a second redox protein is fully enzymatically active when produced in association with an oil body.","In contrast, when the redox protein is prepared without the second redox protein it has reduced enzymatic activity.","In one embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide relative to production as a non-fusion polypeptide.","Accordingly, provided herein are methods for producing an oil body associated with a heteromultimeric redox protein complex, said method comprising: (a) producing in a cell comprising oil bodies, a first redox protein and a second redox protein wherein said first redox protein is capable of interacting with said second redox protein, preferably in the cell, to form said heteromultimeric redox protein complex; and (b) associating said heteromultimeric redox protein complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said heteromultimeric redox protein complex.","In a particular embodiment the first and second redox protein are prepared as a fusion protein to form a redox fusion polypeptide.","Accordingly, provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.","The oil bodies in association with the redox protein may be used to prepare a variety of useful emulsions.","As used herein the phrase “redox proteins” or grammatical variations thereof, refers to any protein or active protein fragment capable of participating in electron transport.","For example, redox proteins are capable of catalyzing the transfer of an electron from an electron donor (also frequently referred to as the reducing agent) to an electron acceptor (also frequently referred to as the oxidizing agent).","In the process of electron transfer, the reducing agent (electron donor) is oxidized and the oxidizing agent (electron acceptor) is reduced.","Exemplary redox proteins for use herein include iron-sulfur proteins, cytochromes, redox active thiol proteins and redox-active flavoproteins.","To carry out their function as conduits for electrons, redox proteins, such as thioredoxin and thioredoxin-reductase for example, are known to function by interacting or associating with one another in multimeric-protein-complexes (e.g., heteromultimeric-protein-complexes).","The term “redox fusion polypeptide” as used herein refers to any fusion polypeptide comprising a first redox protein linked to a second redox protein (e.g., an in-frame translational fusion).","The redox proteins that may be used with the methods and compositions provided herein may be any redox protein.","In one embodiment the first and second redox proteins are a pair of redox proteins that would normally occur together from the same source, in nature.","In a particular embodiment, the first redox protein is a thioredoxin and the second redox protein is a thioredoxin-reductase.","The redox fusion polypeptide may be produced in any cell comprising oil bodies, including any animal cell, plant cell, algae cell, fungal cell or bacterial cell.","In certain embodiments the redox fusion polypeptide is produced in a plant cell and in particular embodiments the redox fusion polypeptide is produced in the seed cells of a seed plant.","In particular embodiments the oil-body-targeting-protein that is used is an oil-body-protein.","In embodiments of the present invention in which an oil-body-protein is used, the first and second redox protein are preferably covalently fused to the oil-body-protein.","Accordingly, provided herein are methods for the preparation of a redox protein In association with an oil body comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a first nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a second nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating said oil bodies comprising said redox fusion polypeptide from said progeny cell.","Redox Proteins In accordance with various methods and compositions provided herein, any nucleic acid sequence encoding a redox protein may be used.","The nucleic acid sequence encoding the first and/or second redox protein may be obtained from any biological source or may be prepared synthetically.","In general, nucleic acid sequences encoding redox proteins are well known in the art and readily available.","See, for example: Cristiano et al.","(1993) Genomics 17: (2) 348-354, Doyama et al.","(1998) Plant Sci.","137: 53-62, Hoeoeg et al.","(1984) Biosci.","Rep. 4: 917-923; as well as the Swiss Protein sequences set forth in Table 5.Known nucleic acid sequences encoding redox proteins may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences Encoding redox proteins, for example by screening cDNA or genomic libraries.","Thus, additional nucleic acid sequences may be discovered and used in accordance with the present invention.","The nucleic acid sequence encoding the first and/or second redox protein may be obtained from separate sources or may be obtained from the same source.","In general however, the nucleic acid sequence encoding a redox-fusion polypeptide comprises nucleic acid sequences encoding a first and a second redox protein obtained from the same or a similar biological source.","In certain embodiments provided herein, wherein the nucleic acid sequence encoding the first and second redox protein is obtained from the same source, the nucleic acid sequence encoding the first redox protein and second redox protein may be naturally fused.","In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second redox protein are preferably obtained from a plant source.","As set forth above, a polypeptide spacer or linker of variable length may separate the first and second redox proteins from each other and/or from the oil-body-targeting-protein; and additional redox proteins (e.g., one or more) may be fused to the first and/or second redox protein.","The nucleic acid sequences encoding the redox proteins may be altered to improve expression levels for example by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the redox proteins, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).","Comparison of the codon usage of the redox protein with codon usage of the host will enable the identification of codons that may be changed.","For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.","By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.","Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The redox proteins may be altered using for example, targeted mutagenesis, random mutagenesis (Shiraishi et al.","(1998) Arch.","Biochem.","Biophys.","358: 104-115; Galkin et al.","(1997) Protein Eng.","10: 687-690; Carugo et al.","(1997) Proteins 28: 10-28; Hurley et al.","(1996) Biochemistry 35: 5670-5678) (and/or by the addition of organic solvent (Holmberg et al.","(1999) Protein Eng.","12: 851-856).","The polypeptide spacer between the first and second redox protein may be altered in similar ways.","The first and second redox protein may be selected by developing a two-dimensional matrix and determining which combination of first and second redox protein is most effective in electron transport using for example, a colorometric reduction assay (Johnson et al (1984) J. of Bact.","Vol.","158 3:1061-1069, Luthman et al (1982) Biochemistry Vol 21 26:6628-2233).","Combinations of thioredoxin and thioredoxin-reductase may be tested by determining the reduction of wheat storage proteins and milk storage protein beta-lactoglobulin in vitro (Del Val et al.","(1999) J. Allerg.","Clin.","Immunol.","103: 690-697).","Using the same strategy polypeptide spacers between the first and second redox proteins may be evaluated for their efficiency.","First and second redox proteins that may be used herein include without limitation any first redox protein and second redox protein selected from the group of redox proteins consisting of cytochromes, such as cytochrome a, cytochrome b and cytochrome c; porphyrin containing proteins, for example hemoglobin; iron-sulfur proteins, such as ferredoxin; flavoproteins such as thioredoxin-reductase, NADH dehydrogenase, succinate dehydrogenase, dihydrolipoyl dehydrogenase, acyl-CoA dehydrogenase, D-amino acid oxidase, xanthine oxidase, orotate reductase and aldehyde oxidase; pyridine-linked dehydrogenases, for example, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and beta-hydroxy-butarate dehydrogenase; and redox active thiol containing proteins such as thioredoxin.","In particular embodiments, the redox proteins provided herein are thioredoxin and its reductant thioredoxin-reductase (which are jointly also referred to herein as “thioredoxin-related” protein(s)).","As used herein, the term “thioredoxin” refers to relatively small proteins (typically approximately 12 kDa) that belong to the family of thioltransferases which catalyze oxido-reductions via the formation or hydrolysis of disulfide bonds and are widely, if not universally, distributed throughout the animal plant and bacterial kingdom.","The reduces form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.","In order to reduce the oxidized thioredoxin, two cellular reductants provide the reduction equivalents: reduced ferredoxin and NADPH.","These reduction equivalents are supplied to thioredoxin via interaction or association with different thioredoxin-reductases including the NADPH thioredoxin-reductase and ferredoxin thioredoxin-reductase.","The supply of these reduction equivalents requires the formation of a heteromultimeric-protein-complex comprising thioredoxin and thioredoxin-reductase.","Ferredoxin thioredoxin-reductase is involved in the reduction of plant thioredoxins designated as Trxf and Trxm, both of which are involved in the regulation of photosynthetic processes in the chloroplast.","The NADPH/thioredoxin active in plant seeds is designated Trxh (also referred to herein as thioredoxin h-type) and is capable of the reduction of a wide range of proteins thereby functioning as an important cellular redox buffer.","Generally, only one kind of thioredoxin, which analogous to the plant Trxh type, is found in bacterial or animal cells.","The h-type thioredoxins are capable of being reduced by NADPH and NADPH-thioredoxin reductase.","Exemplary thioredoxins are further characterized as a protein having a core of 5 beta-sheets surrounded by 4 to 6 alpha helixes.","Exemplary thioredoxins are further characterized by having an active site containing the consensus amino acid sequence: XCYYCZ, wherein Y is any amino acid, such as hydrophobic or non-polar amino acids, wherein X can be any of the 20 amino acids, preferably a hydrophobic amino acid, such as a tryptophan, and Z can be any amino acid, preferably polar amino acids.","In certain embodiments, the thioredoxins for use herein comprise an active site having the amino acid sequence XCGPCZ.","When the cysteines in the active site of thioredoxin or thioredoxin-like proteins are oxidized, they form an intramolecular disulfide bond.","In the reduced state, the same active sites are capable of participating in redox reactions through the reversible oxidation of its active site dithiol, to a disulfide and catalyzes dithioldisulfide exchange reactions.","Exemplary thioredoxins are well-known in the art and can be obtained from several organisms including Arabidopsis thaliana (Riveira Madrid et al.","(1995) Proc.","Natl.","Acad.","Sci.","92: 5620-5624), wheat (Gautier et al.","(1998) Eur.","J. Biochem.","252: 314-324); Escherichia coli (Hoeoeg et al (1984) Biosci.","Rep. 4: 917-923) and thermophylic microorganisms such as Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126).","Thioredoxins have also been recombinantly expressed in several host systems including bacteria (Gautier et al.","(1998) Eur J. Biochem.","252: 314-324) and plants (PCT Patent Application WO 00/58453) Commercial preparations of E. coli sourced Thioredoxins are readily available from for example: Sigma Cat No.","T 0910 Thioredoxin (E. coli, recombinant; expressed in E. coli).","Exemplary nucleic acid sequences encoding thioredoxin polypeptides for use herein are readily available from a variety of diverse biological sources including E. coli (Hoeoeg et al.","(1984) Biosci.","Rep.: 4 917-923); Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126); Arabidopsis thaliana (Rivera-Madrid (1995) Proc.","Natl.","Acad.","Sci.","92: 5620-5624); wheat (Gautier et al (1998) Eur.","J. Biochem.","252(2): 314-324); tobacco (Marty et al.","(1991) Plant Mol.","Biol.","17: 143-148); barley (PCT Patent Application 00/58352); rice (Ishiwatari et al.","(1995) Planta 195: 456-463); soybean (Shi et al.","(1996) Plant Mol.","Biol.","32: 653-662); rapeseed (Bower et al.","Plant Cell 8: 1641-1650) and calf (Terashima et al.","(1999) DNA Seq.","10(3): 203-205); and the like.","In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth as SEQ ID NOs:38, 42, 46 and 50; and those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth in Table 5 as SEQ ID NOs:52-194.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:52-194 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).","As used herein, the term “thioredoxin-reductase” refers to a protein that complexes with a flavin, such as FAD.","The flavin compound serves as an electron donor for the thioredoxin-reductase protein active site.","Thioredoxin reductases have a redox active, disulfide bond site capable of reducing thioredoxin.","The active site of thioredoxin-reductase contains 2 cysteines.","The type of amino acids surrounding the 2 cysteine residues forming the active site can vary as hydrophobic, non-polar or polar.","An exemplary thioredoxin-reductase is NADPH-thioredoxin-reductase (NTR), which is a cytosolic homodimeric enzyme comprising typically 300-500 amino acids.","Crystal structures of both E. coli and plant thioredoxin-reductase have been obtained (Waksman et al.","(1994) J. Mol.","Biol.","236: 800-816; Dai et al.","(1996) J. Mol.","Biol.","264:1044-1057).","NADPH-thioredoxin-reductases have been expressed in heterologous hosts, for example the Arabidopsis NADPH-thioredoxin-reductase has been expressed in E. coli (Jacquot et al.","(1994) J. Mol.","Biol.","235: 1357-1363) and wheat (PCT Patent Application 00/58453).","Exemplary nucleic acid sequences encoding thioredoxin-reductase proteins can readily be obtained from a variety of sources, such as from the sequence set forth in Table 5 and the Sequence Listing provide herein, from Arabidopsis (Riveira Madrid et al.","(1995) Proc.","Natl.","Acad.","Sci.","USA 92: 5620-5624), E. coli (Russel et al.","(1988) J. Biol.","Chem.","263: 9015-9019); barley (PCT Patent Application 00158352 and wheat (Gautier et al., (1998) Eur.","J. Biochem.","252: 314-324); and the like.","In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin-reductase polypeptide chains set forth as SEQ ID NOs:8, 9, 10, 40, 44, 48 and 50; and those encoding the thioredoxin-reductase polypeptide chains set forth in Table 5 as SEQ ID NOs:195-313.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:195-313 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).","Also contemplated for use in the methods and compositions provided herein are nucleic acid and amino acid homologs that are “substantially homologous” to the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein, which includes thioredoxin and thioredoxin-reductase polypeptides encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides encoding the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein (e.g., in the Examples, Sequence Listing and/or Table 5).","As used herein, a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.","By the term “substantially homologous” is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.","The terms “homology” and “identity” are often used interchangeably.","In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program.","The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol.","Biol.","48:443 (1970), as revised by Smith and Waterman (Adv.","Appl.","Math.","2:482 (1981).","Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.","The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl.","Acids Res.","14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.","353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.","By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier.","Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest.","Preferably the two molecules will hybridize under conditions of high stringency.","Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.","Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc.","Natl.","Acad.","Sci.","USA 85:2444 (1988).","Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.","In general, sequences are aligned so that the highest order match is obtained.","“Identity” per se has an art-recognized meaning and can be calculated using published techniques.","(See, e.g.",": Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).","While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).","Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).","Methods to determine identity and similarity are codified in computer programs.","Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).","Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide.","For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.","As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides.","Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared.","No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides.","Similar comparisons may be made between a test and reference polynucleotides.","Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g.","10/100 amino acid difference (approximately 90% identity).","Differences are defined as nucleic acid or amino acid substitutions, or deletions.","As used herein: stringency of hybridization in determining percentage mismatch is as follows: 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C. 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. Those of skill in this art know that the washing step selects for stable hybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), vol.","3, p. B.","13, see, also, numerous catalogs that describe commonly used laboratory solutions).","SSPE is pH 7.4 phosphate-buffered 0.18 NaCl.","Further, those of skill in the art recognize that the stability of hybrids is determined by Tm, which is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6(log10[Na+])+0.41 (% G+C)−600/l)), so that the only parameters in the wash conditions critical to hybrid stability are sodium ion concentration in the SSPE (or SSC) and temperature.","It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.","By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, Proc.","Natl.","Acad.","Sci.","USA, 78:6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate, adjusted to a pH of 7).","In a particular embodiment, a heteromultimeric-protein-complex is produced as a fusion polypeptide between the first and second redox protein, wherein the first redox protein is thioredoxin and the second redox protein is a thioredoxin-reductase.","In one embodiment, the second recombinant polypeptide, e.g., the thioredoxin-reductase is positioned N-terminal relative to the first recombinant polypeptide, e.g., the thioredoxin.","Accordingly, any protein which is classified as thioredoxin, such as the thioredoxin component of the NADPH thioredoxin system and the thioredoxin present in the ferredoxin/thioredoxin system also known as TRx and TRm may be used in combination with any thioredoxin-reductase such as the NADPH thioredoxin-reductase and the ferredoxin-thioredoxin-reductase and any other proteins having the capability of reducing thioredoxin.","In particular embodiments the thioredoxin and thioredoxin-reductase are plant derived.","In an alternate embodiment, the naturally occurring nucleic acid sequence encoding the thioredoxin/thioredoxin-reductase protein fusion obtainable from Mycobacterium leprae (Wieles et al.","(1995) J. Biol.","Chem.","27:25604-25606) is used, as set forth in the Examples herein.","Immunoglobulins In another embodiment of the present invention, the multimeric-protein-complexes are immunoglobulins.","As used herein “immunoglobulin-polypeptide-chain” refers to a first polypeptide that is capable of associating with a second polypeptide to form an immunologically active (i.e.","capable of antigen binding multimeric-protein-complex.","The types of immunoglobulins and immunoglobulin-polypeptide-chains contemplated for use herein include the immunologically active (i.e.","antigen binding) portions of a light and heavy chain.","Exemplary immunoglobulins and immunoglobulin-polypeptide-chains for use herein include substantially intact immunoglobulins, including any IgG, IgA, IgD, IgE and IgM, as well as any portion of an immunoglobulin, including those portions well-known as Fab fragments, Fab′ fragments, F(ab′).sub2.fragments and Fv fragments.","In this embodiment, the first recombinant polypeptide may be any immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain.","Accordingly, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.","As set forth herein, the multimeric immunoglobulin is associated with an oil body through an oil-body-targeting-protein.","In particular embodiments, the oil-body-targeting-protein may be a fusion polypeptide comprising an oil-body-protein and an immunoglobulin binding protein, such as for example protein A, protein L, and protein G. In yet another embodiment involving immunoglobulins, the first and second recombinant polypeptides (immunoglobulins) are separately fused to an oil body protein, for example an oleosin or caleosin.","For example, a) the first recombinant polypeptide may be an immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or b) the first recombinant polypeptide may be the variable and first constant domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or c) the first recombinant polypeptide may be the variable domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be the variable domain from a kappa or lambda immunoglobulin light chain.","In certain embodiments, the fusion polypeptides are designed or selected to allow the heteromultimeric-protein-complex formation between immunoglobulin light and heavy chain sequences on the oil bodies within the cell comprising oil bodies.","Preparation of Expression Vectors Comprising Oil-Body-Targeting-Proteins and the First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, or the First and/or Second Thioredoxin-Related Proteins.","In accordance with the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are conveniently produced in a cell.","In order to produce the recombinant polypeptides or multimeric-protein-complexes, a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein are incorporated in a recombinant expression vector.","Accordingly, provided herein are recombinant expression vectors comprising the chimeric nucleic acids provided herein suitable for expression of the oil-body-targeting-protein and the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, suitable for the selected cell.","The term “suitable for expression in the selected cell” means that the recombinant expression vector contains all nucleic acid sequences required to ensure expression in the selected cell.","Accordingly, the recombinant expression vectors further contain regulatory nucleic acid sequences selected on the basis of the cell which is used for expression and ensuring initiation and termination of transcription operatively linked to the nucleic acid sequence encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein.","Regulatory nucleic acid sequences include promoters, enhancers, silencing elements, ribosome binding sites, Shine-Dalgarno sequences, introns and other expression elements.","“Operatively linked” is intended to mean that the nucleic acid sequences comprising the regulatory regions linked to the nucleic acid sequences encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein allow expression in the cell.","A typical nucleic acid construct comprises in the 5′ to 3′ direction a promoter region capable of directing expression, a coding region comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or an oil-body-targeting-protein and a termination region functional in the selected cell.","The selection of regulatory sequences will depend on the organism and the cell type in which the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein is expressed, and may influence the expression levels of the polypeptide.","Regulatory sequences are art-recognized and selected to direct expression of the oil-body-targeting-protein and the recombinant polypeptides or multimeric-protein-complexes in the cell.","Promoters that may be used in bacterial cells include the lac promoter (Blackman et al.","(1978) Cell: 13: 65-71), the trp promoter (Masuda et al.","(1996) Protein Eng: 9: 101-106) and the T7 promoters (Studier et al.","(1986) J. Mol.","Biol.","189: 113-130).","Promoters functional in plant cells that may be used herein include constitutive promoters such as the 35S CaMV promoter (Rothstein et al.","(1987) Gene: 53: 153-161) the actin promoter (McElroy et al.","(1990) Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).","Other promoters are specific to certain tissues or organs (for example, roots, leaves, flowers or seeds) or cell types (for example, leaf epidermal cells, mesophyll cells or root cortex cells) and or to certain stages of plant development.","Timing of expression may be controlled by selecting an inducible promoter, for example the PR-a promoter described in U.S. Pat.","No.","5,614,395.Selection of the promoter therefore depends on the desired location and timing of the accumulation of the desired polypeptide.","In a particular embodiment, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are expressed in a seed cell and seed specific promoters are utilized.","Seed specific promoters that may be used herein include for example the phaseolin promoter (Sengupta-Gopalan et al.","(1985) Proc.","Natl.","Acad.","Sci.","USA: 82: 3320-3324), and the Arabidopsis 18 kDa oleosin promoter (van Rooijen et al.","(1992) Plant.","Mol.","Biol.","18: 1177-1179).","New promoters useful in various plant cell types are constantly discovered.","Numerous examples of plant promoters may be found in Ohamuro et al.","(Biochem of Pl.","(1989) 15: 1-82).","Genetic elements capable of enhancing expression of the polypeptide may be included in the expression vectors.","In plant cells these include for example, the untranslated leader sequences from viruses such as the AMV leader sequence (Jobling and Gehrke (1987) Nature: 325: 622-625) and the intron associated with the maize ubiquitin promoter (See: U.S. Pat.","No.","5,504,200).","Transcriptional terminators are generally art recognized and besides serving as a signal for transcription termination serve as a protective element serving to extend the mRNA half-life (Guarneros et al.","(1982) Proc.","Natl.","Acad.","Sci.","USA: 79: 238-242).","In nucleic acid sequences for the expression in plant cells, the transcriptional terminator typically is from about 200 nucleotide to about 1000 nucleotides in length.","Terminator sequences that may be used herein include for example, the nopaline synthase termination region (Bevan et al.","(1983) Nucl.","Acid.","Res.",": 11: 369-385), the phaseolin terminator (van der Geest et al.","(1994) Plant J.: δ: 413-423), the terminator for the octopine synthase gene of Agrobacterium tumefaciens or other similarly functioning elements.","Transcriptional terminators can be obtained as described by An (1987) Methods in Enzym.","153: 292).","The selection of the transcriptional terminator may have an effect on the rate of transcription.","Accordingly, provided herein are chimeric nucleic acid sequences encoding a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins.","In one embodiment, said nucleic acid comprises: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide, immunoglobulin-polypeptide-chain, or redox protein; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, immunoglobulin-polypeptide-chain or redox protein, wherein said first and second recombinant polypeptides, immunoglobulin-polypeptide-chains or redox proteins are capable of forming a multimeric-protein-complex.","In another embodiment, provided herein is an expression vector comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a nucleic acid sequence encoding a multimeric-fusion-protein, such as a redox fusion polypeptide or immunoglobulin, comprising a first recombinant polypeptide, such as a redox protein or immunoglobulin-polypeptide-chain, linked to a second recombinant polypeptide, such as a second redox protein or a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.","The recombinant expression vector further may contain a marker gene.","Marker genes that may be used in accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells including all selectable and screenable marker genes.","A marker may be a resistance marker such as an antibiotic resistance marker against for example kanamycin, ampicillin, G418, bleomycin hygromycin, chloramphenicol which allows selection of a trait by chemical means or a tolerance marker against for example a chemical agent such as the normally phytotoxic sugar mannose (Negrotto et al.","(2000) Plant Cell Rep. 19: 798-803).","In plant recombinant expression vectors herbicide resistance markers may conveniently be used for example markers conferring resistance against glyphosate (U.S. Pat.","Nos.","4,940,935 and 5,188,642) or phosphinothricin (White et al.","(1990) Nucl.","Acids Res.","18: 1062; Spencer et al.","(1990) Theor.","Appl.","Genet.","79: 625-631).","Resistance markers to a herbicide when linked in close proximity to the redox protein or oil-body-targeting-protein may be used to maintain selection pressure on a population of plant cells or plants for those plants that have not lost the protein of interest.","Screenable markers that may be employed to identify transformants through visual observation include beta-glucuronidase (GUS) (see U.S. Pat.","No.","5,268,463 and U.S. Pat.","No.","5,599,670) and green fluorescent protein (GFP) (Niedz et al.","(1995) Plant Cell Rep.: 14: 403).","The recombinant expression vectors further may contain nucleic acid sequences encoding targeting signals ensuring targeting to a cell compartment or organelle.","Suitable targeting signals that may be used herein include those that are capable of targeting polypeptides to the endomembrane system.","Exemplary targeting signals that may be used herein include targeting signals capable of directing the protein to the periplasm, the cytoplasm, the golgi apparatus, the apoplast (Sijmons et al., 1990, Bio/Technology, 8:217-221) the chloroplast (Comai et al.","(1988) J. Biol.","Chem.","263: 15104-15109), the mitochondrion, the peroxisome (Unger et al.","(1989) Plant Mol.","Biol.","13: 411-418), the ER, the vacuole (Shinshi et al.","(1990) Plant Mol.","Biol.","14: 357-368 and the oil body.","By the inclusion of the appropriate targeting sequences it is possible to direct the oil-body-targeting-protein or the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, to the desired organelle or cell compartment.","The recombinant expression vectors of the present invention may be prepared in accordance with methodologies well known to those of skill in the art of molecular biology (see for example: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press).","The preparation of these constructs may involve techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing and PCR.","A wide variety of cloning vectors is available to perform the necessary cloning steps resulting in a recombinant expression vector ensuring expression of the polypeptide.","Especially suitable for this purpose are vectors with a replication system that is functional in Escherichia coli such as pBR322, the PUC series of vectors, the M13 mp series of vectors, pBluescript etc.","Typically these vectors contain a marker allowing the selection of transformed cells for example by conferring antibiotic resistance.","Nucleic acid sequences may be introduced in these vectors and the vectors may be introduced in E. coli grown in an appropriate medium.","Vectors may be recovered from cells upon harvesting and lysing the cells.","Recombinant expression vectors suitable for the introduction of nucleic acid sequences in plant cells include Agrobacterium and Rhizobium based vectors such as the Ti and Ri plasmids.","Agrobacterium based vectors typically carry at least one T-DNA border sequence and include vectors such pBIN 19 (Bevan (1984) Nucl Acids Res.","Vol.","12, 22:8711-8721) and other binary vector systems (for example: U.S. Pat.","No.","4,940,838).","Production of Cells Comprising a First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, and/or a First and/or Second Thioredoxin-Related Protein and Oil-Body-Targeting-Proteins In accordance with the present invention, the recombinant expression vectors are introduced into the cell that is selected and the selected cells are grown to produce the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, a first and/or second thioredoxin-related protein; and the oil-body-targeting-protein either directly or in a progeny cell.","Methodologies to introduce recombinant expression vectors into a cell also referred to herein as “transformation” are well known to the art and vary depending on the cell type that is selected.","General techniques to transfer the recombinant expression vectors into the cell include electroporation; chemically mediated techniques, for example CaCl2 mediated nucleic acid uptake; particle bombardment (biolistics); the use of naturally infective nucleic acid sequences for example virally derived nucleic acid sequences or when plant cells are used Agrobacterium or Rhizobium derived nucleic acid sequences; PEG mediated nucleic acid uptake, microinjection, and the use of silicone carbide whiskers (Kaeppler et al.","(1990) Plant Cell Rep. 9:415-418) all of which may be used herein.","Introduction of the recombinant expression vector into the cell may result in integration of its whole or partial uptake into host cell genome including the chromosomal DNA or the plastid genome.","Alternatively the recombinant expression vector may not be integrated into the genome and replicate independently of the host cell's genomic DNA.","Genomic integration of the nucleic acid sequence is typically used as it will allow for stable inheritance of the introduced nucleic acid sequences by subsequent generations of cells and the creation of cell, plant or animal lines.","Particular embodiments involve the use of plant cells.","Particular plant cells used herein include cells obtainable from Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.","); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.","and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.","); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticum aestivum) and sunflower (Helianthus annuus).","Transformation methodologies for dicotelydenous plant species are well known.","Generally Agrobacterium mediated transformation is utilized because of its high efficiency as well as the general susceptibility by many, if not all dicotelydenous plant species.","Agrobacterium transformation generally involves the transfer of a binary vector (e.g.","pBIN19) comprising the DNA of interest to an appropriate Agrobacterium strain (e.g.","CIB542) by for example tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilization of the binary vector to the target Agrobacterium strain, or by DNA transformation of the Agrobacterium strain (Hofgen et al.","Nucl.","Acids.","Res.","(1988) 16: 9877.Other transformation methodologies that may be used to transform dicotelydenous plant species include biolistics (Sanford (1988) Trends in Biotechn.","6: 299-302); electroporation (Fromm et al.","(1985) Proc.","Natl.","Acad.","Sci.","USA 82: 5824-5828); PEG mediated DNA uptake (Potrykus et al.","(1985) Mol.","Gen. Genetics 199: 169-177); microinjection (Reich et al.","Bio/Techn.","(1986) 4: 1001-1004) and silicone carbide whiskers (Kaeppler et al.","(1990) Plant Cell Rep. 9: 415-418).","The exact transformation methodologies typically vary somewhat depending on the plant species that is used.","In a particular embodiment the oil bodies are obtained from safflower and the recombinant proteins are expressed in safflower.","Safflower transformation has been described by Baker and Dyer (Plant Cell Rep. (1996) 16: 106-110).","Monocotelydenous plant species may now also be transformed using a variety of methodologies including particle bombardment (Christou et al.","(1991) Biotechn.","9: 957-962; Weeks et al.","Plant Physiol.","(1993) 102: 1077-1084; Gordon-Kamm et al.","Plant Cell (1990) 2: 603-618) PEG mediated DNA uptake (EP 0 292 435; 0 392 225) or Agrobacterium-mediated transformation (Goto-Fumiyuki et al (1999) Nature-Biotech.","17 (3):282-286).","Plastid transformation is described in U.S. Pat.","Nos.","5,451,513; 5,545,817 and 5,545,818; and PCT Patent Applications 95/16783; 98/11235 and 00/39313) Basic chloroplast transformation involves the introduction of cloned plastid DNA flanking a selectable marker together with the nucleic acid sequence of interest into a suitable target tissue using for example biolistics or protoplast transformation.","Selectable markers that may be used include for example the bacterial aadA gene (Svab et al.","(1993) Proc.","Natl.","Acad.","Sci.","USA 90: 913-917).","Plastid promoters that may be used include for example the tobacco clpP gene promoter (PCT Patent Application 97/06250).","In another embodiment, the invention chimeric nucleic acid constructs provided herein are directly transformed into the plastid genome.","Plastid transformation technology is described extensively in U.S. Pat.","Nos.","5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos.","WO 95/16783 and WO 97/32977; and in McBride et.","al., Proc Natl Acad Sci USA 91: 7301-7305 (1994), the entire disclosures of all of which are hereby incorporated by reference.","In one embodiment, plastid transformation is achieved via biolistics, first carried out in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al.","(1988) Science 240:1534-1537)) and then extended to Nicotiana tabacum (Svab et al.","(1990) Proc Natl Acad Sci USA 87:8526-8530), combined with selection for cis-acting antibiotic resistance loci (spectinomycin or streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes.","In another embodiment, tobacco plastid transformation is carried out by particle bombardment of leaf or callus tissue, or polyethylene glycol (PEG)-mediated uptake of plasmid DNA by protoplasts, using cloned plastid DNA flanking a selectable antibiotic resistance marker.","For example, 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome.","In one embodiment, point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin can be utilized as selectable markers for transformation (Svab et al.","(1990) Proc Natl Acad Sci USA 87:8526-8530; Staub et al.","(1992) Plant Cell 4:39-45, the entire disclosures of which are hereby incorporated by reference), resulting in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.","The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al.","(1993) EMBO J 12:601-606, the entire disclosure of which is hereby incorporated by reference).","In another embodiment, substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al.","(1993) Proc Natl Acad Sci USA 90: 913-917, the entire disclosure of which is hereby incorporated by reference).","This marker has also been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl Acids Res 19, 4083-4089, the entire disclosure of which is hereby incorporated by reference).","In other embodiments, plastid transformation of protoplasts from tobacco and the moss Physcomitrella can be attained using PEG-mediated DNA uptake (O'Neill et al.","(1993) Plant J 3:729-738; Koop et al.","(1996) Planta 199:193-201, the entire disclosures of which are hereby incorporated by reference).","Both particle bombardment and protoplast transformation are also contemplated for use herein.","Plastid transformation of oilseed plants has been successfully carried out in the genera Arabidopsis and Brassica (Sikdar et al.","(1998) Plant Cell Rep 18:20-24; PCT Application WO 00/39313, the entire disclosures of which are hereby incorporated by reference).","A chimeric nucleic sequence construct is inserted into a plastid expression cassette including a promoter capable of expressing the construct in plant plastids.","A particular promoter capable of expression in a plant plastid is, for example, a promoter isolated from the 5′ flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues.","Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (Stern et al.","(1997) Trends in Plant Sci 2:308-315, the entire disclosure of which is hereby incorporated by reference).","Plastid promoters generally contain the −35 and −10 elements typical of prokaryotic promoters, and some plastid promoters called PEP (plastid-encoded RNA polymerase) promoters are recognized by an E. coli-like RNA polymerase mostly encoded in the plastid genome, while other plastid promoters called NEP promoters are recognized by a nuclear-encoded RNA polymerase.","Both types of plastid promoters are suitable for use herein.","Examples of plastid promoters include promoters of clpP genes such as the tobacco clpP gene promoter (WO 97/06250, the entire disclosure of which is hereby incorporated by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser.","No.","09/038,878, the entire disclosure of which is hereby incorporated by reference).","Another promoter capable of driving expression of a chimeric nucleic acid construct in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., (1994) Microbiol Rev 58:700-754; Shinozaki et al.","(1986) EMBO J 5:2043-2049, the entire disclosures of both of which are hereby incorporated by reference).","Other examples of promoters capable of driving expression of a nucleic acid construct in plant plastids include a psbA promoter or am rbcL promoter.","A plastid expression cassette preferably further includes a plastid gene 3′ untranslated sequence (3′ UTR) operatively linked to a chimeric nucleic acid construct of the present invention.","The role of untranslated sequences is preferably to direct the 3′ processing of the transcribed RNA rather than termination of transcription.","An exemplary 3′ UTR is a plastid rps16 gene 3′ untranslated sequence, or the Arabidopsis plastid psbA gene 3′ untranslated sequence.","In a further embodiment, a plastid expression cassette includes a poly-G tract instead of a 3′ untranslated sequence.","A plastid expression cassette also preferably further includes a 5′ untranslated sequence (5′ UTR) functional in plant plastids, operatively linked to a chimeric nucleic acid construct provided herein.","A plastid expression cassette is contained in a plastid transformation vector, which preferably further includes flanking regions for integration into the plastid genome by homologous recombination.","The plastid transformation vector may optionally include at least one plastid origin of replication.","The present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the chimeric nucleic acid construct is expressible in the plant plastid.","Also encompassed herein is a plant or plant cell, including the progeny thereof, including this plant plastid.","In a particular embodiment, the plant or plant cell, including the progeny thereof, is homoplasmic for transgenic plastids.","Other promoters capable of driving expression of a chimeric nucleic acid construct in plant plastids include transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected.","In these cases, the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA.","The transactivator is targeted to plastids using a plastid transit peptide.","The transactivator and the transactivator-driven DNA molecule are brought together either by crossing a selected plastid-transformed line with and a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a chimeric nucleic acid construct encoding the transactivator supplemented with a plastid-targeting sequence operably linked to a nuclear promoter.","If the nuclear promoter is an inducible promoter, in particular a chemically inducible embodiment, expression of the chimeric nucleic acid construct in the plastids of plants is activated by foliar application of a chemical inducer.","Such an inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction.","A particular transactivator is, for example, viral RNA polymerase.","Particular promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase.","The gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide.","Promoters suitable for nuclear expression of a gene, for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described above and elsewhere in this application.","Expression of chimeric nucleic acid constructs in plastids can be constitutive or can be inducible, and such plastid expression can be also organ- or tissue-specific.","Examples of various expression systems are extensively described in WO 98/11235, the entire disclosure of which is hereby incorporated by reference.","Thus, in one aspect, the present invention utilizes coupled expression in the nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under the control of the chemically inducible PR-1a promoter, for example of the PR-1 promoter of tobacco, operably linked with a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences, for example as described in as in U.S. Pat.","No.","5,614,395 the entire disclosure of which is hereby incorporated by reference.","In another embodiment, when plastid transformants homoplasmic for the maternally inherited TR or NTR genes are pollinated by lines expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry both transgene constructs but do not express them until synthesis of large amounts of enzymatically active protein in the plastids is triggered by foliar application of the PR-1a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH).","In a particular embodiment, two or more genes, for example TR and NTR genes, are transcribed from the plastid genome from a single promoter in an operon-like polycistronic gene.","In one embodiment, the operon-like polycistronic gene includes an intervening DNA sequence between two genes in the operon-like polycistronic gene.","In a particular embodiment, the intervening DNA sequence is not present in the plastid genome to avoid homologous recombination with plastid sequences.","In another embodiment, the DNA sequence is derived from the 5′ untranslated (UTR) region of a non-eukaryotic gene, preferably from a viral 5′UTR, preferably from a 6′UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage.","In one embodiment, a portion of the DNA sequence may be modified to prevent the formation of RNA secondary structures in an RNA transcript of the operon-like polycistronic gene, for example between the DNA sequence and the RBS of the downstream gene.","Such secondary structures may inhibit or repress the expression of the downstream gene, particularly the initiation of translation.","Such RNA secondary structures are predicted by determining their melting temperatures using computer models and programs such a the “mfold” program version 3 (available from Zuker and Turner, Washington University School of Medicine, St-Louis, Mo.)","and other methods known to one skilled in the art.","The presence of the intervening DNA sequence in the operon-like polycistronic gene increases the accessibility of the RBS of the downstream gene, thus resulting in higher rates of expression.","Such strategy is applicable to any two or more genes to be transcribed from the plastid genome from a single promoter in an operon-like chimeric heteromultimeric gene.","Following transformation the cells are grown, typically in a selective medium allowing the identification of transformants.","Cells may be harvested in accordance with methodologies known to the art.","In order to associate the oil bodies with the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and/or second thioredoxin-related protein, the integrity of cells may be disrupted using any physical, chemical or biological methodology capable of disrupting the cells' integrity.","These methodologies are generally cell-type dependent and known to the skilled artisan.","Where plants are employed they may be regenerated into mature plants using plant tissue culture techniques generally known to the skilled artisan.","Seeds may be harvested from mature transformed plants and used to propagate the plant line.","Plants may also be crossed and in this manner, contemplated herein is the breeding of cells lines and transgenic plants that vary in genetic background.","It is also possible to cross a plant line comprising the first recombinant polypeptide with a plant line comprising the second recombinant polypeptide.","Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox protein (e.g., a thioredoxin-related protein, and the like) or an immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox protein (e.g., a thioredoxin-related protein, and the like) or a second immunoglobulin-polypeptide-chain; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","The second recombinant polypeptide may also associate with the oil bodies.","Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox (or thioredoxin-related) protein or immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox (thioredoxin-related) protein or a second immunoglobulin-polypeptide-chain, wherein said second recombinant polypeptide is capable of associating with said oil bodies through an oil body targeting protein; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.","Isolation of Oil Bodies The oil bodies provided herein may be obtained from any cell containing oil bodies, including any animal cell; plant cell; fungal cell; for example a yeast cell, algae cell; or bacterial cell.","Any process suitable for the isolation oil bodies from cells may be used herein.","Processes for the isolation of oil bodies from plant seed cells have been described in U.S. Pat.","Nos.","(6,146,645 and 6,183,762) and the isolation of oil bodies from yeast cells has been described by Ting et al.","(1997) J. Biol.","Chem.","272: 3699-3706).","In certain embodiments, the oil bodies are obtained from a plant cell such as for example a pollen cell; a fruit cell; a spore cell; a nut cell; mesocarp cell; for example the mesocarp cells obtainable from olive (Olea europaea) or avocado (Persea americana); or a seed cell.","In particular embodiments the oil bodies are obtained from a plant seed cell.","The seeds can be obtained from a transgenic plant according to the present invention.","In particular embodiments, a seed of a transgenic plant according to the present invention contains the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or first and/or second thioredoxin-related proteins in a concentration of at least about 0.5% of total cellular seed protein.","In further embodiments, a seed of a transgenic plant provided herein contains a recombinant polypeptide or multimeric-protein-complex in a concentration of at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, of total cellular seed protein.","The upper limits of the recombinant polypeptide or multimeric-protein-complex concentration can be up to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.","Thus, the ranges at least about 0.5% up to about 15%; at least about 1.0% up to about 10%; and at least about 5% up to about 8% are among the various ranges contemplated herein.","Among the plant seeds useful in this regard are plant seeds obtainable from the group of plant species consisting of Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.","); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.","and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.","); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); sunflower (Helianthus annuus); barley (Hordeum vulgare); wheat (Traeticum aestivum) and mixtures thereof.","In a particular embodiment, oil bodies are obtainable from the seeds obtainable from safflower (Carthamus tinctorius).","In order to prepare oil bodies from plant seeds, plants are grown and allowed to set seed in accordance with common agricultural practices.","Thus, the present invention also provides seeds comprising oil bodies, wherein said oil bodies further comprise invention multimeric-protein-complexes described herein.","Upon harvesting the seed and, if necessary the removal of large insoluble materials such as stones or seed hulls, by for example sieving or rinsing, any process suitable for the isolation of oil bodies from seeds may be used herein.","A typical process involves grinding of the seeds followed by an aqueous extraction process.","Seed grinding may be accomplished by any comminuting process resulting in a substantial disruption of the seed cell membrane and cell walls without compromising the structural integrity of the oil bodies present in the seed cell.","Suitable grinding processes in this regard include mechanical pressing and milling of the seed.","Wet milling processes such as described for cotton (Lawhon et al.","(1977) J.","Am.","Oil Chem.","Soc.","63: 533-534) and soybean (U.S. Pat.","No.","3,971,856; Carter et al.","(1974) J.","Am.","Oil Chem.","Soc.","51: 137-141) are particularly useful in this regard.","Suitable milling equipment capable of industrial scale seed milling include colloid mills, disc mills, pin mills, orbital mills, IKA mills and industrial scale homogenizers.","The selection of the milling equipment will depend on the seed, which is selected, as well as the throughput requirement.","Solid contaminants such as seed hulls, fibrous materials, undissolved carbohydrates, proteins and other insoluble contaminants are subsequently preferably removed from the ground seed fraction using size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.","Centrifugation may be accomplished using for example a decantation centrifuge such as a HASCO 200 2-phase decantation centrifuge or an NX310B (Alpha Laval).","Operating conditions are selected such that a substantial portion of the insoluble contaminants and sediments and may be separated from the soluble fraction.","Following the removal of insolubles the oil body fraction may be separated from the aqueous fraction.","Gravitational based methods as well as size exclusion based technologies may be used.","Gravitational based methods that may be used include centrifugation using for example a tubular bowl centrifuge such as a Sharples AS-16 or AS-46 (Alpha Laval), a disc stack centrifuge or a hydrocyclone, or separation of the phases under natural gravitation.","Size exclusion methodologies that may be used include membrane ultra filtration and crossflow microfiltration.","Separation of solids and separation of the oil body phase from the aqueous phase may also be carried out concomitantly using gravity based separation methods or size exclusion based methods.","The oil body preparations obtained at this stage in the process are generally relatively crude and depending on the application of the oil bodies, it may be desirable to remove additional contaminants.","Any process capable of removing additional seed contaminants may be used in this regard.","Conveniently the removal of these contaminants from the oil body preparation may be accomplished by resuspending the oil body preparation in an aqueous phase and re-centrifuging the resuspended fraction, a process referred to herein as “washing the oil bodies”.","The washing conditions selected may vary depending on the desired purity of the oil body fractions.","For example where oil bodies are used in pharmaceutical compositions, generally a higher degree of purity may be desirable than when the oil bodies are used in food preparations.","The oil bodies may be washed one or more times depending on the desired purity and the ionic strength, pH and temperature may all be varied.","Analytical techniques may be used to monitor the removal of contaminants.","For example SDS gel electrophoresis may be employed to monitor the removal of seed proteins.","The entire oil body isolation process may be performed in a batch wise fashion or continuous flow.","In a particular embodiment, industrial scale continuous flow processes are utilized.","Through the application of these and similar techniques the skilled artisan is able to obtain oil bodies from any cell comprising oil bodies.","The skilled artisan will recognize that generally the process will vary somewhat depending on the cell type that is selected.","However, such variations may be made without departing from the scope and spirit of the present invention.","Association of the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins with oil bodies.","In accordance with the present invention, the oil bodies are associated with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins through association with an oil-body-targeting-protein capable of association with these multimeric-protein-complexes and the oil bodies.","As used herein the phrase “associating the oil bodies with the multimeric-protein-complex” means that the oil bodies are brought in proximity of the multimeric-protein-complexes in a manner that allows the association of the oil bodies with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.","The association of the oil bodies with the multimeric-protein-complexes is accomplished by association of the oil-body-targeting-protein with both the oil body and with the multimeric-protein-complex.","In particular embodiments, the cells expressing the multimeric-protein-complex associate with the oil bodies that are obtainable from these same cells, which permits the convenient production and isolation of the multimeric-protein-complex, including the first and/or second recombinant polypeptides, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, in an oil body-comprising host cell system.","Accordingly, in one embodiment, the association of the oil body with the multimeric-protein-complex is accomplished intracellularly during the growth of the cell.","For example, a redox fusion polypeptide may be fused to an oil-body-protein and the chimeric protein may be expressed in oil body-containing plant seeds.","Isolation of the oil bodies from the seeds in this case results in isolation of oil bodies comprising either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.","In another embodiment, in which the multimeric-protein-complex associates with oil bodies obtainable from the same cells in which the complex is produced, the association of the oil bodies with the multimeric-protein-complex is accomplished upon disrupting the cell's integrity.","For example, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins may be expressed in such a manner that it is targeted to the endomembrane system of the seed cells.","Oil bodies present in the same seed cells comprising an oil-body-targeting-protein capable of association with these multimeric-protein-complexes, for example an oleosin linked to a single chain antibody capable of association with a recombinant polypeptide or multimeric-protein-complex, may then associate with the recombinant polypeptide or multimeric-protein-complex upon grinding of the seed.","In accordance with this embodiment, plant seed cells comprising a light and heavy chain of an immunoglobulin targeted to the plant apoplast can be prepared.","These particular seed cells are prepared to further comprise oil bodies associated with an oil-body-targeting-protein capable of association with the immunoglobulin, such as for example, an oleosin-protein A fusion protein, and the like.","Upon grinding of the seed, the oil bodies comprising protein A associate with the immunoglobulin through binding.","In yet another embodiment, the oil bodies used to associate with the multimeric-protein-complex are obtained from a cellular source different from the cell comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, such as from a separate plant line.","For example, oil bodies associated with protein A may be prepared from one plant line.","These oil bodies may then be mixed with ground seeds comprising an apoplastically expressed light and heavy chain constituting an immunoglobulin.","Alternatively, a plant line comprising oil bodies associated with protein A may be crossed with a plant line comprising an immunoglobulin.","The first recombinant polypeptide, second recombinant polypeptide and oil-body-targeting-protein may also be prepared in separate cellular compartments.","Association of the first polypeptide, second polypeptide, and oil body then may occur upon disruption of the cell's integrity.","For example, various mechanisms for targeting gene products are known to exist in plants, and the sequences controlling the functioning of these mechanisms have been characterized in some detail.","For example, the targeting of gene products to the chloroplast is controlled by a transit sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (Comai et al.","(1988) J Biol Chem 263: 15104-15109).","Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (Unger et al.","(1989) Plant Mol Biol 13:411-418).","The cDNAs encoding these products can be manipulated to target heterologous gene products to these organelles.","In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments.","Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho (1990) Plant Cell 2:769-783).","Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., (1990) Plant Mol Biol 14:357-368).","By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to the desired organelle or cell compartment.","As hereinbefore mentioned, the redox protein obtained using the methods provided herein is enzymatically active while associated with the oil body.","Preferably the redox protein is at least 5 times more active when produced as a redox fusion polypeptide with a second redox protein relative to its production in association with an oil body as a non-fusion polypeptide (i.e.","without the second redox protein).","More preferably the redox protein is at least 10 times more active when produced as a redox fusion polypeptide.","The activity of the redox fusion polypeptide may be determined in accordance with methodologies generally known to the art (see for example: Johnson et al (1984) J. of Bact.","Vol.","158 3:1061-1069) and may be optimized by for example the addition of detergents, including ionic and non-ionic detergents.","Formulation of Oil Bodies In accordance with a particular embodiment, the oil bodies comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, are preferably formulated into an emulsion.","The emulsion is preferably used in the preparation of a pharmaceutical composition, personal care or a food product.","In emulsified form, the oil body offers certain desirable properties, such as for example excellent compatibility with the human skin.","It particular embodiments, the oil body formulation is stabilized so that a final product may be obtained which may be stored and preserved for longer periods of time.","As used herein, the term “stabilized oil body preparation” refers to an oil body preparation that is prepared so that the formulation does not undergo undesirable physical or chemical alterations when the oil body preparation is stored.","The stabilization requirements may vary depending on the final product.","For example personal care products are preferably stable for at least one year at room temperature while additionally being able to withstand short temperature fluctuations.","Pharmaceutical formulations may in some cases be less stable as they may be stored at lower temperatures thereby preventing the occurrence of undesirable reactions.","In general, stabilization techniques that may be used herein include any and all methods for the preservation of biological material including the addition of chemical agents, temperature modulation based methodologies, radiation-based technologies and combinations thereof.","In particular embodiments small amounts of stabilizing chemical agents are mixed with the oil body formulation to achieve stabilization.","These chemical agents include inter alia preservatives, antioxidants, acids, salts, bases, viscosity modifying agents, emulsifiers, gelling agents and mixtures thereof and may all be used to stabilize the oil body preparation.","In view of the presence of the redox fusion polypeptide the stabilizing agent is generally selected to be compatible with and resulting in good enzymatic function of the redox fusion polypeptide.","Diagnostic parameters to assess the stability of the oil body preparation may be as desired and include all parameters indicative of undesirable qualitative or quantitative changes with respect to chemical or physical stability.","Typical parameters to assess the oil body preparation over time include color, odor, viscosity, texture, pH and microbial growth, and enzymatic activity.","In particular embodiments, the oil body formulation is stabilized prior to the addition of further ingredients that may be used to prepare the final product.","However, in other embodiments, it is nevertheless possible to formulate the final formulation using non-stabilized oil bodies and stabilize the final formulation.","The final preparations may be obtained using one or more additional ingredients and any formulation process suitable for the preparation of a formulation comprising oil bodies.","Ingredients and processes employed will generally vary depending on the desired use of the final product, will be art recognized and may be as desired.","Ingredients and processes that may be used herein include those described in (U.S. Pat.","Nos.","6,146,645 and 6,183,762) which are incorporated by reference herein.","In particular embodiments, the redox fusion polypeptide comprises a thioredoxin and a thioredoxin-reductase.","Accordingly, provided herein are oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.","Also provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of treating or protecting a target against oxidative stress.","The stress of the target is treated or prevented by contacting the target with the formulation.","The target may be any substance susceptible to oxidative stress, including any molecule, molecular complex, cell, tissue or organ.","In another embodiment, provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of chemically reducing a target.","Contacting the target with the formulation reduces the target.","The target may be any substance susceptible to reduction, including any molecule or molecular complex.","Particularly susceptible targets in this regard are the disulfide bonds present in proteins.","The oil bodies comprising thioredoxin/thioredoxin-reductase may be used to prepare formulations used to reduce the allergenicity of food or increase the digestibility of food.","Preferably, the method of reducing the food allergenicity is practiced by mixing the thioredoxin/thioredoxin-reductase comprising oil bodies with food or food ingredients selected from a variety of sources including for example wheat flour, wheat dough, milk, cheese, soya, yogurt and ice cream.","The thioredoxin/thioredoxin-reductase comprising oil bodies may also be used to increase the digestibility of milk as well as other disulfide containing proteins (Jiao, J. et al.","(1992) J. Agric.","Food Chem 40: 2333-2336).","Further food applications include the use of the oil thioredoxin/thioredoxin-reductase comprising oil bodies as a food additive to enhance dough strength and bread quality properties (Wong et al., (1993) J. Cereal Chem.","70: 113-114; Kobrehel et al.","(1994) Gluten Proteins: Association of Cereal Research; Detmold, Germany).","Also provided herein are pharmaceutical compositions comprising, in a pharmaceutically active carrier: oil bodies comprising a thioredoxin/thioredoxin-reductase; oil bodies comprising multimeric-protein-complexes, such as heteromultimeric-protein-complexes; isolated thioredoxin/thioredoxin-reductase fusion proteins; or isolated multimeric-protein-complexes.","These pharmaceutical compositions may be used for the treatment of reperfusion injury (Aota et al.","(1996) J. Cardiov.","Pharmacol.","(1996) 27: 727-732), cataracts (U.S. Pat.","No.","4,771,036), chronic obstructive pulmonary disease (COPD) (MacNee et al.","(1999) Am.","J. Respir.","Crit.","Care Med.","160:S58-S65), diabetes (Hotta et al.","J. Exp.","Med.","188: 1445-1451), envenomation (PCT Patent Application 99/20122; U.S. Pat.","No.","5,792,506), bronchiopulmonary disease (MacNee (2000) Chest 117:3035-3175); malignancies (PCT Patent Application 91/04320) and the alleviation of the allergenic potential of airborne, for example pollen-derived, and contact allergens (PCT Patent Application 00/44781).","Other diseases or conditions that may be treated with the pharmaceutical compositions provided herein include: psoriasis, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, transplantation, GERD (gastro esophageal reflux disease).","In another embodiment, the pharmaceutical compositions provided herein, particularly those comprising one or more redox proteins alone or in combination with oil bodies, can be used in the treatment of inflammatory and viral diseases by reductively inactivating phospholipase A2, one of the contributing factors in inflammatory diseases.","Additionally, the redox fusion polypeptide system has been found to function as a self-defense mechanism in response to environmental stimuli, including oxidative stress caused by UV-generated free radicals.","Consequently, redox proteins, e.g., oleosin-thioredoxin, oleosin-thioredoxin-reductase, the various redox fusion polypeptides described herein, provide beneficial effects in certain skin conditions such as psoriasis, skin cancer, dandruff, diaper rash, dermatitis, acne, sun damage, aging, inflammation, and the like.","In another embodiment, oil-body-thioredoxin-related fusion proteins, e.g., oleosin-Thioredoxin-reductase, can also be used as a venom antidote.","Many animal venoms and other toxins contain disulfide bonds, including all snake venom neurotoxins, some bacterial neurotoxins including tetanus and botulinum A, bee venom phospholipase A2, and scorpion venom.","In a further embodiment, the redox protein related pharmaceutical compositions provided herein can be used to inactivate venom toxins by reduction of disulfide bonds.","A method of treating an individual suffering from the effects of a venom or toxin can include the step of administering an effective dose of a pharmaceutical composition, in a pharmaceutically effective carrier in an amount sufficient to relieve or reverse the effects of the venom toxin on the individual.","The pharmaceutical compositions provided herein are preferably formulated for single dosage administration.","The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment.","Typically, the compositions are formulated for single dosage administration.","To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.","Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.","In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.","Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.","These may be prepared according to methods known to those skilled in the art.","For example, liposome formulations may be prepared as described in U.S. Pat.","No.","4,522,811.The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.","The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.","The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.","Typically a therapeutically effective dosage is contemplated.","The amounts administered may be on the order of 0.001 to 1 mg/ml, preferably about 0.005-0.05 mg/ml, more preferably about 0.01 mg/ml, of blood volume.","Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg, more preferably about 25-75 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form.","The precise dosage can be empirically determined.","The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.","It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.","It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.","It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of the claimed compositions and combinations containing them.","Preferred pharmaceutically acceptable derivatives include acids, salts, esters, hydrates, solvates and prodrug forms.","The derivative is typically selected such that its pharmacokinetic properties are superior to the corresponding neutral compound.","Thus, effective concentrations or amounts of one or more of the compounds provided herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.","Compounds are included in an amount effective for ameliorating or treating the disorder for which treatment is contemplated.","The concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.","Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.","Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.","In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used.","Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as Tween®, or dissolution in aqueous sodium bicarbonate.","Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.","For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable carrier.","For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred.","Time release formulations are also desirable.","Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.","Upon mixing or addition of the compound with the vehicle, the resulting mixture may be a solution, suspension, emulsion or other composition.","The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.","If necessary, pharmaceutically acceptable salts or other derivatives of the compounds are prepared.","The compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.","It is understood that number and degree of side effects depends upon the condition for which the compounds are administered.","For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses that would not be tolerated when treating disorders of lesser consequence.","The compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action known to those of skill in the art.","The formulations of the compounds and agents for use herein include those suitable for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration or any route.","The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.","The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.","The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.","Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.","Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier, vehicle or diluent.","Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules.","Unit-dose forms may be administered in fractions or multiples thereof.","A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form.","Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.","Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.","The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polivinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.","Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.","If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.","Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).","The composition or formulation to be administered will contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject.","Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.","For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).","The tablets may be coated by methods well-known in the art.","The pharmaceutical preparation may also be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.","Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).","Formulations suitable for rectal administration are preferably presented as unit dose suppositories.","These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.","Formulations suitable for topical application to the skin or to the eye preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol and oil.","Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof.","The topical formulations may further advantageously contain 0.05 to 15 percent by weight of thickeners selected from among hydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly(alkylene glycols), poly/hydroxyalkyl, (meth)acrylates or poly(meth)acrylamides.","A topical formulation is often applied by instillation or as an ointment into the conjunctival sac.","It can also be used for irrigation or lubrication of the eye, facial sinuses, and external auditory meatus.","It may also be injected into the anterior eye chamber and other places.","The topical formulations in the liquid state may be also present in a hydrophilic three-dimensional polymer matrix in the form of a strip, contact lens, and the like from which the active components are released.","For administration by inhalation, the compounds for use herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.","In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.","Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.","Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.","The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.","Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.","The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.","Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use.","Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.","Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 to 0.2 M concentration with respect to the active compound.","Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.","The pharmaceutical compositions may also be administered by controlled release means and/or delivery devices (see, e.g., in U.S. Pat.","Nos.","3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).","Desirable blood levels may be maintained by a continuous infusion of the active agent as ascertained by plasma levels.","It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions.","Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).","The efficacy and/or toxicity of the pharmaceutical compositions provided herein, alone or in combination with other agents can also be assessed by the methods known in the art (See generally, O'Reilly, Investigational New Drugs, 15:5-13 (1997)).","The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.","Kits containing the compositions and/or the combinations with instructions for administration thereof are provided.","The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad.","Instructions are optionally included for administration of the active agent by a clinician or by the patient.","Finally, the pharmaceutical compositions provided herein containing any of the preceding agents may be packaged as articles of manufacture containing packaging material, a compound or suitable derivative thereof provided herein, which is effective for treatment of a diseases or disorders contemplated herein, within the packaging material, and a label that indicates that the compound or a suitable derivative thereof is for treating the diseases or disorders contemplated herein.","The label can optionally include the disorders for which the therapy is warranted.","Also provided herein are personal care formulations containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.","Personal care products comprising thioredoxin and thioredoxin-reductase are disclosed in for example Japanese Patent Applications JP9012471A2, JP103743A2, and JP1129785A2 Personal care formulations that may be prepared in accordance with the present invention include formulations capable of improving the physical appearance of skin exposed to detrimental environmental stimuli resulting in oxidative stress for example oxidative stress caused by UV-generated free-radicals.","The oil bodies comprising thioredoxin/thioredoxin-reductase may also be used to prepare hair care products as described in U.S. Pat.","Nos.","4,935,231 and 4,973,475 (incorporated herein by reference in their entirety).","The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.","Example 1 Isolation of Thioredoxin and NADPH Thioredoxin-Reductase Genes An Arabidopsis silique cDNA library CD4-12 was obtained from the Arabidopsis Biological Resource Centre (ABRC, http://aims.cps.msu.edu) Arabidopsis stock centre and used as a template for the isolation of the thioredoxin h (Trxh) and thioredoxin-reductase genes from Arabidopsis.","For the isolation of the Trxh gene the following primers were synthesized: GVR833: 5′ TACCATGGCTTCGGAAGAAGGA 3′ (SEQ ID NO:1) The sequence identical to the 5′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.","Underlined is an NcoI restriction site to facilitate cloning.","GVR834: 5′ GAAAGCTTAAGCCAAGTGTTTG 3′ (SEQ ID NO:2) The sequence complementary to the 3′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.","Underlined is an HindIII restriction site to facilitate cloning.","A Polymerase Chain Reaction (PCR) was carried out using GVR833 and GVR834 as primers and the cDNA library CD4-12 as a template.","The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.","The isolated sequence encoding Trxh was identical to the published Trxh gene sequence (Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328).","The pBluescript vector containing the Trxh gene is called pSBS2500.For the isolation of the thioredoxin-reductase gene the following primers were synthesized: GVR836: 5′ GGCCAGCACACTACCATGAATGGTCTCGAAACTCAC 3′ (SEQ ID NO:3).","The sequence identical to the 5′ end of the thioredoxin-reductase gene as published (Jacquot et al, J Mol Biol.","(1994) 235 (4):1357-63), is indicated in bold).","GVR837: 5′ TTAAGCTTCAATCACTCTTACCTTGCTG 3′.","(SEQ ID NO:4) A Polymerase Chain Reaction (PCR) was carried out using GVR836 and GVR837 as primers and the cDNA library CD4-12 as a template.","The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.","The pBluescript vector containing the thioredoxin-reductase gene is called pSBS2502.A total of three clones were sequenced, the sequence of each of the three clones were identical to each other.","However, as depicted in FIG.","1 this sequence indicated several nucleotide differences compared to the published thioredoxin-reductase gene sequence published (Jacquot et al, J Mol Biol.","(1994) 235 (4):1357-63.).","The complete coding sequence and its deduced amino acid sequence is shown in SEQ ID NO:10.As a result of the nucleotide differences between the published sequence and the sequence isolated in Example 1, several amino acid changes are also predicted.","A comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence thioredoxin-reductase (ATTHIREDB, Jacquot et al, J Mol Biol.","(1994) 235 (4):1357-63.)","with the sequence isolated in Example 1 (TR) is shown in FIG.","3.Example 2 Construction of Plant Expression Vectors Expression vectors were constructed to allow for the seed specific over-expression of thioredoxin and NADPH thioredoxin-reductase in seeds.","Vectors were constructed to allow for over-expression in its natural subcellular location and for accumulation on oil bodies.","Construction of Plant Transformation Vector pSBS2520.The Arabidopsis thioredoxin h gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324)).","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with Pst I and HindIII/KpnI sites respectively (see SEQ ID NO:14).","Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the Trxh gene.","The PstI-phaseolin promoter-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 (pSBS3000 is a derivative from the Agrobacterium binary plasmid pPZP221 (Hajdukiewicz et al., 1994, Plant Molec.","Biol.","25: 989-994).","In pSBS3000, the CaMV35S promoter-gentamycin resistance gene-CAMV 35S terminator of pPZP221 was replaced with parsley ubiquitin promoter-phosphinothricin acetyl transferase gene-parsley ubiquitin termination sequence to confer resistance to the herbicide glufosinate ammonium.)","The resulting plasmid is called pSBS2520.The sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence is shown in SEQ ID NO:14.Construction of Plant Transformation Vector pSBS2510.The 3′ coding sequence of an Arabidopsis oleosin gene (van Rooijen et al (1992) Plant Mol.","Biol.","18: 1177-1179) was altered to contain an NcoI site.","The NcoI-HindIII fragment from vector pSBS2500 (Example 1) containing the Trxh was ligated to the coding sequence of this Arabidopsis oleosin utilizing this NcoI restriction site.","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) containing a synthetic PstI site (see construction of pSBS2520) to the coding sequence of the Arabidopsis oleosin.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the Trxh gene.","The PstI-phaseolin promoter-oleosin-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2510.The sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence is shown in SEQ ID NO:16.Construction of Plant Transformation Vector pSBS2521.This vector contains the same genetic elements as the insert of pSBS2510 except the Trxh gene is fused to the 5′ end of the oleosin gene.","The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al (1992) Plant Mol.","Biol.","18: 1177-1179) was furnished with a HindIII cloning site.","Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene and to fuse the Trxh gene to the oleosin sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.","The PstI-phaseolin promoter-Trxh oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2521.The sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence is shown in SEQ ID NO:19.Construction of Plant Transformation Vector pSBS2527.The Arabidopsis NADPH thioredoxin-reductase gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324).","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with PstI and HindIII/KpnI sites respectively (see SEQ ID NO:14).","Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the thioredoxin-reductase gene.","The PstI-phaseolin promoter-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 The resulting plasmid is called pSBS2527.The sequence of the phaseolin promoter-Arabidopsis thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:22.Construction of Plant Transformation Vector pSBS2531.A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.","The same gene splicing technique was used to fuse the oleosin gene to the thioredoxin-reductase coding sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin downstream of the thioredoxin-reductase gene.","The PstI-phaseolin promoter-oleosin-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2531.The sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:24.Construction of Plant Transformation Vector pSBS2529 This vector contains the same genetic elements as the insert of pSBS2531 except the thioredoxin-reductase gene is fused to the 5′ end of the oleosin gene.","The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al.","(1992) Plant Mol.","Biol, 18: 1177-1179) was furnished with a HindIII cloning site.","Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene and to fuse the thioredoxin-reductase gene to the oleosin sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.","The PstI-phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2529.The sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence is shown in SEQ ID NO:27.Construction of Plant Transformation Vector pSBS2530.A plant transformation was constructed containing the Mycobacterium Leprae thioredoxin-reductase /thioredoxin gene (Mlep TR/Trxh).","A construct called pHIS/TR/Trxh (Wieles et al (1995) J Biol Chem 270:25604-25606) was obtained from the department of Immunohematology and Blood bank, Leiden University, The Netherlands and use as a template for PCR to generate pSBS2530.The construction of pSBS2530 was identical to the construction of pSBS2531 except that the Mlep TR/Trxh gene was used instead of the Arabidopsis thioredoxin-reductase gene.","A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.","Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.","The same gene splicing technique was used to fuse the oleosin gene to the Mlep TR/Trxh coding sequence.","Standard molecular biology laboratory techniques (see eg: Sambrook et al.","(1990) Molecular Cloning, 2nd ed.","Cold Spring Harbor Press) were again used to clone the HindIII-KpnI fragment containing the phaseolin downstream of the Mlep TR/Trxh gene.","The PstI-phaseolin promoter-oleosin-Mlep TR/Trxh -phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2530.The sequence of the phaseolin promoter-oleosin Mlep TR/Trxh -phaseolin terminator sequence is shown in SEQ ID NO:30.Construction of Plant Transformation Vector pSBS2542.From initial activity assays (FIG.","4), it was apparent that oil bodies expressing the oleosin-M. lep TR/Trxh fusion protein contained considerable reducing activity.","It was anticipated that a similar oleosin fusion construct encoding the Arabidopsis thioredoxin-reductase and thioredoxin proteins would behave in an analogous manner.","Molecular modeling was used to aid in the design of such a construct.","Primers were designed (thioredoxin link-L: 5′-ACTGGAGATGTTGACTCGACGGATACTACGGATTGGTCGACGG CTATGGAAGAAGGACAAGTGATCGCCTGC-3′; (SEQ ID NO:5), and thioredoxin link-R: 5′-ATCCGTCGAGTCAACATCTCCAGTTTCCTCGGTGGTCTCGTTAGCCTTCGAT CCAGCAATCTCTTGTAAGAATGCTCTGC-3′; (SEQ ID NO:6) to code for a synthetic linker peptide between the thioredoxin-reductase and thioredoxin proteins.","These primers were used in conjunction with primers GVR 873 (5′-GTGGAAGCT TATGGAGATGGAG-3′; SEQ ID NO:7) and GVR834 (5′-GAAAGCTTAAGCCAAGTGTTTG-3′; SEQ ID NO:2) to amplify a region coding for a thioredoxin-reductase-linker region-thioredoxin utilizing a gene splicing by overlap extension technique (Horton et al (1989) 15:61-68).","The thioredoxin-reductase-linker-thioredoxin encoding sequence was then cloned into a pre-existing pSBS3000 vector using standard molecular biology techniques (Sambrook et al (1990) Molecular Cloning 2nd Edition Cold Spring Harbour Press).","The resulting plasmid was called pSBS2542.The sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region is shown in SEQ ID NO:33.An amino acid sequence comparison between this Arabidopsis thioredoxin-reductase-linker-thioredoxin and the M. leprae TR/Trxh protein is shown in FIG.","12.Plasmids pSBS2510, pSBS2520, pSBS2521, pSBS2527, pSBS2529, pSBS2530, pSBS2531 and pSBS2542 were electroporated into Agrobacterium strain EHA101.These Agrobacterium strains were used to transform Arabidopsis.","Arabidopsis transformation was done essentially as described in “Arabidopsis Protocols; Methods in molecular biology Vol 82.Edited by Martinez-Zapater J M and Salinas J. ISBN 0-89603-391-0 pg 259-266 (1998) except the putative transgenic plants were selected on agarose plates containing 80 μM L-phosphinothricine, after they were transplanted to soil and allowed to set seed.","Example 3 Polyacrylamide Gelelectrophoresis and Immunoblotting of Transgenic Seed Extracts Source of Arabidopsis Thioredoxin, Thioredoxin-Reductase and Oleosin Antibodies.","The Arabidopsis thioredoxin and thioredoxin-reductase genes were cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression of Arabidopsis thioredoxin and thioredoxin-reductase proteins.","These proteins were purified using standard protocols (see eg Invitrogen protocol) and used to raise antibodies in rabbits using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).","The Arabidopsis oleosin gene genes was cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression Arabidopsis oleosin protein.","This protein was purified using standard protocols (see eg Invitrogen protocol) and used to prepare mouse monoclonal antibodies using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).","Preparation of total Arabidopsis seed extracts for PAGE.","Arabidopsis seeds were ground in approximately 20 volumes of 2% SDS, 50 mM Tris-Cl, this extract was boiled, spun and the supernatant was prepared for polyacrylamide gelelectrophoresis (PAGE) using standard protocols.","Preparation of Arabidopsis Oil-Body-Protein Extracts.","Arabidopsis seeds were ground in approximately 20 volumes of water and spun in a microfuge.","The oil bodies were recovered and washed sequentially with approximately 20 volumes of water, a high stringency wash buffer, containing 8M urea and 100 mM sodiumcarbonate and water.","After this last wash the oil bodies are prepared for poly acrylamide gelelectrophoresis (PAGE) using standard protocols.","Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2510 Total seed and oil body protein extracts from plants transformed with pSBS2510 were loaded onto polyacrylamide gels and either stained with coomassie brilliant blue or electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.","Expression of the oleosin-thioredoxin results in an additional band of 31.2 kDa.","The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).","This indicates that oleosin-thioredoxin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.","Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2521 Total seed and oil body protein extracts from plants transformed with pSBS25121 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.","Expression of the thioredoxin-oleosin results in an additional band of 31.2 kDa.","The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).","This indicates that thioredoxin-oleosin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.","Analysis of seed extracts from plants transformed with pSBS2520 Total seed extracts from plants transformed with pSBS2520 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin and visualized using alkaline phosphatase.","The results indicated that the thioredoxin antibodies are immunologically reactive with a band of approximately the right predicted molecular weight (12 kDa).","Untransformed seeds do not show a detectable thioredoxin band.","Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2529 Total seed and oil body protein extracts from plants transformed with pSBS2529 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.","Expression of the thioredoxin-reductase -oleosin results in an additional band of 53.8 kDa.","The results indicate that the thioredoxin-reductase antibodies are immunologically reactive with a band of the right predicted molecular weight for the fusion protein (53.8 kDa), the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight (53.8 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).","This indicates that thioredoxin-reductase-oleosin is expressed in Arabidopsis seeds.","Analysis of seed extracts from plants transformed with pSBS2527 Total seed extracts from plants transformed with pSBS2527 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.","The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase and visualized using alkaline phosphatase.","The thioredoxin-reductase antibodies are immunologically reactive with a band of approximately the right predicted molecular weight for the (35.3 kDa).","Untransformed seeds do not show a detectable thioredoxin band.","Analysis of seed extracts from plants transformed with pSBS2531 A protein gel and immunoblot was prepared assaying the expression of oleosin-DMSR in Arabidopsis T2 seeds and correct targeting to Arabidopsis oil bodies.","The expected molecular weight based on the deduced amino acid sequence is calculated to be 53,817 Da.","In the oil body extract of the transgenic oleosin-thioredoxin-reductase sample an extra band of approximately 54 kDa was observed.","This band was confirmed to be oleosin-thioredoxin-reductase by immunoblotting.","From the polyacrylamide gel it was observed that the expression of the oleosin-Thioredoxin-reductase is about double compared to the expression of the major 18.5 kDa Arabidopsis oleosin.","This represents approximately 2-4% of total seed protein.","Analysis of seed extracts from plants transformed with pSBS2530 A protein gel and immunoblot was prepared assaying the expression of oleosin-M.lep TR/Trxh in Arabidopsis T2 seeds and the correct targeting to Arabidopsis oil bodies.","The expected molecular weight based on the deduced amino acid sequence is calculated to be 67,550 Da.","In the oil body extract of the transgenic oleosin-M.lep TR/Trxh sample an extra band of approximately 68 kDa was observed.","This band was confirmed to be oleosin-M.lep TR/Trxh by immunoblotting.","From the polyacrylamide gel it was observed that the expression of the oleosin-M.lep TR/Trxh is similar to the expression of the major 18.5 kDa Arabidopsis oleosin.","This represents approximately 1-2% of total seed protein.","Analysis of seed extracts from plants transformed with pSBS2542 Crude oil body extracts from pSBS2542 lines were prepared by grinding 100 μg of seed in 1 mL of 100 mM Tris buffer at pH 7.5.The samples were then centrifuged in order to isolate the oil body fraction.","The oil body fraction was then loaded on an SDS polyacrylamide gel for expression analysis.","A Coomassie stained gel revealed that the synthetic fusion accumulated to high levels in crude oil body extracts from 3 of the 4 lines tested.","It was estimated that the fusion protein represented approximately 2-5% of total seed protein.","Furthermore, western blots utilizing either anti-thioredoxin or anti-thioredoxin-reductase antibodies confirmed that the over expressed 70 kDa protein was indeed oleosin-thioredoxin-reductase-linker-thioredoxin.","Example 4 Biological Activity of Thioredoxin and Thioredoxin-Reductase Transformants Initial Reduction Assays: DTNB Assay The activity of the thioredoxin and thioredoxin reductase was determined using a calorimetric DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] assay.","The assay was performed in a 700 μL reaction volume containing 100 mM Tris-Cl pH 8.0, 5 mM EDTA, 200 μM DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] and 200 μM NADPH.","If thioredoxin-reductase and thioredoxin are added, NADPH will reduce the thioredoxin-reductase, which will then reduce thioredoxin, which will, in turn, reduce DTNB (see equations below).","NADPH2+thioredoxin-reductaseox→thioredoxin-reductasered+NADP+thioredoxin-reductasered+thioredoxinox→thioredoxinred+thioredoxin-reductaseox thioredoxinred+DTNBox→2(2-nitro-5-mercaptobenzoic acid)+thioredoxinox The formation of the yellow product was monitored by measuring the OD412 in a spectrophotometer after a set period of time (usually 0.5-2 hours).","The results of initial activity assays are shown in the bar graph in FIG.","4 and described below.","Initially, 100 μg of total seed proteins were added from each of the Arabidopsis transgenic lines, pSBS2520 (cytosolic thioredoxin) and pSBS2527 (cytosolic thioredoxin-reductase), which corresponds to approximately 1 μg of cytosolic thioredoxin and thioredoxin-reductase used in the assay.","In this case, the amount of DTNB reduced was comparable to the reduction caused by 1 μg each of E. coli thioredoxin and thioredoxin-reductase.","In these plant seed samples, background readings were very low when only one of the 2 extracts (either cytosolic thioredoxin or cytosolic thioredoxin-reductase; FIG.","4, bars 3 and 6, respectively) was added to the reaction, along with wild type oil bodies.","Analysis with oil body fractions from transgenic seeds revealed that Arabidopsis thioredoxin and thioredoxin-reductase were substantially less active when fused to oleosins on oil bodies.","Approximately 300 μg of crude, unwashed oil-body-protein was used in the assay (which corresponds to 10-30 μg of thioredoxin-oleosin (pSBS 2521; FIG.","4, bar 2), oleosin-thioredoxin (pSBS 2510, FIG.","4, bar 1), thioredoxin-reductase-oleosin (pSBS 2529, FIG.","4, bar 5), or oleosin-thioredoxin-reductase (pSBS 2531, FIG.","4, bar 4).","The oil-body-proteins were tested in conjunction with 100 μg of total seed protein containing approximately 1 μg of cytosolic thioredoxin (pSBS 2520) or thioredoxin-reductase (pSBS 2527).","In such assays, pSBS2529 (thioredoxin-reductase-oleosin) and pSBS2531 (oleosin-thioredoxin-reductase) do contain reductase activity when combined with cytosolic thioredoxin from pSBS2520 (see FIG.","4, bars 7 and 8, respectively).","Experiments estimated that the reductase activity of oleosin-thioredoxin-reductase was about 10-15% that of the cytosolic thioredoxin-reductase.","The addition of tween at a final concentration of 0.4% could enhance this activity 2 or 3 fold.","Interestingly, oleosin-thioredoxin-reductase (pSBS 2531) appears to be capable of reducing DTNB in the absence of added thioredoxin, although added thioredoxin causes significantly more DTNB reduction (see FIG.","4; compare bar 4 W.T.+oleosin-thioredoxin-reductase to bar 7 thioredoxin+oleosin-thioredoxin-reductase).","Experiments with pSBS2521 (thioredoxin-oleosin) or pSBS2510 (oleosin-thioredoxin) combined with cytosolic thioredoxin-reductase from pSBS2527 (see FIG.","4, bars 10 and 11, respectively) indicate that thioredoxin activity of these fusions is undetectable at these concentrations.","Oil bodies from the transgenic Arabidopsis line, pSBS2530 (oleosin-M.lep TR/Trxh) contain significant thioredoxin/thioredoxin-reductase activity (see FIG.","4, bar 12).","One hundred micrograms of crude oil body protein for pSBS2530 was tested (corresponding to approximately 5 μg of oleosin-M.lep TR/trxh fusion) in the assay.","Based on the assay, it was estimated that this fusion is about 25-40% as active as cytosolic Arabidopsis thioredoxin and thioredoxin-reductase (FIG.","4, bar 9) when comparing specific activity.","Insulin Reduction Assay The results from the DTNB assays were confirmed with insulin reduction assays.","This assay contained insulin at a final concentration of 1 mg/mL in 100 mM KH2PO4 pH 7.0+5 mM EDTA.","In the presence of NADPH (500 μM), thioredoxin, and thioredoxin-reductase, insulin is reduced and precipitates from the solution.","Normally, insulin reduction is followed by measuring turbidity at OD 650.Alternatively, one can measure the conversion of NADPH2 to NADP+by monitoring the decrease in absorbance at 340 nm.","Both of the assays are difficult to measure when oil bodies are present, due to interference with the spectrophotometer readings.","However, qualitative data could be obtained by centrifuging the tubes after a set period of time, and determining if an insulin pellet was present (oil bodies float to the top, while the insulin precipitate pellets out).","Alternatively, samples could be filtered after a set period of time, and the change in absorbance at 340 nm could be measured.","As mentioned previously, the results of the insulin reduction assays agreed with those of the DTNB assay, with the exception of the observation that pSBS2531 (oleosin-thioredoxin-reductase) only reduced insulin in the presence of free thioredoxin from pSBS2520.Assays on Seeds from Arabidopsis Crosses that Co-Express Oleosin-Thioredoxin and Oleosin-Thioredoxin-Reductase.","Based upon initial DTNB and insulin reduction assays, it was apparent that mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase transgenic seeds resulted in very limited reducing activity (Note: the <-> indicates both configurations of oleosin fusions; ie.","oleosin<->thioredoxin would represent oleosin-thioredoxin and thioredoxin-oleosin fusions).","To determine whether having oleosin<->thioredoxin and oleosin<->thioredoxin-reductase proteins present on the same oil body would have a positive effect on the reducing activity of these proteins, crosses were set up to generate double transgenic Arabidopsis lines.","The crosses are illustrated in Table 2.TABLE 2 Confirmed double transgenic lines (PCR and Western Male Female Blot) oleo-thioredoxin X oleo-thioredoxin-reductase 4 oleo-thioredoxin X thioredoxin-reductase-oleo 1 thioredoxin-oleo X oleo-thioredoxin-reductase 0 thioredoxin-oleo X thioredoxin-reductase-oleo 4 oleo-thioredoxin- X oleo-thioredoxin 2 reductase oleo-thioredoxin- X thioredoxin-oleo 0 reductase thioredoxin- X oleo-thioredoxin 7 reductase-oleo thioredoxin- X thioredoxin-oleo 0 reductase-oleo Seeds from Arabidopsis crosses were germinated on PPT plates and the seedlings were transferred to soil after approximately 2 weeks.","PCR experiments on DNA isolated from the seedlings identified a number of plants which contain both an oleosin<->thioredoxin and an oleosin<->thioredoxin-reductase gene construct within their genome.","Seeds were harvested from these plants for expression and activity assays.","Western blots were carried out to confirm expression of both oleosin<->thioredoxin and oleosin<->thioredoxin-reductase in the lines.","DTNB and insulin reduction assays were also performed to compare activity between single transgenic parent lines and the double transgenic offspring and results are summarized in Table 3.Table 3 summarizes DTNB reducing activity of various transgenic lines.","The last 2 rows compare mixing oil bodies from single transgenic parent lines to using oil bodies from double transgenic offspring.","Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.","TABLE 3 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + 100 thioredoxin-reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Based on DTNB and insulin reduction assays, it is evident that double transgenic plants co-expressing oleosin<->thioredoxin and oleosin<->thioredoxin-reductase on the same, single oil body contained significantly more reducing activity compared to mixing oil bodies from single transgenic oleosin<->thioredoxin and oleosin<->thioredoxin-reductase lines.","It was additionally apparent that oil body extracts from co-expressing lines contained more reducing activity compared to line pSBS2530 (oleosin-M. lep TR/Trxh), which was previously identified as the line containing the highest reducing activity from oil bodies.","These results suggest that the creation of double transgenic lines (either through crossing or by co-transforming 2 expression constructs into plants) may represent one means by which we could solve our initial problem of not being able to generate reducing activity by mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase single transgenic lines.","Assays on Seeds from Arabidopsis pSBS2542 Transgenic Lines that Express Oleosin-Thioredoxin-Reductase-Linker-Thioredoxin.","Oil body extracts from four pSBS2542 lines were tested for reducing activity in DTNB and insulin reduction assays, using standard protocols described previously.","Again, oil body extracts containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein possessed significant reducing activity.","Based on such assays, it was revealed that the oleosin-thioredoxin-reductase-linker-thioredoxin synthetic fusion protein was more active than the oleosin-M. lep TR/Trxh fusion.","Furthermore, oil bodies containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein appeared to have more reducing activity compared to oil bodies from double transgenic lines that co-expressed oleosin<->thioredoxin and oleosin<->thioredoxin-reductase.","The results comparing reducing activity for the various thioredoxin-reductase/thioredoxin constructs is summarized in Table 4.Table 4 summarizes DTNB reducing activity of various transgenic lines.","The pSBS2542 line expressing oleosin-thioredoxin-reductase-linker-thioredoxin contains significant reducing activity, comparable to the “free” forms of Arabidopsis thioredoxin and thioredoxin-reductase and the equivalent E. coli proteins.","Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.","TABLE 4 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + thioredoxin- 100 reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Oleosin-thioredoxin-reductase-linker-thioredoxin ~75-100 (pSBS2542) Reduction Assays Comparing the Utilization of NADH Vs. NADPH as a Cofactor (Electron Donor) for the Thioredoxin-Reductase/Thioredoxin System.","DTNB and insulin reduction assays were conducted as described previously, except that NADH was substituted for NADPH as an electron donor in the system utilizing E. coli thioredoxin-reductase and thioredoxin.","Thus, a comparison was conducted of the utilization of NADH versus NADPH as a cofactor for the E. coli thioredoxin-reductase/thioredoxin system.","For the DTNB assay, the reaction mixture consisted of 400 μM DTNB, 10 μg/mL E. coli thioredoxin, and 10 μg/mL E. coli thioredoxin-reductase in 100 mM Tris-Cl buffer pH 8.0.Either NADH or NADPH was then added to the DTNB reaction as follows: Reaction A.","200 μM NADPH (Sigma) Reaction B.","800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 800 μM NADH (no TR or Trxh).","For the insulin reduction assay, the reaction mixture consisted of 1 mg/mL bovine pancreatic insulin, 20 μg/mL E. coli thioredoxin, and 20 μg/mL E. coli thioredoxin-reductase in 100 mM potassium phosphate buffer at pH 7.0.Either NADH or NADPH was then added to the reaction as follows: Reaction A.","800 μM NADPH (Sigma) Reaction B.","800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 2 mM NADH (no TR or Trxh).","The results indicate that NADH, purchased from either Sigma or Roche, could act as an electron donor in both the DTNB and insulin reduction assays.","However, the rate of reduction was lower than the rate observed with NADPH as a cofactor.","It was estimated that the rate of insulin reduction utilizing NADH as an electron donor was approximately 25-50% when compared to the maximum rate using NADPH.","Furthermore, it was estimated that the rate of DTNB reduction utilizing NADH as an electron donor was approximately 5-10% of the maximum rate using NADPH.","Similar results were observed using the oleosin-thioredoxin-reductase-linker thioredoxin fusion protein on Arabidopsis oil bodies instead of the E. coli thioredoxin-reductase and thioredoxin.","Example 5 Production of Multimeric Immunoglobulin Protein in Plant Seed Cells and Capture on Oil Bodies Using Protein A-Oleosin Fusion Proteins 1—Production of Multimeric Immunoglobulin Protein in Plant Seed Cells For expression of multimeric-protein-complexes containing multimeric-immunoglobulin-complexes, the cDNA sequences encoding individual light and heavy chains can be isolated from; 1) cell lines expressing a particular antibody, such as clonal B cell lines, or a hybridoma cell line, or 2) may be a recombinant antibody, assembled by combining select light and heavy chain variable domains and available light and heavy chain constant domain sequences, respectively.","Variable domains with specific binding properties may be isolated from screening populations of such sequences, usually in the form of a single-chain Fv phage display library.","Starting from known nucleic acid sequences and a source of light and heavy chains, the mature polypeptide coding sequences of each chain is isolated with a secretion signal sequence.","The signal sequence can be the native antibody sequence or derived from a known secreted plant sequence (e.g.","a PR sequence from Arabidopsis or tobacco).","The addition of a plant secretion signal sequence to both light and heavy chain mature coding sequences is carried out by standard molecular biology techniques.","PCR fusion is used routinely to make such modifications.","Secretion signal sequences are included to target the light and heavy immunoglobulin polypeptides for secretion from the cell and further assembly of the two chains into a multimeric-immunoglobulin-complex.","For expression in transgenic plant seeds, an expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—light chain sequence, and 3) a regulatory sequence to terminate transcription.","A second expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—heavy chain sequence, and 3) a regulatory sequence to terminate transcription.","Each of the antibody chain expression cassettes is cloned individually into an Agrobacterium plant transformation vector or is combined into a single transformation vector with both expression cassettes.","In both cases, the expression cassettes are cloned into plant transformation vectors, between the left and right delineating border sequences, and adjacent to a plant selectable marker cassette.","Each plant transformation vector is transformed into Agrobacterium.","The resulting Agrobacterium strains are used to infect plant tissues.","Transgenic plant material is regenerated and viable transgenic plants are selected.","When individual transformation vectors are used, the transgenic plant lines that are produced, expressing either light or heavy chain sequences, are crossed to generate a single plant line expressing both chains in the same plant cell.","When a single transformation vector, containing both light and heavy expression cassettes, is used, the initial transgenic plant line produces both light and heavy chain sequences in the same plant cell.","2—Production of Transgenic Oil Bodies which Display Protein a for the Capture of Immunoglobulins To capture and display immunoglobulin protein on oil bodies, oil bodies are engineered to display an immunoglobulin binding protein.","In this example, the well-known antibody-binding domains from Protein A are used.","Based on the known sequence for Protein A from Staphylococcus aureus, PCR primers are designed to isolate the five consecutive Ig-binding domains from the bacterial Protein A sequence.","Primers are designed to allow cloning of the Protein A sequence as either an N-terminal or C-terminal fusion to an oleosin sequence for targeting to oil bodies.","The sequence that encodes an in-frame translational fusion between Protein A and oleosin is cloned into a plant expression cassette for seed-specific expression.","The final cassette consists of a regulatory promoter sequence that provides expression in seeds, the Protein A-oleosin fusion sequence, and a regulatory sequence to terminate transcription.","The Protein A-oleosin expression cassette is cloned into a plant transformation vector compatible with Agrobacterium-mediated plant transformation.","The transformation vector comprises left and right border sequences flanking the Protein A-oleosin expression cassette and an adjacent plant selectable marker cassette.","The Agrobacterium strain containing this vector is used to infect plant tissues and subsequent regeneration and selection from transgenic plant material to create transgenic plants.","3—Capture and Display of Multimeric-Immunoglobulins on Oil Bodies Displaying Protein A Having produced light and heavy chain multimeric immunoglobulin complexes in one transgenic plant line and the display of Protein A on oil bodies through the oil body targeting of a Protein A-oleosin fusion protein in a second plant line, at least two embodiments can be used to capture the immunoglobulin complex on the Protein A oil bodies.","In the first embodiment, transgenic seed from both the immunoglobulin and the Protein A-oleosin expression lines is combined in an optimum ratio and then ground together such that the disrupted material from both seed lines would be combined in the same extract.","The combined seed extracts are mixed and/or incubated under conditions that allow maximum recovery of the immunoglobulin by Protein A.","The oil body fraction is separated using standard phase separation techniques (e.g.","centrifugation).","The recovered oil body fraction contains both native oil bodies, from the immunoglobulin expression line, and transgenic Protein A oil bodies from the Protein A-oleosin expression line.","In a second embodiment, the plant lines expressing the immunoglobulin complex and the Protein A-oleosin fusion are crossed and individual plant lines expressing both components are identified and propagated.","In this approach, the immunoglobulin complex and the Protein A-oleosin fusion are produced in different cellular compartments of the same plant seed cell.","Seed from the double transgenic line is ground to disrupt the cellular material and mix the contents of all cellular compartments, including combining the immunoglobulin in the extracellular compartment and the Protein A-oleosin on the oil body in the cytosolic compartment.","The material is mixed and/or incubated under conditions to allow maximum recovery of the immunoglobulin by Protein A, and the oil body fraction is separated by phase separation techniques.","The recovered oil body fraction contains the displayed Protein A and the capture immunoglobulin complex.","Example 6 Production of Assembled Multimeric-Immunoglobulin-Complexes as Fusions with Oil Body Targeting Domains Individual polypeptides are produced as a fusion protein with oil body targeting sequences (e.g.","oleosin) for display on oil bodies.","It has been found that the individual subunits of naturally associating heterodimeric proteins can be co-produced as individual oleosin fusions and still associate as an active heterodimer on the surface of the oil body.","In this example, the heterodimer is the light and heavy chain subunits, or derived portions thereof, of an immunoglobulin complex.","Production of an Immunoglobulin Fab Complex on Oil Bodies.","The mature light chain sequence, lacking the secretion signal sequence, is attached as an in-frame N-terminal fusion to an oleosin sequence.","This fusion sequence is assembled into a seed-specific expression cassette consisting of a seed-specific promoter sequence, the light chain-oleosin fusion sequence, and a transcriptional terminator sequence.","The expression cassette is inserted between the left and right border markers, adjacent to a plant selectable marker cassette, of a transformation vector.","The transformation vector, in Agrobacterium, is used to infect plants and generate transgenic plants.","An equivalent construct for the heavy chain subunit, comprising the variable and constant heavy chain domains, is also attached as an in-frame fusion to oleosin and assembled into an expression cassette for seed-specific expression.","The expression cassette can be a part of a separate transformation vector for the generation of a separate transgenic line, or the heavy chain expression cassette can be combined together with the light chain cassette into a single transformation vector.","If light and heavy chain expression cassettes are transformed into plants on separate transformation vectors, the individual plant lines are crossed to create a single line expressing both heterodimer subunit-oleosin fusions in the same plant cell.","Seed from the double transgenic line, or a single transgenic line generated from the dual expression vector, is extracted to isolate oil bodies.","The seed material is ground to release the cellular contents and oil bodies are isolated by phase separation.","The targeting of both light and heavy chain sequence to oil bodies, as oleosin fusions, allows the association of the immunoglobulin complex on the surface of the oil body.","Similar configurations, using the entire heavy chain sequence in combination with the entire light chain sequence, or using the variable domains from both the light and heavy chain sequences, are constructed to assemble different types of heteromultimeric-immunoglobulin-complexes (e.g., heterodimers) on the surface of oil bodies.","The present invention should therefore not be seen as limited to the particular embodiments described herein, but rather, it should be understood that the present invention has wide applicability with respect to protein expression generally.","Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.","SUMMARY OF SEQUENCES SEQ ID NOs:1-4 set forth primers which were synthesized for the isolation of the thioredoxin h (Trxh) and thioredoxin reductase genes from Arabidopsis, as described in Example 1.SEQ ID NOs:5-7 set forth primers which were designed to code for a specific linker peptide between thioredoxin reductase and thioredoxin proteins, as described in Example 2.SEQ ID NOs:8, 10 and 11 set forth the nucleotide sequence and the deduced amino acid sequence of the NADPH thioredoxin reductase sequence isolated herein as described in Example 1.SEQ ID NOs:9 and 11, respectively, set forth the nucleotide sequence of the published NADPH thioredoxin reductase sequence (ATTHIREDB) and the deduced amino acid sequence.","SEQ ID NO:12 sets forth the deduced amino acid sequence of the published NADPH thioredoxin reductase sequence.","SEQ ID NO:13 sets forth the deduced amino acid sequence of the NADPH reductase sequence isolated in this report.","SEQ ID NOs:14 and 15 set forth the nucleotide sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequence.","The Trxh coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554, and the phaseolin terminator corresponds to nucleotide sequence 1905-3124.The promoter was furnished with a PstI site (nt 1-6) and the terminator was furnished with a HindIII site (nt 1898-1903) and a KpnI site (nt 3124-3129) to facilitate cloning.","SEQ ID NOs:16, 17 and 18 set forth the nucleotide sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequences.","The oleosin-Trxh coding sequence and the deduced amino acid sequences are indicated in SEQ ID NO:16.As in SEQ ID NO:14, the phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1908-2147) is indicated in italics.","The Trxh coding sequence corresponds to nt 2314-2658.The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:19, 20 and 21 set forth the nucleotide sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The Trxh oleosin-coding sequence and its deduced amino acid sequences are indicated in SEQ ID NO:19.As in SEQ ID NOs:14 and 16, the phaseolin promoter corresponds to nucleotide 6-1554.The Trxh coding sequence corresponds to nt 1555-1896.The sequence encoding oleosin corresponds to nt 1897-2658, the intron in this sequence (nt 2250-2489) is indicated in italics.","The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:22 and 23 set forth the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequence.","The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated in SEQ ID NO:22.The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2556 and the deduced amino acid is set forth in SEQ ID NO:23.The phaseolin terminator corresponds to nucleotide sequence 2563-3782.SEQ ID NOs:24, 25 and 26 show the nucleotide sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The oleosin-thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1980-2147) is indicated in italics.","The thioredoxin-reductase coding sequence corresponds to nt 2314-3315.The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NOs:27, 28 and 29 show the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2553.The sequence encoding oleosin corresponds to nt 2554-3315, the intron in this sequence (nt 2751-3146) is indicated in italics.","The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NO:30, 31 and 32 show the sequence of the phaseolin promoter-oleosin-Mlep thioredoxin-reductase/thioredoxin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.","The oleosin-Mlep thioredoxin-reductase/thioredoxin coding sequence and its deduced amino acid sequence is indicated.","The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt) is indicated in italics.","The Mlep thioredoxin-reductase/thioredoxin coding sequence corresponds to nt 2314-3690.The phaseolin terminator corresponds to nucleotide sequence 3698-4917.SEQ ID NOs:33, 34 and 35 set forth the nucleotide sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region of pSBS2542, and the deduced amino acid sequences.","The deduced amino acid sequence of oleosin-thioredoxin-reductase-linker-thioredoxin is also shown in SEQ ID NO:33.Amino acids representing oleosin are set forth at positions 1-173, those amino acids representing thioredoxin-reductase are set forth at positions 174-501, those amino acids representing the linker or spacer peptide are set forth at positions 501-524, and those representing thioredoxin are set forth at positions 525-636.SEQ ID NOs:38 and 39 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin h (Trx h 1) and the encoded protein, respectively.","SEQ ID NOs:40 and 41 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin Reductase (NTR1) and the encoded protein, respectively.","SEQ ID NOs:42 and 43 set forth the nucleotide sequence of E. Coli Thioredoxin (TrxA) and the encoded protein, respectively.","SEQ ID NOs:44 and 45, set forth the nucleotide sequence of E. Coli Thioredoxin Reductase and the encoded protein, respectively.","SEQ ID NOs:46 and 47 set forth the nucleotide sequence of Human Thioredoxin and the encoded protein, respectively.","SEQ ID NOs:48 and 49, set forth the nucleotide sequence of Human Thioredoxin Reductase and the encoded protein, respectively.","SEQ ID NOs:50 and 51, respectively, set forth the nucleotide sequence of Mycobacterium leprae Thioredoxin-Thioredoxin Reductase and the encoded protein, respectively.","SEQ ID NOs:52-313 are described in Table 5.TABLE 5 EXAMPLES OF REDOX PROTEINS SWISS PROTEIN IDENTIFIER SEQ.","ID NO.","(in parenthesis) PLANT THIOREDOXINS Thioredoxin f-type 52 (Q9XFH8) Thioredoxin F-type 1, chloroplast precursor (TRX- F1).","- Arabidopsis thaliana (Mouse-ear cress) 53 (Q9XFH9) Thioredoxin F-type 2, chloroplast precursor (TRX- F2).","{GENE: AT5G16400 OR MQK4.13} - Arabidopsis thaliana (Mouse-ear cress) 54 (O48897) Thioredoxin F-type, chloroplast precursor (TRX-F).","{GENE: TRXF} - Brassica napus (Rape) 55 (O81332) Thioredoxin F-type, chloroplast precursor (TRX-F).","- Mesembryanthemum crystallinum (Common ice plant) 56 (P29450) Thioredoxin F-type, chloroplast precursor (TRX-F).","- Pisum sativum (Garden pea) 57 (P09856) Thioredoxin F-type, chloroplast precursor (TRX-F).","- Spinacia oleracea (Spinach) Thioredoxin m-type 58 (P06544) Thioredoxin 1 (TRX-1) (Thioredoxin M).","{GENE: TRXA} - Anabaena sp.","(strain PCC 7119) 59 (O48737) Thioredoxin M-type 1, chloroplast precursor (TRX- M1).","{GENE: AT1G03680 OR F21B7_7 OR F21B7.28} - Arabidopsis thaliana (Mouse-ear cress) 60 (Q9SEU8) Thioredoxin M-type 2, chloroplast precursor (TRX- M2).","{GENE: AT4G03520 OR F9H3.15 OR T5L23.1} - Arabidopsis thaliana (Mouse-ear cress) 61 (Q9SEU7) Thioredoxin M-type 3, chloroplast precursor (TRX- M3).","{GENE: AT2G15570 OR F9O13.12} - Arabidopsis thaliana (Mouse-ear cress) 62 (Q9SEU6) Thioredoxin M-type 4, chloroplast precursor (TRX- M4).","- Arabidopsis thaliana (Mouse-ear cress) 63 (Q9XGS0) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Brassica napus (Rape) 64 (P23400) Thioredoxin M-type, chloroplast precursor (TRX-M) (Thioredoxin CH2).","{GENE: TRXM} - Chlamydomonas reinhardtii 65 (Q41864) Thioredoxin M-type, chloroplast precursor (TRX-M).","{GENE: TRM1} - Zea mays (Maize) 66 (Q9ZP20) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Oryza sativa (Rice) 67 (P48384) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Pisum sativum (Garden pea) 68 (P07591) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Spinacia oleracea (Spinach) 69 (Q9ZP21) Thioredoxin M-type, chloroplast precursor (TRX-M).","- Triticum aestivum (Wheat) 70 (P12243) Thioredoxin 1 (TRX-1) (Thioredoxin M).","{GENE: TRXA OR TRXM} - Synechococcus sp.","(strain PCC 7942) (Anacystis nidulans R2) 71 (P37395) Thioredoxin.","{GENE: TRXA OR TRX} - Cyanidium caldarium [Chloroplast] 72 (O22022) Thioredoxin.","{GENE: TRXA OR TRXM} - Cyanidioschyzon merolae [Chloroplast] 73 (P50338) Thioredoxin.","{GENE: TRXA} - Griffithsia pacifica [Chloroplast] 74 (P50254) Thioredoxin.","{GENE: TRXA} - Porphyra yezoensis [Chloroplast] 75 (P51225) Thioredoxin.","{GENE: TRXA} - Porphyra purpurea [Chloroplast] Thioredoxin h-type 76 (P29448) Thioredoxin H-type 1 (TRX-H-1).","{GENE: TRX1 OR AT3G51030 OR F24M12.70} - Arabidopsis thaliana (Mouse- ear cress) 77 (P20857) Thioredoxin 2 (TRX-2).","{GENE: TRXB} - Anabaena sp.","(strain PCC 7120) 78 (Q42388) Thioredoxin H-type 1 (TRX-H-1) (Pollen coat protein).","{GENE: THL-1 OR BOPC17} - Brassica napus (Rape), Brassica oleracea (Cauliflower) 79 (P29449) Thioredoxin H-type 1 (TRX-H1).","- Nicotiana tabacum (Common tobacco) 80 (Q38879) Thioredoxin H-type 2 (TRX-H-2).","{GENE: TRX2 OR AT5G39950 OR MYH19.14} - Arabidopsis thaliana (Mouse-ear cress) 81 (Q39362) Thioredoxin H-type 2 (TRX-H-2).","{GENE: THL-2} - Brassica napus (Rape) 82 (Q07090) Thioredoxin H-type 2 (TRX-H2).","- Nicotiana tabacum (Common tobacco) 83 (Q42403) Thioredoxin H-type 3 (TRX-H-3).","{GENE: TRX3 OR AT5G42980 OR MBD2.18} - Arabidopsis thaliana (Mouse-ear cress) 84 (Q39239) Thioredoxin H-type 4 (TRX-H-4).","{GENE: TRX4} - Arabidopsis thaliana (Mouse-ear cress) 85 (Q39241) Thioredoxin H-type 5 (TRX-H-5).","{GENE: TRX5} - Arabidopsis thaliana (Mouse-ear cress) 86 (O64432) Thioredoxin H-type (TRX-H).","{GENE: PEC-2} - Brassica rapa (Turnip) 87 (P80028) Thioredoxin H-type (TRX-H) (Thioredoxin CH1).","{GENE: TRXH} - Chlamydomonas reinhardtii 88 (Q96419) Thioredoxin H-type (TRX-H).","- Fagopyrum esculentum (Common buckwheat) 89 (Q42443) Thioredoxin H-type (TRX-H) (Phloem sap 13 kDa protein-1).","- Oryza sativa (Rice) 90 (O65049) Thioredoxin H-type (TRX-H).","{GENE: SB09} - Picea mariana (Black spruce) 91 (Q43636) Thioredoxin H-type (TRX-H).","- Ricinus communis (Castor bean) 92 (O64394) Thioredoxin H-type (TRX-H) (TrxTa).","- Triticum aestivum (Wheat) 93 (P29429) Thioredoxin.","- Emericella nidulans (Aspergillus nidulans) VIRUSES, BACTERIA AND FUNGI THIOREDOXINS 94 (P80579) Thioredoxin (TRX).","{GENE: TRXA} - Alicyclobacillus acidocaldarius (Bacillus acidocaldarius) 95 (O28137) Thioredoxin.","{GENE: AF2145} - Archaeoglobus fulgidus 96 (P14949) Thioredoxin (TRX).","{GENE: TRXA OR TRX} - Bacillus subtilis 97 (P00276) Thioredoxin.","{GENE: NRDC} - Bacteriophage T4 98 (O51088) Thioredoxin (TRX).","{GENE: TRXA OR BB0061} - Borrelia burgdorferi (Lyme disease spirochete) 99 (P57653) Thioredoxin (TRX).","{GENE: TRXA OR BU597} - Buchnera aphidicola (subsp.","Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 100 (O51890) Thioredoxin (TRX).","{GENE: TRXA} - Buchnera aphidicola (subsp.","Schizaphis graminum) 101 (P10472) Thioredoxin (TRX).","{GENE: TRXA} - Chlorobium limicola f.sp.","thiosulfatophilum 102 (Q9PJK3) Thioredoxin (TRX).","{GENE: TRXA OR TC0826} - Chlamydia muridarum 103 (Q9Z7P5) Thioredoxin (TRX).","{GENE: TRXA OR CPN0659 OR CP0088} - Chlamydia pneumoniae (Chlamydophila pneumoniae) 104 (P52227) Thioredoxin (TRX).","{GENE: TRXA} - Chlamydia psittaci (Chlamydophila psittaci) 105 (O84544) Thioredoxin (TRX).","{GENE: TRXA OR CT539} - Chlamydia trachomatis 106 (P00275) Thioredoxin C-1.- Corynebacterium nephridii 107 (P07887) Thioredoxin C-2.- Corynebacterium nephridii 108 (P52228) Thioredoxin C-3.- Corynebacterium nephridii 109 (P09857) Thioredoxin (TRX).","{GENE: TRXA} - Chromatium vinosum 110 (P21609) Thioredoxin (TRX).","{GENE: TRXA} - Clostridium litorale (Bacterium W6) 111 (P81108) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Clostridium sporogenes 112 (P81109) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Clostridium sticklandii 113 (Q9UW02) Thioredoxin (Allergen Cop c 2).","- Coprinus comatus (Shaggy mane) 114 (P29445) Thioredoxin 1.","{GENE: TRXA OR TRX1} - Dictyostelium discoideum (Slime mold) 115 (P29446) Thioredoxin 2 (Fragment).","{GENE: TRXB OR TRX2} - Dictyostelium discoideum (Slime mold) 116 (P29447) Thioredoxin 3.","{GENE: TRXC OR TRX3} - Dictyostelium discoideum (Slime mold) 117 (P00274) Thioredoxin 1 (TRX1) (TRX).","{GENE: TRXA OR TSNC OR FIPA OR B3781} - Escherichia coli, Salmonella typhimurium 118 (P52232) Thioredoxin-like protein SLR0233.","{GENE: SLR0233} - Synechocystis sp.","(strain PCC 6803) 119 (P33636) Thioredoxin 2 (Trx2).","{GENE: TRXC OR B2582 OR Z3867 OR ECS3448} - Escherichia coli, Escherichia coli O157:H7 120 (P21610) Thioredoxin (TRX).","{GENE: TRXA} - Eubacterium acidaminophilum 121 (P43785) Thioredoxin (TRX).","{GENE: TRXA OR TRXM OR HI0084} - Haemophilus influenzae 122 (P43787) Thioredoxin-like protein HI1115.","{GENE: HI1115} - Haemophilus influenzae 123 (P56430) Thioredoxin (TRX).","{GENE: TRXA OR HP0824 OR JHP0763} - Helicobacter pylori (Campylobacter pylori), Helicobacter pylori J99 (Campylobacter pylori J99) 124 (Q9S386) Thioredoxin (EC 1.6.4.5) {GENE: TRXA} - Listeria monocytogenes 125 (Q57755) Thioredoxin.","{GENE: TRX OR MJ0307} - Methanococcus jannaschii 126 (P47370) Thioredoxin (TRX).","{GENE: TRXA OR TRX OR MG124} - Mycoplasma genitalium 127 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].","{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 128 (P75512) Thioredoxin (TRX).","{GENE: TRXA OR TRX OR MPN263 OR MP570} - Mycoplasma pneumoniae 129 (O30974) Thioredoxin (TRX).","{GENE: TRXA} - Mycobacterium smegmatis 130 (P52229) Thioredoxin (TRX) (MPT46).","{GENE: TRXA OR TRX OR TRXC OR RV3914 OR MT4033 OR MTV028.05} - Mycobacterium tuberculosis 131 (P42115) Thioredoxin.","{GENE: TRX} - Neurospora crassa 132 (P34723) Thioredoxin.","{GENE: TRXA} - Penicillium chrysogenum 133 (Q9X2T1) Thioredoxin (TRX).","{GENE: TRXA OR TRX OR PA5240} - Pseudomonas aeruginosa 134 (P10473) Thioredoxin (TRX).","{GENE: TRXA} - Rhodospirillum rubrum 135 (P08058) Thioredoxin (TRX).","{GENE: TRXA} - Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) 136 (Q9ZEE0) Thioredoxin (TRX).","{GENE: TRXA OR RP002} - Rickettsia prowazekii 137 (P33791) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Streptomyces aureofaciens 138 (P52230) Thioredoxin (TRX).","{GENE: TRXA OR SCH24.11C} - Streptomyces coelicolor 139 (Q05739) Thioredoxin (TRX).","{GENE: TRXA} - Streptomyces clavuligerus 140 (P52231) Thioredoxin (TRX).","{GENE: TRXA OR SLR0623} - Synechocystis sp.","(strain PCC 6803) 141 (P73263) Thioredoxin-like protein SLR1139.","{GENE: SLR1139} - Synechocystis sp.","(strain PCC 6803) 142 (P52233) Thioredoxin (TRX).","{GENE: TRXA} - Thiobacillus ferrooxidans 143 (P96132) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Thiocapsa roseopersicina 144 (P81110) Thioredoxin (TRX) (Fragment).","{GENE: TRXA} - Tissierella creatinophila 145 (O83889) Thioredoxin (TRX).","{GENE: TRXA OR TP0919} - Treponema pallidum ANIMAL THIOREDOXIN 146 (O97680) Thioredoxin.","{GENE: TXN} - Bos taurus (Bovine) 147 (Q95108) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2} - Bos taurus (Bovine) 148 (Q09433) Thioredoxin.","{GENE: B0228.5} - Caenorhabditis elegans 149 (P99505) Thioredoxin (Fragment).","{GENE: TXN} - Canis familiaris (Dog 150 (P08629) Thioredoxin.","{GENE: TXN} - Gallus gallus (Chicken) 151 (P47938) Thioredoxin (Deadhead protein).","{GENE: DHD OR CG4193} - Drosophila melanogaster (Fruit fly) 152 (P10599) Thioredoxin (ATL-derived factor) (ADF) (Surface associated sulphydryl protein) (SASP).","{GENE: TXN OR TRDX OR TRX} - Homo sapiens (Human) 153 (Q99757) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2} - Homo sapiens (Human) 154 (P29451) Thioredoxin.","{GENE: TXN} - Macaca mulatta (Rhesus macaque) 155 (P10639) Thioredoxin (ATL-derived factor) (ADF).","{GENE: TXN} - Mus musculus (Mouse) 156 (P97493) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2} - Mus musculus (Mouse) 157 (P82460) Thioredoxin (Fragment).","{GENE: TXN} - Sus scrofa (Pig) 158 (P08628) Thioredoxin.","{GENE: TXN} - Oryctolagus cuniculus (Rabbit) 159 (P11232) Thioredoxin.","{GENE: TXN} - Rattus norvegicus (Rat) 160 (P97615) Thioredoxin, mitochondrial precursor (MT-TRX).","{GENE: TXN2 OR TRX2} - Rattus norvegicus (Rat) 161 (P50413) Thioredoxin.","{GENE: TXN} - Ovis aries (Sheep) PLANTS THIOREDOXIN-LIKE PROTEINS 162 (O23166) THIOL-DISULFIDE INTERCHANGE LIKE PROTEIN (THIOREDOXIN-LIKE PROTEIN) {GENE: C7A10.160 OR AT4G37200 OR HCF164} - Arabidopsis thaliana (Mouse-ear cress) 163 (Q9C9Y6) Thioredoxin-like protein {GENE: F17O14.18} - Arabidopsis thaliana (Mouse-ear cress) 164 (Q9FYD5) Thioredoxin-like protein {GENE: F21E1_180} - Arabidopsis thaliana (Mouse-ear cress) 165 (Q38878) THIOREDOXIN-LIKE PROTEIN {GENE: TRX6 OR T7D17.3} - Arabidopsis thaliana (Mouse-ear cress) 166 (Q9LVI2) Thioredoxin-like protein - Arabidopsis thaliana (Mouse-ear cress) 167 (Q9SCN9) Thioredoxin-like protein {GENE: T4D2.150} - Arabidopsis thaliana (Mouse-ear cress) 168 (Q9SRD7) Thioredoxin-like protein, 49720-48645 {GENE: F28O16.13} - Arabidopsis thaliana (Mouse-ear cress) 169 (Q9SU84) THIOREDOXIN-LIKE PROTEIN {GENE: T16L4.180 OR AT4G29670} - Arabidopsis thaliana (Mouse-ear cress) 170 (Q9SWG6) Thioredoxin-like protein {GENE: TRX} - Hordeum bulbosum 171 (Q9SWG4) Thioredoxin-like protein {GENE: TRX} - Lolium perenne (Perennial ryegrass) 172 (Q9AS75) Thioredoxin-like protein {GENE: P0028E10.17} - Oryza sativa (Rice) 173 (O04002) CDSP32 protein (Chloroplast Drought-induced Stress Protein of 32 kDa) - Solanum tuberosum (Potato) 174 (Q9SWG5) Thioredoxin-like protein {GENE: TRX} - Secale cereale (Rye) 175 (Q9SP36) Thioredoxin-like protein (Fragment) {GENE: TRX} - Secale cereale (Rye) 176 (Q9U515) Thioredoxin-like protein - Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-LIKE PROTEINS 177 (P43221) Thiol: disulfide interchange protein tlpA (Cytochrome c biogenesis protein tIpA).","{GENE: TLPA} - Bradyrhizobium japonicum 178 (P43787) Thioredoxin-like protein HI1115.","{GENE: HI1115} - Haemophilus influenzae 179 (Q9GUP7) Thioredoxin-like protein {GENE: TRXLP} - Leishmania major 180 (Q9UVH0) Thioredoxin-like protein - Mortierella alpina 181 (P95355) Thioredoxin-like protein - Neisseria gonorrhoeae 182 (Q98G37) Thioredoxin-like protein {GENE: MLL3505} - Rhizobium loti (Mesorhizobium loti) 183 (P36893) Thiol: disulfide interchange protein helX precursor (Cytochrome c biogenesis protein helX).","{GENE: HELX} - Rhodobacter capsulatus (Rhodopseudomonas capsulata) 184 (P52232) Thioredoxin-like protein SLR0233.","{GENE: SLR0233} - Synechocystis sp.","(strain PCC 6803) 185 (P73263) Thioredoxin-like protein SLR1139.","{GENE: SLR1139} - Synechocystis sp.","(strain PCC 6803) 186 (Q9USR1) Thioredoxin-like protein {GENE: SPBC577.08C} - Schizosaccharomyces pombe (Fission yeast) 187 (Q9R788) Thioredoxin {GENE: TPTRX} - Treponema pallidum ANIMALS THIOREDOXIN-LIKE PROTEINS 188 (Q9UAV4) F46E10.9 PROTEIN (THIOREDOXIN-LIKE PROTEIN DPY-11) {GENE: F46E10.9 OR DPY-11} - Caenorhabditis elegans 189 (Q9N2K6) Thioredoxin-like protein (Y54E10A.3 protein) (Thioredoxin-like protein TXL) {GENE: TXL OR Y54E10A.3} - Caenorhabditis elegans 190 (Q9VRP3) THIOREDOXIN-LIKE PROTEIN TXL (CG5495 PROTEIN) {GENE: TXL OR CG5495} - Drosophila melanogaster (Fruit fly) 191 (O43396) Thioredoxin-like protein (32 kDa thioredoxin-related protein).","{GENE: TXNL OR TRP32 OR TXL} - Homo sapiens (Human) 192 (O76003) Thioredoxin-like protein - Homo sapiens (Human) 193 (Q9S753) THIOREDOXIN-LIKE PROTEIN {GENE: TRX} - Phalaris coerulescens 194 (O77404) TRYPAREDOXIN - Trypanosoma brucei brucei PLANT THIOREDOXIN-REDUCTASES 195 (Q39243) Thioredoxin-reductase 1 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 1) (NTR 1).","{GENE: NTR1 OR AT4G35460 OR F15J1.30} - Arabidopsis thaliana (Mouse-ear cress) 196 (Q39242) Thioredoxin-reductase 2 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 2) (NTR 2).","{GENE: NTR2 OR AT2G17420 OR F5J6.18} - Arabidopsis thaliana (Mouse-ear cress) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 197 (O66790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR AQ_500} - Aquifex aeolicus 198 (P80880) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (General stress protein 35) (GSP35).","{GENE: TRXB} - Bacillus subtilis 199 (P94284) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR BB0515} - Borrelia burgdorferi (Lyme disease spirochete) 200 (P57399) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR BU314} - Buchnera aphidicola (subsp.","Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 201 (P81433) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Buchnera aphidicola (subsp.","Schizaphis graminum) 202 (Q9PKT7) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR TC0375} - Chlamydia muridarum 203 (Q9Z8M4) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR CPN0314 OR CP0444} - Chiamydia pneumoniae (Chlamydophila pneumoniae) 204 (O84101) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR CT099} - Chlamydia trachomatis 205 (P52213) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Clostridium litorale (Bacterium W6) 206 (P39916) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Coxiella burnetii 207 (P09625) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR B0888 OR Z1232 OR ECSO973} - Escherichia coli, Escherichia coli O157: H7 208 (P50971) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Eubacterium acidaminophilum 209 (P43788) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR HI1158} - Haemophilus influenzae 210 (Q9ZL18) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR JHP0764} - Helicobacter pylori J99 (Campylobacter pylori J99) 211 (P56431) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR HP0825} - Helicobacter pylori (Campylobacter pylori) 212 (O32823) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR LMO2478} - Listeria monocytogenes 213 (P47348) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR MG102} - Mycoplasma genitalium 214 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].","{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 215 (P75531) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR MPN240 OR MP591} - Mycoplasma pneumoniae 216 (O30973) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Mycobacterium smegmatis 217 (P52214) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (TR).","{GENE: TRXB OR RV3913 OR MT4032 OR MTV028.04} - Mycobacterium tuberculosis 218 (P51978) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: CYS-9} - Neurospora crassa 219 (P43496) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: TRXB} - Penicillium chrysogenum 220 (Q9ZD97) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR RP445} - Rickettsia prowazekii 221 (Q92375) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: SPBC3F6.03} - Schizosaccharomyces pombe (Fission yeast) 222 (Q05741) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB} - Streptomyces clavuligerus 223 (P52215) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR SCH24.12} - Streptomyces coelicolor 224 (O83790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).","{GENE: TRXB OR TP0814} - Treponema pallidum 225 (P80892) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (Fragment).","{GENE: TRXB} - Vibrio fischeri 226 (P29509) Thioredoxin-reductase 1 (EC 1.6.4.5).","{GENE: TRR1 OR YDR353W OR D9476.5} - Saccharomyces cerevisiae (Baker's yeast) 227 (P38816) Thioredoxin-reductase 2, mitochondrial precursor (EC 1.6.4.5).","{GENE: TRR2 OR YHR106W} - Saccharomyces cerevisiae (Baker's yeast) ANIMAL THIOREDOXIN-REDUCTASES 228 (O62768) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: TXNRD1} - Bos taurus (Bovine) 229 (Q17745) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: C06G3.7} - Caenorhabditis elegans 230 (Q16881) Thioredoxin-reductase (EC 1.6.4.5).","{GENE: TXNRD1} - Homo sapiens (Human) 231 (Q25861) Thioredoxin-reductase (EC 1.6.4.5) (TrxR).","{GENE: TR OR GR} - Plasmodium falciparum (isolate FCH-5) Other thioredoxin-reductases PLANTS THIOREDOXIN-REDUCTASES 232 (O22229) Thioredoxin-reductase {GENE: AT2G41680} - Arabidopsis thaliana (Mouse-ear cress) 233 (Q39951) NADPH thioredoxin-reductase (Fragment} - Helianthus annuus (Common sunflower) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 234 (O28718) THioredoxin-reductase (TRXB) {GENE: AF1554} - Archaeoglobus fulgidus 235 (Q9K703) Thioredoxin-reductase (NADPH) (EC 1.6.4.5) {GENE: TRXB OR BH3571} - Bacillus halodurans 236 (Q9K7F3) Thioredoxin-reductase {GENE: BH3408} - Bacillus halodurans 237 (Q9KCZ0) Thioredoxin-reductase {GENE: BH1429} - Bacillus halodurans 238 (Q9KCZ1) Thioredoxin-reductase {GENE: BH1428} - Bacillus halodurans 239 (Q9PIY1) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR CJ0146} - Campylobacter jejuni 240 (Q9A4G3) Thioredoxin-reductase {GENE: CC2871} - Caulobacter crescentus 241 (Q97EM8) Thioredoxin-reductase {GENE: CAC3082} - Clostridium acetobutylicum 242 (Q97IU2) Thioredoxin-reductase {GENE: CAC1548} - Clostridium acetobutylicum 243 (Q9EV96) Thioredoxin-reductase {GENE: TRXB} - Clostridium sticklandii 244 (Q9RSY7) THioredoxin-reductase {GENE: DR1982} - Deinococcus radiodurans 245 (O30739) Thioredoxin-reductase (Fragment} - Enterococcus faecalis (Streptococcus faecalis) 246 (O54535) Thioredoxin-reductase {GENE: TRXB OR TRXB1_2 OR VNG6452G OR TRXB1_1 OR VNG6074G} - Halobacterium sp.","(strain NRC-1) [Plasmid pNRC100, and Plasmid pNRC200] 247 (P82854) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Halobacterium sp.","(strain NRC-1) 248 (Q9HN08) Thioredoxin-reductase {GENE: TXRB3 OR VNG2301G} - Halobacterium sp.","(strain NRC-1) 249 (O25779) THioredoxin-reductase (TRXB) {GENE: HP1164} - Helicobacter pylori (Campylobacter pylori) 250 (O86255) Thioredoxin-reductase {GENE: TRXB} - Klebsiella oxytoca 251 (Q9AEV9) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis (subsp.","lactis) (Streptococcus lactis) 252 (Q9CF34) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Lactococcus lactis (subsp.","lactis) (Streptococcus lactis) 253 (Q9CH02) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB1} - Lactococcus lactis (subsp.","lactis) (Streptococcus lactis) 254 (Q9ZFC8) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis 255 (O32822) Hypothetical 39.7 kDa protein (Fragment) - Listeria monocytogenes 256 (O26804) THioredoxin-reductase {GENE: MTH708} - Methanothermobacter thermautotrophicus 257 (P94397) Homologue of thioredoxin-reductase of Mycoplama genitalium {GENE: YCGT} - Bacillus subtilis 258 (Q98PK9) THioredoxin-reductase (EC 1.6.4.5) {GENE: MYPU_7130} - Mycoplasma pulmonis 259 (Q9JU23) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR NMA1538} - Neisseria meningitidis (serogroup A) 260 (Q9JZ28) Thioredoxin-reductase {GENE: NMB1324} - Neisseria meningitidis (serogroup B) 261 (Q9I0M2) Thioredoxin-reductase 1 {GENE: TRXB1 OR PA2616} - Pseudomonas aeruginosa 262 (Q9I592) Thioredoxin-reductase 2 {GENE: TRXB2 OR PA0849} - Pseudomonas aeruginosa 263 (Q9V0Q8) THioredoxin-reductase (TRXB) {GENE: TRXB OR PAB0500} - Pyrococcus abyssi 264 (Q9ZD33) THioredoxin-reductase (TRXB2) {GENE: RP514} - Rickettsia prowazekii 265 (O54079) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB} - Staphylococcus aureus 266 (Q9RIS2) Thioredoxin-reductase {GENE: TRXB OR TRXB2} - Streptomyces coelicolor 267 (Q9K4L6) Thioredoxin-reductase {GENE: SC5F8.08C} - Streptomyces coelicolor 268 (Q97PY2) Thioredoxin-reductase {GENE: SP1458} - Streptococcus pneumoniae 269 (Q9A0B5) Thioredoxin-reductase {GENE: SPY0850} - Streptococcus pyogenes 270 (Q97V69) Thioredoxin-reductase (trxB-2) (EC 1.6.4.5) {GENE: TRXB-2} - Sulfolobus solfataricus 271 (Q97W27) Thioredoxin-reductase (trxB-3) (EC 1.6.4.5) {GENE: TRXB-3} - Sulfolobus solfataricus 272 (Q97WJ5) Thioredoxin-reductase (trxB-1) (EC 1.6.4.5) {GENE: TRXB-1} - Sulfolobus solfataricus 273 (Q98I59) Thioredoxin-reductase {GENE: MLL2552} - Rhizobium loti (Mesorhizobium loti) 274 (Q98M06) Thioredoxin-reductase {GENE: MLL0792} - Rhizobium loti (Mesorhizobium loti) 275 (Q9UR80) 35 kDa THioredoxin-reductase HOMOLOG (FRAGMENT) {GENE: TRR1 AND YDR353W} - Saccharomyces cerevisiae (Baker's yeast) 276 (Q9ZEH4) THIOREDOXIN {GENE: TRXA OR SA0992} - Staphylococcus aureus, Staphylococcus aureus subsp.","aureus N315 277 (Q9S1H1) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Staphylococcus xylosus 278 (Q9HJI4) Thioredoxin-reductase {GENE: TA0984} - Thermoplasma acidophilum 279 (Q9WZX3) THioredoxin-reductase {GENE: TM0869} - Thermotoga maritima 280 (Q979K8) Thioredoxin-reductase {GENE: TVG1183005} - Thermoplasma volcanium 281 (Q9PR71) Thioredoxin-reductase {GENE: TRXB OR UU074} - Ureaplasma parvum (Ureaplasma urealyticum biotype 1) 282 (Q9KSS4) Thioredoxin-reductase {GENE: VC1182} - Vibrio cholerae 283 (Q9PDD1) Thioredoxin-reductase {GENE: XF1448} - Xylella fastidiosa 284 (Q9X5F7) Thioredoxin-reductase {GENE: TRXB1} - Zymomonas mobilis ANIMAL THIOREDOXIN-REDUCTASES 285 (Q9GKW9) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Bos taurus (Bovine) 286 (Q9N2I8) Thioredoxin-reductase (EC 1.6.4.5) - Bos taurus (Bovine) 287 (Q9N2K1) Thioredoxin-reductase homolog - Caenorhabditis elegans 288 (Q9NJH3) Thioredoxin-reductase - Caenorhabditis elegans 289 (Q9VNT5) CG11401 PROTEIN (THioredoxin-reductase 2) {GENE: TRXR-2 OR CG11401} - Drosophila melanogaster (Fruit fly) 290 (O95840) Thioredoxin-reductase - Homo sapiens (Human) 291 (Q9UES8) Thioredoxin-reductase GRIM-12 - Homo sapiens (Human) 292 (Q9UH79) Thioredoxin-reductase {GENE: TR} - Homo sapiens (Human) 293 (Q9UQU8) Thioredoxin-reductase - Homo sapiens (Human) 294 (Q9NNW6) Thioredoxin-reductase TR2 (Fragment) - Homo sapiens (Human) 295 (Q9NNW7) Thioredoxin-reductase TR3 - Homo sapiens (Human) 296 (Q9P101) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Homo sapiens (Human) 297 (Q9P2Y0) Thioredoxin-reductase II beta (EC 1.6.4.5) - Homo sapiens (Human) 298 (Q9H2Z5) Mitochondrial thioredoxin-reductase {GENE: TRXR2A} - Homo sapiens (Human) 299 (Q99475) KM-102-DERIVED REDUCTASE-LIKE FACTOR (THioredoxin-reductase) - Homo sapiens (Human) 300 (Q99P49) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 301 (Q9CSV5) Thioredoxin-reductase 1 (Fragment) {GENE: TXNRD1} - Mus musculus (Mouse) 302 (Q9CZE5) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 303 (Q9JHA7) Thioredoxin-reductase TR3 {GENE: TXNRD2 OR TR3} - Mus musculus (Mouse) 304 (Q9JLT4) Thioredoxin-reductase {GENE: TXNRD2 OR TRXR2} - Mus musculus (Mouse) 305 (Q9JMH5) Thioredoxin-reductase 2 {GENE: TXNRD2 OR TXNRD2} - Mus musculus (Mouse) 306 (Q9JMH6) Thioredoxin-reductase 1 {GENE: TXNRD1 OR TXNRD1} - Mus musculus (Mouse) 307 (O89049) Thioredoxin-reductase - Rattus norvegicus (Rat) 308 (Q9JKZ3) Thioredoxin-reductase 1 (Fragment) - Rattus norvegicus (Rat) 309 (Q9JKZ4) Thioredoxin-reductase 1 - Rattus norvegicus (Rat) 310 (Q9JLE6) Thioredoxin-reductase (Fragment) - Rattus norvegicus (Rat) 311 (Q9R1I3) NADPH-dependent thioredoxin-reductase {GENE: TRR1} - Rattus norvegicus (Rat) 312 (Q9Z0J5) Thioredoxin-reductase precursor {GENE: TRXR2} - Rattus norvegicus (Rat) 313 (Q9MYY8) Redox enzyme thioredoxin-reductase - Sus scrofa (Pig)"]],"string":"[\n [\n \"Methods for the production of multimeric proteins and related compositions\",\n \"Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobins, in association with oil bodies are described.\",\n \"The redox protein is enzymatically active when prepared in association with the oil bodies.\",\n \"Also provided are related nucleic acids, proteins, cells, plants, and compositions.\"\n ],\n [\n \"1-266.\",\n \"(canceled) 267.A method of producing an oil body associated with a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.\",\n \"268.A method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.\",\n \"269.A method of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"270.A chimeric nucleic acid sequence encoding a multimeric-fusion-protein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"271.A recombinant multimeric-fusion-protein comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.\",\n \"272.Isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"273.Isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"274.A cell comprising oil bodies and (i) an oil-body-targeting-protein, (ii) a first recombinant polypeptide and (iii) a second recombinant polypeptide wherein (1) said first recombinant polypeptide is capable of associating with said oil-body-targeting-protein; and (2) said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form a multimeric-protein-complex.\",\n \"275.A composition comprising isolated oil bodies, thioredoxin and thioredoxin-reductase.\",\n \"276.A food product, personal care product or pharmaceutical composition comprising the composition of claim 275.277.A method of reducing allergenicity of a food comprising the steps of: providing the isolated oil bodies of claim 273; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.\",\n \"278.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing the recombinant fusion polypeptide of claim 269; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"279.A method for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"280.A method for preparing a redox protein associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide.\",\n \"281.A chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"282.A transgenic plant comprising the chimeric nucleic acid sequence of claim 281.283.A safflower plant comprising the chimeric nucleic acid of anyone of claim 281.284.A plant seed comprising the chimeric nucleic acid of claim 281.285.A safflower seed comprising the chimeric nucleic acid of claim 281.286.An oil body preparation obtained by the method of claim 279.287.A food product comprising an oil body preparation of claim 286.288.A composition comprising an oil body preparation of claim 286 (New).\",\n \"289.A personal care product comprising an oil body preparation of claim 286.290.A product capable of treating oxidative stress in a target comprising an oil body preparation of claim 286.291.A product capable of chemically reducing a target comprising an oil body preparation of claim 286.292.A detergent composition comprising the product of claim 286.293.A method of cleansing an item, comprising administering the product of claim 290 to said item under conditions that promote cleansing.\",\n \"294.An emulsion formulation prepared by the method of claim 279.295.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.\",\n \"296.The seed of the plant of claim 281.297.An extract of the seed of claim 296, wherein the extract comprises an activity of a thioredoxin-related protein.\",\n \"298.An oil body from the seed of claim 296.299.Oil produced from the seed of claim 296.300.A method of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; obtaining seeds from the plant; and recovering the fusion protein by isolating oil bodies from the seeds.\",\n \"301.Oil bodies in association with a fusion protein, obtained by the method of claim 300.302.A method of reducing allergenicity of a food comprising the steps of: providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.\",\n \"303.A composition comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"304.A cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in an acceptable carrier.\",\n \"305.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"306.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one recombinant polypeptide and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.\"\n ],\n [\n \" BACKGROUND Multimeric proteins (i.e.\",\n \"proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.\",\n \"Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.\",\n \"However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.\",\n \"Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.\",\n \"For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.\",\n \"(1996) J. Cardiov.\",\n \"Pharmacol.\",\n \"(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.\",\n \"(1999) J. Allerg.\",\n \"Vlin.\",\n \"Immunol.\",\n \"103: 690-697) and wheat (Buchanan et al.\",\n \"(1997) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 94: 5372-5377).\",\n \"However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.\",\n \"The present invention satisfies this need and provides related advantages as well.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.\",\n \"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.\",\n \"The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.\",\n \"The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.\",\n \"Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-protein can be an oleosin or caleosin.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.\",\n \"In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.\",\n \"These formulations can further comprise the addition of NADP or NADPH.\",\n \"The food product can be a milk or wheat based food product.\",\n \"The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.\",\n \"The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).\",\n \"Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.\",\n \"In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.\",\n \"5 .\",\n \"Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.\",\n \"The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.\",\n \"Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.\",\n \"For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis .\",\n \"In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.\",\n \"Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.\",\n \"Accordingly, an emulsion formulation composition is provided.\",\n \"Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.\",\n \"The chimeric nucleic acids can be contained within a plastid.\",\n \"Accordingly, isolated plastids are provided having chimeric nucleic acids therein.\",\n \"Also provided are plant seeds comprising the chimeric nucleic acids provided herein.\",\n \"Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.\",\n \"The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.\",\n \"Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.\",\n \"Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.\",\n \"In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.\",\n \"In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.\",\n \"The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.\",\n \"The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.\",\n \"The second region can encode a thioredoxin and thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae .\",\n \"In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.\",\n \"The first region can precede, in a 5′ to 3′ direction, the second region.\",\n \"Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.\",\n \"In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.\",\n \"In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.\",\n \"In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.\",\n \"Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.\",\n \"The thioredoxin-related protein can be thioredoxin.\",\n \"The nucleic acid construct can be contained within a plastid.\",\n \"In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.\",\n \"Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.\",\n \"In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.\",\n \"Also provided herein is an extract comprising an activity of a thioredoxin-related protein.\",\n \"Also provided are oil bodies and/or oil obtained from various seeds.\",\n \"Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.\",\n \"In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.\",\n \"The oil bodies can be in association with a fusion protein.\",\n \"The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.\",\n \"The cleaving step can make use of a protease or chemical proteolysis.\",\n \"Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.\",\n \"The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"The oil bodies can be associated with the fusion protein.\",\n \"Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.\",\n \"Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.\",\n \"These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.\",\n \"The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.\",\n \"For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.\",\n \"Other features and advantages of the present invention will become readily apparent from the following detailed description.\",\n \"It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.\"\n ],\n [\n \"RELATED APPLICATIONS Benefit of priority under 35 U.S.C.\",\n \"§119(e) is claimed to U.S. provisional application Ser.\",\n \"No.\",\n \"60/302,885, filed Jul.\",\n \"5, 2001, to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”.\",\n \"This application is also a continuation-in-part of U.S. utility application Ser.\",\n \"No.\",\n \"10/006,038, filed Dec. 4, 2001 to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”; which is a continuation-in-part of U.S. utility application Ser.\",\n \"No.\",\n \"09/742,900, filed Dec. 19, 2000 to Heifetz, et al., entitled “METHOD OF PRODUCTION AND DELIVERY OF THIOREDOXIN”.\",\n \"This application is also a continuation-in-part of U.S. utility application Ser.\",\n \"No.\",\n \"09/742,900.The subject matter of each of the provisional and utility applications is incorporated herein by reference in its entirety.\",\n \"FIELD OF THE INVENTION The present invention relates to multimeric-protein-complexes, redox proteins, and recombinant polypeptides; and improved methods for their production.\",\n \"BACKGROUND Multimeric proteins (i.e.\",\n \"proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.\",\n \"Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.\",\n \"However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.\",\n \"Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.\",\n \"For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.\",\n \"(1996) J. Cardiov.\",\n \"Pharmacol.\",\n \"(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.\",\n \"(1999) J. Allerg.\",\n \"Vlin.\",\n \"Immunol.\",\n \"103: 690-697) and wheat (Buchanan et al.\",\n \"(1997) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 94: 5372-5377).\",\n \"However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.\",\n \"The present invention satisfies this need and provides related advantages as well.\",\n \"SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.\",\n \"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.\",\n \"This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.\",\n \"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-targeting-protein can be an oleosin or caleosin.\",\n \"When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.\",\n \"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.\",\n \"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.\",\n \"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.\",\n \"Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.\",\n \"The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.\",\n \"The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.\",\n \"The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.\",\n \"Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.\",\n \"The oil-body-protein can be an oleosin or caleosin.\",\n \"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.\",\n \"Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.\",\n \"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.\",\n \"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.\",\n \"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.\",\n \"In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.\",\n \"These formulations can further comprise the addition of NADP or NADPH.\",\n \"The food product can be a milk or wheat based food product.\",\n \"The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.\",\n \"The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).\",\n \"Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.\",\n \"In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.\",\n \"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.\",\n \"5.Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.\",\n \"The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.\",\n \"Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.\",\n \"For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.\",\n \"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.\",\n \"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.\",\n \"The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis.\",\n \"In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.\",\n \"Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.\",\n \"Accordingly, an emulsion formulation composition is provided.\",\n \"Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.\",\n \"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.\",\n \"Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.\",\n \"The chimeric nucleic acids can be contained within a plastid.\",\n \"Accordingly, isolated plastids are provided having chimeric nucleic acids therein.\",\n \"Also provided are plant seeds comprising the chimeric nucleic acids provided herein.\",\n \"Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.\",\n \"The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.\",\n \"Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.\",\n \"Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.\",\n \"In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.\",\n \"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.\",\n \"In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.\",\n \"The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.\",\n \"The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.\",\n \"The second region can encode a thioredoxin and thioredoxin-reductase.\",\n \"In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae.\",\n \"In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.\",\n \"The first region can precede, in a 5′ to 3′ direction, the second region.\",\n \"Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.\",\n \"In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.\",\n \"In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.\",\n \"In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.\",\n \"Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.\",\n \"The thioredoxin-related protein can be thioredoxin.\",\n \"The nucleic acid construct can be contained within a plastid.\",\n \"In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.\",\n \"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.\",\n \"The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.\",\n \"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.\",\n \"The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.\",\n \"Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.\",\n \"In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.\",\n \"Also provided herein is an extract comprising an activity of a thioredoxin-related protein.\",\n \"Also provided are oil bodies and/or oil obtained from various seeds.\",\n \"Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.\",\n \"In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.\",\n \"The oil bodies can be in association with a fusion protein.\",\n \"The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.\",\n \"The cleaving step can make use of a protease or chemical proteolysis.\",\n \"Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.\",\n \"The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.\",\n \"In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.\",\n \"Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"The oil bodies can be associated with the fusion protein.\",\n \"Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.\",\n \"Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.\",\n \"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.\",\n \"Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.\",\n \"Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.\",\n \"These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.\",\n \"The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.\",\n \"For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.\",\n \"Other features and advantages of the present invention will become readily apparent from the following detailed description.\",\n \"It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows a ClustalW Formatted Alignment comparison of the published NADPH thioredoxin-reductase nucleic acid sequence (SEQ ID NO:9) (ATTHIREDB-Jacquot et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"(1994) 235 (4):1357-63.)\",\n \"with the sequence isolated herein in Example 1 (TR; SEQ ID NO:8).\",\n \"FIG.\",\n \"2 shows a ClustalW Formatted Alignment comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence (SEQ ID NO:12) (ATTHIREDB Jacquot et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"(1994) 235 (4):1357-63.)\",\n \"with the sequence isolated herein in Example 1 (TR; SEQ ID NO:13).\",\n \"FIG.\",\n \"3 shows a clustal alignment comparing the amino acid sequence of the Arabidopsis thaliana thioredoxin-reductase-linker-thioredoxin synthetic fusion (Arab TR-link-Trxh; SEQ ID NO:37) to the Mycobacterium leprae thioredoxin-reductase-thioredoxin natural fusion (M. lep TR/Trxh; SEQ ID NO:36) natural fusion.\",\n \"Overall, the proteins are approximately 50% identical at the amino acid level.\",\n \"FIG.\",\n \"4 is a bar graph showing the thioredoxin/thioredoxin-reductase activity measurements for the various transgenic Arabidopsis seed fractions.\",\n \"Relative specific activity is expressed as a percentage of the E. coli thioredoxin and thioredoxin-reductase activities.\",\n \"The numbered bars in the graph correspond to the following: 1.W.T.+oleosin-thioredoxin 2.W.T.+thioredoxin-oleosin 3.W.T.+thioredoxin 4.W.T.+oleosin-thioredoxin-reductase 5.W.T.+thioredoxin-reductase-oleosin 6.W.T.+thioredoxin-reductase 7.thioredoxin+oleosin-thioredoxin-reductase 8.thioredoxin+thioredoxin-reductase-oleosin 9.thioredoxin+thioredoxin-reductase 10.thioredoxin-reductase+oleosin-thioredoxin 11.thioredoxin-reductase+thioredoxin-oleosin 12.oleosin-M. lep TR/Trxh 13.E.\",\n \"coli thioredoxin-reductase+thioredoxin FIG.\",\n \"5 provides a listing of exemplary proteins for use in the heteromultimeric-fusion-proteins and heteromultimeric-protein-complexes provided herein.\",\n \"DETAILED DESCRIPTION As hereinbefore mentioned, the present invention relates to novel and improved methods for the production of multimeric proteins, including a first and second recombinant polypeptide, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and a first and second thioredoxin-related protein; and related products.\",\n \"These methods permit the production of active multimeric-protein-complexes in association with oil bodies.\",\n \"The oil bodies in association with the multimeric-protein-complex may be used to prepare various useful emulsions.\",\n \"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex associated with an oil body, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.\",\n \"DEFINITIONS AND TERMS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.\",\n \"Where permitted, all patents, applications, published applications and other publications and sequences from GenBank, SwissPro and other data bases referred to throughout in the disclosure herein are incorporated by reference in their entirety.\",\n \"As used herein, the phrase “multimeric-protein-complex”, refers to two or more polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.\",\n \"It should be noted that the polypeptides may be independently biologically active without interaction or coordination to form the complex.\",\n \"The multimeric-protein-complex may provide a biological structure, or it may be capable of facilitating a chemical or biological reaction.\",\n \"For example, one of the protein regions within the multimeric-protein-complex can repeatedly activate or repeatedly inactivate the biological or metabolic activity of one or more of the other proteins contained within the multimeric-protein-complex.\",\n \"In one embodiment, the first and second recombinant polypeptide contained in a multimeric-protein-complex may either associate or interact as independent non-contiguous polypeptide chains or the multimeric-protein-complex may be prepared as a fusion polypeptide (multimeric-fusion-protein) between the first and second recombinant polypeptide.\",\n \"One example of a repeated (e.g., reoccurring) interaction or association between the two or more polypeptides of a multimeric-protein-complex provided herein is the interaction between two or more non-identical redox proteins to form a heteromultimeric-protein-complex.\",\n \"Exemplary redox proteins for use in this regard are thioredoxin and the thioredoxin-reductase.\",\n \"A further example is the interaction between two or more immunoglobulin-polypeptide-chains to form an immunoglobulin.\",\n \"As used herein, the phrase “heteromultimeric-protein-complex”, refers to two or more non-identical polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.\",\n \"Other examples of multimeric-protein-complexes provided herein include a first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, first and second immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and second thioredoxin-related protein.\",\n \"The recombinant polypeptide or multimeric-protein-complex is associated with an oil body.\",\n \"As used herein, the phrase “oil body” or “oil bodies” refers to any oil or fat storage organelle in any cell type.\",\n \"Accordingly, the oil bodies may be obtained from any cell comprising oil bodies, including plant cells (described in for example: Huang (1992) Ann.\",\n \"Rev.\",\n \"Plant Mol.\",\n \"Biol.\",\n \"43: 177-200), animal cells (described in for example: Murphy (1990) Prog Lipid Res 29(4): 299-324), including adipocytes, hepatocytes, steroidogenic cells, mammary epithelial cells, macrophages, algae cells (described in for example: Rossler (1988) J. Physiol.\",\n \"London, 24: 394-400) fungal cells, including yeast cells (described in for example Leber et al.\",\n \"(1994) Yeast 10: 1421-1428) and bacterial cells (described in for example: Pieper-Furst et al.\",\n \"(1994) J. Bacteriol.\",\n \"176: 4328-4337).\",\n \"Preferably the oil bodies used herein are oil bodies obtainable from plant cells and more preferably the oil bodies obtainable from plant seed cells.\",\n \"As used herein, the phrase “is capable of associating with”, “associate” or grammatical variations thereof, refers to any interaction between two or more polypeptides, including any covalent interactions (e.g.\",\n \"multimeric-fusion-proteins) as well as non-covalent interactions.\",\n \"Exemplary non-covalent interactions can be between the oil-body-targeting-protein and a redox protein or immunoglobulin-polypeptide-chain, as well as between two or more different proteins contained within two or more separate oil-body-protein fusion proteins (e.g., the redox proteins in oleosin-thioredoxin and oleosin-thioredoxin-reductase).\",\n \"As used herein, the term “recombinant” (also referred to as heterologous) in the context of recombinant proteins and amino acids, means “of different natural origin” or represents a non-natural state.\",\n \"For example, if a host cell is transformed with a nucleotide sequence derived from another organism, particularly from another species, that nucleotide sequence and amino acid sequence encoded thereby, is recombinant (heterologous) with respect to that host cell and also with respect to descendants of the host cell which carry that gene.\",\n \"Similarly, recombinant (or heterologous) refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.\",\n \"A transforming nucleotide sequence may include a recombinant coding sequence, or recombinant regulatory elements.\",\n \"Alternatively, the transforming nucleotide sequence may be completely heterologous or may include any possible combination of heterologous and endogenous nucleic acid sequences.\",\n \"In various embodiments of the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, are produced in a cell comprising oil bodies.\",\n \"As used herein the phrase “in a cell”, “in the cell”, or grammatical variations thereof, mean that the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, may be produced in any cellular compartment of that cell, so long as that cell comprises oil bodies therein.\",\n \"In embodiments of the invention in which plant cells are used, the phrase is intended to include the plant apoplast.\",\n \"In various embodiments provided herein, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, associate with an oil body through an oil-body-targeting-protein.\",\n \"As used herein, the phrase “oil-body-targeting-protein” refers to any protein, protein fragment or peptide capable of associating with an oil body.\",\n \"Exemplary oil-body-targeting-proteins for use herein include oil-body-proteins, such as oleosin and caleosin; immunoglobulins, such as bi-specific antibodies; and the like.\",\n \"In embodiments described herein in which an oil-body-protein is used, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, are preferably fused to the oil-body-protein.\",\n \"The term “oil-body-protein” refers to any protein naturally present in cells and having the capability of association with oil bodies, including any oleosin or caleosin.\",\n \"Accordingly, provided herein a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide, such as a redox protein, an immunoglobulin-polypeptide-chain or an thioredoxin-related protein, fused to an oil-body-protein; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide, such as a second redox protein, a second immunoglobulin-polypeptide-chain or a second thioredoxin-related protein; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex, preferably in said progeny cell; and (d) associating said first recombinant polypeptide with an oil body through said oil-body-protein.\",\n \"The term “nucleic acid” as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.\",\n \"The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.\",\n \"The nucleic acid sequences may be ribonucleic acids (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.\",\n \"The sequences also may contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo-uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol-adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8 thiol guanine, 8-thioalkyl guanines, 8 hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.\",\n \"Multimeric-Protein-Complexes In accordance with the methods and compositions provided herein, any two recombinant polypeptides capable of forming a multimeric-protein-complex may be used.\",\n \"The nucleic acid sequences encoding the two recombinant polypeptides may be obtained from any biological source or may be prepared synthetically.\",\n \"In general nucleic acid sequence encoding multimeric proteins are known to the art and readily available.\",\n \"Known nucleic acid sequences encoding multimeric-protein-complexes may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences encoding multimeric-protein-complexes, for example, by screening cDNA or genomic libraries or using 2- or multi-hybrid systems.\",\n \"Thus, additional nucleic acid sequences encoding multimeric-protein-complexes may be discovered and used as described herein.\",\n \"The first and/or second recombinant polypeptides that are comprised within a multimeric-protein-complex provided herein, can themselves be in the form of heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein.\",\n \"The nucleic acid sequence encoding the first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein may be obtained from separate sources or may be obtained from the same source.\",\n \"In general however, such nucleic acid sequence is obtained from the same or a similar biological source.\",\n \"In certain embodiments wherein the nucleic acid sequence encoding the first and second recombinant polypeptide protein are obtained from the same source, the nucleic acid sequence encoding the first recombinant polypeptide and second recombinant polypeptide may be naturally fused.\",\n \"In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second recombinant polypeptide are obtained from a plant source.\",\n \"Oil-Body-Surface-Avoiding Linkers Polypeptide spacers or linkers of variable length and/or negative charge can be used herein to separate the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins from the in-frame oil-body-targeting-protein, to improve activity of and/or the accessibility of the polypeptide or complex.\",\n \"For example, in one embodiment set forth herein, positioned between a nucleic acid sequence encoding a sufficient portion of an oil-body-protein and a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins; is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.\",\n \"Oil-body-surface-avoiding linkers are positioned between the oil-body targeting sequence and an in-frame recombinant polypeptide of interest, e.g., the multimeric-protein-complexes provided herein, serve to increase the distance and or decrease the interaction between the negatively charged oil body surface and the recombinant polypeptide of interest.\",\n \"A negatively charged linker is repelled by the negatively charged oil body surface, in turn increasing the distance or decreasing the interaction of its attached recombinant polypeptide with the oil body surface.\",\n \"As a consequence of the increased distance from the oil body surface, the recombinant polypeptide will be more accessible, e.g.\",\n \"to its target(s) substrate, protein substrate, protein partner, and less affected by the charged oil body surface.\",\n \"Exemplary linker sequences for use herein can be either a negatively charged linker, or a linker having a molecular weight of at least about 35 kd or more.\",\n \"As used herein, a “negatively charged linker” sequence, refers to any amino acid segment, or nucleic acid encoding such, that has a pI less than or equal to the pI of an oil body.\",\n \"In certain embodiments, the pI of the negatively charged linker is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, down to about 25% or more, below that of the pI of an oil body in the particular plant or cell system being used.\",\n \"Exemplary negatively charged linkers can be prepared comprising any combination of the negatively charged amino acid residues.\",\n \"For example, in one embodiment, a negatively charged linker comprises either a poly-glutamate or poly-aspartate sequence, or any combination of both amino acid residues.\",\n \"The negatively charged linker is typically at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.\",\n \"The negatively charged linkers are preferably non-proteolytic (e.g., non-proteolytic linkers), having no site for efficient proteolysis.\",\n \"When linker size rather than charge is used to minimize interaction of the recombinant polypeptide of interest with the oil body surface, then the linker is non-proteolytic and ranges in molecular weight from about 35 kd up to about 100 kd.\",\n \"The upper size limit is chosen such that the expression of, the activity of, the conformation of, and/or the access to target of, the recombinant polypeptide of interest is not significantly affected by the linker.\",\n \"In certain embodiments, described herein where a non-proteolytic linker amino acid sequence is employed, the gene-fusion or protein fusion (multimeric-fusion-protein) can optionally further comprise a linker nucleic or amino acid sequence encoding a sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the non-proteolytic linker sequence and sequence encoding the desired recombinant protein region, e.g., the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins set forth herein.\",\n \"When a cleavable linker sequence is used herein, in a particular embodiment, it is further downstream than the non-proteolytic linker sequence from the oil-body-targeting-protein region of the fusion protein.\",\n \"By virtue of cleavable linker, the recombinant fusion polypeptides provided herein, such as the multimeric-fusion-proteins and redox fusion polypeptides, can be isolated and purified by introducing an enzyme or chemical that cleaves said multimeric-fusion-protein and/or redox fusion polypeptide from said oil body, thereby obtaining and/or isolating the desired protein.\",\n \"It is contemplated herein that the use of cleavable linker sequence downstream of the non-proteolytic linker/spacer sequence will improve the yield of protein recovery when isolating or purifying proteins using the methods provided herein.\",\n \"The nucleic acid sequences encoding the first or second recombinant polypeptide may be altered to improve expression levels for example, by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the first and second recombinant polypeptide, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).\",\n \"Comparison of the codon usage of the first and second recombinant polypeptide with codon usage of the host will enable the identification of codons that may be changed.\",\n \"For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.\",\n \"By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.\",\n \"Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The first and second recombinant polypeptide can be altered using for example targeted mutagenesis, random mutagenesis (Shiraishi et al.\",\n \"(1998) Arch.\",\n \"Biochem.\",\n \"Biophys.\",\n \"358: 104-115; Galkin et al.\",\n \"(1997) Protein Eng.\",\n \"10: 687-690; Carugo et al.\",\n \"(1997) Proteins 28: 10-28; Hurley et al.\",\n \"(1996) Biochemistry 35: 5670-5678), gene shuffling, and/or by the addition of organic solvent (Holmberg et al.\",\n \"(1999) Protein Eng.\",\n \"12: 851-856).\",\n \"Any polypeptide spacers that are used in accordance with the methods and products provided herein may be altered in similar ways.\",\n \"In particular embodiments provided herein, the recombinant polypeptides or thioredoxin-related proteins capable of forming a multimeric-protein-complex are capable of forming a heteromultimeric-protein-complex.\",\n \"Examples of heteromultimeric-protein-complexes that contain polypeptide chains that repeatedly interact, either to activate, inactivate, oxidize, reduce, stabilize, etc., with one another, that can be produced in association with oil bodies using the methods provided herein include those set forth in FIG.\",\n \"5.Accordingly, exemplary proteins for use in the heteromultimeric-protein-complexes and nucleic acid constructs encoding such, provided herein include, among others described herein, those set forth in FIG.\",\n \"5.Other polypeptide regions that can be used in the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, provided herein include, among other, those immunoglobulin regions set forth in Table 1.TABLE 1 ANTIBODY HETERODIMERS Class or molecule Subunits Fab Variable region and first constant region of heavy chain and complete light chain Fv Variable regions of heavy and light antibody chains IgA heavy chains, light chains and J (joining) chain IgG, IgD, IgE heavy and light chains IgM heavy chains, light chains and J (joining) chain Antibody chain(s) and a toxin Antibody chain(s) and a toxin Autoantigens, allergens and Autoantigens, allergens and transplant transplant antigens with an antigens with an adjuvant or tolerogen adjuvant or tolerogen Chimeras using antibody Fc Receptor subunits fused to the constant domain region of antibody heavy chains As set forth above, in one embodiment, exemplary heteromultimeric-protein-complexes and exemplary heteromultimeric-fusion-proteins provided herein comprise redox proteins, such as the thioredoxins and thioredoxin-reductases and immunoglobulins.\",\n \"Oil-Body-Targeting-Proteins The nucleic acid sequence encoding the oil-body-targeting-protein that may be used in the methods and compositions provided herein may be any nucleic acid sequence encoding an oil-body-targeting-protein, protein fragment or peptide capable of association with first recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein and the oil bodies.\",\n \"The nucleic acid sequence encoding the oil body targeting peptide may be synthesized or obtained from any biological source.\",\n \"For example, in one embodiment the oil-body-targeting-protein is an immunoglobulin or an immunoglobulin derived molecule, for example, a bispecific single chain antibody.\",\n \"The generation of single chain antibodies and bi-specific single chain antibodies is known to the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,763,733, U.S. Pat.\",\n \"No.\",\n \"5,767,260 and U.S. Pat.\",\n \"No.\",\n \"5,260,203).\",\n \"Nucleic acid sequences encoding single chain antibodies functioning as oil-body-targeting-proteins may be prepared from hybridoma cell lines expressing monoclonal antibodies raised against an oleosin as described by Alting-Mees et al (2000) IBC's Annual International Conference on Antibody Engineering, Poster #1.In order to attain specificity for the first recombinant polypeptide a nucleic acid sequence encoding a second single chain antibody prepared from a monoclonal raised against the first recombinant polypeptide may be prepared and linked to the anti-oleosin single chain antibody.\",\n \"In this embodiment the oil body associates with the first recombinant polypeptide through non-covalent interactions of the oil-body-targeting-protein with the first recombinant polypeptide and the oil body.\",\n \"Alternatively the first recombinant polypeptide may be prepared as a fusion protein with an oil-body-targeting-protein.\",\n \"For example, a nucleic acid sequence encoding a single chain antibody raised against an oleosin may be fused to a nucleic acid sequence encoding the first recombinant polypeptide Non-immunoglobulin-based oil-body-targeting-proteins capable of association with the first recombinant polypeptide may be discovered and prepared using for example phage display techniques (Pharmacia Biotech Catalogue Number 27-9401-011 Recombinant Phage Antibody System Expression Kit).\",\n \"Oil-body-targeting-proteins may also be chemically modified.\",\n \"For example, oleosins may be modified by changing chemical modification of the lysine residues using chemical agents such as biotinyl-N-hydroxysuccinimide ester resulting in a process referred to as biotinylation.\",\n \"Conveniently this is accomplished by in vitro biotinylation of the oil bodies.\",\n \"In vivo biotinylation may be accomplished using the biotinylation domain peptide from the biotin carboxy carrier protein of E. coli acetyl-CoA carboxylase (Smith et al.\",\n \"(1998) Nucl.\",\n \"Acids.\",\n \"Res.\",\n \"26: 1414-1420).\",\n \"Avidin or streptavidin may subsequently be used to accomplish association of the redox protein with the oil body.\",\n \"In a particular embodiment the oil-body-targeting-protein is an oil-body-protein such as for example an oleosin or a caleosin or a sufficient portion derived thereof capable of targeting to an oil body.\",\n \"Nucleic acid sequences encoding oleosins are known to the art.\",\n \"These include for example the Arabidopsis oleosin (van Rooijen et al (1991) Plant Mol.\",\n \"Bio.\",\n \"18:1177-1179); the maize oleosin (Qu and Huang (1990) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"265 4:2238-2243); rapeseed oleosin (Lee and Huang (1991) Plant Physiol.\",\n \"96:1395-1397); and the carrot oleosin (Hatzopoulos et al (1990) Plant Cell Vol.\",\n \"2, 457-467.).\",\n \"Caleosin nucleic acid sequences are also known to the art (Naested et al (2000) Plant Mol.\",\n \"Biol.\",\n \"44(4):463-476; Chen et al (1999) Plant Cell Physiol.\",\n \"40(10):1079-1086).\",\n \"Animal cell derived oil body proteins that may be used herein include adopihilin (Brasaemle et al, (1997) J. Lipid Res., 38: 2249-2263; Heid et al.\",\n \"(1998) Cell Tissue Research 294: 309-321), perilipin (Blanchette-Mackie et al.\",\n \"(1995), J. Lipid Res.\",\n \"36: 1211-1226; Servetnick et al.\",\n \"(1995) J. Biol.\",\n \"Chem.\",\n \"270: 16970-16973), apolipoproteins such as apo A-I, A-II, A-IV, C-I, C-II, CIII (Segrest et al.\",\n \"(1990), Proteins 8:103-117) and apoB (Chatterton et al.\",\n \"(1995) J. Lipid Res.\",\n \"36: 2027-2037; Davis, R A in: Vance D E, Vance J. editors.\",\n \"Lipoprotein structure and secretion.\",\n \"The Netherlands, Elsevier, 191: 403-426.In one embodiment, the first recombinant polypeptide is fused to an oil-body-protein.\",\n \"The methodology is further described in U.S. Pat.\",\n \"No.\",\n \"5,650,554, which is incorporated herein by reference in its entirety.\",\n \"The first recombinant polypeptide may be fused to the N-terminus as well as to the C-terminus of the oil-body-protein (as described in: Moloney and van Rooijen (1996) INFORM 7:107-113) and fragments of the oil-body-protein such as for example the central domain of an oleosin molecule, or modified versions of the oil-body-protein may be used.\",\n \"In this embodiment, the second recombinant polypeptide is expressed intracellularly and then intracellularly associates with the first recombinant polypeptide to form the multimeric-protein-complex in the cell.\",\n \"Oil bodies comprising the multimeric-protein-complex are then conveniently isolated from the cells.\",\n \"In a further embodiment both the first and second recombinant polypeptide are separately fused to an oil-body-protein.\",\n \"In this embodiment nucleic acid sequences encoding the first and second polypeptides may be prepared separately and introduced in separate cell lines or they may be introduced in the same cell lines.\",\n \"Where the nucleic acid sequences are introduced in the same cell line, these nucleic acid sequence may be prepared using two separate expression vectors, or they may be prepared using a single vector comprising nucleic acid sequences encoding both the first polypeptide fused to an oil body protein and the second polypeptide fused to an oil-body-protein.\",\n \"Where separate cell lines are used subsequent mating of the offspring (e.g.\",\n \"mating of plants) is used to prepare a generation of cells comprising oil bodies which comprise both the first and second recombinant polypeptide fused to an oil-body-protein.\",\n \"In further alternate embodiment, the first and second recombinant polypeptide are fused to form a multimeric-fusion-protein comprising the multimeric-protein-complex.\",\n \"In such an embodiment, the first and second polypeptide is associated with the oil body through an oil-body-targeting-protein capable of associating with both the fusion protein and with the oil body.\",\n \"In a particular embodiment, the fusion protein comprising the multimeric-protein-complex is fused to an oil-body-protein, for example, an oleosin or caleosin.\",\n \"In embodiments provided herein in which the multimeric-protein-complex is an immunoglobulin (e.g., a multimeric-immunoglobulin-complex), a particularly preferred oil body targeting protein is an oleosin or caleosin associated with an immunoglobulin binding protein, such as for example protein A (U.S. Pat.\",\n \"No.\",\n \"5,151,350), protein L (U.S. Pat.\",\n \"No.\",\n \"5,965,390) and protein G (U.S. Pat.\",\n \"No.\",\n \"4,954,618), or active fragments of such immunoglobulin binding proteins.\",\n \"New oil-body-proteins may be discovered for example by preparing oil bodies (described in further detail below) and identifying proteins in these preparations using for example SDS gel electrophoresis.\",\n \"Polyclonal antibodies may be raised against these proteins and used to screen cDNA libraries in order to identify nucleic acid sequences encoding oil-body-proteins.\",\n \"The methodologies are familiar to the skilled artisan (Huynh et al.\",\n \"(1985) in DNA Cloning Vol.\",\n \"1.a Practical Approach ed.\",\n \"D M Glover, IRL Press, pp 49-78).\",\n \"New oil-body-proteins may further be discovered using known nucleic acid sequences encoding oil-body-proteins (e.g.\",\n \"the Arabidopsis, rapeseed, carrot and corn nucleic acid sequences) to probe for example cDNA and genomic libraries for the presence of nucleic acid sequences encoding oil-body-proteins.\",\n \"In one embodiment, the first and second polypeptide are a first and second redox protein.\",\n \"Accordingly, one embodiment provided herein relates to novel and improved methods for the production of redox proteins.\",\n \"It has unexpectedly been found that a redox protein when prepared as a fusion protein with a second redox protein is fully enzymatically active when produced in association with an oil body.\",\n \"In contrast, when the redox protein is prepared without the second redox protein it has reduced enzymatic activity.\",\n \"In one embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide relative to production as a non-fusion polypeptide.\",\n \"Accordingly, provided herein are methods for producing an oil body associated with a heteromultimeric redox protein complex, said method comprising: (a) producing in a cell comprising oil bodies, a first redox protein and a second redox protein wherein said first redox protein is capable of interacting with said second redox protein, preferably in the cell, to form said heteromultimeric redox protein complex; and (b) associating said heteromultimeric redox protein complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said heteromultimeric redox protein complex.\",\n \"In a particular embodiment the first and second redox protein are prepared as a fusion protein to form a redox fusion polypeptide.\",\n \"Accordingly, provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.\",\n \"The oil bodies in association with the redox protein may be used to prepare a variety of useful emulsions.\",\n \"As used herein the phrase “redox proteins” or grammatical variations thereof, refers to any protein or active protein fragment capable of participating in electron transport.\",\n \"For example, redox proteins are capable of catalyzing the transfer of an electron from an electron donor (also frequently referred to as the reducing agent) to an electron acceptor (also frequently referred to as the oxidizing agent).\",\n \"In the process of electron transfer, the reducing agent (electron donor) is oxidized and the oxidizing agent (electron acceptor) is reduced.\",\n \"Exemplary redox proteins for use herein include iron-sulfur proteins, cytochromes, redox active thiol proteins and redox-active flavoproteins.\",\n \"To carry out their function as conduits for electrons, redox proteins, such as thioredoxin and thioredoxin-reductase for example, are known to function by interacting or associating with one another in multimeric-protein-complexes (e.g., heteromultimeric-protein-complexes).\",\n \"The term “redox fusion polypeptide” as used herein refers to any fusion polypeptide comprising a first redox protein linked to a second redox protein (e.g., an in-frame translational fusion).\",\n \"The redox proteins that may be used with the methods and compositions provided herein may be any redox protein.\",\n \"In one embodiment the first and second redox proteins are a pair of redox proteins that would normally occur together from the same source, in nature.\",\n \"In a particular embodiment, the first redox protein is a thioredoxin and the second redox protein is a thioredoxin-reductase.\",\n \"The redox fusion polypeptide may be produced in any cell comprising oil bodies, including any animal cell, plant cell, algae cell, fungal cell or bacterial cell.\",\n \"In certain embodiments the redox fusion polypeptide is produced in a plant cell and in particular embodiments the redox fusion polypeptide is produced in the seed cells of a seed plant.\",\n \"In particular embodiments the oil-body-targeting-protein that is used is an oil-body-protein.\",\n \"In embodiments of the present invention in which an oil-body-protein is used, the first and second redox protein are preferably covalently fused to the oil-body-protein.\",\n \"Accordingly, provided herein are methods for the preparation of a redox protein In association with an oil body comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a first nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a second nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating said oil bodies comprising said redox fusion polypeptide from said progeny cell.\",\n \"Redox Proteins In accordance with various methods and compositions provided herein, any nucleic acid sequence encoding a redox protein may be used.\",\n \"The nucleic acid sequence encoding the first and/or second redox protein may be obtained from any biological source or may be prepared synthetically.\",\n \"In general, nucleic acid sequences encoding redox proteins are well known in the art and readily available.\",\n \"See, for example: Cristiano et al.\",\n \"(1993) Genomics 17: (2) 348-354, Doyama et al.\",\n \"(1998) Plant Sci.\",\n \"137: 53-62, Hoeoeg et al.\",\n \"(1984) Biosci.\",\n \"Rep. 4: 917-923; as well as the Swiss Protein sequences set forth in Table 5.Known nucleic acid sequences encoding redox proteins may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences Encoding redox proteins, for example by screening cDNA or genomic libraries.\",\n \"Thus, additional nucleic acid sequences may be discovered and used in accordance with the present invention.\",\n \"The nucleic acid sequence encoding the first and/or second redox protein may be obtained from separate sources or may be obtained from the same source.\",\n \"In general however, the nucleic acid sequence encoding a redox-fusion polypeptide comprises nucleic acid sequences encoding a first and a second redox protein obtained from the same or a similar biological source.\",\n \"In certain embodiments provided herein, wherein the nucleic acid sequence encoding the first and second redox protein is obtained from the same source, the nucleic acid sequence encoding the first redox protein and second redox protein may be naturally fused.\",\n \"In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second redox protein are preferably obtained from a plant source.\",\n \"As set forth above, a polypeptide spacer or linker of variable length may separate the first and second redox proteins from each other and/or from the oil-body-targeting-protein; and additional redox proteins (e.g., one or more) may be fused to the first and/or second redox protein.\",\n \"The nucleic acid sequences encoding the redox proteins may be altered to improve expression levels for example by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the redox proteins, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).\",\n \"Comparison of the codon usage of the redox protein with codon usage of the host will enable the identification of codons that may be changed.\",\n \"For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.\",\n \"By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.\",\n \"Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The redox proteins may be altered using for example, targeted mutagenesis, random mutagenesis (Shiraishi et al.\",\n \"(1998) Arch.\",\n \"Biochem.\",\n \"Biophys.\",\n \"358: 104-115; Galkin et al.\",\n \"(1997) Protein Eng.\",\n \"10: 687-690; Carugo et al.\",\n \"(1997) Proteins 28: 10-28; Hurley et al.\",\n \"(1996) Biochemistry 35: 5670-5678) (and/or by the addition of organic solvent (Holmberg et al.\",\n \"(1999) Protein Eng.\",\n \"12: 851-856).\",\n \"The polypeptide spacer between the first and second redox protein may be altered in similar ways.\",\n \"The first and second redox protein may be selected by developing a two-dimensional matrix and determining which combination of first and second redox protein is most effective in electron transport using for example, a colorometric reduction assay (Johnson et al (1984) J. of Bact.\",\n \"Vol.\",\n \"158 3:1061-1069, Luthman et al (1982) Biochemistry Vol 21 26:6628-2233).\",\n \"Combinations of thioredoxin and thioredoxin-reductase may be tested by determining the reduction of wheat storage proteins and milk storage protein beta-lactoglobulin in vitro (Del Val et al.\",\n \"(1999) J. Allerg.\",\n \"Clin.\",\n \"Immunol.\",\n \"103: 690-697).\",\n \"Using the same strategy polypeptide spacers between the first and second redox proteins may be evaluated for their efficiency.\",\n \"First and second redox proteins that may be used herein include without limitation any first redox protein and second redox protein selected from the group of redox proteins consisting of cytochromes, such as cytochrome a, cytochrome b and cytochrome c; porphyrin containing proteins, for example hemoglobin; iron-sulfur proteins, such as ferredoxin; flavoproteins such as thioredoxin-reductase, NADH dehydrogenase, succinate dehydrogenase, dihydrolipoyl dehydrogenase, acyl-CoA dehydrogenase, D-amino acid oxidase, xanthine oxidase, orotate reductase and aldehyde oxidase; pyridine-linked dehydrogenases, for example, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and beta-hydroxy-butarate dehydrogenase; and redox active thiol containing proteins such as thioredoxin.\",\n \"In particular embodiments, the redox proteins provided herein are thioredoxin and its reductant thioredoxin-reductase (which are jointly also referred to herein as “thioredoxin-related” protein(s)).\",\n \"As used herein, the term “thioredoxin” refers to relatively small proteins (typically approximately 12 kDa) that belong to the family of thioltransferases which catalyze oxido-reductions via the formation or hydrolysis of disulfide bonds and are widely, if not universally, distributed throughout the animal plant and bacterial kingdom.\",\n \"The reduces form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.\",\n \"In order to reduce the oxidized thioredoxin, two cellular reductants provide the reduction equivalents: reduced ferredoxin and NADPH.\",\n \"These reduction equivalents are supplied to thioredoxin via interaction or association with different thioredoxin-reductases including the NADPH thioredoxin-reductase and ferredoxin thioredoxin-reductase.\",\n \"The supply of these reduction equivalents requires the formation of a heteromultimeric-protein-complex comprising thioredoxin and thioredoxin-reductase.\",\n \"Ferredoxin thioredoxin-reductase is involved in the reduction of plant thioredoxins designated as Trxf and Trxm, both of which are involved in the regulation of photosynthetic processes in the chloroplast.\",\n \"The NADPH/thioredoxin active in plant seeds is designated Trxh (also referred to herein as thioredoxin h-type) and is capable of the reduction of a wide range of proteins thereby functioning as an important cellular redox buffer.\",\n \"Generally, only one kind of thioredoxin, which analogous to the plant Trxh type, is found in bacterial or animal cells.\",\n \"The h-type thioredoxins are capable of being reduced by NADPH and NADPH-thioredoxin reductase.\",\n \"Exemplary thioredoxins are further characterized as a protein having a core of 5 beta-sheets surrounded by 4 to 6 alpha helixes.\",\n \"Exemplary thioredoxins are further characterized by having an active site containing the consensus amino acid sequence: XCYYCZ, wherein Y is any amino acid, such as hydrophobic or non-polar amino acids, wherein X can be any of the 20 amino acids, preferably a hydrophobic amino acid, such as a tryptophan, and Z can be any amino acid, preferably polar amino acids.\",\n \"In certain embodiments, the thioredoxins for use herein comprise an active site having the amino acid sequence XCGPCZ.\",\n \"When the cysteines in the active site of thioredoxin or thioredoxin-like proteins are oxidized, they form an intramolecular disulfide bond.\",\n \"In the reduced state, the same active sites are capable of participating in redox reactions through the reversible oxidation of its active site dithiol, to a disulfide and catalyzes dithioldisulfide exchange reactions.\",\n \"Exemplary thioredoxins are well-known in the art and can be obtained from several organisms including Arabidopsis thaliana (Riveira Madrid et al.\",\n \"(1995) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"92: 5620-5624), wheat (Gautier et al.\",\n \"(1998) Eur.\",\n \"J. Biochem.\",\n \"252: 314-324); Escherichia coli (Hoeoeg et al (1984) Biosci.\",\n \"Rep. 4: 917-923) and thermophylic microorganisms such as Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126).\",\n \"Thioredoxins have also been recombinantly expressed in several host systems including bacteria (Gautier et al.\",\n \"(1998) Eur J. Biochem.\",\n \"252: 314-324) and plants (PCT Patent Application WO 00/58453) Commercial preparations of E. coli sourced Thioredoxins are readily available from for example: Sigma Cat No.\",\n \"T 0910 Thioredoxin (E. coli, recombinant; expressed in E. coli).\",\n \"Exemplary nucleic acid sequences encoding thioredoxin polypeptides for use herein are readily available from a variety of diverse biological sources including E. coli (Hoeoeg et al.\",\n \"(1984) Biosci.\",\n \"Rep.: 4 917-923); Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126); Arabidopsis thaliana (Rivera-Madrid (1995) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"92: 5620-5624); wheat (Gautier et al (1998) Eur.\",\n \"J. Biochem.\",\n \"252(2): 314-324); tobacco (Marty et al.\",\n \"(1991) Plant Mol.\",\n \"Biol.\",\n \"17: 143-148); barley (PCT Patent Application 00/58352); rice (Ishiwatari et al.\",\n \"(1995) Planta 195: 456-463); soybean (Shi et al.\",\n \"(1996) Plant Mol.\",\n \"Biol.\",\n \"32: 653-662); rapeseed (Bower et al.\",\n \"Plant Cell 8: 1641-1650) and calf (Terashima et al.\",\n \"(1999) DNA Seq.\",\n \"10(3): 203-205); and the like.\",\n \"In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth as SEQ ID NOs:38, 42, 46 and 50; and those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth in Table 5 as SEQ ID NOs:52-194.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:52-194 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).\",\n \"As used herein, the term “thioredoxin-reductase” refers to a protein that complexes with a flavin, such as FAD.\",\n \"The flavin compound serves as an electron donor for the thioredoxin-reductase protein active site.\",\n \"Thioredoxin reductases have a redox active, disulfide bond site capable of reducing thioredoxin.\",\n \"The active site of thioredoxin-reductase contains 2 cysteines.\",\n \"The type of amino acids surrounding the 2 cysteine residues forming the active site can vary as hydrophobic, non-polar or polar.\",\n \"An exemplary thioredoxin-reductase is NADPH-thioredoxin-reductase (NTR), which is a cytosolic homodimeric enzyme comprising typically 300-500 amino acids.\",\n \"Crystal structures of both E. coli and plant thioredoxin-reductase have been obtained (Waksman et al.\",\n \"(1994) J. Mol.\",\n \"Biol.\",\n \"236: 800-816; Dai et al.\",\n \"(1996) J. Mol.\",\n \"Biol.\",\n \"264:1044-1057).\",\n \"NADPH-thioredoxin-reductases have been expressed in heterologous hosts, for example the Arabidopsis NADPH-thioredoxin-reductase has been expressed in E. coli (Jacquot et al.\",\n \"(1994) J. Mol.\",\n \"Biol.\",\n \"235: 1357-1363) and wheat (PCT Patent Application 00/58453).\",\n \"Exemplary nucleic acid sequences encoding thioredoxin-reductase proteins can readily be obtained from a variety of sources, such as from the sequence set forth in Table 5 and the Sequence Listing provide herein, from Arabidopsis (Riveira Madrid et al.\",\n \"(1995) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 92: 5620-5624), E. coli (Russel et al.\",\n \"(1988) J. Biol.\",\n \"Chem.\",\n \"263: 9015-9019); barley (PCT Patent Application 00158352 and wheat (Gautier et al., (1998) Eur.\",\n \"J. Biochem.\",\n \"252: 314-324); and the like.\",\n \"In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin-reductase polypeptide chains set forth as SEQ ID NOs:8, 9, 10, 40, 44, 48 and 50; and those encoding the thioredoxin-reductase polypeptide chains set forth in Table 5 as SEQ ID NOs:195-313.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:195-313 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).\",\n \"Also contemplated for use in the methods and compositions provided herein are nucleic acid and amino acid homologs that are “substantially homologous” to the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein, which includes thioredoxin and thioredoxin-reductase polypeptides encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides encoding the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein (e.g., in the Examples, Sequence Listing and/or Table 5).\",\n \"As used herein, a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.\",\n \"By the term “substantially homologous” is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.\",\n \"The terms “homology” and “identity” are often used interchangeably.\",\n \"In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program.\",\n \"The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol.\",\n \"Biol.\",\n \"48:443 (1970), as revised by Smith and Waterman (Adv.\",\n \"Appl.\",\n \"Math.\",\n \"2:482 (1981).\",\n \"Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.\",\n \"The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl.\",\n \"Acids Res.\",\n \"14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.\",\n \"353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.\",\n \"By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier.\",\n \"Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest.\",\n \"Preferably the two molecules will hybridize under conditions of high stringency.\",\n \"Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.\",\n \"Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 85:2444 (1988).\",\n \"Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.\",\n \"In general, sequences are aligned so that the highest order match is obtained.\",\n \"“Identity” per se has an art-recognized meaning and can be calculated using published techniques.\",\n \"(See, e.g.\",\n \": Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).\",\n \"While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).\",\n \"Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).\",\n \"Methods to determine identity and similarity are codified in computer programs.\",\n \"Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).\",\n \"Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide.\",\n \"For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.\",\n \"As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides.\",\n \"Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared.\",\n \"No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides.\",\n \"Similar comparisons may be made between a test and reference polynucleotides.\",\n \"Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g.\",\n \"10/100 amino acid difference (approximately 90% identity).\",\n \"Differences are defined as nucleic acid or amino acid substitutions, or deletions.\",\n \"As used herein: stringency of hybridization in determining percentage mismatch is as follows: 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C. 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. Those of skill in this art know that the washing step selects for stable hybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), vol.\",\n \"3, p. B.\",\n \"13, see, also, numerous catalogs that describe commonly used laboratory solutions).\",\n \"SSPE is pH 7.4 phosphate-buffered 0.18 NaCl.\",\n \"Further, those of skill in the art recognize that the stability of hybrids is determined by Tm, which is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6(log10[Na+])+0.41 (% G+C)−600/l)), so that the only parameters in the wash conditions critical to hybrid stability are sodium ion concentration in the SSPE (or SSC) and temperature.\",\n \"It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.\",\n \"By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 78:6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate, adjusted to a pH of 7).\",\n \"In a particular embodiment, a heteromultimeric-protein-complex is produced as a fusion polypeptide between the first and second redox protein, wherein the first redox protein is thioredoxin and the second redox protein is a thioredoxin-reductase.\",\n \"In one embodiment, the second recombinant polypeptide, e.g., the thioredoxin-reductase is positioned N-terminal relative to the first recombinant polypeptide, e.g., the thioredoxin.\",\n \"Accordingly, any protein which is classified as thioredoxin, such as the thioredoxin component of the NADPH thioredoxin system and the thioredoxin present in the ferredoxin/thioredoxin system also known as TRx and TRm may be used in combination with any thioredoxin-reductase such as the NADPH thioredoxin-reductase and the ferredoxin-thioredoxin-reductase and any other proteins having the capability of reducing thioredoxin.\",\n \"In particular embodiments the thioredoxin and thioredoxin-reductase are plant derived.\",\n \"In an alternate embodiment, the naturally occurring nucleic acid sequence encoding the thioredoxin/thioredoxin-reductase protein fusion obtainable from Mycobacterium leprae (Wieles et al.\",\n \"(1995) J. Biol.\",\n \"Chem.\",\n \"27:25604-25606) is used, as set forth in the Examples herein.\",\n \"Immunoglobulins In another embodiment of the present invention, the multimeric-protein-complexes are immunoglobulins.\",\n \"As used herein “immunoglobulin-polypeptide-chain” refers to a first polypeptide that is capable of associating with a second polypeptide to form an immunologically active (i.e.\",\n \"capable of antigen binding multimeric-protein-complex.\",\n \"The types of immunoglobulins and immunoglobulin-polypeptide-chains contemplated for use herein include the immunologically active (i.e.\",\n \"antigen binding) portions of a light and heavy chain.\",\n \"Exemplary immunoglobulins and immunoglobulin-polypeptide-chains for use herein include substantially intact immunoglobulins, including any IgG, IgA, IgD, IgE and IgM, as well as any portion of an immunoglobulin, including those portions well-known as Fab fragments, Fab′ fragments, F(ab′).sub2.fragments and Fv fragments.\",\n \"In this embodiment, the first recombinant polypeptide may be any immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain.\",\n \"Accordingly, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.\",\n \"As set forth herein, the multimeric immunoglobulin is associated with an oil body through an oil-body-targeting-protein.\",\n \"In particular embodiments, the oil-body-targeting-protein may be a fusion polypeptide comprising an oil-body-protein and an immunoglobulin binding protein, such as for example protein A, protein L, and protein G. In yet another embodiment involving immunoglobulins, the first and second recombinant polypeptides (immunoglobulins) are separately fused to an oil body protein, for example an oleosin or caleosin.\",\n \"For example, a) the first recombinant polypeptide may be an immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or b) the first recombinant polypeptide may be the variable and first constant domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or c) the first recombinant polypeptide may be the variable domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be the variable domain from a kappa or lambda immunoglobulin light chain.\",\n \"In certain embodiments, the fusion polypeptides are designed or selected to allow the heteromultimeric-protein-complex formation between immunoglobulin light and heavy chain sequences on the oil bodies within the cell comprising oil bodies.\",\n \"Preparation of Expression Vectors Comprising Oil-Body-Targeting-Proteins and the First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, or the First and/or Second Thioredoxin-Related Proteins.\",\n \"In accordance with the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are conveniently produced in a cell.\",\n \"In order to produce the recombinant polypeptides or multimeric-protein-complexes, a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein are incorporated in a recombinant expression vector.\",\n \"Accordingly, provided herein are recombinant expression vectors comprising the chimeric nucleic acids provided herein suitable for expression of the oil-body-targeting-protein and the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, suitable for the selected cell.\",\n \"The term “suitable for expression in the selected cell” means that the recombinant expression vector contains all nucleic acid sequences required to ensure expression in the selected cell.\",\n \"Accordingly, the recombinant expression vectors further contain regulatory nucleic acid sequences selected on the basis of the cell which is used for expression and ensuring initiation and termination of transcription operatively linked to the nucleic acid sequence encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein.\",\n \"Regulatory nucleic acid sequences include promoters, enhancers, silencing elements, ribosome binding sites, Shine-Dalgarno sequences, introns and other expression elements.\",\n \"“Operatively linked” is intended to mean that the nucleic acid sequences comprising the regulatory regions linked to the nucleic acid sequences encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein allow expression in the cell.\",\n \"A typical nucleic acid construct comprises in the 5′ to 3′ direction a promoter region capable of directing expression, a coding region comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or an oil-body-targeting-protein and a termination region functional in the selected cell.\",\n \"The selection of regulatory sequences will depend on the organism and the cell type in which the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein is expressed, and may influence the expression levels of the polypeptide.\",\n \"Regulatory sequences are art-recognized and selected to direct expression of the oil-body-targeting-protein and the recombinant polypeptides or multimeric-protein-complexes in the cell.\",\n \"Promoters that may be used in bacterial cells include the lac promoter (Blackman et al.\",\n \"(1978) Cell: 13: 65-71), the trp promoter (Masuda et al.\",\n \"(1996) Protein Eng: 9: 101-106) and the T7 promoters (Studier et al.\",\n \"(1986) J. Mol.\",\n \"Biol.\",\n \"189: 113-130).\",\n \"Promoters functional in plant cells that may be used herein include constitutive promoters such as the 35S CaMV promoter (Rothstein et al.\",\n \"(1987) Gene: 53: 153-161) the actin promoter (McElroy et al.\",\n \"(1990) Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).\",\n \"Other promoters are specific to certain tissues or organs (for example, roots, leaves, flowers or seeds) or cell types (for example, leaf epidermal cells, mesophyll cells or root cortex cells) and or to certain stages of plant development.\",\n \"Timing of expression may be controlled by selecting an inducible promoter, for example the PR-a promoter described in U.S. Pat.\",\n \"No.\",\n \"5,614,395.Selection of the promoter therefore depends on the desired location and timing of the accumulation of the desired polypeptide.\",\n \"In a particular embodiment, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are expressed in a seed cell and seed specific promoters are utilized.\",\n \"Seed specific promoters that may be used herein include for example the phaseolin promoter (Sengupta-Gopalan et al.\",\n \"(1985) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA: 82: 3320-3324), and the Arabidopsis 18 kDa oleosin promoter (van Rooijen et al.\",\n \"(1992) Plant.\",\n \"Mol.\",\n \"Biol.\",\n \"18: 1177-1179).\",\n \"New promoters useful in various plant cell types are constantly discovered.\",\n \"Numerous examples of plant promoters may be found in Ohamuro et al.\",\n \"(Biochem of Pl.\",\n \"(1989) 15: 1-82).\",\n \"Genetic elements capable of enhancing expression of the polypeptide may be included in the expression vectors.\",\n \"In plant cells these include for example, the untranslated leader sequences from viruses such as the AMV leader sequence (Jobling and Gehrke (1987) Nature: 325: 622-625) and the intron associated with the maize ubiquitin promoter (See: U.S. Pat.\",\n \"No.\",\n \"5,504,200).\",\n \"Transcriptional terminators are generally art recognized and besides serving as a signal for transcription termination serve as a protective element serving to extend the mRNA half-life (Guarneros et al.\",\n \"(1982) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA: 79: 238-242).\",\n \"In nucleic acid sequences for the expression in plant cells, the transcriptional terminator typically is from about 200 nucleotide to about 1000 nucleotides in length.\",\n \"Terminator sequences that may be used herein include for example, the nopaline synthase termination region (Bevan et al.\",\n \"(1983) Nucl.\",\n \"Acid.\",\n \"Res.\",\n \": 11: 369-385), the phaseolin terminator (van der Geest et al.\",\n \"(1994) Plant J.: δ: 413-423), the terminator for the octopine synthase gene of Agrobacterium tumefaciens or other similarly functioning elements.\",\n \"Transcriptional terminators can be obtained as described by An (1987) Methods in Enzym.\",\n \"153: 292).\",\n \"The selection of the transcriptional terminator may have an effect on the rate of transcription.\",\n \"Accordingly, provided herein are chimeric nucleic acid sequences encoding a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins.\",\n \"In one embodiment, said nucleic acid comprises: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide, immunoglobulin-polypeptide-chain, or redox protein; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, immunoglobulin-polypeptide-chain or redox protein, wherein said first and second recombinant polypeptides, immunoglobulin-polypeptide-chains or redox proteins are capable of forming a multimeric-protein-complex.\",\n \"In another embodiment, provided herein is an expression vector comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a nucleic acid sequence encoding a multimeric-fusion-protein, such as a redox fusion polypeptide or immunoglobulin, comprising a first recombinant polypeptide, such as a redox protein or immunoglobulin-polypeptide-chain, linked to a second recombinant polypeptide, such as a second redox protein or a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.\",\n \"The recombinant expression vector further may contain a marker gene.\",\n \"Marker genes that may be used in accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells including all selectable and screenable marker genes.\",\n \"A marker may be a resistance marker such as an antibiotic resistance marker against for example kanamycin, ampicillin, G418, bleomycin hygromycin, chloramphenicol which allows selection of a trait by chemical means or a tolerance marker against for example a chemical agent such as the normally phytotoxic sugar mannose (Negrotto et al.\",\n \"(2000) Plant Cell Rep. 19: 798-803).\",\n \"In plant recombinant expression vectors herbicide resistance markers may conveniently be used for example markers conferring resistance against glyphosate (U.S. Pat.\",\n \"Nos.\",\n \"4,940,935 and 5,188,642) or phosphinothricin (White et al.\",\n \"(1990) Nucl.\",\n \"Acids Res.\",\n \"18: 1062; Spencer et al.\",\n \"(1990) Theor.\",\n \"Appl.\",\n \"Genet.\",\n \"79: 625-631).\",\n \"Resistance markers to a herbicide when linked in close proximity to the redox protein or oil-body-targeting-protein may be used to maintain selection pressure on a population of plant cells or plants for those plants that have not lost the protein of interest.\",\n \"Screenable markers that may be employed to identify transformants through visual observation include beta-glucuronidase (GUS) (see U.S. Pat.\",\n \"No.\",\n \"5,268,463 and U.S. Pat.\",\n \"No.\",\n \"5,599,670) and green fluorescent protein (GFP) (Niedz et al.\",\n \"(1995) Plant Cell Rep.: 14: 403).\",\n \"The recombinant expression vectors further may contain nucleic acid sequences encoding targeting signals ensuring targeting to a cell compartment or organelle.\",\n \"Suitable targeting signals that may be used herein include those that are capable of targeting polypeptides to the endomembrane system.\",\n \"Exemplary targeting signals that may be used herein include targeting signals capable of directing the protein to the periplasm, the cytoplasm, the golgi apparatus, the apoplast (Sijmons et al., 1990, Bio/Technology, 8:217-221) the chloroplast (Comai et al.\",\n \"(1988) J. Biol.\",\n \"Chem.\",\n \"263: 15104-15109), the mitochondrion, the peroxisome (Unger et al.\",\n \"(1989) Plant Mol.\",\n \"Biol.\",\n \"13: 411-418), the ER, the vacuole (Shinshi et al.\",\n \"(1990) Plant Mol.\",\n \"Biol.\",\n \"14: 357-368 and the oil body.\",\n \"By the inclusion of the appropriate targeting sequences it is possible to direct the oil-body-targeting-protein or the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, to the desired organelle or cell compartment.\",\n \"The recombinant expression vectors of the present invention may be prepared in accordance with methodologies well known to those of skill in the art of molecular biology (see for example: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press).\",\n \"The preparation of these constructs may involve techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing and PCR.\",\n \"A wide variety of cloning vectors is available to perform the necessary cloning steps resulting in a recombinant expression vector ensuring expression of the polypeptide.\",\n \"Especially suitable for this purpose are vectors with a replication system that is functional in Escherichia coli such as pBR322, the PUC series of vectors, the M13 mp series of vectors, pBluescript etc.\",\n \"Typically these vectors contain a marker allowing the selection of transformed cells for example by conferring antibiotic resistance.\",\n \"Nucleic acid sequences may be introduced in these vectors and the vectors may be introduced in E. coli grown in an appropriate medium.\",\n \"Vectors may be recovered from cells upon harvesting and lysing the cells.\",\n \"Recombinant expression vectors suitable for the introduction of nucleic acid sequences in plant cells include Agrobacterium and Rhizobium based vectors such as the Ti and Ri plasmids.\",\n \"Agrobacterium based vectors typically carry at least one T-DNA border sequence and include vectors such pBIN 19 (Bevan (1984) Nucl Acids Res.\",\n \"Vol.\",\n \"12, 22:8711-8721) and other binary vector systems (for example: U.S. Pat.\",\n \"No.\",\n \"4,940,838).\",\n \"Production of Cells Comprising a First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, and/or a First and/or Second Thioredoxin-Related Protein and Oil-Body-Targeting-Proteins In accordance with the present invention, the recombinant expression vectors are introduced into the cell that is selected and the selected cells are grown to produce the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, a first and/or second thioredoxin-related protein; and the oil-body-targeting-protein either directly or in a progeny cell.\",\n \"Methodologies to introduce recombinant expression vectors into a cell also referred to herein as “transformation” are well known to the art and vary depending on the cell type that is selected.\",\n \"General techniques to transfer the recombinant expression vectors into the cell include electroporation; chemically mediated techniques, for example CaCl2 mediated nucleic acid uptake; particle bombardment (biolistics); the use of naturally infective nucleic acid sequences for example virally derived nucleic acid sequences or when plant cells are used Agrobacterium or Rhizobium derived nucleic acid sequences; PEG mediated nucleic acid uptake, microinjection, and the use of silicone carbide whiskers (Kaeppler et al.\",\n \"(1990) Plant Cell Rep. 9:415-418) all of which may be used herein.\",\n \"Introduction of the recombinant expression vector into the cell may result in integration of its whole or partial uptake into host cell genome including the chromosomal DNA or the plastid genome.\",\n \"Alternatively the recombinant expression vector may not be integrated into the genome and replicate independently of the host cell's genomic DNA.\",\n \"Genomic integration of the nucleic acid sequence is typically used as it will allow for stable inheritance of the introduced nucleic acid sequences by subsequent generations of cells and the creation of cell, plant or animal lines.\",\n \"Particular embodiments involve the use of plant cells.\",\n \"Particular plant cells used herein include cells obtainable from Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.\",\n \"); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.\",\n \"and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.\",\n \"); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticum aestivum) and sunflower (Helianthus annuus).\",\n \"Transformation methodologies for dicotelydenous plant species are well known.\",\n \"Generally Agrobacterium mediated transformation is utilized because of its high efficiency as well as the general susceptibility by many, if not all dicotelydenous plant species.\",\n \"Agrobacterium transformation generally involves the transfer of a binary vector (e.g.\",\n \"pBIN19) comprising the DNA of interest to an appropriate Agrobacterium strain (e.g.\",\n \"CIB542) by for example tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilization of the binary vector to the target Agrobacterium strain, or by DNA transformation of the Agrobacterium strain (Hofgen et al.\",\n \"Nucl.\",\n \"Acids.\",\n \"Res.\",\n \"(1988) 16: 9877.Other transformation methodologies that may be used to transform dicotelydenous plant species include biolistics (Sanford (1988) Trends in Biotechn.\",\n \"6: 299-302); electroporation (Fromm et al.\",\n \"(1985) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 82: 5824-5828); PEG mediated DNA uptake (Potrykus et al.\",\n \"(1985) Mol.\",\n \"Gen. Genetics 199: 169-177); microinjection (Reich et al.\",\n \"Bio/Techn.\",\n \"(1986) 4: 1001-1004) and silicone carbide whiskers (Kaeppler et al.\",\n \"(1990) Plant Cell Rep. 9: 415-418).\",\n \"The exact transformation methodologies typically vary somewhat depending on the plant species that is used.\",\n \"In a particular embodiment the oil bodies are obtained from safflower and the recombinant proteins are expressed in safflower.\",\n \"Safflower transformation has been described by Baker and Dyer (Plant Cell Rep. (1996) 16: 106-110).\",\n \"Monocotelydenous plant species may now also be transformed using a variety of methodologies including particle bombardment (Christou et al.\",\n \"(1991) Biotechn.\",\n \"9: 957-962; Weeks et al.\",\n \"Plant Physiol.\",\n \"(1993) 102: 1077-1084; Gordon-Kamm et al.\",\n \"Plant Cell (1990) 2: 603-618) PEG mediated DNA uptake (EP 0 292 435; 0 392 225) or Agrobacterium-mediated transformation (Goto-Fumiyuki et al (1999) Nature-Biotech.\",\n \"17 (3):282-286).\",\n \"Plastid transformation is described in U.S. Pat.\",\n \"Nos.\",\n \"5,451,513; 5,545,817 and 5,545,818; and PCT Patent Applications 95/16783; 98/11235 and 00/39313) Basic chloroplast transformation involves the introduction of cloned plastid DNA flanking a selectable marker together with the nucleic acid sequence of interest into a suitable target tissue using for example biolistics or protoplast transformation.\",\n \"Selectable markers that may be used include for example the bacterial aadA gene (Svab et al.\",\n \"(1993) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90: 913-917).\",\n \"Plastid promoters that may be used include for example the tobacco clpP gene promoter (PCT Patent Application 97/06250).\",\n \"In another embodiment, the invention chimeric nucleic acid constructs provided herein are directly transformed into the plastid genome.\",\n \"Plastid transformation technology is described extensively in U.S. Pat.\",\n \"Nos.\",\n \"5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos.\",\n \"WO 95/16783 and WO 97/32977; and in McBride et.\",\n \"al., Proc Natl Acad Sci USA 91: 7301-7305 (1994), the entire disclosures of all of which are hereby incorporated by reference.\",\n \"In one embodiment, plastid transformation is achieved via biolistics, first carried out in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al.\",\n \"(1988) Science 240:1534-1537)) and then extended to Nicotiana tabacum (Svab et al.\",\n \"(1990) Proc Natl Acad Sci USA 87:8526-8530), combined with selection for cis-acting antibiotic resistance loci (spectinomycin or streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes.\",\n \"In another embodiment, tobacco plastid transformation is carried out by particle bombardment of leaf or callus tissue, or polyethylene glycol (PEG)-mediated uptake of plasmid DNA by protoplasts, using cloned plastid DNA flanking a selectable antibiotic resistance marker.\",\n \"For example, 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome.\",\n \"In one embodiment, point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin can be utilized as selectable markers for transformation (Svab et al.\",\n \"(1990) Proc Natl Acad Sci USA 87:8526-8530; Staub et al.\",\n \"(1992) Plant Cell 4:39-45, the entire disclosures of which are hereby incorporated by reference), resulting in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.\",\n \"The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al.\",\n \"(1993) EMBO J 12:601-606, the entire disclosure of which is hereby incorporated by reference).\",\n \"In another embodiment, substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al.\",\n \"(1993) Proc Natl Acad Sci USA 90: 913-917, the entire disclosure of which is hereby incorporated by reference).\",\n \"This marker has also been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl Acids Res 19, 4083-4089, the entire disclosure of which is hereby incorporated by reference).\",\n \"In other embodiments, plastid transformation of protoplasts from tobacco and the moss Physcomitrella can be attained using PEG-mediated DNA uptake (O'Neill et al.\",\n \"(1993) Plant J 3:729-738; Koop et al.\",\n \"(1996) Planta 199:193-201, the entire disclosures of which are hereby incorporated by reference).\",\n \"Both particle bombardment and protoplast transformation are also contemplated for use herein.\",\n \"Plastid transformation of oilseed plants has been successfully carried out in the genera Arabidopsis and Brassica (Sikdar et al.\",\n \"(1998) Plant Cell Rep 18:20-24; PCT Application WO 00/39313, the entire disclosures of which are hereby incorporated by reference).\",\n \"A chimeric nucleic sequence construct is inserted into a plastid expression cassette including a promoter capable of expressing the construct in plant plastids.\",\n \"A particular promoter capable of expression in a plant plastid is, for example, a promoter isolated from the 5′ flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues.\",\n \"Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (Stern et al.\",\n \"(1997) Trends in Plant Sci 2:308-315, the entire disclosure of which is hereby incorporated by reference).\",\n \"Plastid promoters generally contain the −35 and −10 elements typical of prokaryotic promoters, and some plastid promoters called PEP (plastid-encoded RNA polymerase) promoters are recognized by an E. coli-like RNA polymerase mostly encoded in the plastid genome, while other plastid promoters called NEP promoters are recognized by a nuclear-encoded RNA polymerase.\",\n \"Both types of plastid promoters are suitable for use herein.\",\n \"Examples of plastid promoters include promoters of clpP genes such as the tobacco clpP gene promoter (WO 97/06250, the entire disclosure of which is hereby incorporated by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser.\",\n \"No.\",\n \"09/038,878, the entire disclosure of which is hereby incorporated by reference).\",\n \"Another promoter capable of driving expression of a chimeric nucleic acid construct in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., (1994) Microbiol Rev 58:700-754; Shinozaki et al.\",\n \"(1986) EMBO J 5:2043-2049, the entire disclosures of both of which are hereby incorporated by reference).\",\n \"Other examples of promoters capable of driving expression of a nucleic acid construct in plant plastids include a psbA promoter or am rbcL promoter.\",\n \"A plastid expression cassette preferably further includes a plastid gene 3′ untranslated sequence (3′ UTR) operatively linked to a chimeric nucleic acid construct of the present invention.\",\n \"The role of untranslated sequences is preferably to direct the 3′ processing of the transcribed RNA rather than termination of transcription.\",\n \"An exemplary 3′ UTR is a plastid rps16 gene 3′ untranslated sequence, or the Arabidopsis plastid psbA gene 3′ untranslated sequence.\",\n \"In a further embodiment, a plastid expression cassette includes a poly-G tract instead of a 3′ untranslated sequence.\",\n \"A plastid expression cassette also preferably further includes a 5′ untranslated sequence (5′ UTR) functional in plant plastids, operatively linked to a chimeric nucleic acid construct provided herein.\",\n \"A plastid expression cassette is contained in a plastid transformation vector, which preferably further includes flanking regions for integration into the plastid genome by homologous recombination.\",\n \"The plastid transformation vector may optionally include at least one plastid origin of replication.\",\n \"The present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the chimeric nucleic acid construct is expressible in the plant plastid.\",\n \"Also encompassed herein is a plant or plant cell, including the progeny thereof, including this plant plastid.\",\n \"In a particular embodiment, the plant or plant cell, including the progeny thereof, is homoplasmic for transgenic plastids.\",\n \"Other promoters capable of driving expression of a chimeric nucleic acid construct in plant plastids include transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected.\",\n \"In these cases, the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA.\",\n \"The transactivator is targeted to plastids using a plastid transit peptide.\",\n \"The transactivator and the transactivator-driven DNA molecule are brought together either by crossing a selected plastid-transformed line with and a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a chimeric nucleic acid construct encoding the transactivator supplemented with a plastid-targeting sequence operably linked to a nuclear promoter.\",\n \"If the nuclear promoter is an inducible promoter, in particular a chemically inducible embodiment, expression of the chimeric nucleic acid construct in the plastids of plants is activated by foliar application of a chemical inducer.\",\n \"Such an inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction.\",\n \"A particular transactivator is, for example, viral RNA polymerase.\",\n \"Particular promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase.\",\n \"The gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide.\",\n \"Promoters suitable for nuclear expression of a gene, for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described above and elsewhere in this application.\",\n \"Expression of chimeric nucleic acid constructs in plastids can be constitutive or can be inducible, and such plastid expression can be also organ- or tissue-specific.\",\n \"Examples of various expression systems are extensively described in WO 98/11235, the entire disclosure of which is hereby incorporated by reference.\",\n \"Thus, in one aspect, the present invention utilizes coupled expression in the nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under the control of the chemically inducible PR-1a promoter, for example of the PR-1 promoter of tobacco, operably linked with a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences, for example as described in as in U.S. Pat.\",\n \"No.\",\n \"5,614,395 the entire disclosure of which is hereby incorporated by reference.\",\n \"In another embodiment, when plastid transformants homoplasmic for the maternally inherited TR or NTR genes are pollinated by lines expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry both transgene constructs but do not express them until synthesis of large amounts of enzymatically active protein in the plastids is triggered by foliar application of the PR-1a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH).\",\n \"In a particular embodiment, two or more genes, for example TR and NTR genes, are transcribed from the plastid genome from a single promoter in an operon-like polycistronic gene.\",\n \"In one embodiment, the operon-like polycistronic gene includes an intervening DNA sequence between two genes in the operon-like polycistronic gene.\",\n \"In a particular embodiment, the intervening DNA sequence is not present in the plastid genome to avoid homologous recombination with plastid sequences.\",\n \"In another embodiment, the DNA sequence is derived from the 5′ untranslated (UTR) region of a non-eukaryotic gene, preferably from a viral 5′UTR, preferably from a 6′UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage.\",\n \"In one embodiment, a portion of the DNA sequence may be modified to prevent the formation of RNA secondary structures in an RNA transcript of the operon-like polycistronic gene, for example between the DNA sequence and the RBS of the downstream gene.\",\n \"Such secondary structures may inhibit or repress the expression of the downstream gene, particularly the initiation of translation.\",\n \"Such RNA secondary structures are predicted by determining their melting temperatures using computer models and programs such a the “mfold” program version 3 (available from Zuker and Turner, Washington University School of Medicine, St-Louis, Mo.)\",\n \"and other methods known to one skilled in the art.\",\n \"The presence of the intervening DNA sequence in the operon-like polycistronic gene increases the accessibility of the RBS of the downstream gene, thus resulting in higher rates of expression.\",\n \"Such strategy is applicable to any two or more genes to be transcribed from the plastid genome from a single promoter in an operon-like chimeric heteromultimeric gene.\",\n \"Following transformation the cells are grown, typically in a selective medium allowing the identification of transformants.\",\n \"Cells may be harvested in accordance with methodologies known to the art.\",\n \"In order to associate the oil bodies with the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and/or second thioredoxin-related protein, the integrity of cells may be disrupted using any physical, chemical or biological methodology capable of disrupting the cells' integrity.\",\n \"These methodologies are generally cell-type dependent and known to the skilled artisan.\",\n \"Where plants are employed they may be regenerated into mature plants using plant tissue culture techniques generally known to the skilled artisan.\",\n \"Seeds may be harvested from mature transformed plants and used to propagate the plant line.\",\n \"Plants may also be crossed and in this manner, contemplated herein is the breeding of cells lines and transgenic plants that vary in genetic background.\",\n \"It is also possible to cross a plant line comprising the first recombinant polypeptide with a plant line comprising the second recombinant polypeptide.\",\n \"Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox protein (e.g., a thioredoxin-related protein, and the like) or an immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox protein (e.g., a thioredoxin-related protein, and the like) or a second immunoglobulin-polypeptide-chain; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"The second recombinant polypeptide may also associate with the oil bodies.\",\n \"Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox (or thioredoxin-related) protein or immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox (thioredoxin-related) protein or a second immunoglobulin-polypeptide-chain, wherein said second recombinant polypeptide is capable of associating with said oil bodies through an oil body targeting protein; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.\",\n \"Isolation of Oil Bodies The oil bodies provided herein may be obtained from any cell containing oil bodies, including any animal cell; plant cell; fungal cell; for example a yeast cell, algae cell; or bacterial cell.\",\n \"Any process suitable for the isolation oil bodies from cells may be used herein.\",\n \"Processes for the isolation of oil bodies from plant seed cells have been described in U.S. Pat.\",\n \"Nos.\",\n \"(6,146,645 and 6,183,762) and the isolation of oil bodies from yeast cells has been described by Ting et al.\",\n \"(1997) J. Biol.\",\n \"Chem.\",\n \"272: 3699-3706).\",\n \"In certain embodiments, the oil bodies are obtained from a plant cell such as for example a pollen cell; a fruit cell; a spore cell; a nut cell; mesocarp cell; for example the mesocarp cells obtainable from olive (Olea europaea) or avocado (Persea americana); or a seed cell.\",\n \"In particular embodiments the oil bodies are obtained from a plant seed cell.\",\n \"The seeds can be obtained from a transgenic plant according to the present invention.\",\n \"In particular embodiments, a seed of a transgenic plant according to the present invention contains the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or first and/or second thioredoxin-related proteins in a concentration of at least about 0.5% of total cellular seed protein.\",\n \"In further embodiments, a seed of a transgenic plant provided herein contains a recombinant polypeptide or multimeric-protein-complex in a concentration of at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, of total cellular seed protein.\",\n \"The upper limits of the recombinant polypeptide or multimeric-protein-complex concentration can be up to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.\",\n \"Thus, the ranges at least about 0.5% up to about 15%; at least about 1.0% up to about 10%; and at least about 5% up to about 8% are among the various ranges contemplated herein.\",\n \"Among the plant seeds useful in this regard are plant seeds obtainable from the group of plant species consisting of Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.\",\n \"); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.\",\n \"and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.\",\n \"); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); sunflower (Helianthus annuus); barley (Hordeum vulgare); wheat (Traeticum aestivum) and mixtures thereof.\",\n \"In a particular embodiment, oil bodies are obtainable from the seeds obtainable from safflower (Carthamus tinctorius).\",\n \"In order to prepare oil bodies from plant seeds, plants are grown and allowed to set seed in accordance with common agricultural practices.\",\n \"Thus, the present invention also provides seeds comprising oil bodies, wherein said oil bodies further comprise invention multimeric-protein-complexes described herein.\",\n \"Upon harvesting the seed and, if necessary the removal of large insoluble materials such as stones or seed hulls, by for example sieving or rinsing, any process suitable for the isolation of oil bodies from seeds may be used herein.\",\n \"A typical process involves grinding of the seeds followed by an aqueous extraction process.\",\n \"Seed grinding may be accomplished by any comminuting process resulting in a substantial disruption of the seed cell membrane and cell walls without compromising the structural integrity of the oil bodies present in the seed cell.\",\n \"Suitable grinding processes in this regard include mechanical pressing and milling of the seed.\",\n \"Wet milling processes such as described for cotton (Lawhon et al.\",\n \"(1977) J.\",\n \"Am.\",\n \"Oil Chem.\",\n \"Soc.\",\n \"63: 533-534) and soybean (U.S. Pat.\",\n \"No.\",\n \"3,971,856; Carter et al.\",\n \"(1974) J.\",\n \"Am.\",\n \"Oil Chem.\",\n \"Soc.\",\n \"51: 137-141) are particularly useful in this regard.\",\n \"Suitable milling equipment capable of industrial scale seed milling include colloid mills, disc mills, pin mills, orbital mills, IKA mills and industrial scale homogenizers.\",\n \"The selection of the milling equipment will depend on the seed, which is selected, as well as the throughput requirement.\",\n \"Solid contaminants such as seed hulls, fibrous materials, undissolved carbohydrates, proteins and other insoluble contaminants are subsequently preferably removed from the ground seed fraction using size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.\",\n \"Centrifugation may be accomplished using for example a decantation centrifuge such as a HASCO 200 2-phase decantation centrifuge or an NX310B (Alpha Laval).\",\n \"Operating conditions are selected such that a substantial portion of the insoluble contaminants and sediments and may be separated from the soluble fraction.\",\n \"Following the removal of insolubles the oil body fraction may be separated from the aqueous fraction.\",\n \"Gravitational based methods as well as size exclusion based technologies may be used.\",\n \"Gravitational based methods that may be used include centrifugation using for example a tubular bowl centrifuge such as a Sharples AS-16 or AS-46 (Alpha Laval), a disc stack centrifuge or a hydrocyclone, or separation of the phases under natural gravitation.\",\n \"Size exclusion methodologies that may be used include membrane ultra filtration and crossflow microfiltration.\",\n \"Separation of solids and separation of the oil body phase from the aqueous phase may also be carried out concomitantly using gravity based separation methods or size exclusion based methods.\",\n \"The oil body preparations obtained at this stage in the process are generally relatively crude and depending on the application of the oil bodies, it may be desirable to remove additional contaminants.\",\n \"Any process capable of removing additional seed contaminants may be used in this regard.\",\n \"Conveniently the removal of these contaminants from the oil body preparation may be accomplished by resuspending the oil body preparation in an aqueous phase and re-centrifuging the resuspended fraction, a process referred to herein as “washing the oil bodies”.\",\n \"The washing conditions selected may vary depending on the desired purity of the oil body fractions.\",\n \"For example where oil bodies are used in pharmaceutical compositions, generally a higher degree of purity may be desirable than when the oil bodies are used in food preparations.\",\n \"The oil bodies may be washed one or more times depending on the desired purity and the ionic strength, pH and temperature may all be varied.\",\n \"Analytical techniques may be used to monitor the removal of contaminants.\",\n \"For example SDS gel electrophoresis may be employed to monitor the removal of seed proteins.\",\n \"The entire oil body isolation process may be performed in a batch wise fashion or continuous flow.\",\n \"In a particular embodiment, industrial scale continuous flow processes are utilized.\",\n \"Through the application of these and similar techniques the skilled artisan is able to obtain oil bodies from any cell comprising oil bodies.\",\n \"The skilled artisan will recognize that generally the process will vary somewhat depending on the cell type that is selected.\",\n \"However, such variations may be made without departing from the scope and spirit of the present invention.\",\n \"Association of the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins with oil bodies.\",\n \"In accordance with the present invention, the oil bodies are associated with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins through association with an oil-body-targeting-protein capable of association with these multimeric-protein-complexes and the oil bodies.\",\n \"As used herein the phrase “associating the oil bodies with the multimeric-protein-complex” means that the oil bodies are brought in proximity of the multimeric-protein-complexes in a manner that allows the association of the oil bodies with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.\",\n \"The association of the oil bodies with the multimeric-protein-complexes is accomplished by association of the oil-body-targeting-protein with both the oil body and with the multimeric-protein-complex.\",\n \"In particular embodiments, the cells expressing the multimeric-protein-complex associate with the oil bodies that are obtainable from these same cells, which permits the convenient production and isolation of the multimeric-protein-complex, including the first and/or second recombinant polypeptides, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, in an oil body-comprising host cell system.\",\n \"Accordingly, in one embodiment, the association of the oil body with the multimeric-protein-complex is accomplished intracellularly during the growth of the cell.\",\n \"For example, a redox fusion polypeptide may be fused to an oil-body-protein and the chimeric protein may be expressed in oil body-containing plant seeds.\",\n \"Isolation of the oil bodies from the seeds in this case results in isolation of oil bodies comprising either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.\",\n \"In another embodiment, in which the multimeric-protein-complex associates with oil bodies obtainable from the same cells in which the complex is produced, the association of the oil bodies with the multimeric-protein-complex is accomplished upon disrupting the cell's integrity.\",\n \"For example, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins may be expressed in such a manner that it is targeted to the endomembrane system of the seed cells.\",\n \"Oil bodies present in the same seed cells comprising an oil-body-targeting-protein capable of association with these multimeric-protein-complexes, for example an oleosin linked to a single chain antibody capable of association with a recombinant polypeptide or multimeric-protein-complex, may then associate with the recombinant polypeptide or multimeric-protein-complex upon grinding of the seed.\",\n \"In accordance with this embodiment, plant seed cells comprising a light and heavy chain of an immunoglobulin targeted to the plant apoplast can be prepared.\",\n \"These particular seed cells are prepared to further comprise oil bodies associated with an oil-body-targeting-protein capable of association with the immunoglobulin, such as for example, an oleosin-protein A fusion protein, and the like.\",\n \"Upon grinding of the seed, the oil bodies comprising protein A associate with the immunoglobulin through binding.\",\n \"In yet another embodiment, the oil bodies used to associate with the multimeric-protein-complex are obtained from a cellular source different from the cell comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, such as from a separate plant line.\",\n \"For example, oil bodies associated with protein A may be prepared from one plant line.\",\n \"These oil bodies may then be mixed with ground seeds comprising an apoplastically expressed light and heavy chain constituting an immunoglobulin.\",\n \"Alternatively, a plant line comprising oil bodies associated with protein A may be crossed with a plant line comprising an immunoglobulin.\",\n \"The first recombinant polypeptide, second recombinant polypeptide and oil-body-targeting-protein may also be prepared in separate cellular compartments.\",\n \"Association of the first polypeptide, second polypeptide, and oil body then may occur upon disruption of the cell's integrity.\",\n \"For example, various mechanisms for targeting gene products are known to exist in plants, and the sequences controlling the functioning of these mechanisms have been characterized in some detail.\",\n \"For example, the targeting of gene products to the chloroplast is controlled by a transit sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (Comai et al.\",\n \"(1988) J Biol Chem 263: 15104-15109).\",\n \"Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (Unger et al.\",\n \"(1989) Plant Mol Biol 13:411-418).\",\n \"The cDNAs encoding these products can be manipulated to target heterologous gene products to these organelles.\",\n \"In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments.\",\n \"Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho (1990) Plant Cell 2:769-783).\",\n \"Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., (1990) Plant Mol Biol 14:357-368).\",\n \"By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to the desired organelle or cell compartment.\",\n \"As hereinbefore mentioned, the redox protein obtained using the methods provided herein is enzymatically active while associated with the oil body.\",\n \"Preferably the redox protein is at least 5 times more active when produced as a redox fusion polypeptide with a second redox protein relative to its production in association with an oil body as a non-fusion polypeptide (i.e.\",\n \"without the second redox protein).\",\n \"More preferably the redox protein is at least 10 times more active when produced as a redox fusion polypeptide.\",\n \"The activity of the redox fusion polypeptide may be determined in accordance with methodologies generally known to the art (see for example: Johnson et al (1984) J. of Bact.\",\n \"Vol.\",\n \"158 3:1061-1069) and may be optimized by for example the addition of detergents, including ionic and non-ionic detergents.\",\n \"Formulation of Oil Bodies In accordance with a particular embodiment, the oil bodies comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, are preferably formulated into an emulsion.\",\n \"The emulsion is preferably used in the preparation of a pharmaceutical composition, personal care or a food product.\",\n \"In emulsified form, the oil body offers certain desirable properties, such as for example excellent compatibility with the human skin.\",\n \"It particular embodiments, the oil body formulation is stabilized so that a final product may be obtained which may be stored and preserved for longer periods of time.\",\n \"As used herein, the term “stabilized oil body preparation” refers to an oil body preparation that is prepared so that the formulation does not undergo undesirable physical or chemical alterations when the oil body preparation is stored.\",\n \"The stabilization requirements may vary depending on the final product.\",\n \"For example personal care products are preferably stable for at least one year at room temperature while additionally being able to withstand short temperature fluctuations.\",\n \"Pharmaceutical formulations may in some cases be less stable as they may be stored at lower temperatures thereby preventing the occurrence of undesirable reactions.\",\n \"In general, stabilization techniques that may be used herein include any and all methods for the preservation of biological material including the addition of chemical agents, temperature modulation based methodologies, radiation-based technologies and combinations thereof.\",\n \"In particular embodiments small amounts of stabilizing chemical agents are mixed with the oil body formulation to achieve stabilization.\",\n \"These chemical agents include inter alia preservatives, antioxidants, acids, salts, bases, viscosity modifying agents, emulsifiers, gelling agents and mixtures thereof and may all be used to stabilize the oil body preparation.\",\n \"In view of the presence of the redox fusion polypeptide the stabilizing agent is generally selected to be compatible with and resulting in good enzymatic function of the redox fusion polypeptide.\",\n \"Diagnostic parameters to assess the stability of the oil body preparation may be as desired and include all parameters indicative of undesirable qualitative or quantitative changes with respect to chemical or physical stability.\",\n \"Typical parameters to assess the oil body preparation over time include color, odor, viscosity, texture, pH and microbial growth, and enzymatic activity.\",\n \"In particular embodiments, the oil body formulation is stabilized prior to the addition of further ingredients that may be used to prepare the final product.\",\n \"However, in other embodiments, it is nevertheless possible to formulate the final formulation using non-stabilized oil bodies and stabilize the final formulation.\",\n \"The final preparations may be obtained using one or more additional ingredients and any formulation process suitable for the preparation of a formulation comprising oil bodies.\",\n \"Ingredients and processes employed will generally vary depending on the desired use of the final product, will be art recognized and may be as desired.\",\n \"Ingredients and processes that may be used herein include those described in (U.S. Pat.\",\n \"Nos.\",\n \"6,146,645 and 6,183,762) which are incorporated by reference herein.\",\n \"In particular embodiments, the redox fusion polypeptide comprises a thioredoxin and a thioredoxin-reductase.\",\n \"Accordingly, provided herein are oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.\",\n \"Also provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of treating or protecting a target against oxidative stress.\",\n \"The stress of the target is treated or prevented by contacting the target with the formulation.\",\n \"The target may be any substance susceptible to oxidative stress, including any molecule, molecular complex, cell, tissue or organ.\",\n \"In another embodiment, provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of chemically reducing a target.\",\n \"Contacting the target with the formulation reduces the target.\",\n \"The target may be any substance susceptible to reduction, including any molecule or molecular complex.\",\n \"Particularly susceptible targets in this regard are the disulfide bonds present in proteins.\",\n \"The oil bodies comprising thioredoxin/thioredoxin-reductase may be used to prepare formulations used to reduce the allergenicity of food or increase the digestibility of food.\",\n \"Preferably, the method of reducing the food allergenicity is practiced by mixing the thioredoxin/thioredoxin-reductase comprising oil bodies with food or food ingredients selected from a variety of sources including for example wheat flour, wheat dough, milk, cheese, soya, yogurt and ice cream.\",\n \"The thioredoxin/thioredoxin-reductase comprising oil bodies may also be used to increase the digestibility of milk as well as other disulfide containing proteins (Jiao, J. et al.\",\n \"(1992) J. Agric.\",\n \"Food Chem 40: 2333-2336).\",\n \"Further food applications include the use of the oil thioredoxin/thioredoxin-reductase comprising oil bodies as a food additive to enhance dough strength and bread quality properties (Wong et al., (1993) J. Cereal Chem.\",\n \"70: 113-114; Kobrehel et al.\",\n \"(1994) Gluten Proteins: Association of Cereal Research; Detmold, Germany).\",\n \"Also provided herein are pharmaceutical compositions comprising, in a pharmaceutically active carrier: oil bodies comprising a thioredoxin/thioredoxin-reductase; oil bodies comprising multimeric-protein-complexes, such as heteromultimeric-protein-complexes; isolated thioredoxin/thioredoxin-reductase fusion proteins; or isolated multimeric-protein-complexes.\",\n \"These pharmaceutical compositions may be used for the treatment of reperfusion injury (Aota et al.\",\n \"(1996) J. Cardiov.\",\n \"Pharmacol.\",\n \"(1996) 27: 727-732), cataracts (U.S. Pat.\",\n \"No.\",\n \"4,771,036), chronic obstructive pulmonary disease (COPD) (MacNee et al.\",\n \"(1999) Am.\",\n \"J. Respir.\",\n \"Crit.\",\n \"Care Med.\",\n \"160:S58-S65), diabetes (Hotta et al.\",\n \"J. Exp.\",\n \"Med.\",\n \"188: 1445-1451), envenomation (PCT Patent Application 99/20122; U.S. Pat.\",\n \"No.\",\n \"5,792,506), bronchiopulmonary disease (MacNee (2000) Chest 117:3035-3175); malignancies (PCT Patent Application 91/04320) and the alleviation of the allergenic potential of airborne, for example pollen-derived, and contact allergens (PCT Patent Application 00/44781).\",\n \"Other diseases or conditions that may be treated with the pharmaceutical compositions provided herein include: psoriasis, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, transplantation, GERD (gastro esophageal reflux disease).\",\n \"In another embodiment, the pharmaceutical compositions provided herein, particularly those comprising one or more redox proteins alone or in combination with oil bodies, can be used in the treatment of inflammatory and viral diseases by reductively inactivating phospholipase A2, one of the contributing factors in inflammatory diseases.\",\n \"Additionally, the redox fusion polypeptide system has been found to function as a self-defense mechanism in response to environmental stimuli, including oxidative stress caused by UV-generated free radicals.\",\n \"Consequently, redox proteins, e.g., oleosin-thioredoxin, oleosin-thioredoxin-reductase, the various redox fusion polypeptides described herein, provide beneficial effects in certain skin conditions such as psoriasis, skin cancer, dandruff, diaper rash, dermatitis, acne, sun damage, aging, inflammation, and the like.\",\n \"In another embodiment, oil-body-thioredoxin-related fusion proteins, e.g., oleosin-Thioredoxin-reductase, can also be used as a venom antidote.\",\n \"Many animal venoms and other toxins contain disulfide bonds, including all snake venom neurotoxins, some bacterial neurotoxins including tetanus and botulinum A, bee venom phospholipase A2, and scorpion venom.\",\n \"In a further embodiment, the redox protein related pharmaceutical compositions provided herein can be used to inactivate venom toxins by reduction of disulfide bonds.\",\n \"A method of treating an individual suffering from the effects of a venom or toxin can include the step of administering an effective dose of a pharmaceutical composition, in a pharmaceutically effective carrier in an amount sufficient to relieve or reverse the effects of the venom toxin on the individual.\",\n \"The pharmaceutical compositions provided herein are preferably formulated for single dosage administration.\",\n \"The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment.\",\n \"Typically, the compositions are formulated for single dosage administration.\",\n \"To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.\",\n \"Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.\",\n \"In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.\",\n \"Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.\",\n \"These may be prepared according to methods known to those skilled in the art.\",\n \"For example, liposome formulations may be prepared as described in U.S. Pat.\",\n \"No.\",\n \"4,522,811.The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.\",\n \"The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.\",\n \"The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.\",\n \"Typically a therapeutically effective dosage is contemplated.\",\n \"The amounts administered may be on the order of 0.001 to 1 mg/ml, preferably about 0.005-0.05 mg/ml, more preferably about 0.01 mg/ml, of blood volume.\",\n \"Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg, more preferably about 25-75 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form.\",\n \"The precise dosage can be empirically determined.\",\n \"The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.\",\n \"It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.\",\n \"It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.\",\n \"It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of the claimed compositions and combinations containing them.\",\n \"Preferred pharmaceutically acceptable derivatives include acids, salts, esters, hydrates, solvates and prodrug forms.\",\n \"The derivative is typically selected such that its pharmacokinetic properties are superior to the corresponding neutral compound.\",\n \"Thus, effective concentrations or amounts of one or more of the compounds provided herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.\",\n \"Compounds are included in an amount effective for ameliorating or treating the disorder for which treatment is contemplated.\",\n \"The concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.\",\n \"Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.\",\n \"Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.\",\n \"In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used.\",\n \"Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as Tween®, or dissolution in aqueous sodium bicarbonate.\",\n \"Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.\",\n \"For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable carrier.\",\n \"For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred.\",\n \"Time release formulations are also desirable.\",\n \"Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.\",\n \"Upon mixing or addition of the compound with the vehicle, the resulting mixture may be a solution, suspension, emulsion or other composition.\",\n \"The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.\",\n \"If necessary, pharmaceutically acceptable salts or other derivatives of the compounds are prepared.\",\n \"The compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.\",\n \"It is understood that number and degree of side effects depends upon the condition for which the compounds are administered.\",\n \"For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses that would not be tolerated when treating disorders of lesser consequence.\",\n \"The compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action known to those of skill in the art.\",\n \"The formulations of the compounds and agents for use herein include those suitable for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration or any route.\",\n \"The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.\",\n \"The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.\",\n \"The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.\",\n \"Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.\",\n \"Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier, vehicle or diluent.\",\n \"Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules.\",\n \"Unit-dose forms may be administered in fractions or multiples thereof.\",\n \"A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form.\",\n \"Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.\",\n \"Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.\",\n \"The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polivinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.\",\n \"Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.\",\n \"If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.\",\n \"Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).\",\n \"The composition or formulation to be administered will contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject.\",\n \"Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.\",\n \"For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).\",\n \"The tablets may be coated by methods well-known in the art.\",\n \"The pharmaceutical preparation may also be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.\",\n \"Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).\",\n \"Formulations suitable for rectal administration are preferably presented as unit dose suppositories.\",\n \"These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.\",\n \"Formulations suitable for topical application to the skin or to the eye preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol and oil.\",\n \"Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof.\",\n \"The topical formulations may further advantageously contain 0.05 to 15 percent by weight of thickeners selected from among hydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly(alkylene glycols), poly/hydroxyalkyl, (meth)acrylates or poly(meth)acrylamides.\",\n \"A topical formulation is often applied by instillation or as an ointment into the conjunctival sac.\",\n \"It can also be used for irrigation or lubrication of the eye, facial sinuses, and external auditory meatus.\",\n \"It may also be injected into the anterior eye chamber and other places.\",\n \"The topical formulations in the liquid state may be also present in a hydrophilic three-dimensional polymer matrix in the form of a strip, contact lens, and the like from which the active components are released.\",\n \"For administration by inhalation, the compounds for use herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.\",\n \"In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.\",\n \"Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.\",\n \"Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.\",\n \"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.\",\n \"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.\",\n \"The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.\",\n \"Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use.\",\n \"Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.\",\n \"Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 to 0.2 M concentration with respect to the active compound.\",\n \"Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.\",\n \"The pharmaceutical compositions may also be administered by controlled release means and/or delivery devices (see, e.g., in U.S. Pat.\",\n \"Nos.\",\n \"3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).\",\n \"Desirable blood levels may be maintained by a continuous infusion of the active agent as ascertained by plasma levels.\",\n \"It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions.\",\n \"Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).\",\n \"The efficacy and/or toxicity of the pharmaceutical compositions provided herein, alone or in combination with other agents can also be assessed by the methods known in the art (See generally, O'Reilly, Investigational New Drugs, 15:5-13 (1997)).\",\n \"The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.\",\n \"Kits containing the compositions and/or the combinations with instructions for administration thereof are provided.\",\n \"The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad.\",\n \"Instructions are optionally included for administration of the active agent by a clinician or by the patient.\",\n \"Finally, the pharmaceutical compositions provided herein containing any of the preceding agents may be packaged as articles of manufacture containing packaging material, a compound or suitable derivative thereof provided herein, which is effective for treatment of a diseases or disorders contemplated herein, within the packaging material, and a label that indicates that the compound or a suitable derivative thereof is for treating the diseases or disorders contemplated herein.\",\n \"The label can optionally include the disorders for which the therapy is warranted.\",\n \"Also provided herein are personal care formulations containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.\",\n \"Personal care products comprising thioredoxin and thioredoxin-reductase are disclosed in for example Japanese Patent Applications JP9012471A2, JP103743A2, and JP1129785A2 Personal care formulations that may be prepared in accordance with the present invention include formulations capable of improving the physical appearance of skin exposed to detrimental environmental stimuli resulting in oxidative stress for example oxidative stress caused by UV-generated free-radicals.\",\n \"The oil bodies comprising thioredoxin/thioredoxin-reductase may also be used to prepare hair care products as described in U.S. Pat.\",\n \"Nos.\",\n \"4,935,231 and 4,973,475 (incorporated herein by reference in their entirety).\",\n \"The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.\",\n \"Example 1 Isolation of Thioredoxin and NADPH Thioredoxin-Reductase Genes An Arabidopsis silique cDNA library CD4-12 was obtained from the Arabidopsis Biological Resource Centre (ABRC, http://aims.cps.msu.edu) Arabidopsis stock centre and used as a template for the isolation of the thioredoxin h (Trxh) and thioredoxin-reductase genes from Arabidopsis.\",\n \"For the isolation of the Trxh gene the following primers were synthesized: GVR833: 5′ TACCATGGCTTCGGAAGAAGGA 3′ (SEQ ID NO:1) The sequence identical to the 5′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.\",\n \"Underlined is an NcoI restriction site to facilitate cloning.\",\n \"GVR834: 5′ GAAAGCTTAAGCCAAGTGTTTG 3′ (SEQ ID NO:2) The sequence complementary to the 3′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.\",\n \"Underlined is an HindIII restriction site to facilitate cloning.\",\n \"A Polymerase Chain Reaction (PCR) was carried out using GVR833 and GVR834 as primers and the cDNA library CD4-12 as a template.\",\n \"The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.\",\n \"The isolated sequence encoding Trxh was identical to the published Trxh gene sequence (Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328).\",\n \"The pBluescript vector containing the Trxh gene is called pSBS2500.For the isolation of the thioredoxin-reductase gene the following primers were synthesized: GVR836: 5′ GGCCAGCACACTACCATGAATGGTCTCGAAACTCAC 3′ (SEQ ID NO:3).\",\n \"The sequence identical to the 5′ end of the thioredoxin-reductase gene as published (Jacquot et al, J Mol Biol.\",\n \"(1994) 235 (4):1357-63), is indicated in bold).\",\n \"GVR837: 5′ TTAAGCTTCAATCACTCTTACCTTGCTG 3′.\",\n \"(SEQ ID NO:4) A Polymerase Chain Reaction (PCR) was carried out using GVR836 and GVR837 as primers and the cDNA library CD4-12 as a template.\",\n \"The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.\",\n \"The pBluescript vector containing the thioredoxin-reductase gene is called pSBS2502.A total of three clones were sequenced, the sequence of each of the three clones were identical to each other.\",\n \"However, as depicted in FIG.\",\n \"1 this sequence indicated several nucleotide differences compared to the published thioredoxin-reductase gene sequence published (Jacquot et al, J Mol Biol.\",\n \"(1994) 235 (4):1357-63.).\",\n \"The complete coding sequence and its deduced amino acid sequence is shown in SEQ ID NO:10.As a result of the nucleotide differences between the published sequence and the sequence isolated in Example 1, several amino acid changes are also predicted.\",\n \"A comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence thioredoxin-reductase (ATTHIREDB, Jacquot et al, J Mol Biol.\",\n \"(1994) 235 (4):1357-63.)\",\n \"with the sequence isolated in Example 1 (TR) is shown in FIG.\",\n \"3.Example 2 Construction of Plant Expression Vectors Expression vectors were constructed to allow for the seed specific over-expression of thioredoxin and NADPH thioredoxin-reductase in seeds.\",\n \"Vectors were constructed to allow for over-expression in its natural subcellular location and for accumulation on oil bodies.\",\n \"Construction of Plant Transformation Vector pSBS2520.The Arabidopsis thioredoxin h gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324)).\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with Pst I and HindIII/KpnI sites respectively (see SEQ ID NO:14).\",\n \"Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the Trxh gene.\",\n \"The PstI-phaseolin promoter-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 (pSBS3000 is a derivative from the Agrobacterium binary plasmid pPZP221 (Hajdukiewicz et al., 1994, Plant Molec.\",\n \"Biol.\",\n \"25: 989-994).\",\n \"In pSBS3000, the CaMV35S promoter-gentamycin resistance gene-CAMV 35S terminator of pPZP221 was replaced with parsley ubiquitin promoter-phosphinothricin acetyl transferase gene-parsley ubiquitin termination sequence to confer resistance to the herbicide glufosinate ammonium.)\",\n \"The resulting plasmid is called pSBS2520.The sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence is shown in SEQ ID NO:14.Construction of Plant Transformation Vector pSBS2510.The 3′ coding sequence of an Arabidopsis oleosin gene (van Rooijen et al (1992) Plant Mol.\",\n \"Biol.\",\n \"18: 1177-1179) was altered to contain an NcoI site.\",\n \"The NcoI-HindIII fragment from vector pSBS2500 (Example 1) containing the Trxh was ligated to the coding sequence of this Arabidopsis oleosin utilizing this NcoI restriction site.\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) containing a synthetic PstI site (see construction of pSBS2520) to the coding sequence of the Arabidopsis oleosin.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the Trxh gene.\",\n \"The PstI-phaseolin promoter-oleosin-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2510.The sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence is shown in SEQ ID NO:16.Construction of Plant Transformation Vector pSBS2521.This vector contains the same genetic elements as the insert of pSBS2510 except the Trxh gene is fused to the 5′ end of the oleosin gene.\",\n \"The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al (1992) Plant Mol.\",\n \"Biol.\",\n \"18: 1177-1179) was furnished with a HindIII cloning site.\",\n \"Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene and to fuse the Trxh gene to the oleosin sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.\",\n \"The PstI-phaseolin promoter-Trxh oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2521.The sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence is shown in SEQ ID NO:19.Construction of Plant Transformation Vector pSBS2527.The Arabidopsis NADPH thioredoxin-reductase gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324).\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with PstI and HindIII/KpnI sites respectively (see SEQ ID NO:14).\",\n \"Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the thioredoxin-reductase gene.\",\n \"The PstI-phaseolin promoter-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 The resulting plasmid is called pSBS2527.The sequence of the phaseolin promoter-Arabidopsis thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:22.Construction of Plant Transformation Vector pSBS2531.A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.\",\n \"The same gene splicing technique was used to fuse the oleosin gene to the thioredoxin-reductase coding sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin downstream of the thioredoxin-reductase gene.\",\n \"The PstI-phaseolin promoter-oleosin-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2531.The sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:24.Construction of Plant Transformation Vector pSBS2529 This vector contains the same genetic elements as the insert of pSBS2531 except the thioredoxin-reductase gene is fused to the 5′ end of the oleosin gene.\",\n \"The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al.\",\n \"(1992) Plant Mol.\",\n \"Biol, 18: 1177-1179) was furnished with a HindIII cloning site.\",\n \"Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene and to fuse the thioredoxin-reductase gene to the oleosin sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.\",\n \"The PstI-phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2529.The sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence is shown in SEQ ID NO:27.Construction of Plant Transformation Vector pSBS2530.A plant transformation was constructed containing the Mycobacterium Leprae thioredoxin-reductase /thioredoxin gene (Mlep TR/Trxh).\",\n \"A construct called pHIS/TR/Trxh (Wieles et al (1995) J Biol Chem 270:25604-25606) was obtained from the department of Immunohematology and Blood bank, Leiden University, The Netherlands and use as a template for PCR to generate pSBS2530.The construction of pSBS2530 was identical to the construction of pSBS2531 except that the Mlep TR/Trxh gene was used instead of the Arabidopsis thioredoxin-reductase gene.\",\n \"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.\",\n \"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.\",\n \"The same gene splicing technique was used to fuse the oleosin gene to the Mlep TR/Trxh coding sequence.\",\n \"Standard molecular biology laboratory techniques (see eg: Sambrook et al.\",\n \"(1990) Molecular Cloning, 2nd ed.\",\n \"Cold Spring Harbor Press) were again used to clone the HindIII-KpnI fragment containing the phaseolin downstream of the Mlep TR/Trxh gene.\",\n \"The PstI-phaseolin promoter-oleosin-Mlep TR/Trxh -phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2530.The sequence of the phaseolin promoter-oleosin Mlep TR/Trxh -phaseolin terminator sequence is shown in SEQ ID NO:30.Construction of Plant Transformation Vector pSBS2542.From initial activity assays (FIG.\",\n \"4), it was apparent that oil bodies expressing the oleosin-M. lep TR/Trxh fusion protein contained considerable reducing activity.\",\n \"It was anticipated that a similar oleosin fusion construct encoding the Arabidopsis thioredoxin-reductase and thioredoxin proteins would behave in an analogous manner.\",\n \"Molecular modeling was used to aid in the design of such a construct.\",\n \"Primers were designed (thioredoxin link-L: 5′-ACTGGAGATGTTGACTCGACGGATACTACGGATTGGTCGACGG CTATGGAAGAAGGACAAGTGATCGCCTGC-3′; (SEQ ID NO:5), and thioredoxin link-R: 5′-ATCCGTCGAGTCAACATCTCCAGTTTCCTCGGTGGTCTCGTTAGCCTTCGAT CCAGCAATCTCTTGTAAGAATGCTCTGC-3′; (SEQ ID NO:6) to code for a synthetic linker peptide between the thioredoxin-reductase and thioredoxin proteins.\",\n \"These primers were used in conjunction with primers GVR 873 (5′-GTGGAAGCT TATGGAGATGGAG-3′; SEQ ID NO:7) and GVR834 (5′-GAAAGCTTAAGCCAAGTGTTTG-3′; SEQ ID NO:2) to amplify a region coding for a thioredoxin-reductase-linker region-thioredoxin utilizing a gene splicing by overlap extension technique (Horton et al (1989) 15:61-68).\",\n \"The thioredoxin-reductase-linker-thioredoxin encoding sequence was then cloned into a pre-existing pSBS3000 vector using standard molecular biology techniques (Sambrook et al (1990) Molecular Cloning 2nd Edition Cold Spring Harbour Press).\",\n \"The resulting plasmid was called pSBS2542.The sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region is shown in SEQ ID NO:33.An amino acid sequence comparison between this Arabidopsis thioredoxin-reductase-linker-thioredoxin and the M. leprae TR/Trxh protein is shown in FIG.\",\n \"12.Plasmids pSBS2510, pSBS2520, pSBS2521, pSBS2527, pSBS2529, pSBS2530, pSBS2531 and pSBS2542 were electroporated into Agrobacterium strain EHA101.These Agrobacterium strains were used to transform Arabidopsis.\",\n \"Arabidopsis transformation was done essentially as described in “Arabidopsis Protocols; Methods in molecular biology Vol 82.Edited by Martinez-Zapater J M and Salinas J. ISBN 0-89603-391-0 pg 259-266 (1998) except the putative transgenic plants were selected on agarose plates containing 80 μM L-phosphinothricine, after they were transplanted to soil and allowed to set seed.\",\n \"Example 3 Polyacrylamide Gelelectrophoresis and Immunoblotting of Transgenic Seed Extracts Source of Arabidopsis Thioredoxin, Thioredoxin-Reductase and Oleosin Antibodies.\",\n \"The Arabidopsis thioredoxin and thioredoxin-reductase genes were cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression of Arabidopsis thioredoxin and thioredoxin-reductase proteins.\",\n \"These proteins were purified using standard protocols (see eg Invitrogen protocol) and used to raise antibodies in rabbits using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).\",\n \"The Arabidopsis oleosin gene genes was cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression Arabidopsis oleosin protein.\",\n \"This protein was purified using standard protocols (see eg Invitrogen protocol) and used to prepare mouse monoclonal antibodies using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).\",\n \"Preparation of total Arabidopsis seed extracts for PAGE.\",\n \"Arabidopsis seeds were ground in approximately 20 volumes of 2% SDS, 50 mM Tris-Cl, this extract was boiled, spun and the supernatant was prepared for polyacrylamide gelelectrophoresis (PAGE) using standard protocols.\",\n \"Preparation of Arabidopsis Oil-Body-Protein Extracts.\",\n \"Arabidopsis seeds were ground in approximately 20 volumes of water and spun in a microfuge.\",\n \"The oil bodies were recovered and washed sequentially with approximately 20 volumes of water, a high stringency wash buffer, containing 8M urea and 100 mM sodiumcarbonate and water.\",\n \"After this last wash the oil bodies are prepared for poly acrylamide gelelectrophoresis (PAGE) using standard protocols.\",\n \"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2510 Total seed and oil body protein extracts from plants transformed with pSBS2510 were loaded onto polyacrylamide gels and either stained with coomassie brilliant blue or electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.\",\n \"Expression of the oleosin-thioredoxin results in an additional band of 31.2 kDa.\",\n \"The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).\",\n \"This indicates that oleosin-thioredoxin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.\",\n \"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2521 Total seed and oil body protein extracts from plants transformed with pSBS25121 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.\",\n \"Expression of the thioredoxin-oleosin results in an additional band of 31.2 kDa.\",\n \"The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).\",\n \"This indicates that thioredoxin-oleosin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.\",\n \"Analysis of seed extracts from plants transformed with pSBS2520 Total seed extracts from plants transformed with pSBS2520 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin and visualized using alkaline phosphatase.\",\n \"The results indicated that the thioredoxin antibodies are immunologically reactive with a band of approximately the right predicted molecular weight (12 kDa).\",\n \"Untransformed seeds do not show a detectable thioredoxin band.\",\n \"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2529 Total seed and oil body protein extracts from plants transformed with pSBS2529 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.\",\n \"Expression of the thioredoxin-reductase -oleosin results in an additional band of 53.8 kDa.\",\n \"The results indicate that the thioredoxin-reductase antibodies are immunologically reactive with a band of the right predicted molecular weight for the fusion protein (53.8 kDa), the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight (53.8 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).\",\n \"This indicates that thioredoxin-reductase-oleosin is expressed in Arabidopsis seeds.\",\n \"Analysis of seed extracts from plants transformed with pSBS2527 Total seed extracts from plants transformed with pSBS2527 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.\",\n \"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase and visualized using alkaline phosphatase.\",\n \"The thioredoxin-reductase antibodies are immunologically reactive with a band of approximately the right predicted molecular weight for the (35.3 kDa).\",\n \"Untransformed seeds do not show a detectable thioredoxin band.\",\n \"Analysis of seed extracts from plants transformed with pSBS2531 A protein gel and immunoblot was prepared assaying the expression of oleosin-DMSR in Arabidopsis T2 seeds and correct targeting to Arabidopsis oil bodies.\",\n \"The expected molecular weight based on the deduced amino acid sequence is calculated to be 53,817 Da.\",\n \"In the oil body extract of the transgenic oleosin-thioredoxin-reductase sample an extra band of approximately 54 kDa was observed.\",\n \"This band was confirmed to be oleosin-thioredoxin-reductase by immunoblotting.\",\n \"From the polyacrylamide gel it was observed that the expression of the oleosin-Thioredoxin-reductase is about double compared to the expression of the major 18.5 kDa Arabidopsis oleosin.\",\n \"This represents approximately 2-4% of total seed protein.\",\n \"Analysis of seed extracts from plants transformed with pSBS2530 A protein gel and immunoblot was prepared assaying the expression of oleosin-M.lep TR/Trxh in Arabidopsis T2 seeds and the correct targeting to Arabidopsis oil bodies.\",\n \"The expected molecular weight based on the deduced amino acid sequence is calculated to be 67,550 Da.\",\n \"In the oil body extract of the transgenic oleosin-M.lep TR/Trxh sample an extra band of approximately 68 kDa was observed.\",\n \"This band was confirmed to be oleosin-M.lep TR/Trxh by immunoblotting.\",\n \"From the polyacrylamide gel it was observed that the expression of the oleosin-M.lep TR/Trxh is similar to the expression of the major 18.5 kDa Arabidopsis oleosin.\",\n \"This represents approximately 1-2% of total seed protein.\",\n \"Analysis of seed extracts from plants transformed with pSBS2542 Crude oil body extracts from pSBS2542 lines were prepared by grinding 100 μg of seed in 1 mL of 100 mM Tris buffer at pH 7.5.The samples were then centrifuged in order to isolate the oil body fraction.\",\n \"The oil body fraction was then loaded on an SDS polyacrylamide gel for expression analysis.\",\n \"A Coomassie stained gel revealed that the synthetic fusion accumulated to high levels in crude oil body extracts from 3 of the 4 lines tested.\",\n \"It was estimated that the fusion protein represented approximately 2-5% of total seed protein.\",\n \"Furthermore, western blots utilizing either anti-thioredoxin or anti-thioredoxin-reductase antibodies confirmed that the over expressed 70 kDa protein was indeed oleosin-thioredoxin-reductase-linker-thioredoxin.\",\n \"Example 4 Biological Activity of Thioredoxin and Thioredoxin-Reductase Transformants Initial Reduction Assays: DTNB Assay The activity of the thioredoxin and thioredoxin reductase was determined using a calorimetric DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] assay.\",\n \"The assay was performed in a 700 μL reaction volume containing 100 mM Tris-Cl pH 8.0, 5 mM EDTA, 200 μM DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] and 200 μM NADPH.\",\n \"If thioredoxin-reductase and thioredoxin are added, NADPH will reduce the thioredoxin-reductase, which will then reduce thioredoxin, which will, in turn, reduce DTNB (see equations below).\",\n \"NADPH2+thioredoxin-reductaseox→thioredoxin-reductasered+NADP+thioredoxin-reductasered+thioredoxinox→thioredoxinred+thioredoxin-reductaseox thioredoxinred+DTNBox→2(2-nitro-5-mercaptobenzoic acid)+thioredoxinox The formation of the yellow product was monitored by measuring the OD412 in a spectrophotometer after a set period of time (usually 0.5-2 hours).\",\n \"The results of initial activity assays are shown in the bar graph in FIG.\",\n \"4 and described below.\",\n \"Initially, 100 μg of total seed proteins were added from each of the Arabidopsis transgenic lines, pSBS2520 (cytosolic thioredoxin) and pSBS2527 (cytosolic thioredoxin-reductase), which corresponds to approximately 1 μg of cytosolic thioredoxin and thioredoxin-reductase used in the assay.\",\n \"In this case, the amount of DTNB reduced was comparable to the reduction caused by 1 μg each of E. coli thioredoxin and thioredoxin-reductase.\",\n \"In these plant seed samples, background readings were very low when only one of the 2 extracts (either cytosolic thioredoxin or cytosolic thioredoxin-reductase; FIG.\",\n \"4, bars 3 and 6, respectively) was added to the reaction, along with wild type oil bodies.\",\n \"Analysis with oil body fractions from transgenic seeds revealed that Arabidopsis thioredoxin and thioredoxin-reductase were substantially less active when fused to oleosins on oil bodies.\",\n \"Approximately 300 μg of crude, unwashed oil-body-protein was used in the assay (which corresponds to 10-30 μg of thioredoxin-oleosin (pSBS 2521; FIG.\",\n \"4, bar 2), oleosin-thioredoxin (pSBS 2510, FIG.\",\n \"4, bar 1), thioredoxin-reductase-oleosin (pSBS 2529, FIG.\",\n \"4, bar 5), or oleosin-thioredoxin-reductase (pSBS 2531, FIG.\",\n \"4, bar 4).\",\n \"The oil-body-proteins were tested in conjunction with 100 μg of total seed protein containing approximately 1 μg of cytosolic thioredoxin (pSBS 2520) or thioredoxin-reductase (pSBS 2527).\",\n \"In such assays, pSBS2529 (thioredoxin-reductase-oleosin) and pSBS2531 (oleosin-thioredoxin-reductase) do contain reductase activity when combined with cytosolic thioredoxin from pSBS2520 (see FIG.\",\n \"4, bars 7 and 8, respectively).\",\n \"Experiments estimated that the reductase activity of oleosin-thioredoxin-reductase was about 10-15% that of the cytosolic thioredoxin-reductase.\",\n \"The addition of tween at a final concentration of 0.4% could enhance this activity 2 or 3 fold.\",\n \"Interestingly, oleosin-thioredoxin-reductase (pSBS 2531) appears to be capable of reducing DTNB in the absence of added thioredoxin, although added thioredoxin causes significantly more DTNB reduction (see FIG.\",\n \"4; compare bar 4 W.T.+oleosin-thioredoxin-reductase to bar 7 thioredoxin+oleosin-thioredoxin-reductase).\",\n \"Experiments with pSBS2521 (thioredoxin-oleosin) or pSBS2510 (oleosin-thioredoxin) combined with cytosolic thioredoxin-reductase from pSBS2527 (see FIG.\",\n \"4, bars 10 and 11, respectively) indicate that thioredoxin activity of these fusions is undetectable at these concentrations.\",\n \"Oil bodies from the transgenic Arabidopsis line, pSBS2530 (oleosin-M.lep TR/Trxh) contain significant thioredoxin/thioredoxin-reductase activity (see FIG.\",\n \"4, bar 12).\",\n \"One hundred micrograms of crude oil body protein for pSBS2530 was tested (corresponding to approximately 5 μg of oleosin-M.lep TR/trxh fusion) in the assay.\",\n \"Based on the assay, it was estimated that this fusion is about 25-40% as active as cytosolic Arabidopsis thioredoxin and thioredoxin-reductase (FIG.\",\n \"4, bar 9) when comparing specific activity.\",\n \"Insulin Reduction Assay The results from the DTNB assays were confirmed with insulin reduction assays.\",\n \"This assay contained insulin at a final concentration of 1 mg/mL in 100 mM KH2PO4 pH 7.0+5 mM EDTA.\",\n \"In the presence of NADPH (500 μM), thioredoxin, and thioredoxin-reductase, insulin is reduced and precipitates from the solution.\",\n \"Normally, insulin reduction is followed by measuring turbidity at OD 650.Alternatively, one can measure the conversion of NADPH2 to NADP+by monitoring the decrease in absorbance at 340 nm.\",\n \"Both of the assays are difficult to measure when oil bodies are present, due to interference with the spectrophotometer readings.\",\n \"However, qualitative data could be obtained by centrifuging the tubes after a set period of time, and determining if an insulin pellet was present (oil bodies float to the top, while the insulin precipitate pellets out).\",\n \"Alternatively, samples could be filtered after a set period of time, and the change in absorbance at 340 nm could be measured.\",\n \"As mentioned previously, the results of the insulin reduction assays agreed with those of the DTNB assay, with the exception of the observation that pSBS2531 (oleosin-thioredoxin-reductase) only reduced insulin in the presence of free thioredoxin from pSBS2520.Assays on Seeds from Arabidopsis Crosses that Co-Express Oleosin-Thioredoxin and Oleosin-Thioredoxin-Reductase.\",\n \"Based upon initial DTNB and insulin reduction assays, it was apparent that mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase transgenic seeds resulted in very limited reducing activity (Note: the <-> indicates both configurations of oleosin fusions; ie.\",\n \"oleosin<->thioredoxin would represent oleosin-thioredoxin and thioredoxin-oleosin fusions).\",\n \"To determine whether having oleosin<->thioredoxin and oleosin<->thioredoxin-reductase proteins present on the same oil body would have a positive effect on the reducing activity of these proteins, crosses were set up to generate double transgenic Arabidopsis lines.\",\n \"The crosses are illustrated in Table 2.TABLE 2 Confirmed double transgenic lines (PCR and Western Male Female Blot) oleo-thioredoxin X oleo-thioredoxin-reductase 4 oleo-thioredoxin X thioredoxin-reductase-oleo 1 thioredoxin-oleo X oleo-thioredoxin-reductase 0 thioredoxin-oleo X thioredoxin-reductase-oleo 4 oleo-thioredoxin- X oleo-thioredoxin 2 reductase oleo-thioredoxin- X thioredoxin-oleo 0 reductase thioredoxin- X oleo-thioredoxin 7 reductase-oleo thioredoxin- X thioredoxin-oleo 0 reductase-oleo Seeds from Arabidopsis crosses were germinated on PPT plates and the seedlings were transferred to soil after approximately 2 weeks.\",\n \"PCR experiments on DNA isolated from the seedlings identified a number of plants which contain both an oleosin<->thioredoxin and an oleosin<->thioredoxin-reductase gene construct within their genome.\",\n \"Seeds were harvested from these plants for expression and activity assays.\",\n \"Western blots were carried out to confirm expression of both oleosin<->thioredoxin and oleosin<->thioredoxin-reductase in the lines.\",\n \"DTNB and insulin reduction assays were also performed to compare activity between single transgenic parent lines and the double transgenic offspring and results are summarized in Table 3.Table 3 summarizes DTNB reducing activity of various transgenic lines.\",\n \"The last 2 rows compare mixing oil bodies from single transgenic parent lines to using oil bodies from double transgenic offspring.\",\n \"Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.\",\n \"TABLE 3 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + 100 thioredoxin-reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Based on DTNB and insulin reduction assays, it is evident that double transgenic plants co-expressing oleosin<->thioredoxin and oleosin<->thioredoxin-reductase on the same, single oil body contained significantly more reducing activity compared to mixing oil bodies from single transgenic oleosin<->thioredoxin and oleosin<->thioredoxin-reductase lines.\",\n \"It was additionally apparent that oil body extracts from co-expressing lines contained more reducing activity compared to line pSBS2530 (oleosin-M. lep TR/Trxh), which was previously identified as the line containing the highest reducing activity from oil bodies.\",\n \"These results suggest that the creation of double transgenic lines (either through crossing or by co-transforming 2 expression constructs into plants) may represent one means by which we could solve our initial problem of not being able to generate reducing activity by mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase single transgenic lines.\",\n \"Assays on Seeds from Arabidopsis pSBS2542 Transgenic Lines that Express Oleosin-Thioredoxin-Reductase-Linker-Thioredoxin.\",\n \"Oil body extracts from four pSBS2542 lines were tested for reducing activity in DTNB and insulin reduction assays, using standard protocols described previously.\",\n \"Again, oil body extracts containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein possessed significant reducing activity.\",\n \"Based on such assays, it was revealed that the oleosin-thioredoxin-reductase-linker-thioredoxin synthetic fusion protein was more active than the oleosin-M. lep TR/Trxh fusion.\",\n \"Furthermore, oil bodies containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein appeared to have more reducing activity compared to oil bodies from double transgenic lines that co-expressed oleosin<->thioredoxin and oleosin<->thioredoxin-reductase.\",\n \"The results comparing reducing activity for the various thioredoxin-reductase/thioredoxin constructs is summarized in Table 4.Table 4 summarizes DTNB reducing activity of various transgenic lines.\",\n \"The pSBS2542 line expressing oleosin-thioredoxin-reductase-linker-thioredoxin contains significant reducing activity, comparable to the “free” forms of Arabidopsis thioredoxin and thioredoxin-reductase and the equivalent E. coli proteins.\",\n \"Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.\",\n \"TABLE 4 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + thioredoxin- 100 reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Oleosin-thioredoxin-reductase-linker-thioredoxin ~75-100 (pSBS2542) Reduction Assays Comparing the Utilization of NADH Vs. NADPH as a Cofactor (Electron Donor) for the Thioredoxin-Reductase/Thioredoxin System.\",\n \"DTNB and insulin reduction assays were conducted as described previously, except that NADH was substituted for NADPH as an electron donor in the system utilizing E. coli thioredoxin-reductase and thioredoxin.\",\n \"Thus, a comparison was conducted of the utilization of NADH versus NADPH as a cofactor for the E. coli thioredoxin-reductase/thioredoxin system.\",\n \"For the DTNB assay, the reaction mixture consisted of 400 μM DTNB, 10 μg/mL E. coli thioredoxin, and 10 μg/mL E. coli thioredoxin-reductase in 100 mM Tris-Cl buffer pH 8.0.Either NADH or NADPH was then added to the DTNB reaction as follows: Reaction A.\",\n \"200 μM NADPH (Sigma) Reaction B.\",\n \"800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 800 μM NADH (no TR or Trxh).\",\n \"For the insulin reduction assay, the reaction mixture consisted of 1 mg/mL bovine pancreatic insulin, 20 μg/mL E. coli thioredoxin, and 20 μg/mL E. coli thioredoxin-reductase in 100 mM potassium phosphate buffer at pH 7.0.Either NADH or NADPH was then added to the reaction as follows: Reaction A.\",\n \"800 μM NADPH (Sigma) Reaction B.\",\n \"800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 2 mM NADH (no TR or Trxh).\",\n \"The results indicate that NADH, purchased from either Sigma or Roche, could act as an electron donor in both the DTNB and insulin reduction assays.\",\n \"However, the rate of reduction was lower than the rate observed with NADPH as a cofactor.\",\n \"It was estimated that the rate of insulin reduction utilizing NADH as an electron donor was approximately 25-50% when compared to the maximum rate using NADPH.\",\n \"Furthermore, it was estimated that the rate of DTNB reduction utilizing NADH as an electron donor was approximately 5-10% of the maximum rate using NADPH.\",\n \"Similar results were observed using the oleosin-thioredoxin-reductase-linker thioredoxin fusion protein on Arabidopsis oil bodies instead of the E. coli thioredoxin-reductase and thioredoxin.\",\n \"Example 5 Production of Multimeric Immunoglobulin Protein in Plant Seed Cells and Capture on Oil Bodies Using Protein A-Oleosin Fusion Proteins 1—Production of Multimeric Immunoglobulin Protein in Plant Seed Cells For expression of multimeric-protein-complexes containing multimeric-immunoglobulin-complexes, the cDNA sequences encoding individual light and heavy chains can be isolated from; 1) cell lines expressing a particular antibody, such as clonal B cell lines, or a hybridoma cell line, or 2) may be a recombinant antibody, assembled by combining select light and heavy chain variable domains and available light and heavy chain constant domain sequences, respectively.\",\n \"Variable domains with specific binding properties may be isolated from screening populations of such sequences, usually in the form of a single-chain Fv phage display library.\",\n \"Starting from known nucleic acid sequences and a source of light and heavy chains, the mature polypeptide coding sequences of each chain is isolated with a secretion signal sequence.\",\n \"The signal sequence can be the native antibody sequence or derived from a known secreted plant sequence (e.g.\",\n \"a PR sequence from Arabidopsis or tobacco).\",\n \"The addition of a plant secretion signal sequence to both light and heavy chain mature coding sequences is carried out by standard molecular biology techniques.\",\n \"PCR fusion is used routinely to make such modifications.\",\n \"Secretion signal sequences are included to target the light and heavy immunoglobulin polypeptides for secretion from the cell and further assembly of the two chains into a multimeric-immunoglobulin-complex.\",\n \"For expression in transgenic plant seeds, an expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—light chain sequence, and 3) a regulatory sequence to terminate transcription.\",\n \"A second expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—heavy chain sequence, and 3) a regulatory sequence to terminate transcription.\",\n \"Each of the antibody chain expression cassettes is cloned individually into an Agrobacterium plant transformation vector or is combined into a single transformation vector with both expression cassettes.\",\n \"In both cases, the expression cassettes are cloned into plant transformation vectors, between the left and right delineating border sequences, and adjacent to a plant selectable marker cassette.\",\n \"Each plant transformation vector is transformed into Agrobacterium.\",\n \"The resulting Agrobacterium strains are used to infect plant tissues.\",\n \"Transgenic plant material is regenerated and viable transgenic plants are selected.\",\n \"When individual transformation vectors are used, the transgenic plant lines that are produced, expressing either light or heavy chain sequences, are crossed to generate a single plant line expressing both chains in the same plant cell.\",\n \"When a single transformation vector, containing both light and heavy expression cassettes, is used, the initial transgenic plant line produces both light and heavy chain sequences in the same plant cell.\",\n \"2—Production of Transgenic Oil Bodies which Display Protein a for the Capture of Immunoglobulins To capture and display immunoglobulin protein on oil bodies, oil bodies are engineered to display an immunoglobulin binding protein.\",\n \"In this example, the well-known antibody-binding domains from Protein A are used.\",\n \"Based on the known sequence for Protein A from Staphylococcus aureus, PCR primers are designed to isolate the five consecutive Ig-binding domains from the bacterial Protein A sequence.\",\n \"Primers are designed to allow cloning of the Protein A sequence as either an N-terminal or C-terminal fusion to an oleosin sequence for targeting to oil bodies.\",\n \"The sequence that encodes an in-frame translational fusion between Protein A and oleosin is cloned into a plant expression cassette for seed-specific expression.\",\n \"The final cassette consists of a regulatory promoter sequence that provides expression in seeds, the Protein A-oleosin fusion sequence, and a regulatory sequence to terminate transcription.\",\n \"The Protein A-oleosin expression cassette is cloned into a plant transformation vector compatible with Agrobacterium-mediated plant transformation.\",\n \"The transformation vector comprises left and right border sequences flanking the Protein A-oleosin expression cassette and an adjacent plant selectable marker cassette.\",\n \"The Agrobacterium strain containing this vector is used to infect plant tissues and subsequent regeneration and selection from transgenic plant material to create transgenic plants.\",\n \"3—Capture and Display of Multimeric-Immunoglobulins on Oil Bodies Displaying Protein A Having produced light and heavy chain multimeric immunoglobulin complexes in one transgenic plant line and the display of Protein A on oil bodies through the oil body targeting of a Protein A-oleosin fusion protein in a second plant line, at least two embodiments can be used to capture the immunoglobulin complex on the Protein A oil bodies.\",\n \"In the first embodiment, transgenic seed from both the immunoglobulin and the Protein A-oleosin expression lines is combined in an optimum ratio and then ground together such that the disrupted material from both seed lines would be combined in the same extract.\",\n \"The combined seed extracts are mixed and/or incubated under conditions that allow maximum recovery of the immunoglobulin by Protein A.\",\n \"The oil body fraction is separated using standard phase separation techniques (e.g.\",\n \"centrifugation).\",\n \"The recovered oil body fraction contains both native oil bodies, from the immunoglobulin expression line, and transgenic Protein A oil bodies from the Protein A-oleosin expression line.\",\n \"In a second embodiment, the plant lines expressing the immunoglobulin complex and the Protein A-oleosin fusion are crossed and individual plant lines expressing both components are identified and propagated.\",\n \"In this approach, the immunoglobulin complex and the Protein A-oleosin fusion are produced in different cellular compartments of the same plant seed cell.\",\n \"Seed from the double transgenic line is ground to disrupt the cellular material and mix the contents of all cellular compartments, including combining the immunoglobulin in the extracellular compartment and the Protein A-oleosin on the oil body in the cytosolic compartment.\",\n \"The material is mixed and/or incubated under conditions to allow maximum recovery of the immunoglobulin by Protein A, and the oil body fraction is separated by phase separation techniques.\",\n \"The recovered oil body fraction contains the displayed Protein A and the capture immunoglobulin complex.\",\n \"Example 6 Production of Assembled Multimeric-Immunoglobulin-Complexes as Fusions with Oil Body Targeting Domains Individual polypeptides are produced as a fusion protein with oil body targeting sequences (e.g.\",\n \"oleosin) for display on oil bodies.\",\n \"It has been found that the individual subunits of naturally associating heterodimeric proteins can be co-produced as individual oleosin fusions and still associate as an active heterodimer on the surface of the oil body.\",\n \"In this example, the heterodimer is the light and heavy chain subunits, or derived portions thereof, of an immunoglobulin complex.\",\n \"Production of an Immunoglobulin Fab Complex on Oil Bodies.\",\n \"The mature light chain sequence, lacking the secretion signal sequence, is attached as an in-frame N-terminal fusion to an oleosin sequence.\",\n \"This fusion sequence is assembled into a seed-specific expression cassette consisting of a seed-specific promoter sequence, the light chain-oleosin fusion sequence, and a transcriptional terminator sequence.\",\n \"The expression cassette is inserted between the left and right border markers, adjacent to a plant selectable marker cassette, of a transformation vector.\",\n \"The transformation vector, in Agrobacterium, is used to infect plants and generate transgenic plants.\",\n \"An equivalent construct for the heavy chain subunit, comprising the variable and constant heavy chain domains, is also attached as an in-frame fusion to oleosin and assembled into an expression cassette for seed-specific expression.\",\n \"The expression cassette can be a part of a separate transformation vector for the generation of a separate transgenic line, or the heavy chain expression cassette can be combined together with the light chain cassette into a single transformation vector.\",\n \"If light and heavy chain expression cassettes are transformed into plants on separate transformation vectors, the individual plant lines are crossed to create a single line expressing both heterodimer subunit-oleosin fusions in the same plant cell.\",\n \"Seed from the double transgenic line, or a single transgenic line generated from the dual expression vector, is extracted to isolate oil bodies.\",\n \"The seed material is ground to release the cellular contents and oil bodies are isolated by phase separation.\",\n \"The targeting of both light and heavy chain sequence to oil bodies, as oleosin fusions, allows the association of the immunoglobulin complex on the surface of the oil body.\",\n \"Similar configurations, using the entire heavy chain sequence in combination with the entire light chain sequence, or using the variable domains from both the light and heavy chain sequences, are constructed to assemble different types of heteromultimeric-immunoglobulin-complexes (e.g., heterodimers) on the surface of oil bodies.\",\n \"The present invention should therefore not be seen as limited to the particular embodiments described herein, but rather, it should be understood that the present invention has wide applicability with respect to protein expression generally.\",\n \"Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.\",\n \"SUMMARY OF SEQUENCES SEQ ID NOs:1-4 set forth primers which were synthesized for the isolation of the thioredoxin h (Trxh) and thioredoxin reductase genes from Arabidopsis, as described in Example 1.SEQ ID NOs:5-7 set forth primers which were designed to code for a specific linker peptide between thioredoxin reductase and thioredoxin proteins, as described in Example 2.SEQ ID NOs:8, 10 and 11 set forth the nucleotide sequence and the deduced amino acid sequence of the NADPH thioredoxin reductase sequence isolated herein as described in Example 1.SEQ ID NOs:9 and 11, respectively, set forth the nucleotide sequence of the published NADPH thioredoxin reductase sequence (ATTHIREDB) and the deduced amino acid sequence.\",\n \"SEQ ID NO:12 sets forth the deduced amino acid sequence of the published NADPH thioredoxin reductase sequence.\",\n \"SEQ ID NO:13 sets forth the deduced amino acid sequence of the NADPH reductase sequence isolated in this report.\",\n \"SEQ ID NOs:14 and 15 set forth the nucleotide sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequence.\",\n \"The Trxh coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554, and the phaseolin terminator corresponds to nucleotide sequence 1905-3124.The promoter was furnished with a PstI site (nt 1-6) and the terminator was furnished with a HindIII site (nt 1898-1903) and a KpnI site (nt 3124-3129) to facilitate cloning.\",\n \"SEQ ID NOs:16, 17 and 18 set forth the nucleotide sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequences.\",\n \"The oleosin-Trxh coding sequence and the deduced amino acid sequences are indicated in SEQ ID NO:16.As in SEQ ID NO:14, the phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1908-2147) is indicated in italics.\",\n \"The Trxh coding sequence corresponds to nt 2314-2658.The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:19, 20 and 21 set forth the nucleotide sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The Trxh oleosin-coding sequence and its deduced amino acid sequences are indicated in SEQ ID NO:19.As in SEQ ID NOs:14 and 16, the phaseolin promoter corresponds to nucleotide 6-1554.The Trxh coding sequence corresponds to nt 1555-1896.The sequence encoding oleosin corresponds to nt 1897-2658, the intron in this sequence (nt 2250-2489) is indicated in italics.\",\n \"The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:22 and 23 set forth the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequence.\",\n \"The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated in SEQ ID NO:22.The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2556 and the deduced amino acid is set forth in SEQ ID NO:23.The phaseolin terminator corresponds to nucleotide sequence 2563-3782.SEQ ID NOs:24, 25 and 26 show the nucleotide sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The oleosin-thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1980-2147) is indicated in italics.\",\n \"The thioredoxin-reductase coding sequence corresponds to nt 2314-3315.The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NOs:27, 28 and 29 show the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2553.The sequence encoding oleosin corresponds to nt 2554-3315, the intron in this sequence (nt 2751-3146) is indicated in italics.\",\n \"The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NO:30, 31 and 32 show the sequence of the phaseolin promoter-oleosin-Mlep thioredoxin-reductase/thioredoxin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.\",\n \"The oleosin-Mlep thioredoxin-reductase/thioredoxin coding sequence and its deduced amino acid sequence is indicated.\",\n \"The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt) is indicated in italics.\",\n \"The Mlep thioredoxin-reductase/thioredoxin coding sequence corresponds to nt 2314-3690.The phaseolin terminator corresponds to nucleotide sequence 3698-4917.SEQ ID NOs:33, 34 and 35 set forth the nucleotide sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region of pSBS2542, and the deduced amino acid sequences.\",\n \"The deduced amino acid sequence of oleosin-thioredoxin-reductase-linker-thioredoxin is also shown in SEQ ID NO:33.Amino acids representing oleosin are set forth at positions 1-173, those amino acids representing thioredoxin-reductase are set forth at positions 174-501, those amino acids representing the linker or spacer peptide are set forth at positions 501-524, and those representing thioredoxin are set forth at positions 525-636.SEQ ID NOs:38 and 39 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin h (Trx h 1) and the encoded protein, respectively.\",\n \"SEQ ID NOs:40 and 41 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin Reductase (NTR1) and the encoded protein, respectively.\",\n \"SEQ ID NOs:42 and 43 set forth the nucleotide sequence of E. Coli Thioredoxin (TrxA) and the encoded protein, respectively.\",\n \"SEQ ID NOs:44 and 45, set forth the nucleotide sequence of E. Coli Thioredoxin Reductase and the encoded protein, respectively.\",\n \"SEQ ID NOs:46 and 47 set forth the nucleotide sequence of Human Thioredoxin and the encoded protein, respectively.\",\n \"SEQ ID NOs:48 and 49, set forth the nucleotide sequence of Human Thioredoxin Reductase and the encoded protein, respectively.\",\n \"SEQ ID NOs:50 and 51, respectively, set forth the nucleotide sequence of Mycobacterium leprae Thioredoxin-Thioredoxin Reductase and the encoded protein, respectively.\",\n \"SEQ ID NOs:52-313 are described in Table 5.TABLE 5 EXAMPLES OF REDOX PROTEINS SWISS PROTEIN IDENTIFIER SEQ.\",\n \"ID NO.\",\n \"(in parenthesis) PLANT THIOREDOXINS Thioredoxin f-type 52 (Q9XFH8) Thioredoxin F-type 1, chloroplast precursor (TRX- F1).\",\n \"- Arabidopsis thaliana (Mouse-ear cress) 53 (Q9XFH9) Thioredoxin F-type 2, chloroplast precursor (TRX- F2).\",\n \"{GENE: AT5G16400 OR MQK4.13} - Arabidopsis thaliana (Mouse-ear cress) 54 (O48897) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"{GENE: TRXF} - Brassica napus (Rape) 55 (O81332) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"- Mesembryanthemum crystallinum (Common ice plant) 56 (P29450) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"- Pisum sativum (Garden pea) 57 (P09856) Thioredoxin F-type, chloroplast precursor (TRX-F).\",\n \"- Spinacia oleracea (Spinach) Thioredoxin m-type 58 (P06544) Thioredoxin 1 (TRX-1) (Thioredoxin M).\",\n \"{GENE: TRXA} - Anabaena sp.\",\n \"(strain PCC 7119) 59 (O48737) Thioredoxin M-type 1, chloroplast precursor (TRX- M1).\",\n \"{GENE: AT1G03680 OR F21B7_7 OR F21B7.28} - Arabidopsis thaliana (Mouse-ear cress) 60 (Q9SEU8) Thioredoxin M-type 2, chloroplast precursor (TRX- M2).\",\n \"{GENE: AT4G03520 OR F9H3.15 OR T5L23.1} - Arabidopsis thaliana (Mouse-ear cress) 61 (Q9SEU7) Thioredoxin M-type 3, chloroplast precursor (TRX- M3).\",\n \"{GENE: AT2G15570 OR F9O13.12} - Arabidopsis thaliana (Mouse-ear cress) 62 (Q9SEU6) Thioredoxin M-type 4, chloroplast precursor (TRX- M4).\",\n \"- Arabidopsis thaliana (Mouse-ear cress) 63 (Q9XGS0) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Brassica napus (Rape) 64 (P23400) Thioredoxin M-type, chloroplast precursor (TRX-M) (Thioredoxin CH2).\",\n \"{GENE: TRXM} - Chlamydomonas reinhardtii 65 (Q41864) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"{GENE: TRM1} - Zea mays (Maize) 66 (Q9ZP20) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Oryza sativa (Rice) 67 (P48384) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Pisum sativum (Garden pea) 68 (P07591) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Spinacia oleracea (Spinach) 69 (Q9ZP21) Thioredoxin M-type, chloroplast precursor (TRX-M).\",\n \"- Triticum aestivum (Wheat) 70 (P12243) Thioredoxin 1 (TRX-1) (Thioredoxin M).\",\n \"{GENE: TRXA OR TRXM} - Synechococcus sp.\",\n \"(strain PCC 7942) (Anacystis nidulans R2) 71 (P37395) Thioredoxin.\",\n \"{GENE: TRXA OR TRX} - Cyanidium caldarium [Chloroplast] 72 (O22022) Thioredoxin.\",\n \"{GENE: TRXA OR TRXM} - Cyanidioschyzon merolae [Chloroplast] 73 (P50338) Thioredoxin.\",\n \"{GENE: TRXA} - Griffithsia pacifica [Chloroplast] 74 (P50254) Thioredoxin.\",\n \"{GENE: TRXA} - Porphyra yezoensis [Chloroplast] 75 (P51225) Thioredoxin.\",\n \"{GENE: TRXA} - Porphyra purpurea [Chloroplast] Thioredoxin h-type 76 (P29448) Thioredoxin H-type 1 (TRX-H-1).\",\n \"{GENE: TRX1 OR AT3G51030 OR F24M12.70} - Arabidopsis thaliana (Mouse- ear cress) 77 (P20857) Thioredoxin 2 (TRX-2).\",\n \"{GENE: TRXB} - Anabaena sp.\",\n \"(strain PCC 7120) 78 (Q42388) Thioredoxin H-type 1 (TRX-H-1) (Pollen coat protein).\",\n \"{GENE: THL-1 OR BOPC17} - Brassica napus (Rape), Brassica oleracea (Cauliflower) 79 (P29449) Thioredoxin H-type 1 (TRX-H1).\",\n \"- Nicotiana tabacum (Common tobacco) 80 (Q38879) Thioredoxin H-type 2 (TRX-H-2).\",\n \"{GENE: TRX2 OR AT5G39950 OR MYH19.14} - Arabidopsis thaliana (Mouse-ear cress) 81 (Q39362) Thioredoxin H-type 2 (TRX-H-2).\",\n \"{GENE: THL-2} - Brassica napus (Rape) 82 (Q07090) Thioredoxin H-type 2 (TRX-H2).\",\n \"- Nicotiana tabacum (Common tobacco) 83 (Q42403) Thioredoxin H-type 3 (TRX-H-3).\",\n \"{GENE: TRX3 OR AT5G42980 OR MBD2.18} - Arabidopsis thaliana (Mouse-ear cress) 84 (Q39239) Thioredoxin H-type 4 (TRX-H-4).\",\n \"{GENE: TRX4} - Arabidopsis thaliana (Mouse-ear cress) 85 (Q39241) Thioredoxin H-type 5 (TRX-H-5).\",\n \"{GENE: TRX5} - Arabidopsis thaliana (Mouse-ear cress) 86 (O64432) Thioredoxin H-type (TRX-H).\",\n \"{GENE: PEC-2} - Brassica rapa (Turnip) 87 (P80028) Thioredoxin H-type (TRX-H) (Thioredoxin CH1).\",\n \"{GENE: TRXH} - Chlamydomonas reinhardtii 88 (Q96419) Thioredoxin H-type (TRX-H).\",\n \"- Fagopyrum esculentum (Common buckwheat) 89 (Q42443) Thioredoxin H-type (TRX-H) (Phloem sap 13 kDa protein-1).\",\n \"- Oryza sativa (Rice) 90 (O65049) Thioredoxin H-type (TRX-H).\",\n \"{GENE: SB09} - Picea mariana (Black spruce) 91 (Q43636) Thioredoxin H-type (TRX-H).\",\n \"- Ricinus communis (Castor bean) 92 (O64394) Thioredoxin H-type (TRX-H) (TrxTa).\",\n \"- Triticum aestivum (Wheat) 93 (P29429) Thioredoxin.\",\n \"- Emericella nidulans (Aspergillus nidulans) VIRUSES, BACTERIA AND FUNGI THIOREDOXINS 94 (P80579) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Alicyclobacillus acidocaldarius (Bacillus acidocaldarius) 95 (O28137) Thioredoxin.\",\n \"{GENE: AF2145} - Archaeoglobus fulgidus 96 (P14949) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX} - Bacillus subtilis 97 (P00276) Thioredoxin.\",\n \"{GENE: NRDC} - Bacteriophage T4 98 (O51088) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR BB0061} - Borrelia burgdorferi (Lyme disease spirochete) 99 (P57653) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR BU597} - Buchnera aphidicola (subsp.\",\n \"Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 100 (O51890) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Buchnera aphidicola (subsp.\",\n \"Schizaphis graminum) 101 (P10472) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Chlorobium limicola f.sp.\",\n \"thiosulfatophilum 102 (Q9PJK3) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TC0826} - Chlamydia muridarum 103 (Q9Z7P5) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR CPN0659 OR CP0088} - Chlamydia pneumoniae (Chlamydophila pneumoniae) 104 (P52227) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Chlamydia psittaci (Chlamydophila psittaci) 105 (O84544) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR CT539} - Chlamydia trachomatis 106 (P00275) Thioredoxin C-1.- Corynebacterium nephridii 107 (P07887) Thioredoxin C-2.- Corynebacterium nephridii 108 (P52228) Thioredoxin C-3.- Corynebacterium nephridii 109 (P09857) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Chromatium vinosum 110 (P21609) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Clostridium litorale (Bacterium W6) 111 (P81108) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Clostridium sporogenes 112 (P81109) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Clostridium sticklandii 113 (Q9UW02) Thioredoxin (Allergen Cop c 2).\",\n \"- Coprinus comatus (Shaggy mane) 114 (P29445) Thioredoxin 1.\",\n \"{GENE: TRXA OR TRX1} - Dictyostelium discoideum (Slime mold) 115 (P29446) Thioredoxin 2 (Fragment).\",\n \"{GENE: TRXB OR TRX2} - Dictyostelium discoideum (Slime mold) 116 (P29447) Thioredoxin 3.\",\n \"{GENE: TRXC OR TRX3} - Dictyostelium discoideum (Slime mold) 117 (P00274) Thioredoxin 1 (TRX1) (TRX).\",\n \"{GENE: TRXA OR TSNC OR FIPA OR B3781} - Escherichia coli, Salmonella typhimurium 118 (P52232) Thioredoxin-like protein SLR0233.\",\n \"{GENE: SLR0233} - Synechocystis sp.\",\n \"(strain PCC 6803) 119 (P33636) Thioredoxin 2 (Trx2).\",\n \"{GENE: TRXC OR B2582 OR Z3867 OR ECS3448} - Escherichia coli, Escherichia coli O157:H7 120 (P21610) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Eubacterium acidaminophilum 121 (P43785) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRXM OR HI0084} - Haemophilus influenzae 122 (P43787) Thioredoxin-like protein HI1115.\",\n \"{GENE: HI1115} - Haemophilus influenzae 123 (P56430) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR HP0824 OR JHP0763} - Helicobacter pylori (Campylobacter pylori), Helicobacter pylori J99 (Campylobacter pylori J99) 124 (Q9S386) Thioredoxin (EC 1.6.4.5) {GENE: TRXA} - Listeria monocytogenes 125 (Q57755) Thioredoxin.\",\n \"{GENE: TRX OR MJ0307} - Methanococcus jannaschii 126 (P47370) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX OR MG124} - Mycoplasma genitalium 127 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].\",\n \"{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 128 (P75512) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX OR MPN263 OR MP570} - Mycoplasma pneumoniae 129 (O30974) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Mycobacterium smegmatis 130 (P52229) Thioredoxin (TRX) (MPT46).\",\n \"{GENE: TRXA OR TRX OR TRXC OR RV3914 OR MT4033 OR MTV028.05} - Mycobacterium tuberculosis 131 (P42115) Thioredoxin.\",\n \"{GENE: TRX} - Neurospora crassa 132 (P34723) Thioredoxin.\",\n \"{GENE: TRXA} - Penicillium chrysogenum 133 (Q9X2T1) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TRX OR PA5240} - Pseudomonas aeruginosa 134 (P10473) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Rhodospirillum rubrum 135 (P08058) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) 136 (Q9ZEE0) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR RP002} - Rickettsia prowazekii 137 (P33791) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Streptomyces aureofaciens 138 (P52230) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR SCH24.11C} - Streptomyces coelicolor 139 (Q05739) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Streptomyces clavuligerus 140 (P52231) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR SLR0623} - Synechocystis sp.\",\n \"(strain PCC 6803) 141 (P73263) Thioredoxin-like protein SLR1139.\",\n \"{GENE: SLR1139} - Synechocystis sp.\",\n \"(strain PCC 6803) 142 (P52233) Thioredoxin (TRX).\",\n \"{GENE: TRXA} - Thiobacillus ferrooxidans 143 (P96132) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Thiocapsa roseopersicina 144 (P81110) Thioredoxin (TRX) (Fragment).\",\n \"{GENE: TRXA} - Tissierella creatinophila 145 (O83889) Thioredoxin (TRX).\",\n \"{GENE: TRXA OR TP0919} - Treponema pallidum ANIMAL THIOREDOXIN 146 (O97680) Thioredoxin.\",\n \"{GENE: TXN} - Bos taurus (Bovine) 147 (Q95108) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2} - Bos taurus (Bovine) 148 (Q09433) Thioredoxin.\",\n \"{GENE: B0228.5} - Caenorhabditis elegans 149 (P99505) Thioredoxin (Fragment).\",\n \"{GENE: TXN} - Canis familiaris (Dog 150 (P08629) Thioredoxin.\",\n \"{GENE: TXN} - Gallus gallus (Chicken) 151 (P47938) Thioredoxin (Deadhead protein).\",\n \"{GENE: DHD OR CG4193} - Drosophila melanogaster (Fruit fly) 152 (P10599) Thioredoxin (ATL-derived factor) (ADF) (Surface associated sulphydryl protein) (SASP).\",\n \"{GENE: TXN OR TRDX OR TRX} - Homo sapiens (Human) 153 (Q99757) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2} - Homo sapiens (Human) 154 (P29451) Thioredoxin.\",\n \"{GENE: TXN} - Macaca mulatta (Rhesus macaque) 155 (P10639) Thioredoxin (ATL-derived factor) (ADF).\",\n \"{GENE: TXN} - Mus musculus (Mouse) 156 (P97493) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2} - Mus musculus (Mouse) 157 (P82460) Thioredoxin (Fragment).\",\n \"{GENE: TXN} - Sus scrofa (Pig) 158 (P08628) Thioredoxin.\",\n \"{GENE: TXN} - Oryctolagus cuniculus (Rabbit) 159 (P11232) Thioredoxin.\",\n \"{GENE: TXN} - Rattus norvegicus (Rat) 160 (P97615) Thioredoxin, mitochondrial precursor (MT-TRX).\",\n \"{GENE: TXN2 OR TRX2} - Rattus norvegicus (Rat) 161 (P50413) Thioredoxin.\",\n \"{GENE: TXN} - Ovis aries (Sheep) PLANTS THIOREDOXIN-LIKE PROTEINS 162 (O23166) THIOL-DISULFIDE INTERCHANGE LIKE PROTEIN (THIOREDOXIN-LIKE PROTEIN) {GENE: C7A10.160 OR AT4G37200 OR HCF164} - Arabidopsis thaliana (Mouse-ear cress) 163 (Q9C9Y6) Thioredoxin-like protein {GENE: F17O14.18} - Arabidopsis thaliana (Mouse-ear cress) 164 (Q9FYD5) Thioredoxin-like protein {GENE: F21E1_180} - Arabidopsis thaliana (Mouse-ear cress) 165 (Q38878) THIOREDOXIN-LIKE PROTEIN {GENE: TRX6 OR T7D17.3} - Arabidopsis thaliana (Mouse-ear cress) 166 (Q9LVI2) Thioredoxin-like protein - Arabidopsis thaliana (Mouse-ear cress) 167 (Q9SCN9) Thioredoxin-like protein {GENE: T4D2.150} - Arabidopsis thaliana (Mouse-ear cress) 168 (Q9SRD7) Thioredoxin-like protein, 49720-48645 {GENE: F28O16.13} - Arabidopsis thaliana (Mouse-ear cress) 169 (Q9SU84) THIOREDOXIN-LIKE PROTEIN {GENE: T16L4.180 OR AT4G29670} - Arabidopsis thaliana (Mouse-ear cress) 170 (Q9SWG6) Thioredoxin-like protein {GENE: TRX} - Hordeum bulbosum 171 (Q9SWG4) Thioredoxin-like protein {GENE: TRX} - Lolium perenne (Perennial ryegrass) 172 (Q9AS75) Thioredoxin-like protein {GENE: P0028E10.17} - Oryza sativa (Rice) 173 (O04002) CDSP32 protein (Chloroplast Drought-induced Stress Protein of 32 kDa) - Solanum tuberosum (Potato) 174 (Q9SWG5) Thioredoxin-like protein {GENE: TRX} - Secale cereale (Rye) 175 (Q9SP36) Thioredoxin-like protein (Fragment) {GENE: TRX} - Secale cereale (Rye) 176 (Q9U515) Thioredoxin-like protein - Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-LIKE PROTEINS 177 (P43221) Thiol: disulfide interchange protein tlpA (Cytochrome c biogenesis protein tIpA).\",\n \"{GENE: TLPA} - Bradyrhizobium japonicum 178 (P43787) Thioredoxin-like protein HI1115.\",\n \"{GENE: HI1115} - Haemophilus influenzae 179 (Q9GUP7) Thioredoxin-like protein {GENE: TRXLP} - Leishmania major 180 (Q9UVH0) Thioredoxin-like protein - Mortierella alpina 181 (P95355) Thioredoxin-like protein - Neisseria gonorrhoeae 182 (Q98G37) Thioredoxin-like protein {GENE: MLL3505} - Rhizobium loti (Mesorhizobium loti) 183 (P36893) Thiol: disulfide interchange protein helX precursor (Cytochrome c biogenesis protein helX).\",\n \"{GENE: HELX} - Rhodobacter capsulatus (Rhodopseudomonas capsulata) 184 (P52232) Thioredoxin-like protein SLR0233.\",\n \"{GENE: SLR0233} - Synechocystis sp.\",\n \"(strain PCC 6803) 185 (P73263) Thioredoxin-like protein SLR1139.\",\n \"{GENE: SLR1139} - Synechocystis sp.\",\n \"(strain PCC 6803) 186 (Q9USR1) Thioredoxin-like protein {GENE: SPBC577.08C} - Schizosaccharomyces pombe (Fission yeast) 187 (Q9R788) Thioredoxin {GENE: TPTRX} - Treponema pallidum ANIMALS THIOREDOXIN-LIKE PROTEINS 188 (Q9UAV4) F46E10.9 PROTEIN (THIOREDOXIN-LIKE PROTEIN DPY-11) {GENE: F46E10.9 OR DPY-11} - Caenorhabditis elegans 189 (Q9N2K6) Thioredoxin-like protein (Y54E10A.3 protein) (Thioredoxin-like protein TXL) {GENE: TXL OR Y54E10A.3} - Caenorhabditis elegans 190 (Q9VRP3) THIOREDOXIN-LIKE PROTEIN TXL (CG5495 PROTEIN) {GENE: TXL OR CG5495} - Drosophila melanogaster (Fruit fly) 191 (O43396) Thioredoxin-like protein (32 kDa thioredoxin-related protein).\",\n \"{GENE: TXNL OR TRP32 OR TXL} - Homo sapiens (Human) 192 (O76003) Thioredoxin-like protein - Homo sapiens (Human) 193 (Q9S753) THIOREDOXIN-LIKE PROTEIN {GENE: TRX} - Phalaris coerulescens 194 (O77404) TRYPAREDOXIN - Trypanosoma brucei brucei PLANT THIOREDOXIN-REDUCTASES 195 (Q39243) Thioredoxin-reductase 1 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 1) (NTR 1).\",\n \"{GENE: NTR1 OR AT4G35460 OR F15J1.30} - Arabidopsis thaliana (Mouse-ear cress) 196 (Q39242) Thioredoxin-reductase 2 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 2) (NTR 2).\",\n \"{GENE: NTR2 OR AT2G17420 OR F5J6.18} - Arabidopsis thaliana (Mouse-ear cress) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 197 (O66790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR AQ_500} - Aquifex aeolicus 198 (P80880) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (General stress protein 35) (GSP35).\",\n \"{GENE: TRXB} - Bacillus subtilis 199 (P94284) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR BB0515} - Borrelia burgdorferi (Lyme disease spirochete) 200 (P57399) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR BU314} - Buchnera aphidicola (subsp.\",\n \"Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 201 (P81433) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Buchnera aphidicola (subsp.\",\n \"Schizaphis graminum) 202 (Q9PKT7) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR TC0375} - Chlamydia muridarum 203 (Q9Z8M4) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR CPN0314 OR CP0444} - Chiamydia pneumoniae (Chlamydophila pneumoniae) 204 (O84101) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR CT099} - Chlamydia trachomatis 205 (P52213) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Clostridium litorale (Bacterium W6) 206 (P39916) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Coxiella burnetii 207 (P09625) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR B0888 OR Z1232 OR ECSO973} - Escherichia coli, Escherichia coli O157: H7 208 (P50971) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Eubacterium acidaminophilum 209 (P43788) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR HI1158} - Haemophilus influenzae 210 (Q9ZL18) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR JHP0764} - Helicobacter pylori J99 (Campylobacter pylori J99) 211 (P56431) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR HP0825} - Helicobacter pylori (Campylobacter pylori) 212 (O32823) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR LMO2478} - Listeria monocytogenes 213 (P47348) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR MG102} - Mycoplasma genitalium 214 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].\",\n \"{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 215 (P75531) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR MPN240 OR MP591} - Mycoplasma pneumoniae 216 (O30973) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Mycobacterium smegmatis 217 (P52214) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (TR).\",\n \"{GENE: TRXB OR RV3913 OR MT4032 OR MTV028.04} - Mycobacterium tuberculosis 218 (P51978) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: CYS-9} - Neurospora crassa 219 (P43496) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: TRXB} - Penicillium chrysogenum 220 (Q9ZD97) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR RP445} - Rickettsia prowazekii 221 (Q92375) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: SPBC3F6.03} - Schizosaccharomyces pombe (Fission yeast) 222 (Q05741) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB} - Streptomyces clavuligerus 223 (P52215) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR SCH24.12} - Streptomyces coelicolor 224 (O83790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).\",\n \"{GENE: TRXB OR TP0814} - Treponema pallidum 225 (P80892) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (Fragment).\",\n \"{GENE: TRXB} - Vibrio fischeri 226 (P29509) Thioredoxin-reductase 1 (EC 1.6.4.5).\",\n \"{GENE: TRR1 OR YDR353W OR D9476.5} - Saccharomyces cerevisiae (Baker's yeast) 227 (P38816) Thioredoxin-reductase 2, mitochondrial precursor (EC 1.6.4.5).\",\n \"{GENE: TRR2 OR YHR106W} - Saccharomyces cerevisiae (Baker's yeast) ANIMAL THIOREDOXIN-REDUCTASES 228 (O62768) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: TXNRD1} - Bos taurus (Bovine) 229 (Q17745) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: C06G3.7} - Caenorhabditis elegans 230 (Q16881) Thioredoxin-reductase (EC 1.6.4.5).\",\n \"{GENE: TXNRD1} - Homo sapiens (Human) 231 (Q25861) Thioredoxin-reductase (EC 1.6.4.5) (TrxR).\",\n \"{GENE: TR OR GR} - Plasmodium falciparum (isolate FCH-5) Other thioredoxin-reductases PLANTS THIOREDOXIN-REDUCTASES 232 (O22229) Thioredoxin-reductase {GENE: AT2G41680} - Arabidopsis thaliana (Mouse-ear cress) 233 (Q39951) NADPH thioredoxin-reductase (Fragment} - Helianthus annuus (Common sunflower) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 234 (O28718) THioredoxin-reductase (TRXB) {GENE: AF1554} - Archaeoglobus fulgidus 235 (Q9K703) Thioredoxin-reductase (NADPH) (EC 1.6.4.5) {GENE: TRXB OR BH3571} - Bacillus halodurans 236 (Q9K7F3) Thioredoxin-reductase {GENE: BH3408} - Bacillus halodurans 237 (Q9KCZ0) Thioredoxin-reductase {GENE: BH1429} - Bacillus halodurans 238 (Q9KCZ1) Thioredoxin-reductase {GENE: BH1428} - Bacillus halodurans 239 (Q9PIY1) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR CJ0146} - Campylobacter jejuni 240 (Q9A4G3) Thioredoxin-reductase {GENE: CC2871} - Caulobacter crescentus 241 (Q97EM8) Thioredoxin-reductase {GENE: CAC3082} - Clostridium acetobutylicum 242 (Q97IU2) Thioredoxin-reductase {GENE: CAC1548} - Clostridium acetobutylicum 243 (Q9EV96) Thioredoxin-reductase {GENE: TRXB} - Clostridium sticklandii 244 (Q9RSY7) THioredoxin-reductase {GENE: DR1982} - Deinococcus radiodurans 245 (O30739) Thioredoxin-reductase (Fragment} - Enterococcus faecalis (Streptococcus faecalis) 246 (O54535) Thioredoxin-reductase {GENE: TRXB OR TRXB1_2 OR VNG6452G OR TRXB1_1 OR VNG6074G} - Halobacterium sp.\",\n \"(strain NRC-1) [Plasmid pNRC100, and Plasmid pNRC200] 247 (P82854) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Halobacterium sp.\",\n \"(strain NRC-1) 248 (Q9HN08) Thioredoxin-reductase {GENE: TXRB3 OR VNG2301G} - Halobacterium sp.\",\n \"(strain NRC-1) 249 (O25779) THioredoxin-reductase (TRXB) {GENE: HP1164} - Helicobacter pylori (Campylobacter pylori) 250 (O86255) Thioredoxin-reductase {GENE: TRXB} - Klebsiella oxytoca 251 (Q9AEV9) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis (subsp.\",\n \"lactis) (Streptococcus lactis) 252 (Q9CF34) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Lactococcus lactis (subsp.\",\n \"lactis) (Streptococcus lactis) 253 (Q9CH02) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB1} - Lactococcus lactis (subsp.\",\n \"lactis) (Streptococcus lactis) 254 (Q9ZFC8) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis 255 (O32822) Hypothetical 39.7 kDa protein (Fragment) - Listeria monocytogenes 256 (O26804) THioredoxin-reductase {GENE: MTH708} - Methanothermobacter thermautotrophicus 257 (P94397) Homologue of thioredoxin-reductase of Mycoplama genitalium {GENE: YCGT} - Bacillus subtilis 258 (Q98PK9) THioredoxin-reductase (EC 1.6.4.5) {GENE: MYPU_7130} - Mycoplasma pulmonis 259 (Q9JU23) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR NMA1538} - Neisseria meningitidis (serogroup A) 260 (Q9JZ28) Thioredoxin-reductase {GENE: NMB1324} - Neisseria meningitidis (serogroup B) 261 (Q9I0M2) Thioredoxin-reductase 1 {GENE: TRXB1 OR PA2616} - Pseudomonas aeruginosa 262 (Q9I592) Thioredoxin-reductase 2 {GENE: TRXB2 OR PA0849} - Pseudomonas aeruginosa 263 (Q9V0Q8) THioredoxin-reductase (TRXB) {GENE: TRXB OR PAB0500} - Pyrococcus abyssi 264 (Q9ZD33) THioredoxin-reductase (TRXB2) {GENE: RP514} - Rickettsia prowazekii 265 (O54079) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB} - Staphylococcus aureus 266 (Q9RIS2) Thioredoxin-reductase {GENE: TRXB OR TRXB2} - Streptomyces coelicolor 267 (Q9K4L6) Thioredoxin-reductase {GENE: SC5F8.08C} - Streptomyces coelicolor 268 (Q97PY2) Thioredoxin-reductase {GENE: SP1458} - Streptococcus pneumoniae 269 (Q9A0B5) Thioredoxin-reductase {GENE: SPY0850} - Streptococcus pyogenes 270 (Q97V69) Thioredoxin-reductase (trxB-2) (EC 1.6.4.5) {GENE: TRXB-2} - Sulfolobus solfataricus 271 (Q97W27) Thioredoxin-reductase (trxB-3) (EC 1.6.4.5) {GENE: TRXB-3} - Sulfolobus solfataricus 272 (Q97WJ5) Thioredoxin-reductase (trxB-1) (EC 1.6.4.5) {GENE: TRXB-1} - Sulfolobus solfataricus 273 (Q98I59) Thioredoxin-reductase {GENE: MLL2552} - Rhizobium loti (Mesorhizobium loti) 274 (Q98M06) Thioredoxin-reductase {GENE: MLL0792} - Rhizobium loti (Mesorhizobium loti) 275 (Q9UR80) 35 kDa THioredoxin-reductase HOMOLOG (FRAGMENT) {GENE: TRR1 AND YDR353W} - Saccharomyces cerevisiae (Baker's yeast) 276 (Q9ZEH4) THIOREDOXIN {GENE: TRXA OR SA0992} - Staphylococcus aureus, Staphylococcus aureus subsp.\",\n \"aureus N315 277 (Q9S1H1) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Staphylococcus xylosus 278 (Q9HJI4) Thioredoxin-reductase {GENE: TA0984} - Thermoplasma acidophilum 279 (Q9WZX3) THioredoxin-reductase {GENE: TM0869} - Thermotoga maritima 280 (Q979K8) Thioredoxin-reductase {GENE: TVG1183005} - Thermoplasma volcanium 281 (Q9PR71) Thioredoxin-reductase {GENE: TRXB OR UU074} - Ureaplasma parvum (Ureaplasma urealyticum biotype 1) 282 (Q9KSS4) Thioredoxin-reductase {GENE: VC1182} - Vibrio cholerae 283 (Q9PDD1) Thioredoxin-reductase {GENE: XF1448} - Xylella fastidiosa 284 (Q9X5F7) Thioredoxin-reductase {GENE: TRXB1} - Zymomonas mobilis ANIMAL THIOREDOXIN-REDUCTASES 285 (Q9GKW9) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Bos taurus (Bovine) 286 (Q9N2I8) Thioredoxin-reductase (EC 1.6.4.5) - Bos taurus (Bovine) 287 (Q9N2K1) Thioredoxin-reductase homolog - Caenorhabditis elegans 288 (Q9NJH3) Thioredoxin-reductase - Caenorhabditis elegans 289 (Q9VNT5) CG11401 PROTEIN (THioredoxin-reductase 2) {GENE: TRXR-2 OR CG11401} - Drosophila melanogaster (Fruit fly) 290 (O95840) Thioredoxin-reductase - Homo sapiens (Human) 291 (Q9UES8) Thioredoxin-reductase GRIM-12 - Homo sapiens (Human) 292 (Q9UH79) Thioredoxin-reductase {GENE: TR} - Homo sapiens (Human) 293 (Q9UQU8) Thioredoxin-reductase - Homo sapiens (Human) 294 (Q9NNW6) Thioredoxin-reductase TR2 (Fragment) - Homo sapiens (Human) 295 (Q9NNW7) Thioredoxin-reductase TR3 - Homo sapiens (Human) 296 (Q9P101) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Homo sapiens (Human) 297 (Q9P2Y0) Thioredoxin-reductase II beta (EC 1.6.4.5) - Homo sapiens (Human) 298 (Q9H2Z5) Mitochondrial thioredoxin-reductase {GENE: TRXR2A} - Homo sapiens (Human) 299 (Q99475) KM-102-DERIVED REDUCTASE-LIKE FACTOR (THioredoxin-reductase) - Homo sapiens (Human) 300 (Q99P49) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 301 (Q9CSV5) Thioredoxin-reductase 1 (Fragment) {GENE: TXNRD1} - Mus musculus (Mouse) 302 (Q9CZE5) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 303 (Q9JHA7) Thioredoxin-reductase TR3 {GENE: TXNRD2 OR TR3} - Mus musculus (Mouse) 304 (Q9JLT4) Thioredoxin-reductase {GENE: TXNRD2 OR TRXR2} - Mus musculus (Mouse) 305 (Q9JMH5) Thioredoxin-reductase 2 {GENE: TXNRD2 OR TXNRD2} - Mus musculus (Mouse) 306 (Q9JMH6) Thioredoxin-reductase 1 {GENE: TXNRD1 OR TXNRD1} - Mus musculus (Mouse) 307 (O89049) Thioredoxin-reductase - Rattus norvegicus (Rat) 308 (Q9JKZ3) Thioredoxin-reductase 1 (Fragment) - Rattus norvegicus (Rat) 309 (Q9JKZ4) Thioredoxin-reductase 1 - Rattus norvegicus (Rat) 310 (Q9JLE6) Thioredoxin-reductase (Fragment) - Rattus norvegicus (Rat) 311 (Q9R1I3) NADPH-dependent thioredoxin-reductase {GENE: TRR1} - Rattus norvegicus (Rat) 312 (Q9Z0J5) Thioredoxin-reductase precursor {GENE: TRXR2} - Rattus norvegicus (Rat) 313 (Q9MYY8) Redox enzyme thioredoxin-reductase - Sus scrofa (Pig)\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450903"}}},{"rowIdx":287,"cells":{"text":{"kind":"list like","value":[["Adaptive comparator circuit and acoustic distance sensor comprising said circuit","The invention relates to an adaptive comparator circuit comprising a comparator with a threshold value input which receives a threshold voltage and a signal input which receives a voltage signal.","The threshold value input is connected to one pole by means of a switch and connected to the other pole of a first voltage source by means of a capacitor.","The threshold value input is also connected to the signal input of the comparator by means of a diode or a second voltage source.","The switch is controlled by the control signal of a signal transmitter.","When the switch is in a closed position, the capacitor is charged with the voltage of the first voltage source.","When the switch is in an open position, the capacitor is discharged in such a way that the threshold voltage of the signal voltage is corrected at a given time interval."],["1.An adaptive comparator circuit, particularly for an acoustic distance sensor, comprising a first comparator (1), having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage (Uref1), said comparator emits a first switching signal (S1), characterized by a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).","2.The adaptive comparator according to claim 1, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).","3.The adaptive comparator according to claim 2, characterized in that the voltage supplied by the second voltage source (7) can be regulated or adjusted.","4.The adaptive comparator according to claim 1, characterized in that the signal transmitter (2) emits an electric control signal (SSt) at regular intervals.","5.The adaptive comparator according to claim 1, characterized in that the first switch (3, 103) is a switch that can be electrically or electronically controlled by the control means (3a, 103a) and the control signal (SSt) is an electric signal that is transmitted by the signal transmitter (2) via a control output (2a) and fed to the control input (3a, 103a).","6.The adaptive comparator according to claim 5, characterized in that the first switch (3, 103) is a transistor (103) and the control-current terminal (3a, 103a) is the base (103) or the gate terminal of the transistor (103).","7.The adaptive comparator according to claim 1, characterized in that a first resistor (11) is connected between the first pole (4a) and the first threshold voltage input (1a), and a second resistor (12) is connected between the first threshold voltage input (1a) and the second pole (4b), so that the first resistor (11) and the second resistor (12) form a first voltage divider (11, 12).","8.The adaptive comparator according to claim 7, characterized in that first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the switch output (3c, 103c).","9.The adaptive comparator according to claim 1, characterized by a second comparator (20) that has a second threshold voltage input (20a) and a second signal input (20b), and that emits a second switching signal S2 when a greater voltage is present at the second signal input (20b) than at the second threshold voltage input (20a), a third resistor (13) via which the first pole (4a) is connected to the second threshold voltage input (20a), a fourth resistor (14) that, on the one hand, is connected to the second threshold voltage input (20a) and, on the other hand, to the second pole (4b) of the first voltage source, so that the third resistor (13) and the fourth resistor (14) form a second voltage divider (13, 14), a second switch (23) that is connected in series to the diode (5) and that can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator (20) emits the second switching signal S2, and otherwise it is in the open state.","10.The adaptive comparator according to claim 9, characterized in that a fifth resistor (15) is connected between the switch output (3c, 103c) and the second threshold voltage input (20a).","11.The adaptive comparator according to claim 9, characterized in that the second voltage divider (13, 14) is a potentiometer.","12.The adaptive comparator according to claim 1, characterized in that a sixth resistor (16) is connected between the control output (2a) and the control-current terminal (3a, 103a).","13.The adaptive comparator according to claim 6, characterized in that a seventh resistor (17) is connected between the control-current terminal (3a, 103a) and the first pole (4a).","14.The adaptive comparator according to claim 1, characterized in that the diode (5) or the second voltage source (7) is connected in series to an eighth resistor (18).","15.The adaptive comparator according to claim 1, characterized in that, before reaching the first signal input (1b), the voltage signal Usignal passes through a voltage follower or impedance transformer (21).","16.The adaptive comparator according to claim 1, characterized in that the voltage supplied by the first voltage source (4a, 4b) is greater in magnitude than the voltage of the maximum of the voltage signal Usignal.","17.The adaptive comparator according to claim 1, characterized in that the diode (5) is connected to at least another diode codirectionally in series.","18.An acoustic distance sensor, comprising a control unit (31) that at times transmits signal pulses (41) to an oscillator (32) which, during the presence of a signal pulse (41), transmits an alternating voltage to a sound transducer (34) that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer (34) as an echo as a result of reflection, and of converting them into an electric received signal, and also comprising an envelope curve shaper (37) to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a voltage signal Usignal, characterized in that the voltage signal is fed to an adaptive comparator circuit comprising a first comparator (1) having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage Uref1, said comparator (1) emits a first switching signal (S1), a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output (3c, 103c) is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).","19.The acoustic distance sensor according to claim 18, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).","20.The acoustic distance sensor according to claim 18 characterized in that the alternating voltage emitted by the oscillator (32) reaches the sound transducer (34) after passing through a driver stage (33).","21.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through an amplifier (36c).","22.The acoustic distance sensor according to claim 21, characterized in that the amplifier (36c) is a logarithmic amplifier.","23.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through a voltage limiter (35) that limits the amplitude of the received signal to a maximum value.","24.The acoustic distance sensor according to claim 18, characterized in that the signal transmitter (2) is integrated into the control unit (31) or is a component of the control unit (31).","25.The acoustic distance sensor according to claim 18, characterized in that the first switching signal (S1) is transmitted to the control unit (31) for evaluation purposes.","26.The acoustic distance sensor according to claim 18, characterized in that the signal pulses (41) are synchronized with the control signals (SSt) in such a way that the control signals (SSt) each begin before the signal pulses (41).","27.The acoustic distance sensor according to claim 18, characterized in that the signal pulses 41 are synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses (41).","28.An adaptive comparator circuit for an acoustic distance sensor, comprising a first comparator (1) having a first signal input and a first threshold voltage input; a first direct voltage source (4) for delivering a threshold voltage to the first threshold voltage input, wherein the first direct voltage source comprises a first and a second pole; a first switch (3), wherein the first switch is connected to the first direct voltage source and to the first threshold voltage input; a signal transmitter (2), wherein the signal transmitter controls the first switch through an input of the switch; a capacitor (6), wherein a first plate of the capacitor is connected to the a first threshold voltage input and wherein a second plate of the capacitor is connected to second pole the first direct voltage source; a diode (5) connected the first signal input and to the first threshold voltage input of the first comparator.","29.The adaptive comparator circuit according to claim 28, wherein the diode is connected in series to a first resistor.","30.The adaptive comparator circuit according to claim 1, wherein the signal transmitter emits an electric control signal at regular intervals.","31.The adaptive comparator circuit according to claim 28 further comprising a second resistor (11) connected between the first pole of the first direct voltage source and the first threshold voltage input, and a third resistor (12) connected between the first threshold voltage input and the second pole of the first direct voltage source, so that the second resistor (11) and the third resistor (12) form a first voltage divider (11, 12).","32.The adaptive comparator circuit according to claim 31, wherein the first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the first threshold voltage input.","33.The adaptive comparator circuit according to claim 28 further comprising a fourth resistor (17) connected between the first pole of the first direct voltage source and the input of the first switch; a fifth resistor (16) connected between the input of the first switch and the output of the signal transmitter (2)."],[" BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.","1 —a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.","2 —a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","1 , FIG.","3 —a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.","4 —a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","3 , FIG.","5 —a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.","6 —a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.","7 and 8 —a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.","9 —a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo occurs during the quiescent phase, FIG.","10 —a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo additionally occurs during the relaxation phase, and FIGS.","11 to 13 —circuit diagrams of further embodiments of comparator circuits according to the invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?","FIGS.","1 to 4 serve to further illustrate the state of the art.","FIG.","1 shows a schematic block diagram of a variant of an acoustic distance sensor 30 a with which a measure according to the state of the art has been taken in order to reduce the short range.","The distance sensor 30 a shown in FIG.","1 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , an amplifier 36 a with regulatable amplification factor, an envelope curve shaper 37 , a comparator 1 as well as an amplification factor actuation stage 38 a.","The control unit 31 transmits short signal pulses 41 to the oscillator 32 .","The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.","to emit sound waves.","Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30 a.","During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.","This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.","In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.","The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36 a with a regulatable amplification factor.","The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36 a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.","The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.","In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.","The amplification factor of the amplifier 36 a is regulated by the amplification factor actuation stage 38 a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35 .","Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto ( FIG.","2 ).","The amplification factor actuation stage 38 a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31 .","In this manner a reduction of the short range can be achieved.","The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.","FIG.","2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36 a , c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.","1 .","The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.","As was already explained with reference to FIG.","1 , during the signal pulse 41 , the amplification factor 42 reaches a minimum 42 a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.","During the signal pulse 41 , the envelope curve voltage 43 reaches a maximum (envelope curve segment 43 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .","Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43 b ), whereby the course of the envelope curve 43 b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.","The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.","The advantageous result of this is a reduction of the short range.","In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43 b ).","The threshold voltage 44 is constant over time.","Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44 , in a disadvantageous manner, at the beginning of the signal pulse 41 , a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44 .","An echo signal (envelope curve segment 43 d ) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41 .","The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.","Further drawbacks associated with a distance sensor of the type illustrated in FIG.","1 were already explained above.","As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.","For purposes of further illustrating the state of the art, FIG.","3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30 b .","The distance sensor 30 b shown in FIG.","3 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , a preferably logarithmic amplifier 36 b , an envelope curve shaper 37 , a comparator 1 as well as a threshold value actuation stage 38 b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1 .","The essential difference from the distance sensor 30 a illustrated in FIG.","1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.","FIG.","4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30 b shown in FIG.","3 .","The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.","During a signal pulse 41 , the envelope curve voltage 53 reaches a maximum (envelope curve segment 53 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .","Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53 b ), since the amplifier 36 b is a logarithmic amplifier.","Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53 c ).","The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38 b in such a way that it rises before every signal pulse 41 , remains at a maximum value for a certain period of time (envelope curve segment 54 a ), then drops (curve segment 54 b ) and finally reaches a constant level (envelope curve segment 54 c ) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53 , as long as no echo is received.","The threshold value actuation stage 38 b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31 .","The advantageous result is a reduction of the short range.","Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41 .","Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53 d ) associated with an echo, so that the switching signal 56 is a useful switching signal 56 .","Drawbacks associated with a distance sensor of the type illustrated in FIG.","3 were already explained above.","Now reference is made to FIG.","5 , which illustrates a schematic circuit diagram of an embodiment 10 a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2 , a first regulatable switch 3 , a capacitor 6 , a diode 5 and a first comparator 1 , that has a first threshold value input 1 a and a first signal input 1 b.","At the first threshold value input 1 a , a threshold voltage U ref1 , is present that is emitted internally in the adaptive comparator circuit according to the invention.","At the signal input 1 b , a voltage signal U signal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.","The first comparator 1 emits a first switching signal S 1 when the voltage signal U signal is greater than the first threshold voltage U ref1 .","The first switching signal S 1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.","Via a control output 2 a , the signal transmitter 2 emits an electric control signal S St at times, for example, at regular intervals.","In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.","Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.","Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal S St having a time duration of typically, for example, 20 microseconds.","The first switch 3 has a control means 3 a , a switch input 3 b and a switch output 3 c .","The control means 3 a is connected to the control output 2 a and can be regulated by the control signal S St in such a way that it is in the closed state when the signal transmitter 2 emits a control signal S St , and otherwise it is in the open state.","The first switch 3 can be, for example, a transistor.","The switch input 3 b is connected to the positive first pole 4 a of a first direct voltage source 4 a , 4 b (not shown here).","The switch output 3 c is connected to the first threshold value input 1 a of the comparator 1 , so that the first threshold voltage U ref1 is equal to the voltage present at the switch output 3 c .","Thus, when the switch 3 is closed, the potential of the positive first pole 4 a is present at the first threshold value input 1 a of the comparator 1 .","The switch output 3 c is also applied via a capacitor 6 to the negative second pole 4 b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.","The switch output 3 c is also connected to the anode 5 a of a diode 5 .","The cathode 5 b of the diode 5 is connected to the first signal input 1 b of the comparator.","As soon as the signal transmitter 2 emits a control signal S St , the first switch 3 is in the closed state.","Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.","When the switch 3 is closed, this voltage is also present at the threshold value input 1 a.","After the end of the control signal S St , the switch 3 is in the open state.","The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first U ref1 reaches a value that corresponds to the sum of the voltage signal U signal and the diode flow voltage dU.","This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal U signal : the smaller the voltage signal U signal , the more the voltage and thus the threshold voltage U ref1 drop according to the invention at the capacitor.","Hence, a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the fundamental voltage distance dU.","According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage U ref1 for as long as and only until the difference of U ref1 −U signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal U signal increases.","If the voltage signal U signal drops, then the threshold voltage U ref1 follows the voltage signal U signal at the distance dU.","In contrast, if the voltage signal U signal increases, then the threshold voltage U ref1 remains constant because of the blocking effect of the diode 5 .","Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.","1 , so that the first pole 4 a is negative and the second pole 4 b is positive.","In this case, the diode 5 in the circuit shown in FIG.","1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3 c and its anode to the second pole 4 b.","In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7 .","According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5 a of the diode 5 and whose negative pole is in the place of the cathode 5 b of the diode 5 .","FIG.","6 shows an embodiment 10 b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.","1 .","The diode shown in FIG.","5 has been replaced by a second voltage source 7 , whereby its positive pole 7 a has been put in the place of the anode 5 a and the negative pole 7 b has been put in the place of the cathode 5 b .","The fundamental voltage distance dU shown in FIG.","1 has now been replaced by the voltage of the voltage source 7 , that is to say, the fundamental voltage distance dU between U ref1 and U signal is now defined by the voltage supplied by the second direct voltage source.","If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.","FIG.","7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10 a shown in FIG.","5 is used.","A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32 .","When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.","The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.","The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10 a shown in FIG.","5 .","In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals S St that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals S St each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.","9 a and 9 b .","In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage U ref1 is greater than the voltage signal U signal , so that no dummy switching signal is triggered.","In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals S St in such a way that the control signals S St each begin before or simultaneously with the end of the signal pulses 41 .","This ensures that, without any time lag, the threshold voltage U ref1 is supplied to the dropping voltage signal U signal at the fundamental voltage distance dU already at the beginning of the relaxation phase.","The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.","FIG.","8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10 a shown in FIG.","5 is likewise used.","The distance sensor shown in FIG.","8 —in comparison to that shown in FIG.","7 —additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34 .","Moreover, before the received signal is fed into the envelope curve shaper 37 , it is passed through a voltage limiter 35 and through a logarithmic amplifier 36 c. Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.","The amplifier 36 c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.","In FIG.","8 , in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31 .","For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31 .","In FIG.","8 , the first switching signal S 1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.","FIGS.","9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.","FIG.","9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo occurs during the quiescent phase.","The control unit 31 transmits short signal pulses 41 to the oscillator 32 , as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet ( FIG.","9 , Curve a).","Moreover, the signal transmitter 2 transmits a control signal S St to the switch 3 , which is synchronized with the signal pulse 41 in such a way that the control signal S St begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former ( FIG.","9 , Curve b).","FIG.","9 , Curve c, shows the course of the voltage signal U signal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage U ref1 (dotted curve).","The course of the U signal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53 , which was explained with reference to FIG.","4 .","While the control signal S St is present at the switch 3 , the latter is opened, so that a threshold voltage U ref1 is established which is identical to the voltage supplied by the first direct voltage source 4 a , 4 b ( FIG.","9 , Curve c).","Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3 .","In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36 c ( FIG.","8 ).","According to the invention, after the end of the control signal S St , a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the voltage distance dU.","Therefore, the threshold voltage U ref1 drops immediately after the end of the control signal by a certain quantity.","From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal U signal as long as the latter does not increase.","A dummy switching signal is not triggered.","If, however, the voltage signal U signal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal U signal leads to a triggering of the comparator 1 as soon as the voltage signal U signal exceeds the threshold voltage U ref1 .","In this case, the comparator 1 emits a first switching signal S 1 ( FIG.","9 , Curve d).","FIG.","10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8 , whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.","The courses over time of the signal pulse 41 and of the control signal S St are identical to those shown in FIG.","9 .","During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal U signal ( FIG.","10 , Curve a).","Up until the beginning of the second echo peak 61 , the curves correspond to those shown in FIG.","9 , Curve c. According to the invention, however, the decrease of the threshold voltage U ref1 comes to a halt as soon as the voltage signal U signal is no longer dropping, and a first switching signal S 1a is triggered by the second echo peak 61 .","After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage U ref1 and the voltage signal U signal .","During the quiescent phase, another, third echo peak 62 of the voltage signal U signal occurs, as a result of which another switching signal S 1b is triggered.","Now reference will be made to FIGS.","11 to 13 , which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.","FIG.","11 shows a preferred embodiment 10 c of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.","6 as follows: a) The first switch is a transistor 103 .","The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.","b) A first resistor 11 is connected between the first pole 4 a and the first threshold value input 1 a , and a second resistor 12 is connected between the first threshold value input 1 a and the second pole 4 b , so that the first resistor 11 and the second resistor 12 form a first voltage divider 11 , 12 .","Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the first voltage divider.","Thus, through the use of the first voltage divider 11 , 12 , the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal U signal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dU a .","When this embodiment 10 c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.","This is advantageous for many applications such as, for example, for the detection of small objects.","The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.","c) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage U ref1 is adapted to the voltage signal U signal .","The eighth resistor also serves to smooth the course of the threshold voltage U ref1 with respect to very rapid fluctuations of the voltage signal U signal .","FIG.","12 shows another preferred embodiment 10 d of the invention, which differs from the adaptive comparator circuit 10 c shown in FIG.","11 in that, before the voltage signal U signal reaches the first signal input 1 b , it passes through an impedance transformer.","This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.","FIG.","13 shows another preferred embodiment 10 e of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.","6 as follows: a) The first switch is a transistor 103 .","The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.","b) A second comparator 20 is used that has a second threshold value input 20 a and a second signal input 20 b , and that emits a second switching signal S 2 when a greater voltage is present at the second signal input 20 b than at the second threshold value input 20 a. c) The first pole 4 a of the first voltage source 4 a , 4 b is connected to the second threshold value input 20 a via a third resistor 13 .","The second threshold value input 20 a is connected to the second pole 4 b via a fourth resistor 14 , so that the third and the fourth resistors 13 , 14 form a second voltage divider which can be configured as a potentiometer.","d) The collector 103 c that serves as the switch output and the second threshold value input 20 a are connected via a fifth resistor 15 .","e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S 2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S 2 , and otherwise it is in the open state.","The second switch 23 can be a transistor.","f) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","g) The diode 5 is connected in series to an eighth resistor 18 .","The purpose of this resistor was already explained with reference to FIG.","11 .","h) Before reaching the first signal input 1 b , the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.","12 .","Due to the second voltage divider 13 , 14 , a voltage U ref2 is present at the second threshold value input 20 a .","As long as the second switch 23 is closed, the first threshold voltage U ref1 follows the dropping voltage signal U signal , parallel-offset by the fundamental voltage distance dU, as explained above.","According to the invention, however, the second switch opens as soon as the voltage signal U signal exceeds the second threshold voltage U ref2 .","As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage U ref1 are ended.","Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage U ref1 has dropped to the magnitude of the second threshold voltage U ref2 supplied by the second potentiometer 13 , 14 .","Thus, through the use of the second voltage divider 13 , 14 , the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal U signal with respect to voltage signal U signal , from the fundamental voltage distance dU to a value dU b .","The second voltage divider 13 , 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20 a.","The fifth resistor 15 is connected between the switch output 103 c and the second threshold value input 20 a .","Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the second voltage divider 13 , 14 .","In particular, through the use of the fifth resistor 15 , it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal U signal strives towards a value dU b that—depending on the voltage supplied by the second voltage divider 13 , 14 —can advantageously be not only larger but also smaller than the fundamental voltage distance dU.","When this embodiment 10 e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13 , 14 .","All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10 b , 10 c , 10 d and 10 e illustrated in FIG.","6 and FIGS.","11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.","8 instead of the adaptive comparator circuit 10 a shown there by way of an example."],["TECHNICAL AREA The invention relates to an adaptive comparator circuit that can be used particularly for reducing the short range of an acoustic distance sensor, as well as to an acoustic distance sensor with such a circuit.","STATE OF THE ART Acoustic distance sensors are used to measure distances by means of the pulse-propagation time method.","In this process, a sound transducer emits a short sound pulse that propagates in a medium (gas, liquid or solid).","This pulse is reflected, for example, on an object and comes back as an echo.","The distance to the object can be determined on the basis of the propagation time of the sound pulse and the sound velocity in the medium.","As a rule, the frequency of the sound used for such measurements lies in the ultra-sound range, typically in the range between 20 kHz and 500 kHz, for example, at 400 kHz.","The sound transducer, which in particular can be a piezoelectric ceramic element, is normally used to transmit the sound pulse as well as to receive the echo.","Typically, sound pulses of about 3 to 50 oscillations periods each are emitted.","This results in time durations of typically about 6 to 2050 μs for the emitted sound pulses.","During this time, i.e.","the transmitting phase, the sound transducer oscillates at a high oscillation amplitude.","This time duration is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.","The relaxation time is typically, for example, 300 μs to 3 ms.","The time interval between the end of the relaxation time and the emission of the next sound pulse will be called “quiescent phase” below.","Due to noise and other interferences, however, the oscillation amplitude does not drop completely to zero even during the quiescent phase, but rather remains at a certain constant mean disturbance level.","Therefore, during the receiving phase that follows the transmitting phase, even in the absence of a useful echo, the oscillation amplitude that dies out during the relaxation time and the mean disturbance level consistently form a finite background signal that is very large immediately after the end of the transmitting phase, that generally decreases exponentially within the relaxation time and that finally remains at the disturbance level.","A useful echo signal is superimposed on this background and, as a rule, is detected by means of a comparator that emits a switching signal when the received signal is greater than a certain reference value that will be called “threshold voltage” hereinafter.","On the one hand, the threshold voltage should be as low as possible so that even weak useful echo signals, e.g.","of small objects, can be detected, which translates into high sensitivity.","On the other hand, the threshold voltage has to lie above the disturbance level by a certain safety margin so as to avoid erroneous triggering of the comparator during the quiescent phase.","When the threshold voltage is constant over time, useful echo signals can be detected as soon as the background signal has dropped below the threshold voltage as a result of the relaxation of the sound transducer.","Before this time, however, that is to say, during the transmitting phase and during part of the relaxation time, the background signal is greater than the threshold voltage so that the comparator—irrespective of the presence of an echo—is triggered in any case during this time.","Thus, during this time, called dead time, the system is fundamentally insensitive to the reception of echoes.","Therefore, the measurement of distances below a certain minimum distance is impossible.","This minimum distance is called the “short range” and is typically a few centimeters.","The existence of the short range often has a detrimental effect, for example, wherever due to space constraints, the sensor is to be situated as close as possible to the object to be detected.","Various methods are known for reducing the short range.","The simplest method is to increase the threshold voltage.","This causes the background signal, which dies out after the transmitting phase, to drop below the threshold voltage at an earlier point in time within the relaxation time.","However, this method entails the serious drawback that an increase in the threshold voltage translates into a reduction of the system sensitivity, as a result of which weak echoes can no longer be detected.","Another method lies in conveying the received signal through an amplifier whose amplification factor can be changed over time.","The amplification factor is minimal immediately after the transmittal pulse and is then boosted, for example, by means of a suitable electronic circuit as a function of the time.","In this manner, despite a constant threshold voltage over time, a reduction of the short range can be combined with a high sensitivity during the quiescent phase.","Of course, for this purpose, the course over time of the amplification factor has to be adapted to the relaxation time.","The drawback is that a one-time optimal adaptation is not sufficient here, since the relaxation time of the sound transducer is not constant but rather, it depends on the medium on the temperature, on the age of the sound transducer and on other influencing factors.","Therefore, in actual practice, either complex compensation measures have to be undertaken or re-adaptations have to be made repeatedly, or else a large safety margin has to be maintained right from the start for purposes of optimal adaptation, which diminishes the system sensitivity, at least during the relaxation time.","Another method is to provide a threshold voltage that can be changed over time.","In the ideal case, this threshold voltage drops at a gradient that is similar to the decaying background signal and, at the beginning of the quiescent phase, turns into a constant value, whereby the threshold voltage nevertheless always remains sufficiently greater than the background signal.","This method can be additionally improved by subjecting the background signal to a logarithmic amplification so that the decrease is linear instead of exponential.","The drawback here, however, is that a one-time adaptation of the time behavior of the threshold voltage is not sufficient to ensure optimal operation over the long run, since here, too, the individual dying-out behavior of the sound transducer is not taken into account on a case-to-case basis.","Another method for reducing the short range lies in the propagation time-dependent control of the length of the emitted sound pulses.","In the case of a short propagation time, that is to say, in the case of an object that is close, very short sound pulses are used to that the oscillation amplitude of the sound transducer does not fully develop and thus the short range is reduced.","Another method lies in the use of threshold voltage that is dynamically changed by means of a microprocessor and a D/A transducer.","The microprocessor regulates the D/A transducer in such a way that a dynamic threshold is created that always lies above the dying-out signal.","Here, an A/D transducer measures the decaying transmission signal.","These methods, however, call for considerable hardware and software resources.","Therefore, the invention is based on the objective of simply and inexpensively providing a comparator circuit with which, in every single case, the course over time of the threshold voltage is automatically adapted to the individual decay behavior of the sound transducer and the magnitude of the disturbance level.","This objective is achieved according to the invention by an adaptive comparator circuit, especially for an acoustic distance sensor, comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and having a first signal input to which a voltage signal (Usignal) is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, characterized by a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.","This is the first basic form of an adaptive comparator circuit according to the invention.","According to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.","This is the second basic form of an adaptive comparator circuit according to the invention.","These two basic forms can be augmented advantageously as will still be explained below.","An acoustic distance sensor according to the invention comprises a control unit that at times transmits signal pulses to an oscillator which, during the presence of a signal pulse, transmits an alternating voltage to a sound transducer that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer in the form of an echo as a result of reflection, and of converting them into an electric received signal, and it also comprises an envelope curve shaper to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a signal voltage, characterized in that the signal voltage is fed to an adaptive comparator circuit comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and a first signal input to which a voltage signal is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.","An acoustic distance sensor according to the invention thus comprises the adaptive comparator circuit according to the invention.","Preferably, the control signal emitted by the signal transmitter is an electric control signal and the first switch is an electrically or electronically controlled switch, for example, a transistor, whose base terminal or gate terminal function as a control-current terminal or, for example, a relay.","In another embodiment of the invention, the control signal emitted by the signal transmitter is an optical control signal that is emitted, for example, by a light diode, and the first switch is an optically controlled switch, e.g.","a photoresistor.","As soon as the signal transmitter emits a control signal, the first switch is in the closed state, so that the capacitor is charged with the voltage supplied by the first voltage source.","After the end of the control signal, the switch is in the open state.","The capacitor voltage and thus the first threshold voltage present at the first threshold value input now assumes a value that corresponds to the sum of the voltage signal and the diode flow voltage or else to the sum of the voltage signal and the voltage of the second direct voltage source.","The diode flow voltage or the voltage of the second direct voltage source are referred to below as “fundamental voltage distance” dU.","The task of the diode or of the second direct voltage source is to allow the discharging of the capacitor and thus a drop in the threshold voltage for as long as and only until the difference of the threshold voltage minus the voltage signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal increases.","Therefore, the time behavior of the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal as long as the voltage signal does not increase.","In contrast, if the voltage signal increases, the threshold voltage remains constant.","Consequently, a maximum, e.g.","a peak, of the voltage signal leads to the triggering of the comparator as soon as the voltage signal exceeds the threshold voltage.","If the voltage signal drops again after having increased, then the threshold voltage follows the voltage signal, once again parallel-offset, as soon as the fundamental, voltage distance dU has been reached again.","Hence, according to the invention, the threshold voltage is specified by the magnitude of the voltage signal at every point in time of the receiving phase.","If the course over time of the voltage signal is changed, then there is automatically a correspondingly changed course over time of the threshold voltage.","Thus, the course over time of the threshold voltage is automatically adapted to that of the voltage signal.","The diode flow voltage is temperature-dependent.","It decreases as the temperature rises.","Consequently, an acoustic distance sensor according to the invention becomes more sensitive as the temperature rises.","On the other hand, the attenuation of ultrasound waves generally increases as the temperature rises.","Therefore, the resultant sensitivity loss is advantageously compensated for partially with the use of a diode to create the fundamental voltage distance dU.","As already explained above, according to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.","In this case, the fundamental voltage distance dU is defined by the voltage supplied by the second direct voltage source.","The basic forms described above of adaptive comparator circuits according to the invention can be advantageously augmented as will be explained below.","In a preferred embodiment of the invention, one of the above-described basic forms has been augmented by a first resistor that is connected between the first pole and the first threshold value input, and by a second resistor that is connected between the first threshold value input and the second pole, so that the first resistor and the second resistor form a first voltage divider.","Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.","Thus, through the use of the first voltage divider, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.","When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.","The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.","Another preferred embodiment of an adaptive comparator circuit according to the invention comprises the following in addition to the basic forms described above: a second comparator that has a second threshold value input and a second signal input, and that emits a second switching signal when a greater voltage is present at the second signal input than at the second threshold value input, a third resistor via which the first pole of the first direct voltage source is connected to the second threshold value input, a fourth resistor that, on the one hand, is connected to the second threshold value input and, on the other hand, to the second pole of the first voltage source, so that the third resistor and the fourth resistor form a second voltage divider, and a second switch that is connected in series to the diode and that can be regulated or activated by the second switching signal in such a way that it is in the closed state when the second comparator emits the second switching signal, and otherwise it is in the open state.","Due to the second voltage divider, a voltage is present at the second threshold value input that will be called “second threshold voltage” Uref2 below.","As long as the second switch is closed, the first threshold voltage follows the dropping voltage signal Usignal with the fundamental voltage distance dU, as explained above.","According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor and thus the dropping of the first threshold voltage are ended.","Hence, in this embodiment of the invention, the capacitor can only discharge until the first threshold voltage Uref1 has dropped to the value of the second threshold voltage Uref2.Thus, through the use of the second voltage divider, the voltage distance by which the first threshold voltage is changed parallel-offset with respect to voltage signal Usignal after the decay of the voltage signal Usignal, advantageously by the fundamental voltage distance dU to a value dUb.","When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor according to the invention, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be reduced.","The second voltage divider can be an adjustable potentiometer whose pick-up is connected to the second threshold value input.","In another configuration of this embodiment of the invention, a fifth resistor is connected between the switch output and the second threshold value input.","Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider.","In particular, through the use of the fifth resistor, it can be additionally achieved that the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.","When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased.","In another advantageous embodiment of the invention, the voltage signal passes through a voltage follower or impedance transformer before reaching the first signal input.","In another embodiment of the invention, a sixth resistor is connected between the control output and the control-current terminal for purposes of limiting the current flow.","In another embodiment of the invention, the first switch is a transistor whose base functions as a control-current terminal, whereby a seventh resistor is connected between the base and the first pole of the first direct voltage source.","In another embodiment of the invention, the diode or the second direct voltage source is connected in series with an eighth resistor.","This brings about a slowing down of the discharging of the capacitor as well as a smoothing of the threshold voltage, which is advantageous for many applications.","The voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.","In this manner, it is prevented that the first comparator is triggered while the first switch is still closed, that is to say, while the threshold voltage cannot yet adapt to the voltage signal.","Moreover, in this manner, it is advantageously achieved that, in any case, the adaptation of the threshold voltage to the voltage signal can begin immediately after the opening of the first switch.","An adaptive comparator circuit according to the invention can be used especially advantageously in an acoustic distance sensor.","In this case, the voltage signal is advantageously formed by the envelope curve of the received signal supplied by the sound transducer.","This received signal consists of a superimposition of the background signal with the useful echo signal.","The formation of the envelope curve, for example, by means of a rectifier circuit, is known from the state of the art and will thus not be elaborated upon in greater depth here.","In the absence of an echo, the envelope curve dies out exponentially during the relaxation phase and, at the beginning of the quiescent phase, turns into a horizontal straight line.","Such an envelope curve will be called “background envelope curve” below.","The threshold voltage adapts according to the invention to the individual course of the background envelope curve in that it follows said background envelope curve parallel-offset by a voltage distance dU or dUa or dUb.","An echo causes a peak to be superimposed on the background envelope curve so that the voltage signal increases.","According to the invention, the threshold voltage does not follow this increase, so that the comparator is triggered and the echo can thus be detected.","As already mentioned above, the voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.","Since in this case, the threshold voltage already follows the background envelope curve at the voltage distance dU at the beginning of the relaxation phase, the voltage signal in the absence of an echo is not greater than the threshold voltage at any point in time, especially not during the entire relaxation phase.","Therefore, throughout the entire receiving phase, the comparator can only be triggered by an echo, but not by the decay behavior of the sound transducer.","Thus, an acoustic distance sensor according to the invention is fundamentally able to detect an echo during the quiescent phase as well as especially during the entire relaxation phase.","As a result, the undesired short range of the sensor is considerably reduced according to the invention.","BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.","1—a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.","2—a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","1, FIG.","3—a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.","4—a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.","3, FIG.","5—a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.","6—a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.","7 and 8—a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.","9—a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo occurs during the quiescent phase, FIG.","10—a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo additionally occurs during the relaxation phase, and FIGS.","11 to 13—circuit diagrams of further embodiments of comparator circuits according to the invention.","FIGS.","1 to 4 serve to further illustrate the state of the art.","FIG.","1 shows a schematic block diagram of a variant of an acoustic distance sensor 30a with which a measure according to the state of the art has been taken in order to reduce the short range.","The distance sensor 30a shown in FIG.","1 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, an amplifier 36a with regulatable amplification factor, an envelope curve shaper 37, a comparator 1 as well as an amplification factor actuation stage 38a.","The control unit 31 transmits short signal pulses 41 to the oscillator 32.The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.","to emit sound waves.","Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30a.","During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.","This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.","In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.","The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36a with a regulatable amplification factor.","The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.","The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.","In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.","The amplification factor of the amplifier 36a is regulated by the amplification factor actuation stage 38a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35.Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto (FIG.","2).","The amplification factor actuation stage 38a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31.In this manner a reduction of the short range can be achieved.","The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.","FIG.","2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36a, c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.","1.The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.","As was already explained with reference to FIG.","1, during the signal pulse 41, the amplification factor 42 reaches a minimum 42a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.","During the signal pulse 41, the envelope curve voltage 43 reaches a maximum (envelope curve segment 43a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43b), whereby the course of the envelope curve 43b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.","The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.","The advantageous result of this is a reduction of the short range.","In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43b).","The threshold voltage 44 is constant over time.","Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44, in a disadvantageous manner, at the beginning of the signal pulse 41, a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44.An echo signal (envelope curve segment 43d) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41.The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.","Further drawbacks associated with a distance sensor of the type illustrated in FIG.","1 were already explained above.","As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.","For purposes of further illustrating the state of the art, FIG.","3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30b.","The distance sensor 30b shown in FIG.","3 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, a preferably logarithmic amplifier 36b, an envelope curve shaper 37, a comparator 1 as well as a threshold value actuation stage 38b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1.The essential difference from the distance sensor 30a illustrated in FIG.","1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.","FIG.","4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30b shown in FIG.","3.The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.","During a signal pulse 41, the envelope curve voltage 53 reaches a maximum (envelope curve segment 53a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53b), since the amplifier 36b is a logarithmic amplifier.","Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53c).","The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38b in such a way that it rises before every signal pulse 41, remains at a maximum value for a certain period of time (envelope curve segment 54a), then drops (curve segment 54b) and finally reaches a constant level (envelope curve segment 54c) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53, as long as no echo is received.","The threshold value actuation stage 38b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31.The advantageous result is a reduction of the short range.","Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41.Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53d) associated with an echo, so that the switching signal 56 is a useful switching signal 56.Drawbacks associated with a distance sensor of the type illustrated in FIG.","3 were already explained above.","Now reference is made to FIG.","5, which illustrates a schematic circuit diagram of an embodiment 10a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2, a first regulatable switch 3, a capacitor 6, a diode 5 and a first comparator 1, that has a first threshold value input 1a and a first signal input 1b.","At the first threshold value input 1a, a threshold voltage Uref1, is present that is emitted internally in the adaptive comparator circuit according to the invention.","At the signal input 1b, a voltage signal Usignal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.","The first comparator 1 emits a first switching signal S1 when the voltage signal Usignal is greater than the first threshold voltage Uref1.The first switching signal S1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.","Via a control output 2a, the signal transmitter 2 emits an electric control signal SSt at times, for example, at regular intervals.","In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.","Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.","Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal SSt having a time duration of typically, for example, 20 microseconds.","The first switch 3 has a control means 3a, a switch input 3b and a switch output 3c.","The control means 3a is connected to the control output 2a and can be regulated by the control signal SSt in such a way that it is in the closed state when the signal transmitter 2 emits a control signal SSt, and otherwise it is in the open state.","The first switch 3 can be, for example, a transistor.","The switch input 3b is connected to the positive first pole 4a of a first direct voltage source 4a, 4b (not shown here).","The switch output 3c is connected to the first threshold value input 1a of the comparator 1, so that the first threshold voltage Uref1 is equal to the voltage present at the switch output 3c.","Thus, when the switch 3 is closed, the potential of the positive first pole 4a is present at the first threshold value input 1a of the comparator 1.The switch output 3c is also applied via a capacitor 6 to the negative second pole 4b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.","The switch output 3c is also connected to the anode 5a of a diode 5.The cathode 5b of the diode 5 is connected to the first signal input 1b of the comparator.","As soon as the signal transmitter 2 emits a control signal SSt, the first switch 3 is in the closed state.","Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.","When the switch 3 is closed, this voltage is also present at the threshold value input 1a.","After the end of the control signal SSt, the switch 3 is in the open state.","The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first Uref1 reaches a value that corresponds to the sum of the voltage signal Usignal and the diode flow voltage dU.","This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal Usignal: the smaller the voltage signal Usignal, the more the voltage and thus the threshold voltage Uref1 drop according to the invention at the capacitor.","Hence, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the fundamental voltage distance dU.","According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage Uref1 for as long as and only until the difference of Uref1−Usignal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal Usignal increases.","If the voltage signal Usignal drops, then the threshold voltage Uref1 follows the voltage signal Usignal at the distance dU.","In contrast, if the voltage signal Usignal increases, then the threshold voltage Uref1 remains constant because of the blocking effect of the diode 5.Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.","1, so that the first pole 4a is negative and the second pole 4b is positive.","In this case, the diode 5 in the circuit shown in FIG.","1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3c and its anode to the second pole 4b.","In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7.According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5a of the diode 5 and whose negative pole is in the place of the cathode 5b of the diode 5.FIG.","6 shows an embodiment 10b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.","1.The diode shown in FIG.","5 has been replaced by a second voltage source 7, whereby its positive pole 7a has been put in the place of the anode 5a and the negative pole 7b has been put in the place of the cathode 5b.","The fundamental voltage distance dU shown in FIG.","1 has now been replaced by the voltage of the voltage source 7, that is to say, the fundamental voltage distance dU between Uref1 and Usignal is now defined by the voltage supplied by the second direct voltage source.","If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.","FIG.","7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10a shown in FIG.","5 is used.","A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32.When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.","The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.","The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10a shown in FIG.","5.In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals SSt that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals SSt each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.","9a and 9b.","In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage Uref1 is greater than the voltage signal Usignal, so that no dummy switching signal is triggered.","In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses 41.This ensures that, without any time lag, the threshold voltage Uref1 is supplied to the dropping voltage signal Usignal at the fundamental voltage distance dU already at the beginning of the relaxation phase.","The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.","FIG.","8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10a shown in FIG.","5 is likewise used.","The distance sensor shown in FIG.","8—in comparison to that shown in FIG.","7—additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34.Moreover, before the received signal is fed into the envelope curve shaper 37, it is passed through a voltage limiter 35 and through a logarithmic amplifier 36c.","Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.","The amplifier 36c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.","In FIG.","8, in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31.For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31.In FIG.","8, the first switching signal S1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.","FIGS.","9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.","FIG.","9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo occurs during the quiescent phase.","The control unit 31 transmits short signal pulses 41 to the oscillator 32, as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet (FIG.","9, Curve a).","Moreover, the signal transmitter 2 transmits a control signal SSt to the switch 3, which is synchronized with the signal pulse 41 in such a way that the control signal SSt begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former (FIG.","9, Curve b).","FIG.","9, Curve c, shows the course of the voltage signal Usignal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage Uref1 (dotted curve).","The course of the Usignal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53, which was explained with reference to FIG.","4.While the control signal SSt is present at the switch 3, the latter is opened, so that a threshold voltage Uref1 is established which is identical to the voltage supplied by the first direct voltage source 4a, 4b (FIG.","9, Curve c).","Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3.In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36c (FIG.","8).","According to the invention, after the end of the control signal SSt, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the voltage distance dU.","Therefore, the threshold voltage Uref1 drops immediately after the end of the control signal by a certain quantity.","From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal Usignal as long as the latter does not increase.","A dummy switching signal is not triggered.","If, however, the voltage signal Usignal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal Usignal leads to a triggering of the comparator 1 as soon as the voltage signal Usignal exceeds the threshold voltage Uref1.In this case, the comparator 1 emits a first switching signal S1 (FIG.","9, Curve d).","FIG.","10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.","8, whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.","The courses over time of the signal pulse 41 and of the control signal SSt are identical to those shown in FIG.","9.During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal Usignal (FIG.","10, Curve a).","Up until the beginning of the second echo peak 61, the curves correspond to those shown in FIG.","9, Curve c. According to the invention, however, the decrease of the threshold voltage Uref1 comes to a halt as soon as the voltage signal Usignal is no longer dropping, and a first switching signal S1a is triggered by the second echo peak 61.After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage Uref1 and the voltage signal Usignal.","During the quiescent phase, another, third echo peak 62 of the voltage signal Usignal occurs, as a result of which another switching signal S1b is triggered.","Now reference will be made to FIGS.","11 to 13, which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.","FIG.","11 shows a preferred embodiment 10c of the invention which differs from the adaptive comparator circuit 10a shown in FIG.","6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.","b) A first resistor 11 is connected between the first pole 4a and the first threshold value input 1a, and a second resistor 12 is connected between the first threshold value input 1a and the second pole 4b, so that the first resistor 11 and the second resistor 12 form a first voltage divider 11, 12.Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.","Thus, through the use of the first voltage divider 11, 12, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.","When this embodiment 10c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.","This is advantageous for many applications such as, for example, for the detection of small objects.","The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.","c) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage Uref1 is adapted to the voltage signal Usignal.","The eighth resistor also serves to smooth the course of the threshold voltage Uref1 with respect to very rapid fluctuations of the voltage signal Usignal.","FIG.","12 shows another preferred embodiment 10d of the invention, which differs from the adaptive comparator circuit 10c shown in FIG.","11 in that, before the voltage signal Usignal reaches the first signal input 1b, it passes through an impedance transformer.","This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.","FIG.","13 shows another preferred embodiment 10e of the invention which differs from the adaptive comparator circuit 10a shown in FIG.","6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.","b) A second comparator 20 is used that has a second threshold value input 20a and a second signal input 20b, and that emits a second switching signal S2 when a greater voltage is present at the second signal input 20b than at the second threshold value input 20a.","c) The first pole 4a of the first voltage source 4a, 4b is connected to the second threshold value input 20a via a third resistor 13.The second threshold value input 20a is connected to the second pole 4b via a fourth resistor 14, so that the third and the fourth resistors 13, 14 form a second voltage divider which can be configured as a potentiometer.","d) The collector 103c that serves as the switch output and the second threshold value input 20a are connected via a fifth resistor 15.e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S2, and otherwise it is in the open state.","The second switch 23 can be a transistor.","f) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.","The purpose of these resistors was already explained above.","g) The diode 5 is connected in series to an eighth resistor 18.The purpose of this resistor was already explained with reference to FIG.","11.h) Before reaching the first signal input 1b, the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.","12.Due to the second voltage divider 13, 14, a voltage Uref2 is present at the second threshold value input 20a.","As long as the second switch 23 is closed, the first threshold voltage Uref1 follows the dropping voltage signal Usignal, parallel-offset by the fundamental voltage distance dU, as explained above.","According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage Uref1 are ended.","Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage Uref1 has dropped to the magnitude of the second threshold voltage Uref2 supplied by the second potentiometer 13, 14.Thus, through the use of the second voltage divider 13, 14, the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal Usignal with respect to voltage signal Usignal, from the fundamental voltage distance dU to a value dUb.","The second voltage divider 13, 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20a.","The fifth resistor 15 is connected between the switch output 103c and the second threshold value input 20a.","Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider 13, 14.In particular, through the use of the fifth resistor 15, it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal Usignal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider 13, 14—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.","When this embodiment 10e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13, 14.All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10b, 10c, 10d and 10e illustrated in FIG.","6 and FIGS.","11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.","8 instead of the adaptive comparator circuit 10a shown there by way of an example.","Commercial Utilization The invention can be utilized commercially, for example, in distance measuring technology, in filling level measuring technology and in automation technology.","The main figure is FIG.","5.List of Reference Numerals and Letters 1 first comparator 1a first threshold value input 1b first signal input 2 signal transmitter 2a control output of 2 2b input of 2 3 first switch 3a control-current terminal of 3 3b switch input of 3 3c switch output of 3 4a, 4b first, second pole of the first direct voltage source 5 diode 5a, 5b anode, cathode of 5 6 capacitor 7a, 7b positive, negative pole of 7 7 second direct voltage source 8, 9 first, second electric terminal 10a,b,c,d,e adaptive comparator circuits 11-18 first to eighth resistor 20 second comparator 20a second threshold value input 20b second signal input 21 voltage follower 23 second switch 30a distance sensor with constant threshold voltage 30b distance sensor with variable threshold voltage 31 control unit 32 oscillator 33 driver 34 sound transducer 35 voltage limiter 36a amplifier with regulatable amplification factor 36b, c logarithmic amplifier 37 envelope curve shaper 38a amplification factor actuation stage 38b threshold value actuation stage 41 signal pulses 42 course over time of the amplification factor of 36a 42a minimum of 42 43, 53 courses over time of the envelope curve voltage 43a, 53a segments of 43, 53 during the signal pulses 41, 51 43b, 53b segments of 43, 53 during the relaxation time of 34 43c, 53c segments of 43, 53 during the quiescent phase 43d, 53d segments of 43, 53 during an echo 44, 54 threshold values of 30a, 30b 45 dummy switching signal 46, 56 useful switching signals 60 first echo peak 61 second echo peak 62 third echo peak 103 transistor 103a base of 103 103b emitter of 103 103c collector of 103 S1, S1a, S1b switching signals of 1 S2 switching signal of 20 dU fundamental voltage distance Uref1 first threshold voltage Usignal voltage signal"]],"string":"[\n [\n \"Adaptive comparator circuit and acoustic distance sensor comprising said circuit\",\n \"The invention relates to an adaptive comparator circuit comprising a comparator with a threshold value input which receives a threshold voltage and a signal input which receives a voltage signal.\",\n \"The threshold value input is connected to one pole by means of a switch and connected to the other pole of a first voltage source by means of a capacitor.\",\n \"The threshold value input is also connected to the signal input of the comparator by means of a diode or a second voltage source.\",\n \"The switch is controlled by the control signal of a signal transmitter.\",\n \"When the switch is in a closed position, the capacitor is charged with the voltage of the first voltage source.\",\n \"When the switch is in an open position, the capacitor is discharged in such a way that the threshold voltage of the signal voltage is corrected at a given time interval.\"\n ],\n [\n \"1.An adaptive comparator circuit, particularly for an acoustic distance sensor, comprising a first comparator (1), having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage (Uref1), said comparator emits a first switching signal (S1), characterized by a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).\",\n \"2.The adaptive comparator according to claim 1, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).\",\n \"3.The adaptive comparator according to claim 2, characterized in that the voltage supplied by the second voltage source (7) can be regulated or adjusted.\",\n \"4.The adaptive comparator according to claim 1, characterized in that the signal transmitter (2) emits an electric control signal (SSt) at regular intervals.\",\n \"5.The adaptive comparator according to claim 1, characterized in that the first switch (3, 103) is a switch that can be electrically or electronically controlled by the control means (3a, 103a) and the control signal (SSt) is an electric signal that is transmitted by the signal transmitter (2) via a control output (2a) and fed to the control input (3a, 103a).\",\n \"6.The adaptive comparator according to claim 5, characterized in that the first switch (3, 103) is a transistor (103) and the control-current terminal (3a, 103a) is the base (103) or the gate terminal of the transistor (103).\",\n \"7.The adaptive comparator according to claim 1, characterized in that a first resistor (11) is connected between the first pole (4a) and the first threshold voltage input (1a), and a second resistor (12) is connected between the first threshold voltage input (1a) and the second pole (4b), so that the first resistor (11) and the second resistor (12) form a first voltage divider (11, 12).\",\n \"8.The adaptive comparator according to claim 7, characterized in that first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the switch output (3c, 103c).\",\n \"9.The adaptive comparator according to claim 1, characterized by a second comparator (20) that has a second threshold voltage input (20a) and a second signal input (20b), and that emits a second switching signal S2 when a greater voltage is present at the second signal input (20b) than at the second threshold voltage input (20a), a third resistor (13) via which the first pole (4a) is connected to the second threshold voltage input (20a), a fourth resistor (14) that, on the one hand, is connected to the second threshold voltage input (20a) and, on the other hand, to the second pole (4b) of the first voltage source, so that the third resistor (13) and the fourth resistor (14) form a second voltage divider (13, 14), a second switch (23) that is connected in series to the diode (5) and that can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator (20) emits the second switching signal S2, and otherwise it is in the open state.\",\n \"10.The adaptive comparator according to claim 9, characterized in that a fifth resistor (15) is connected between the switch output (3c, 103c) and the second threshold voltage input (20a).\",\n \"11.The adaptive comparator according to claim 9, characterized in that the second voltage divider (13, 14) is a potentiometer.\",\n \"12.The adaptive comparator according to claim 1, characterized in that a sixth resistor (16) is connected between the control output (2a) and the control-current terminal (3a, 103a).\",\n \"13.The adaptive comparator according to claim 6, characterized in that a seventh resistor (17) is connected between the control-current terminal (3a, 103a) and the first pole (4a).\",\n \"14.The adaptive comparator according to claim 1, characterized in that the diode (5) or the second voltage source (7) is connected in series to an eighth resistor (18).\",\n \"15.The adaptive comparator according to claim 1, characterized in that, before reaching the first signal input (1b), the voltage signal Usignal passes through a voltage follower or impedance transformer (21).\",\n \"16.The adaptive comparator according to claim 1, characterized in that the voltage supplied by the first voltage source (4a, 4b) is greater in magnitude than the voltage of the maximum of the voltage signal Usignal.\",\n \"17.The adaptive comparator according to claim 1, characterized in that the diode (5) is connected to at least another diode codirectionally in series.\",\n \"18.An acoustic distance sensor, comprising a control unit (31) that at times transmits signal pulses (41) to an oscillator (32) which, during the presence of a signal pulse (41), transmits an alternating voltage to a sound transducer (34) that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer (34) as an echo as a result of reflection, and of converting them into an electric received signal, and also comprising an envelope curve shaper (37) to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a voltage signal Usignal, characterized in that the voltage signal is fed to an adaptive comparator circuit comprising a first comparator (1) having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage Uref1, said comparator (1) emits a first switching signal (S1), a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output (3c, 103c) is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).\",\n \"19.The acoustic distance sensor according to claim 18, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).\",\n \"20.The acoustic distance sensor according to claim 18 characterized in that the alternating voltage emitted by the oscillator (32) reaches the sound transducer (34) after passing through a driver stage (33).\",\n \"21.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through an amplifier (36c).\",\n \"22.The acoustic distance sensor according to claim 21, characterized in that the amplifier (36c) is a logarithmic amplifier.\",\n \"23.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through a voltage limiter (35) that limits the amplitude of the received signal to a maximum value.\",\n \"24.The acoustic distance sensor according to claim 18, characterized in that the signal transmitter (2) is integrated into the control unit (31) or is a component of the control unit (31).\",\n \"25.The acoustic distance sensor according to claim 18, characterized in that the first switching signal (S1) is transmitted to the control unit (31) for evaluation purposes.\",\n \"26.The acoustic distance sensor according to claim 18, characterized in that the signal pulses (41) are synchronized with the control signals (SSt) in such a way that the control signals (SSt) each begin before the signal pulses (41).\",\n \"27.The acoustic distance sensor according to claim 18, characterized in that the signal pulses 41 are synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses (41).\",\n \"28.An adaptive comparator circuit for an acoustic distance sensor, comprising a first comparator (1) having a first signal input and a first threshold voltage input; a first direct voltage source (4) for delivering a threshold voltage to the first threshold voltage input, wherein the first direct voltage source comprises a first and a second pole; a first switch (3), wherein the first switch is connected to the first direct voltage source and to the first threshold voltage input; a signal transmitter (2), wherein the signal transmitter controls the first switch through an input of the switch; a capacitor (6), wherein a first plate of the capacitor is connected to the a first threshold voltage input and wherein a second plate of the capacitor is connected to second pole the first direct voltage source; a diode (5) connected the first signal input and to the first threshold voltage input of the first comparator.\",\n \"29.The adaptive comparator circuit according to claim 28, wherein the diode is connected in series to a first resistor.\",\n \"30.The adaptive comparator circuit according to claim 1, wherein the signal transmitter emits an electric control signal at regular intervals.\",\n \"31.The adaptive comparator circuit according to claim 28 further comprising a second resistor (11) connected between the first pole of the first direct voltage source and the first threshold voltage input, and a third resistor (12) connected between the first threshold voltage input and the second pole of the first direct voltage source, so that the second resistor (11) and the third resistor (12) form a first voltage divider (11, 12).\",\n \"32.The adaptive comparator circuit according to claim 31, wherein the first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the first threshold voltage input.\",\n \"33.The adaptive comparator circuit according to claim 28 further comprising a fourth resistor (17) connected between the first pole of the first direct voltage source and the input of the first switch; a fifth resistor (16) connected between the input of the first switch and the output of the signal transmitter (2).\"\n ],\n [\n \" BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.\",\n \"1 —a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"2 —a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"1 , FIG.\",\n \"3 —a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"4 —a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"3 , FIG.\",\n \"5 —a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.\",\n \"6 —a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.\",\n \"7 and 8 —a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.\",\n \"9 —a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo occurs during the quiescent phase, FIG.\",\n \"10 —a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo additionally occurs during the relaxation phase, and FIGS.\",\n \"11 to 13 —circuit diagrams of further embodiments of comparator circuits according to the invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\",\n \"FIGS.\",\n \"1 to 4 serve to further illustrate the state of the art.\",\n \"FIG.\",\n \"1 shows a schematic block diagram of a variant of an acoustic distance sensor 30 a with which a measure according to the state of the art has been taken in order to reduce the short range.\",\n \"The distance sensor 30 a shown in FIG.\",\n \"1 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , an amplifier 36 a with regulatable amplification factor, an envelope curve shaper 37 , a comparator 1 as well as an amplification factor actuation stage 38 a.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32 .\",\n \"The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.\",\n \"to emit sound waves.\",\n \"Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30 a.\",\n \"During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.\",\n \"This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.\",\n \"In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.\",\n \"The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36 a with a regulatable amplification factor.\",\n \"The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36 a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.\",\n \"The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.\",\n \"In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.\",\n \"The amplification factor of the amplifier 36 a is regulated by the amplification factor actuation stage 38 a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35 .\",\n \"Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto ( FIG.\",\n \"2 ).\",\n \"The amplification factor actuation stage 38 a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31 .\",\n \"In this manner a reduction of the short range can be achieved.\",\n \"The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.\",\n \"FIG.\",\n \"2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36 a , c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.\",\n \"1 .\",\n \"The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.\",\n \"As was already explained with reference to FIG.\",\n \"1 , during the signal pulse 41 , the amplification factor 42 reaches a minimum 42 a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.\",\n \"During the signal pulse 41 , the envelope curve voltage 43 reaches a maximum (envelope curve segment 43 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .\",\n \"Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43 b ), whereby the course of the envelope curve 43 b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.\",\n \"The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.\",\n \"The advantageous result of this is a reduction of the short range.\",\n \"In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43 b ).\",\n \"The threshold voltage 44 is constant over time.\",\n \"Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44 , in a disadvantageous manner, at the beginning of the signal pulse 41 , a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44 .\",\n \"An echo signal (envelope curve segment 43 d ) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41 .\",\n \"The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.\",\n \"Further drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"1 were already explained above.\",\n \"As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.\",\n \"For purposes of further illustrating the state of the art, FIG.\",\n \"3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30 b .\",\n \"The distance sensor 30 b shown in FIG.\",\n \"3 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , a preferably logarithmic amplifier 36 b , an envelope curve shaper 37 , a comparator 1 as well as a threshold value actuation stage 38 b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1 .\",\n \"The essential difference from the distance sensor 30 a illustrated in FIG.\",\n \"1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.\",\n \"FIG.\",\n \"4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30 b shown in FIG.\",\n \"3 .\",\n \"The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.\",\n \"During a signal pulse 41 , the envelope curve voltage 53 reaches a maximum (envelope curve segment 53 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .\",\n \"Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53 b ), since the amplifier 36 b is a logarithmic amplifier.\",\n \"Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53 c ).\",\n \"The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38 b in such a way that it rises before every signal pulse 41 , remains at a maximum value for a certain period of time (envelope curve segment 54 a ), then drops (curve segment 54 b ) and finally reaches a constant level (envelope curve segment 54 c ) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53 , as long as no echo is received.\",\n \"The threshold value actuation stage 38 b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31 .\",\n \"The advantageous result is a reduction of the short range.\",\n \"Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41 .\",\n \"Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53 d ) associated with an echo, so that the switching signal 56 is a useful switching signal 56 .\",\n \"Drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"3 were already explained above.\",\n \"Now reference is made to FIG.\",\n \"5 , which illustrates a schematic circuit diagram of an embodiment 10 a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2 , a first regulatable switch 3 , a capacitor 6 , a diode 5 and a first comparator 1 , that has a first threshold value input 1 a and a first signal input 1 b.\",\n \"At the first threshold value input 1 a , a threshold voltage U ref1 , is present that is emitted internally in the adaptive comparator circuit according to the invention.\",\n \"At the signal input 1 b , a voltage signal U signal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.\",\n \"The first comparator 1 emits a first switching signal S 1 when the voltage signal U signal is greater than the first threshold voltage U ref1 .\",\n \"The first switching signal S 1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.\",\n \"Via a control output 2 a , the signal transmitter 2 emits an electric control signal S St at times, for example, at regular intervals.\",\n \"In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.\",\n \"Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.\",\n \"Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal S St having a time duration of typically, for example, 20 microseconds.\",\n \"The first switch 3 has a control means 3 a , a switch input 3 b and a switch output 3 c .\",\n \"The control means 3 a is connected to the control output 2 a and can be regulated by the control signal S St in such a way that it is in the closed state when the signal transmitter 2 emits a control signal S St , and otherwise it is in the open state.\",\n \"The first switch 3 can be, for example, a transistor.\",\n \"The switch input 3 b is connected to the positive first pole 4 a of a first direct voltage source 4 a , 4 b (not shown here).\",\n \"The switch output 3 c is connected to the first threshold value input 1 a of the comparator 1 , so that the first threshold voltage U ref1 is equal to the voltage present at the switch output 3 c .\",\n \"Thus, when the switch 3 is closed, the potential of the positive first pole 4 a is present at the first threshold value input 1 a of the comparator 1 .\",\n \"The switch output 3 c is also applied via a capacitor 6 to the negative second pole 4 b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.\",\n \"The switch output 3 c is also connected to the anode 5 a of a diode 5 .\",\n \"The cathode 5 b of the diode 5 is connected to the first signal input 1 b of the comparator.\",\n \"As soon as the signal transmitter 2 emits a control signal S St , the first switch 3 is in the closed state.\",\n \"Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.\",\n \"When the switch 3 is closed, this voltage is also present at the threshold value input 1 a.\",\n \"After the end of the control signal S St , the switch 3 is in the open state.\",\n \"The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first U ref1 reaches a value that corresponds to the sum of the voltage signal U signal and the diode flow voltage dU.\",\n \"This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal U signal : the smaller the voltage signal U signal , the more the voltage and thus the threshold voltage U ref1 drop according to the invention at the capacitor.\",\n \"Hence, a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the fundamental voltage distance dU.\",\n \"According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage U ref1 for as long as and only until the difference of U ref1 −U signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal U signal increases.\",\n \"If the voltage signal U signal drops, then the threshold voltage U ref1 follows the voltage signal U signal at the distance dU.\",\n \"In contrast, if the voltage signal U signal increases, then the threshold voltage U ref1 remains constant because of the blocking effect of the diode 5 .\",\n \"Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.\",\n \"1 , so that the first pole 4 a is negative and the second pole 4 b is positive.\",\n \"In this case, the diode 5 in the circuit shown in FIG.\",\n \"1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3 c and its anode to the second pole 4 b.\",\n \"In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7 .\",\n \"According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5 a of the diode 5 and whose negative pole is in the place of the cathode 5 b of the diode 5 .\",\n \"FIG.\",\n \"6 shows an embodiment 10 b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.\",\n \"1 .\",\n \"The diode shown in FIG.\",\n \"5 has been replaced by a second voltage source 7 , whereby its positive pole 7 a has been put in the place of the anode 5 a and the negative pole 7 b has been put in the place of the cathode 5 b .\",\n \"The fundamental voltage distance dU shown in FIG.\",\n \"1 has now been replaced by the voltage of the voltage source 7 , that is to say, the fundamental voltage distance dU between U ref1 and U signal is now defined by the voltage supplied by the second direct voltage source.\",\n \"If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.\",\n \"FIG.\",\n \"7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10 a shown in FIG.\",\n \"5 is used.\",\n \"A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32 .\",\n \"When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.\",\n \"The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.\",\n \"The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10 a shown in FIG.\",\n \"5 .\",\n \"In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals S St that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals S St each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.\",\n \"9 a and 9 b .\",\n \"In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage U ref1 is greater than the voltage signal U signal , so that no dummy switching signal is triggered.\",\n \"In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals S St in such a way that the control signals S St each begin before or simultaneously with the end of the signal pulses 41 .\",\n \"This ensures that, without any time lag, the threshold voltage U ref1 is supplied to the dropping voltage signal U signal at the fundamental voltage distance dU already at the beginning of the relaxation phase.\",\n \"The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.\",\n \"FIG.\",\n \"8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10 a shown in FIG.\",\n \"5 is likewise used.\",\n \"The distance sensor shown in FIG.\",\n \"8 —in comparison to that shown in FIG.\",\n \"7 —additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34 .\",\n \"Moreover, before the received signal is fed into the envelope curve shaper 37 , it is passed through a voltage limiter 35 and through a logarithmic amplifier 36 c. Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.\",\n \"The amplifier 36 c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.\",\n \"In FIG.\",\n \"8 , in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31 .\",\n \"For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31 .\",\n \"In FIG.\",\n \"8 , the first switching signal S 1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.\",\n \"FIGS.\",\n \"9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.\",\n \"FIG.\",\n \"9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo occurs during the quiescent phase.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32 , as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet ( FIG.\",\n \"9 , Curve a).\",\n \"Moreover, the signal transmitter 2 transmits a control signal S St to the switch 3 , which is synchronized with the signal pulse 41 in such a way that the control signal S St begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former ( FIG.\",\n \"9 , Curve b).\",\n \"FIG.\",\n \"9 , Curve c, shows the course of the voltage signal U signal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage U ref1 (dotted curve).\",\n \"The course of the U signal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53 , which was explained with reference to FIG.\",\n \"4 .\",\n \"While the control signal S St is present at the switch 3 , the latter is opened, so that a threshold voltage U ref1 is established which is identical to the voltage supplied by the first direct voltage source 4 a , 4 b ( FIG.\",\n \"9 , Curve c).\",\n \"Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3 .\",\n \"In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36 c ( FIG.\",\n \"8 ).\",\n \"According to the invention, after the end of the control signal S St , a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the voltage distance dU.\",\n \"Therefore, the threshold voltage U ref1 drops immediately after the end of the control signal by a certain quantity.\",\n \"From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal U signal as long as the latter does not increase.\",\n \"A dummy switching signal is not triggered.\",\n \"If, however, the voltage signal U signal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal U signal leads to a triggering of the comparator 1 as soon as the voltage signal U signal exceeds the threshold voltage U ref1 .\",\n \"In this case, the comparator 1 emits a first switching signal S 1 ( FIG.\",\n \"9 , Curve d).\",\n \"FIG.\",\n \"10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8 , whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.\",\n \"The courses over time of the signal pulse 41 and of the control signal S St are identical to those shown in FIG.\",\n \"9 .\",\n \"During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal U signal ( FIG.\",\n \"10 , Curve a).\",\n \"Up until the beginning of the second echo peak 61 , the curves correspond to those shown in FIG.\",\n \"9 , Curve c. According to the invention, however, the decrease of the threshold voltage U ref1 comes to a halt as soon as the voltage signal U signal is no longer dropping, and a first switching signal S 1a is triggered by the second echo peak 61 .\",\n \"After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage U ref1 and the voltage signal U signal .\",\n \"During the quiescent phase, another, third echo peak 62 of the voltage signal U signal occurs, as a result of which another switching signal S 1b is triggered.\",\n \"Now reference will be made to FIGS.\",\n \"11 to 13 , which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.\",\n \"FIG.\",\n \"11 shows a preferred embodiment 10 c of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103 .\",\n \"The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.\",\n \"b) A first resistor 11 is connected between the first pole 4 a and the first threshold value input 1 a , and a second resistor 12 is connected between the first threshold value input 1 a and the second pole 4 b , so that the first resistor 11 and the second resistor 12 form a first voltage divider 11 , 12 .\",\n \"Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the first voltage divider.\",\n \"Thus, through the use of the first voltage divider 11 , 12 , the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal U signal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dU a .\",\n \"When this embodiment 10 c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.\",\n \"This is advantageous for many applications such as, for example, for the detection of small objects.\",\n \"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.\",\n \"c) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage U ref1 is adapted to the voltage signal U signal .\",\n \"The eighth resistor also serves to smooth the course of the threshold voltage U ref1 with respect to very rapid fluctuations of the voltage signal U signal .\",\n \"FIG.\",\n \"12 shows another preferred embodiment 10 d of the invention, which differs from the adaptive comparator circuit 10 c shown in FIG.\",\n \"11 in that, before the voltage signal U signal reaches the first signal input 1 b , it passes through an impedance transformer.\",\n \"This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.\",\n \"FIG.\",\n \"13 shows another preferred embodiment 10 e of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103 .\",\n \"The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.\",\n \"b) A second comparator 20 is used that has a second threshold value input 20 a and a second signal input 20 b , and that emits a second switching signal S 2 when a greater voltage is present at the second signal input 20 b than at the second threshold value input 20 a. c) The first pole 4 a of the first voltage source 4 a , 4 b is connected to the second threshold value input 20 a via a third resistor 13 .\",\n \"The second threshold value input 20 a is connected to the second pole 4 b via a fourth resistor 14 , so that the third and the fourth resistors 13 , 14 form a second voltage divider which can be configured as a potentiometer.\",\n \"d) The collector 103 c that serves as the switch output and the second threshold value input 20 a are connected via a fifth resistor 15 .\",\n \"e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S 2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S 2 , and otherwise it is in the open state.\",\n \"The second switch 23 can be a transistor.\",\n \"f) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"g) The diode 5 is connected in series to an eighth resistor 18 .\",\n \"The purpose of this resistor was already explained with reference to FIG.\",\n \"11 .\",\n \"h) Before reaching the first signal input 1 b , the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.\",\n \"12 .\",\n \"Due to the second voltage divider 13 , 14 , a voltage U ref2 is present at the second threshold value input 20 a .\",\n \"As long as the second switch 23 is closed, the first threshold voltage U ref1 follows the dropping voltage signal U signal , parallel-offset by the fundamental voltage distance dU, as explained above.\",\n \"According to the invention, however, the second switch opens as soon as the voltage signal U signal exceeds the second threshold voltage U ref2 .\",\n \"As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage U ref1 are ended.\",\n \"Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage U ref1 has dropped to the magnitude of the second threshold voltage U ref2 supplied by the second potentiometer 13 , 14 .\",\n \"Thus, through the use of the second voltage divider 13 , 14 , the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal U signal with respect to voltage signal U signal , from the fundamental voltage distance dU to a value dU b .\",\n \"The second voltage divider 13 , 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20 a.\",\n \"The fifth resistor 15 is connected between the switch output 103 c and the second threshold value input 20 a .\",\n \"Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the second voltage divider 13 , 14 .\",\n \"In particular, through the use of the fifth resistor 15 , it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal U signal strives towards a value dU b that—depending on the voltage supplied by the second voltage divider 13 , 14 —can advantageously be not only larger but also smaller than the fundamental voltage distance dU.\",\n \"When this embodiment 10 e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13 , 14 .\",\n \"All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10 b , 10 c , 10 d and 10 e illustrated in FIG.\",\n \"6 and FIGS.\",\n \"11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.\",\n \"8 instead of the adaptive comparator circuit 10 a shown there by way of an example.\"\n ],\n [\n \"TECHNICAL AREA The invention relates to an adaptive comparator circuit that can be used particularly for reducing the short range of an acoustic distance sensor, as well as to an acoustic distance sensor with such a circuit.\",\n \"STATE OF THE ART Acoustic distance sensors are used to measure distances by means of the pulse-propagation time method.\",\n \"In this process, a sound transducer emits a short sound pulse that propagates in a medium (gas, liquid or solid).\",\n \"This pulse is reflected, for example, on an object and comes back as an echo.\",\n \"The distance to the object can be determined on the basis of the propagation time of the sound pulse and the sound velocity in the medium.\",\n \"As a rule, the frequency of the sound used for such measurements lies in the ultra-sound range, typically in the range between 20 kHz and 500 kHz, for example, at 400 kHz.\",\n \"The sound transducer, which in particular can be a piezoelectric ceramic element, is normally used to transmit the sound pulse as well as to receive the echo.\",\n \"Typically, sound pulses of about 3 to 50 oscillations periods each are emitted.\",\n \"This results in time durations of typically about 6 to 2050 μs for the emitted sound pulses.\",\n \"During this time, i.e.\",\n \"the transmitting phase, the sound transducer oscillates at a high oscillation amplitude.\",\n \"This time duration is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.\",\n \"The relaxation time is typically, for example, 300 μs to 3 ms.\",\n \"The time interval between the end of the relaxation time and the emission of the next sound pulse will be called “quiescent phase” below.\",\n \"Due to noise and other interferences, however, the oscillation amplitude does not drop completely to zero even during the quiescent phase, but rather remains at a certain constant mean disturbance level.\",\n \"Therefore, during the receiving phase that follows the transmitting phase, even in the absence of a useful echo, the oscillation amplitude that dies out during the relaxation time and the mean disturbance level consistently form a finite background signal that is very large immediately after the end of the transmitting phase, that generally decreases exponentially within the relaxation time and that finally remains at the disturbance level.\",\n \"A useful echo signal is superimposed on this background and, as a rule, is detected by means of a comparator that emits a switching signal when the received signal is greater than a certain reference value that will be called “threshold voltage” hereinafter.\",\n \"On the one hand, the threshold voltage should be as low as possible so that even weak useful echo signals, e.g.\",\n \"of small objects, can be detected, which translates into high sensitivity.\",\n \"On the other hand, the threshold voltage has to lie above the disturbance level by a certain safety margin so as to avoid erroneous triggering of the comparator during the quiescent phase.\",\n \"When the threshold voltage is constant over time, useful echo signals can be detected as soon as the background signal has dropped below the threshold voltage as a result of the relaxation of the sound transducer.\",\n \"Before this time, however, that is to say, during the transmitting phase and during part of the relaxation time, the background signal is greater than the threshold voltage so that the comparator—irrespective of the presence of an echo—is triggered in any case during this time.\",\n \"Thus, during this time, called dead time, the system is fundamentally insensitive to the reception of echoes.\",\n \"Therefore, the measurement of distances below a certain minimum distance is impossible.\",\n \"This minimum distance is called the “short range” and is typically a few centimeters.\",\n \"The existence of the short range often has a detrimental effect, for example, wherever due to space constraints, the sensor is to be situated as close as possible to the object to be detected.\",\n \"Various methods are known for reducing the short range.\",\n \"The simplest method is to increase the threshold voltage.\",\n \"This causes the background signal, which dies out after the transmitting phase, to drop below the threshold voltage at an earlier point in time within the relaxation time.\",\n \"However, this method entails the serious drawback that an increase in the threshold voltage translates into a reduction of the system sensitivity, as a result of which weak echoes can no longer be detected.\",\n \"Another method lies in conveying the received signal through an amplifier whose amplification factor can be changed over time.\",\n \"The amplification factor is minimal immediately after the transmittal pulse and is then boosted, for example, by means of a suitable electronic circuit as a function of the time.\",\n \"In this manner, despite a constant threshold voltage over time, a reduction of the short range can be combined with a high sensitivity during the quiescent phase.\",\n \"Of course, for this purpose, the course over time of the amplification factor has to be adapted to the relaxation time.\",\n \"The drawback is that a one-time optimal adaptation is not sufficient here, since the relaxation time of the sound transducer is not constant but rather, it depends on the medium on the temperature, on the age of the sound transducer and on other influencing factors.\",\n \"Therefore, in actual practice, either complex compensation measures have to be undertaken or re-adaptations have to be made repeatedly, or else a large safety margin has to be maintained right from the start for purposes of optimal adaptation, which diminishes the system sensitivity, at least during the relaxation time.\",\n \"Another method is to provide a threshold voltage that can be changed over time.\",\n \"In the ideal case, this threshold voltage drops at a gradient that is similar to the decaying background signal and, at the beginning of the quiescent phase, turns into a constant value, whereby the threshold voltage nevertheless always remains sufficiently greater than the background signal.\",\n \"This method can be additionally improved by subjecting the background signal to a logarithmic amplification so that the decrease is linear instead of exponential.\",\n \"The drawback here, however, is that a one-time adaptation of the time behavior of the threshold voltage is not sufficient to ensure optimal operation over the long run, since here, too, the individual dying-out behavior of the sound transducer is not taken into account on a case-to-case basis.\",\n \"Another method for reducing the short range lies in the propagation time-dependent control of the length of the emitted sound pulses.\",\n \"In the case of a short propagation time, that is to say, in the case of an object that is close, very short sound pulses are used to that the oscillation amplitude of the sound transducer does not fully develop and thus the short range is reduced.\",\n \"Another method lies in the use of threshold voltage that is dynamically changed by means of a microprocessor and a D/A transducer.\",\n \"The microprocessor regulates the D/A transducer in such a way that a dynamic threshold is created that always lies above the dying-out signal.\",\n \"Here, an A/D transducer measures the decaying transmission signal.\",\n \"These methods, however, call for considerable hardware and software resources.\",\n \"Therefore, the invention is based on the objective of simply and inexpensively providing a comparator circuit with which, in every single case, the course over time of the threshold voltage is automatically adapted to the individual decay behavior of the sound transducer and the magnitude of the disturbance level.\",\n \"This objective is achieved according to the invention by an adaptive comparator circuit, especially for an acoustic distance sensor, comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and having a first signal input to which a voltage signal (Usignal) is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, characterized by a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.\",\n \"This is the first basic form of an adaptive comparator circuit according to the invention.\",\n \"According to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.\",\n \"This is the second basic form of an adaptive comparator circuit according to the invention.\",\n \"These two basic forms can be augmented advantageously as will still be explained below.\",\n \"An acoustic distance sensor according to the invention comprises a control unit that at times transmits signal pulses to an oscillator which, during the presence of a signal pulse, transmits an alternating voltage to a sound transducer that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer in the form of an echo as a result of reflection, and of converting them into an electric received signal, and it also comprises an envelope curve shaper to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a signal voltage, characterized in that the signal voltage is fed to an adaptive comparator circuit comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and a first signal input to which a voltage signal is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.\",\n \"An acoustic distance sensor according to the invention thus comprises the adaptive comparator circuit according to the invention.\",\n \"Preferably, the control signal emitted by the signal transmitter is an electric control signal and the first switch is an electrically or electronically controlled switch, for example, a transistor, whose base terminal or gate terminal function as a control-current terminal or, for example, a relay.\",\n \"In another embodiment of the invention, the control signal emitted by the signal transmitter is an optical control signal that is emitted, for example, by a light diode, and the first switch is an optically controlled switch, e.g.\",\n \"a photoresistor.\",\n \"As soon as the signal transmitter emits a control signal, the first switch is in the closed state, so that the capacitor is charged with the voltage supplied by the first voltage source.\",\n \"After the end of the control signal, the switch is in the open state.\",\n \"The capacitor voltage and thus the first threshold voltage present at the first threshold value input now assumes a value that corresponds to the sum of the voltage signal and the diode flow voltage or else to the sum of the voltage signal and the voltage of the second direct voltage source.\",\n \"The diode flow voltage or the voltage of the second direct voltage source are referred to below as “fundamental voltage distance” dU.\",\n \"The task of the diode or of the second direct voltage source is to allow the discharging of the capacitor and thus a drop in the threshold voltage for as long as and only until the difference of the threshold voltage minus the voltage signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal increases.\",\n \"Therefore, the time behavior of the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal as long as the voltage signal does not increase.\",\n \"In contrast, if the voltage signal increases, the threshold voltage remains constant.\",\n \"Consequently, a maximum, e.g.\",\n \"a peak, of the voltage signal leads to the triggering of the comparator as soon as the voltage signal exceeds the threshold voltage.\",\n \"If the voltage signal drops again after having increased, then the threshold voltage follows the voltage signal, once again parallel-offset, as soon as the fundamental, voltage distance dU has been reached again.\",\n \"Hence, according to the invention, the threshold voltage is specified by the magnitude of the voltage signal at every point in time of the receiving phase.\",\n \"If the course over time of the voltage signal is changed, then there is automatically a correspondingly changed course over time of the threshold voltage.\",\n \"Thus, the course over time of the threshold voltage is automatically adapted to that of the voltage signal.\",\n \"The diode flow voltage is temperature-dependent.\",\n \"It decreases as the temperature rises.\",\n \"Consequently, an acoustic distance sensor according to the invention becomes more sensitive as the temperature rises.\",\n \"On the other hand, the attenuation of ultrasound waves generally increases as the temperature rises.\",\n \"Therefore, the resultant sensitivity loss is advantageously compensated for partially with the use of a diode to create the fundamental voltage distance dU.\",\n \"As already explained above, according to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.\",\n \"In this case, the fundamental voltage distance dU is defined by the voltage supplied by the second direct voltage source.\",\n \"The basic forms described above of adaptive comparator circuits according to the invention can be advantageously augmented as will be explained below.\",\n \"In a preferred embodiment of the invention, one of the above-described basic forms has been augmented by a first resistor that is connected between the first pole and the first threshold value input, and by a second resistor that is connected between the first threshold value input and the second pole, so that the first resistor and the second resistor form a first voltage divider.\",\n \"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.\",\n \"Thus, through the use of the first voltage divider, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.\",\n \"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.\",\n \"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.\",\n \"Another preferred embodiment of an adaptive comparator circuit according to the invention comprises the following in addition to the basic forms described above: a second comparator that has a second threshold value input and a second signal input, and that emits a second switching signal when a greater voltage is present at the second signal input than at the second threshold value input, a third resistor via which the first pole of the first direct voltage source is connected to the second threshold value input, a fourth resistor that, on the one hand, is connected to the second threshold value input and, on the other hand, to the second pole of the first voltage source, so that the third resistor and the fourth resistor form a second voltage divider, and a second switch that is connected in series to the diode and that can be regulated or activated by the second switching signal in such a way that it is in the closed state when the second comparator emits the second switching signal, and otherwise it is in the open state.\",\n \"Due to the second voltage divider, a voltage is present at the second threshold value input that will be called “second threshold voltage” Uref2 below.\",\n \"As long as the second switch is closed, the first threshold voltage follows the dropping voltage signal Usignal with the fundamental voltage distance dU, as explained above.\",\n \"According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor and thus the dropping of the first threshold voltage are ended.\",\n \"Hence, in this embodiment of the invention, the capacitor can only discharge until the first threshold voltage Uref1 has dropped to the value of the second threshold voltage Uref2.Thus, through the use of the second voltage divider, the voltage distance by which the first threshold voltage is changed parallel-offset with respect to voltage signal Usignal after the decay of the voltage signal Usignal, advantageously by the fundamental voltage distance dU to a value dUb.\",\n \"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor according to the invention, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be reduced.\",\n \"The second voltage divider can be an adjustable potentiometer whose pick-up is connected to the second threshold value input.\",\n \"In another configuration of this embodiment of the invention, a fifth resistor is connected between the switch output and the second threshold value input.\",\n \"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider.\",\n \"In particular, through the use of the fifth resistor, it can be additionally achieved that the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.\",\n \"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased.\",\n \"In another advantageous embodiment of the invention, the voltage signal passes through a voltage follower or impedance transformer before reaching the first signal input.\",\n \"In another embodiment of the invention, a sixth resistor is connected between the control output and the control-current terminal for purposes of limiting the current flow.\",\n \"In another embodiment of the invention, the first switch is a transistor whose base functions as a control-current terminal, whereby a seventh resistor is connected between the base and the first pole of the first direct voltage source.\",\n \"In another embodiment of the invention, the diode or the second direct voltage source is connected in series with an eighth resistor.\",\n \"This brings about a slowing down of the discharging of the capacitor as well as a smoothing of the threshold voltage, which is advantageous for many applications.\",\n \"The voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.\",\n \"In this manner, it is prevented that the first comparator is triggered while the first switch is still closed, that is to say, while the threshold voltage cannot yet adapt to the voltage signal.\",\n \"Moreover, in this manner, it is advantageously achieved that, in any case, the adaptation of the threshold voltage to the voltage signal can begin immediately after the opening of the first switch.\",\n \"An adaptive comparator circuit according to the invention can be used especially advantageously in an acoustic distance sensor.\",\n \"In this case, the voltage signal is advantageously formed by the envelope curve of the received signal supplied by the sound transducer.\",\n \"This received signal consists of a superimposition of the background signal with the useful echo signal.\",\n \"The formation of the envelope curve, for example, by means of a rectifier circuit, is known from the state of the art and will thus not be elaborated upon in greater depth here.\",\n \"In the absence of an echo, the envelope curve dies out exponentially during the relaxation phase and, at the beginning of the quiescent phase, turns into a horizontal straight line.\",\n \"Such an envelope curve will be called “background envelope curve” below.\",\n \"The threshold voltage adapts according to the invention to the individual course of the background envelope curve in that it follows said background envelope curve parallel-offset by a voltage distance dU or dUa or dUb.\",\n \"An echo causes a peak to be superimposed on the background envelope curve so that the voltage signal increases.\",\n \"According to the invention, the threshold voltage does not follow this increase, so that the comparator is triggered and the echo can thus be detected.\",\n \"As already mentioned above, the voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.\",\n \"Since in this case, the threshold voltage already follows the background envelope curve at the voltage distance dU at the beginning of the relaxation phase, the voltage signal in the absence of an echo is not greater than the threshold voltage at any point in time, especially not during the entire relaxation phase.\",\n \"Therefore, throughout the entire receiving phase, the comparator can only be triggered by an echo, but not by the decay behavior of the sound transducer.\",\n \"Thus, an acoustic distance sensor according to the invention is fundamentally able to detect an echo during the quiescent phase as well as especially during the entire relaxation phase.\",\n \"As a result, the undesired short range of the sensor is considerably reduced according to the invention.\",\n \"BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.\",\n \"1—a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"2—a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"1, FIG.\",\n \"3—a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.\",\n \"4—a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.\",\n \"3, FIG.\",\n \"5—a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.\",\n \"6—a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.\",\n \"7 and 8—a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.\",\n \"9—a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo occurs during the quiescent phase, FIG.\",\n \"10—a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo additionally occurs during the relaxation phase, and FIGS.\",\n \"11 to 13—circuit diagrams of further embodiments of comparator circuits according to the invention.\",\n \"FIGS.\",\n \"1 to 4 serve to further illustrate the state of the art.\",\n \"FIG.\",\n \"1 shows a schematic block diagram of a variant of an acoustic distance sensor 30a with which a measure according to the state of the art has been taken in order to reduce the short range.\",\n \"The distance sensor 30a shown in FIG.\",\n \"1 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, an amplifier 36a with regulatable amplification factor, an envelope curve shaper 37, a comparator 1 as well as an amplification factor actuation stage 38a.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32.The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.\",\n \"to emit sound waves.\",\n \"Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30a.\",\n \"During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.\",\n \"This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.\",\n \"In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.\",\n \"The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36a with a regulatable amplification factor.\",\n \"The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.\",\n \"The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.\",\n \"In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.\",\n \"The amplification factor of the amplifier 36a is regulated by the amplification factor actuation stage 38a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35.Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto (FIG.\",\n \"2).\",\n \"The amplification factor actuation stage 38a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31.In this manner a reduction of the short range can be achieved.\",\n \"The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.\",\n \"FIG.\",\n \"2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36a, c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.\",\n \"1.The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.\",\n \"As was already explained with reference to FIG.\",\n \"1, during the signal pulse 41, the amplification factor 42 reaches a minimum 42a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.\",\n \"During the signal pulse 41, the envelope curve voltage 43 reaches a maximum (envelope curve segment 43a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43b), whereby the course of the envelope curve 43b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.\",\n \"The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.\",\n \"The advantageous result of this is a reduction of the short range.\",\n \"In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43b).\",\n \"The threshold voltage 44 is constant over time.\",\n \"Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44, in a disadvantageous manner, at the beginning of the signal pulse 41, a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44.An echo signal (envelope curve segment 43d) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41.The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.\",\n \"Further drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"1 were already explained above.\",\n \"As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.\",\n \"For purposes of further illustrating the state of the art, FIG.\",\n \"3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30b.\",\n \"The distance sensor 30b shown in FIG.\",\n \"3 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, a preferably logarithmic amplifier 36b, an envelope curve shaper 37, a comparator 1 as well as a threshold value actuation stage 38b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1.The essential difference from the distance sensor 30a illustrated in FIG.\",\n \"1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.\",\n \"FIG.\",\n \"4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30b shown in FIG.\",\n \"3.The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.\",\n \"During a signal pulse 41, the envelope curve voltage 53 reaches a maximum (envelope curve segment 53a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53b), since the amplifier 36b is a logarithmic amplifier.\",\n \"Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53c).\",\n \"The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38b in such a way that it rises before every signal pulse 41, remains at a maximum value for a certain period of time (envelope curve segment 54a), then drops (curve segment 54b) and finally reaches a constant level (envelope curve segment 54c) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53, as long as no echo is received.\",\n \"The threshold value actuation stage 38b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31.The advantageous result is a reduction of the short range.\",\n \"Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41.Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53d) associated with an echo, so that the switching signal 56 is a useful switching signal 56.Drawbacks associated with a distance sensor of the type illustrated in FIG.\",\n \"3 were already explained above.\",\n \"Now reference is made to FIG.\",\n \"5, which illustrates a schematic circuit diagram of an embodiment 10a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2, a first regulatable switch 3, a capacitor 6, a diode 5 and a first comparator 1, that has a first threshold value input 1a and a first signal input 1b.\",\n \"At the first threshold value input 1a, a threshold voltage Uref1, is present that is emitted internally in the adaptive comparator circuit according to the invention.\",\n \"At the signal input 1b, a voltage signal Usignal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.\",\n \"The first comparator 1 emits a first switching signal S1 when the voltage signal Usignal is greater than the first threshold voltage Uref1.The first switching signal S1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.\",\n \"Via a control output 2a, the signal transmitter 2 emits an electric control signal SSt at times, for example, at regular intervals.\",\n \"In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.\",\n \"Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.\",\n \"Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal SSt having a time duration of typically, for example, 20 microseconds.\",\n \"The first switch 3 has a control means 3a, a switch input 3b and a switch output 3c.\",\n \"The control means 3a is connected to the control output 2a and can be regulated by the control signal SSt in such a way that it is in the closed state when the signal transmitter 2 emits a control signal SSt, and otherwise it is in the open state.\",\n \"The first switch 3 can be, for example, a transistor.\",\n \"The switch input 3b is connected to the positive first pole 4a of a first direct voltage source 4a, 4b (not shown here).\",\n \"The switch output 3c is connected to the first threshold value input 1a of the comparator 1, so that the first threshold voltage Uref1 is equal to the voltage present at the switch output 3c.\",\n \"Thus, when the switch 3 is closed, the potential of the positive first pole 4a is present at the first threshold value input 1a of the comparator 1.The switch output 3c is also applied via a capacitor 6 to the negative second pole 4b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.\",\n \"The switch output 3c is also connected to the anode 5a of a diode 5.The cathode 5b of the diode 5 is connected to the first signal input 1b of the comparator.\",\n \"As soon as the signal transmitter 2 emits a control signal SSt, the first switch 3 is in the closed state.\",\n \"Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.\",\n \"When the switch 3 is closed, this voltage is also present at the threshold value input 1a.\",\n \"After the end of the control signal SSt, the switch 3 is in the open state.\",\n \"The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first Uref1 reaches a value that corresponds to the sum of the voltage signal Usignal and the diode flow voltage dU.\",\n \"This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal Usignal: the smaller the voltage signal Usignal, the more the voltage and thus the threshold voltage Uref1 drop according to the invention at the capacitor.\",\n \"Hence, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the fundamental voltage distance dU.\",\n \"According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage Uref1 for as long as and only until the difference of Uref1−Usignal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal Usignal increases.\",\n \"If the voltage signal Usignal drops, then the threshold voltage Uref1 follows the voltage signal Usignal at the distance dU.\",\n \"In contrast, if the voltage signal Usignal increases, then the threshold voltage Uref1 remains constant because of the blocking effect of the diode 5.Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.\",\n \"1, so that the first pole 4a is negative and the second pole 4b is positive.\",\n \"In this case, the diode 5 in the circuit shown in FIG.\",\n \"1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3c and its anode to the second pole 4b.\",\n \"In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7.According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5a of the diode 5 and whose negative pole is in the place of the cathode 5b of the diode 5.FIG.\",\n \"6 shows an embodiment 10b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.\",\n \"1.The diode shown in FIG.\",\n \"5 has been replaced by a second voltage source 7, whereby its positive pole 7a has been put in the place of the anode 5a and the negative pole 7b has been put in the place of the cathode 5b.\",\n \"The fundamental voltage distance dU shown in FIG.\",\n \"1 has now been replaced by the voltage of the voltage source 7, that is to say, the fundamental voltage distance dU between Uref1 and Usignal is now defined by the voltage supplied by the second direct voltage source.\",\n \"If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.\",\n \"FIG.\",\n \"7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10a shown in FIG.\",\n \"5 is used.\",\n \"A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32.When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.\",\n \"The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.\",\n \"The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10a shown in FIG.\",\n \"5.In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals SSt that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals SSt each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.\",\n \"9a and 9b.\",\n \"In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage Uref1 is greater than the voltage signal Usignal, so that no dummy switching signal is triggered.\",\n \"In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses 41.This ensures that, without any time lag, the threshold voltage Uref1 is supplied to the dropping voltage signal Usignal at the fundamental voltage distance dU already at the beginning of the relaxation phase.\",\n \"The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.\",\n \"FIG.\",\n \"8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10a shown in FIG.\",\n \"5 is likewise used.\",\n \"The distance sensor shown in FIG.\",\n \"8—in comparison to that shown in FIG.\",\n \"7—additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34.Moreover, before the received signal is fed into the envelope curve shaper 37, it is passed through a voltage limiter 35 and through a logarithmic amplifier 36c.\",\n \"Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.\",\n \"The amplifier 36c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.\",\n \"In FIG.\",\n \"8, in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31.For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31.In FIG.\",\n \"8, the first switching signal S1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.\",\n \"FIGS.\",\n \"9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.\",\n \"FIG.\",\n \"9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo occurs during the quiescent phase.\",\n \"The control unit 31 transmits short signal pulses 41 to the oscillator 32, as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet (FIG.\",\n \"9, Curve a).\",\n \"Moreover, the signal transmitter 2 transmits a control signal SSt to the switch 3, which is synchronized with the signal pulse 41 in such a way that the control signal SSt begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former (FIG.\",\n \"9, Curve b).\",\n \"FIG.\",\n \"9, Curve c, shows the course of the voltage signal Usignal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage Uref1 (dotted curve).\",\n \"The course of the Usignal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53, which was explained with reference to FIG.\",\n \"4.While the control signal SSt is present at the switch 3, the latter is opened, so that a threshold voltage Uref1 is established which is identical to the voltage supplied by the first direct voltage source 4a, 4b (FIG.\",\n \"9, Curve c).\",\n \"Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3.In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36c (FIG.\",\n \"8).\",\n \"According to the invention, after the end of the control signal SSt, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the voltage distance dU.\",\n \"Therefore, the threshold voltage Uref1 drops immediately after the end of the control signal by a certain quantity.\",\n \"From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal Usignal as long as the latter does not increase.\",\n \"A dummy switching signal is not triggered.\",\n \"If, however, the voltage signal Usignal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal Usignal leads to a triggering of the comparator 1 as soon as the voltage signal Usignal exceeds the threshold voltage Uref1.In this case, the comparator 1 emits a first switching signal S1 (FIG.\",\n \"9, Curve d).\",\n \"FIG.\",\n \"10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.\",\n \"8, whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.\",\n \"The courses over time of the signal pulse 41 and of the control signal SSt are identical to those shown in FIG.\",\n \"9.During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal Usignal (FIG.\",\n \"10, Curve a).\",\n \"Up until the beginning of the second echo peak 61, the curves correspond to those shown in FIG.\",\n \"9, Curve c. According to the invention, however, the decrease of the threshold voltage Uref1 comes to a halt as soon as the voltage signal Usignal is no longer dropping, and a first switching signal S1a is triggered by the second echo peak 61.After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage Uref1 and the voltage signal Usignal.\",\n \"During the quiescent phase, another, third echo peak 62 of the voltage signal Usignal occurs, as a result of which another switching signal S1b is triggered.\",\n \"Now reference will be made to FIGS.\",\n \"11 to 13, which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.\",\n \"FIG.\",\n \"11 shows a preferred embodiment 10c of the invention which differs from the adaptive comparator circuit 10a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.\",\n \"b) A first resistor 11 is connected between the first pole 4a and the first threshold value input 1a, and a second resistor 12 is connected between the first threshold value input 1a and the second pole 4b, so that the first resistor 11 and the second resistor 12 form a first voltage divider 11, 12.Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.\",\n \"Thus, through the use of the first voltage divider 11, 12, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.\",\n \"When this embodiment 10c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.\",\n \"This is advantageous for many applications such as, for example, for the detection of small objects.\",\n \"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.\",\n \"c) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage Uref1 is adapted to the voltage signal Usignal.\",\n \"The eighth resistor also serves to smooth the course of the threshold voltage Uref1 with respect to very rapid fluctuations of the voltage signal Usignal.\",\n \"FIG.\",\n \"12 shows another preferred embodiment 10d of the invention, which differs from the adaptive comparator circuit 10c shown in FIG.\",\n \"11 in that, before the voltage signal Usignal reaches the first signal input 1b, it passes through an impedance transformer.\",\n \"This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.\",\n \"FIG.\",\n \"13 shows another preferred embodiment 10e of the invention which differs from the adaptive comparator circuit 10a shown in FIG.\",\n \"6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.\",\n \"b) A second comparator 20 is used that has a second threshold value input 20a and a second signal input 20b, and that emits a second switching signal S2 when a greater voltage is present at the second signal input 20b than at the second threshold value input 20a.\",\n \"c) The first pole 4a of the first voltage source 4a, 4b is connected to the second threshold value input 20a via a third resistor 13.The second threshold value input 20a is connected to the second pole 4b via a fourth resistor 14, so that the third and the fourth resistors 13, 14 form a second voltage divider which can be configured as a potentiometer.\",\n \"d) The collector 103c that serves as the switch output and the second threshold value input 20a are connected via a fifth resistor 15.e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S2, and otherwise it is in the open state.\",\n \"The second switch 23 can be a transistor.\",\n \"f) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.\",\n \"The purpose of these resistors was already explained above.\",\n \"g) The diode 5 is connected in series to an eighth resistor 18.The purpose of this resistor was already explained with reference to FIG.\",\n \"11.h) Before reaching the first signal input 1b, the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.\",\n \"12.Due to the second voltage divider 13, 14, a voltage Uref2 is present at the second threshold value input 20a.\",\n \"As long as the second switch 23 is closed, the first threshold voltage Uref1 follows the dropping voltage signal Usignal, parallel-offset by the fundamental voltage distance dU, as explained above.\",\n \"According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage Uref1 are ended.\",\n \"Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage Uref1 has dropped to the magnitude of the second threshold voltage Uref2 supplied by the second potentiometer 13, 14.Thus, through the use of the second voltage divider 13, 14, the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal Usignal with respect to voltage signal Usignal, from the fundamental voltage distance dU to a value dUb.\",\n \"The second voltage divider 13, 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20a.\",\n \"The fifth resistor 15 is connected between the switch output 103c and the second threshold value input 20a.\",\n \"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider 13, 14.In particular, through the use of the fifth resistor 15, it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal Usignal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider 13, 14—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.\",\n \"When this embodiment 10e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13, 14.All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10b, 10c, 10d and 10e illustrated in FIG.\",\n \"6 and FIGS.\",\n \"11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.\",\n \"8 instead of the adaptive comparator circuit 10a shown there by way of an example.\",\n \"Commercial Utilization The invention can be utilized commercially, for example, in distance measuring technology, in filling level measuring technology and in automation technology.\",\n \"The main figure is FIG.\",\n \"5.List of Reference Numerals and Letters 1 first comparator 1a first threshold value input 1b first signal input 2 signal transmitter 2a control output of 2 2b input of 2 3 first switch 3a control-current terminal of 3 3b switch input of 3 3c switch output of 3 4a, 4b first, second pole of the first direct voltage source 5 diode 5a, 5b anode, cathode of 5 6 capacitor 7a, 7b positive, negative pole of 7 7 second direct voltage source 8, 9 first, second electric terminal 10a,b,c,d,e adaptive comparator circuits 11-18 first to eighth resistor 20 second comparator 20a second threshold value input 20b second signal input 21 voltage follower 23 second switch 30a distance sensor with constant threshold voltage 30b distance sensor with variable threshold voltage 31 control unit 32 oscillator 33 driver 34 sound transducer 35 voltage limiter 36a amplifier with regulatable amplification factor 36b, c logarithmic amplifier 37 envelope curve shaper 38a amplification factor actuation stage 38b threshold value actuation stage 41 signal pulses 42 course over time of the amplification factor of 36a 42a minimum of 42 43, 53 courses over time of the envelope curve voltage 43a, 53a segments of 43, 53 during the signal pulses 41, 51 43b, 53b segments of 43, 53 during the relaxation time of 34 43c, 53c segments of 43, 53 during the quiescent phase 43d, 53d segments of 43, 53 during an echo 44, 54 threshold values of 30a, 30b 45 dummy switching signal 46, 56 useful switching signals 60 first echo peak 61 second echo peak 62 third echo peak 103 transistor 103a base of 103 103b emitter of 103 103c collector of 103 S1, S1a, S1b switching signals of 1 S2 switching signal of 20 dU fundamental voltage distance Uref1 first threshold voltage Usignal voltage signal\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450932"}}},{"rowIdx":288,"cells":{"text":{"kind":"list like","value":[["Fuel injector and method for installing a fuel injector in a valve seat","A fuel injector, which, at its inflow-side end, has an intake connector at whose outer circumference at least two sealing rings are provided, which seal the fuel injector from a valve seat.","Due to an intermediate ring, the two sealing rings are axially set apart from one another in a respective ring chamber.","The intermediate ring is embodied independently of the intake connector and is made up of two parts.","A flexible outer ring is locked in place on an inner extruded support ring, the outer ring only being mounted once the first sealing ring has already been moved across the support ring and has been stripped off.","The fuel injector is particularly suited for the use in mixture-compressing internal combustion engines having external ignition."],["1-13.","(canceled) 14.A fuel injector for injecting fuel into a combustion chamber of a mixture-compressing internal combustion engine having external ignition, the fuel injector having a longitudinal valve axis, the fuel injector comprising: an intake connector situated at an inflow-side end, and having at least two sealing rings provided at an outer periphery of the intake connector to seal the fuel injector from a valve seat; and an intermediate ring, wherein, via the intermediate ring, the two sealing rings are axially set apart from one another, being situated in each case in a ring chamber, the intermediate ring being embodied independently of the intake connector and including two parts.","15.The fuel injector of claim 14, wherein the intermediate ring includes an inner support ring and an outer ring.","16.The fuel injector of claim 15, wherein the inner support ring, via an inwardly projecting lip, engages in a groove of the intake connector.","17.The fuel injector of claim 15, wherein the inner support ring is made of plastic.","18.The fuel injector of claim 17, wherein the inner support ring is made of the same plastic as a plastic extrusion coat partially surrounding the fuel injector.","19.The fuel injector of claim 14, further comprising: an outwardly projecting locking bead, at an outer diameter of the support ring, at which the outer ring is able to lock into place.","20.The fuel injector of claim 19, wherein an upper locking surface of the locking bead extends largely perpendicularly to the longitudinal valve axis and a lower locking surface has a slanted arrangement.","21.The fuel injector of claim 15, wherein the outer ring is made of flexible plastic.","22.The fuel injector of claim 14, wherein the intermediate ring forms a support shoulder for at least one of the at least two sealing rings.","23.A method for installing a fuel injector in a valve seat, the fuel injector, at the inflow-side end, having an intake connector at whose outer circumference at least two sealing rings are provided, which are axially set apart from one another by an intermediate ring and which seal the fuel injector from the valve seat, the method comprising: affixing an inner support ring of the intermediate ring on a circumference of the intake connector; spreading open a first one of the at least two sealing rings, mounting it on the intake connector, moving it across the inner support ring and stripping it into a ring chamber, and mounting an outer ring on the inner support ring to complete the intermediate ring; spreading open a second sealing ring, mounting it on the intake connector and stripping it into an additional ring chamber; and subsequently inserting the fuel injector in the valve seat together with a sealing system thus mounted on the intake connector.","24.The method of claim 23, wherein the inner support ring of the intermediate ring is extruded on a circumference of the intake connector in a same extrusion procedure as a plastic extrusion coat at least partially surrounding the fuel injector.","25.The method of claim 23, wherein the outer ring of the intermediate ring is locked into place on a locking bead of the inner support ring.","26.The method of claim 23, wherein the fuel injector is secured after having been inserted in the valve seat with corresponding affixation elements provided on the valve seat and on the fuel injector."],[" BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.","For instance, German Patent Application No.","28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.","These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.","German Patent Application No.","28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.","Furthermore, German Patent Application No.","28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.","Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.","37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.","Moreover, German Patent Application No.","197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.","In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another."],[" SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.","Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.","Of particular advantage may be the simple handling of the two partial components of the intermediate ring.","While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.","The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.","The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.","The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.","In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.","It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.","Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings."],["FIELD OF THE INVENTION The present invention relates to a fuel injector, and a method for installing a fuel injector in a valve seat.","BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.","For instance, German Patent Application No.","28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.","These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.","German Patent Application No.","28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.","Furthermore, German Patent Application No.","28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.","Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.","37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.","Moreover, German Patent Application No.","197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.","In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.","SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.","Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.","Of particular advantage may be the simple handling of the two partial components of the intermediate ring.","While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.","The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.","The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.","The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.","In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.","It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.","Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows an inflow-side end of a fuel injector having a sealing system configured according to the present invention for sealing the fuel injector from a valve seat.","FIG.","2 shows an enlargement of the cut-out section II in FIG.","1.DETAILED DESCRIPTION FIG.","1 shows a partial representation of a fuel injector 1, which, for instance, is embodied as a fuel injector for the direct injection of fuel into the combustion chamber of a mixture-compressing internal combustion engine having external ignition.","By way of an inflow-side end 2, fuel injector 1 projects into a corresponding valve seat 3.Valve seat 3 is embodied, for instance, as a cup-shaped intake connecting piece 4, which constitutes part of a fuel-supply line (not shown further), such as a fuel rail.","The fuel-supply line usually has a plurality of intake connecting pieces 4, so that the fuel conveyed via the fuel-supply line may be distributed to a plurality of fuel injectors 1.Intake connecting piece 4 has a stepped arrangement or configuration, for example, and, at its end facing away from the fuel-supply line, has one or a plurality of affixation elements 5 projecting in the manner of a lip.","These affixation elements 5 engage in openings 6 of an securing clamp 7, which is used to reliably secure fuel injector 1 in valve seat 3.Securing clamp 7 is inserted, for instance, in a circumferential groove 8 at the outer periphery of a plastic extrusion coat 9 of fuel injector 1.Fuel injector 1 is largely surrounded by plastic extrusion coat 9, for example.","However, at inflow-side end 2 of fuel injector 1, a metal intake connector 11 for the supply of fuel to the interior of fuel injector 1 projects beyond plastic extrusion coat 9.Above plastic extrusion coat 9, two sealing rings 13, 14, configured as O-rings, are situated at the outer periphery of intake connector 11.Due to an intermediate ring 15 configured according to the exemplary embodiment of the present invention, the two sealing rings 13, 14, which follow each other closely in the axial direction, are positioned at a spatial distance from one another.","Forming the upstream end of fuel injector 1 is an upper retaining ring 16, which is made of plastic and mounted on intake connector 11.In this manner, both sealing rings 13, 14 are embedded between retaining ring 16 and upper end face 17 of plastic extrusion coat 9, intermediate ring 15 forming two ring chambers 18, 19 for sealing rings 13, 14.In contrast to the sealing rings having so-called emergency-sealing properties known in part from the related art, the two sealing rings 13, 14 are two “full-fledged” sealing rings made of known elastomer sealing material.","The necessity of two sealing rings immediately following one another at the periphery of fuel injector 1 results from the composition of certain fuels.","In long-term use of sealing rings it has become apparent that hydrocarbons permeate through the elastomers normally used for sealing rings and may even permeate them completely.","In order to fully prevent the emission of hydrocarbons into the environment in accordance with ever stricter environmental regulations, it may be advantageous to situate an additional sealing ring immediately behind a first sealing ring.","In this manner, the second sealing ring following a sealing ring permeated in the manner described above, offers a substantially improved sealing reliability.","Intermediate ring 15, which partitions the sealing chamber into two ring chambers 18, 19, is made up of two sub-components.","An inner support ring 22 engages, for instance, in a circumferential groove 23 at the periphery of intake connector 11.Ideally, support ring 22 is extruded (injected) on the metal valve body, together with plastic extrusion coat 9 and retaining ring 16, in one plastic-extrusion procedure.","On its inner side, support ring 22 has a slanted lip 24, which projects toward the inside and is able to be inserted in groove 23 with form accuracy.","Provided at the outer diameter of support ring 22 is a locking bead 25, which projects radially outward and at which an outer ring 26, which also belongs to intermediate ring 15, is able to be snapped into place.","The region of locking bead 25 is shown in FIG.","2 in an enlarged view.","Locking bead 25 at support ring 22 is arranged or configured such that the high compression forces of second sealing ring 13 during the installation of fuel injector 1 in intake connecting piece 4 are absorbed by an upper locking surface 28, which mostly extends perpendicularly to a longitudinal valve axis 30.The lower axial demounting forces of first sealing ring 14 are sufficiently absorbed by a lower locking surface 29 of locking bead 25, locking surface 29 having a slanted arrangement or configuration.","Outer ring 26 is made of flexible plastic.","When outer ring 26 is mounted on inner support ring 22, this plastic expands and then locks into place in the limit position above locking bead 25.Intermediate ring 15, made up of outer ring 26 and support ring 22, is used as support shoulder for second sealing ring 13.The mounting of the sealing system on intake connector 11 of fuel injector 1 is briefly explained in the following.","As already described above, plastic extrusion coat 9, inner support ring 22 of intermediate ring 15, and retaining ring 16 are, for instance, simultaneously extruded on the metal valve body in one extrusion procedure.","Subsequently, first sealing ring 14 is spread open, mounted on intake connector 11, moved across support ring 22 and stripped into ring chamber 19.Then, outer ring 26 is clipped onto inner support ring 22 to complete intermediate ring 15.Second sealing ring 13 is then spread open, mounted on intake connector 11 and stripped into ring chamber 18.With the aid of the sealing system mounted in this manner on intake connector 11, fuel injector 1 may be inserted in valve seat 3.The exemplary embodiment and/or method of the present invention is not limited to the described exemplary embodiment or method.","Thus, intake connector 11, for instance, may have a stepped arrangement or configuration in the region of sealing rings 13, 14 and/or sealing rings 13, 14 may have different cross sections or diameters."]],"string":"[\n [\n \"Fuel injector and method for installing a fuel injector in a valve seat\",\n \"A fuel injector, which, at its inflow-side end, has an intake connector at whose outer circumference at least two sealing rings are provided, which seal the fuel injector from a valve seat.\",\n \"Due to an intermediate ring, the two sealing rings are axially set apart from one another in a respective ring chamber.\",\n \"The intermediate ring is embodied independently of the intake connector and is made up of two parts.\",\n \"A flexible outer ring is locked in place on an inner extruded support ring, the outer ring only being mounted once the first sealing ring has already been moved across the support ring and has been stripped off.\",\n \"The fuel injector is particularly suited for the use in mixture-compressing internal combustion engines having external ignition.\"\n ],\n [\n \"1-13.\",\n \"(canceled) 14.A fuel injector for injecting fuel into a combustion chamber of a mixture-compressing internal combustion engine having external ignition, the fuel injector having a longitudinal valve axis, the fuel injector comprising: an intake connector situated at an inflow-side end, and having at least two sealing rings provided at an outer periphery of the intake connector to seal the fuel injector from a valve seat; and an intermediate ring, wherein, via the intermediate ring, the two sealing rings are axially set apart from one another, being situated in each case in a ring chamber, the intermediate ring being embodied independently of the intake connector and including two parts.\",\n \"15.The fuel injector of claim 14, wherein the intermediate ring includes an inner support ring and an outer ring.\",\n \"16.The fuel injector of claim 15, wherein the inner support ring, via an inwardly projecting lip, engages in a groove of the intake connector.\",\n \"17.The fuel injector of claim 15, wherein the inner support ring is made of plastic.\",\n \"18.The fuel injector of claim 17, wherein the inner support ring is made of the same plastic as a plastic extrusion coat partially surrounding the fuel injector.\",\n \"19.The fuel injector of claim 14, further comprising: an outwardly projecting locking bead, at an outer diameter of the support ring, at which the outer ring is able to lock into place.\",\n \"20.The fuel injector of claim 19, wherein an upper locking surface of the locking bead extends largely perpendicularly to the longitudinal valve axis and a lower locking surface has a slanted arrangement.\",\n \"21.The fuel injector of claim 15, wherein the outer ring is made of flexible plastic.\",\n \"22.The fuel injector of claim 14, wherein the intermediate ring forms a support shoulder for at least one of the at least two sealing rings.\",\n \"23.A method for installing a fuel injector in a valve seat, the fuel injector, at the inflow-side end, having an intake connector at whose outer circumference at least two sealing rings are provided, which are axially set apart from one another by an intermediate ring and which seal the fuel injector from the valve seat, the method comprising: affixing an inner support ring of the intermediate ring on a circumference of the intake connector; spreading open a first one of the at least two sealing rings, mounting it on the intake connector, moving it across the inner support ring and stripping it into a ring chamber, and mounting an outer ring on the inner support ring to complete the intermediate ring; spreading open a second sealing ring, mounting it on the intake connector and stripping it into an additional ring chamber; and subsequently inserting the fuel injector in the valve seat together with a sealing system thus mounted on the intake connector.\",\n \"24.The method of claim 23, wherein the inner support ring of the intermediate ring is extruded on a circumference of the intake connector in a same extrusion procedure as a plastic extrusion coat at least partially surrounding the fuel injector.\",\n \"25.The method of claim 23, wherein the outer ring of the intermediate ring is locked into place on a locking bead of the inner support ring.\",\n \"26.The method of claim 23, wherein the fuel injector is secured after having been inserted in the valve seat with corresponding affixation elements provided on the valve seat and on the fuel injector.\"\n ],\n [\n \" BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.\",\n \"For instance, German Patent Application No.\",\n \"28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.\",\n \"These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.\",\n \"German Patent Application No.\",\n \"28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.\",\n \"Furthermore, German Patent Application No.\",\n \"28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.\",\n \"Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.\",\n \"37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.\",\n \"Moreover, German Patent Application No.\",\n \"197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.\",\n \"In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.\",\n \"Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.\",\n \"Of particular advantage may be the simple handling of the two partial components of the intermediate ring.\",\n \"While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.\",\n \"The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.\",\n \"The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.\",\n \"The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.\",\n \"In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.\",\n \"It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.\",\n \"Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a fuel injector, and a method for installing a fuel injector in a valve seat.\",\n \"BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.\",\n \"For instance, German Patent Application No.\",\n \"28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.\",\n \"These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.\",\n \"German Patent Application No.\",\n \"28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.\",\n \"Furthermore, German Patent Application No.\",\n \"28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.\",\n \"Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.\",\n \"37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.\",\n \"Moreover, German Patent Application No.\",\n \"197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.\",\n \"In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.\",\n \"SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.\",\n \"Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.\",\n \"Of particular advantage may be the simple handling of the two partial components of the intermediate ring.\",\n \"While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.\",\n \"The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.\",\n \"The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.\",\n \"The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.\",\n \"In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.\",\n \"It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.\",\n \"Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows an inflow-side end of a fuel injector having a sealing system configured according to the present invention for sealing the fuel injector from a valve seat.\",\n \"FIG.\",\n \"2 shows an enlargement of the cut-out section II in FIG.\",\n \"1.DETAILED DESCRIPTION FIG.\",\n \"1 shows a partial representation of a fuel injector 1, which, for instance, is embodied as a fuel injector for the direct injection of fuel into the combustion chamber of a mixture-compressing internal combustion engine having external ignition.\",\n \"By way of an inflow-side end 2, fuel injector 1 projects into a corresponding valve seat 3.Valve seat 3 is embodied, for instance, as a cup-shaped intake connecting piece 4, which constitutes part of a fuel-supply line (not shown further), such as a fuel rail.\",\n \"The fuel-supply line usually has a plurality of intake connecting pieces 4, so that the fuel conveyed via the fuel-supply line may be distributed to a plurality of fuel injectors 1.Intake connecting piece 4 has a stepped arrangement or configuration, for example, and, at its end facing away from the fuel-supply line, has one or a plurality of affixation elements 5 projecting in the manner of a lip.\",\n \"These affixation elements 5 engage in openings 6 of an securing clamp 7, which is used to reliably secure fuel injector 1 in valve seat 3.Securing clamp 7 is inserted, for instance, in a circumferential groove 8 at the outer periphery of a plastic extrusion coat 9 of fuel injector 1.Fuel injector 1 is largely surrounded by plastic extrusion coat 9, for example.\",\n \"However, at inflow-side end 2 of fuel injector 1, a metal intake connector 11 for the supply of fuel to the interior of fuel injector 1 projects beyond plastic extrusion coat 9.Above plastic extrusion coat 9, two sealing rings 13, 14, configured as O-rings, are situated at the outer periphery of intake connector 11.Due to an intermediate ring 15 configured according to the exemplary embodiment of the present invention, the two sealing rings 13, 14, which follow each other closely in the axial direction, are positioned at a spatial distance from one another.\",\n \"Forming the upstream end of fuel injector 1 is an upper retaining ring 16, which is made of plastic and mounted on intake connector 11.In this manner, both sealing rings 13, 14 are embedded between retaining ring 16 and upper end face 17 of plastic extrusion coat 9, intermediate ring 15 forming two ring chambers 18, 19 for sealing rings 13, 14.In contrast to the sealing rings having so-called emergency-sealing properties known in part from the related art, the two sealing rings 13, 14 are two “full-fledged” sealing rings made of known elastomer sealing material.\",\n \"The necessity of two sealing rings immediately following one another at the periphery of fuel injector 1 results from the composition of certain fuels.\",\n \"In long-term use of sealing rings it has become apparent that hydrocarbons permeate through the elastomers normally used for sealing rings and may even permeate them completely.\",\n \"In order to fully prevent the emission of hydrocarbons into the environment in accordance with ever stricter environmental regulations, it may be advantageous to situate an additional sealing ring immediately behind a first sealing ring.\",\n \"In this manner, the second sealing ring following a sealing ring permeated in the manner described above, offers a substantially improved sealing reliability.\",\n \"Intermediate ring 15, which partitions the sealing chamber into two ring chambers 18, 19, is made up of two sub-components.\",\n \"An inner support ring 22 engages, for instance, in a circumferential groove 23 at the periphery of intake connector 11.Ideally, support ring 22 is extruded (injected) on the metal valve body, together with plastic extrusion coat 9 and retaining ring 16, in one plastic-extrusion procedure.\",\n \"On its inner side, support ring 22 has a slanted lip 24, which projects toward the inside and is able to be inserted in groove 23 with form accuracy.\",\n \"Provided at the outer diameter of support ring 22 is a locking bead 25, which projects radially outward and at which an outer ring 26, which also belongs to intermediate ring 15, is able to be snapped into place.\",\n \"The region of locking bead 25 is shown in FIG.\",\n \"2 in an enlarged view.\",\n \"Locking bead 25 at support ring 22 is arranged or configured such that the high compression forces of second sealing ring 13 during the installation of fuel injector 1 in intake connecting piece 4 are absorbed by an upper locking surface 28, which mostly extends perpendicularly to a longitudinal valve axis 30.The lower axial demounting forces of first sealing ring 14 are sufficiently absorbed by a lower locking surface 29 of locking bead 25, locking surface 29 having a slanted arrangement or configuration.\",\n \"Outer ring 26 is made of flexible plastic.\",\n \"When outer ring 26 is mounted on inner support ring 22, this plastic expands and then locks into place in the limit position above locking bead 25.Intermediate ring 15, made up of outer ring 26 and support ring 22, is used as support shoulder for second sealing ring 13.The mounting of the sealing system on intake connector 11 of fuel injector 1 is briefly explained in the following.\",\n \"As already described above, plastic extrusion coat 9, inner support ring 22 of intermediate ring 15, and retaining ring 16 are, for instance, simultaneously extruded on the metal valve body in one extrusion procedure.\",\n \"Subsequently, first sealing ring 14 is spread open, mounted on intake connector 11, moved across support ring 22 and stripped into ring chamber 19.Then, outer ring 26 is clipped onto inner support ring 22 to complete intermediate ring 15.Second sealing ring 13 is then spread open, mounted on intake connector 11 and stripped into ring chamber 18.With the aid of the sealing system mounted in this manner on intake connector 11, fuel injector 1 may be inserted in valve seat 3.The exemplary embodiment and/or method of the present invention is not limited to the described exemplary embodiment or method.\",\n \"Thus, intake connector 11, for instance, may have a stepped arrangement or configuration in the region of sealing rings 13, 14 and/or sealing rings 13, 14 may have different cross sections or diameters.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450961"}}},{"rowIdx":289,"cells":{"text":{"kind":"list like","value":[["PROCESS FOR THE PRODUCTION OF GRAIN ORIENTED ELECTRICAL STEEL STRIPS","A process for the production of grain oriented electrical Fe—Si strips in which a Si-containing alloy is directly cast as a strip between 2.5-5.0 mm thick and cold rolled in one stage, or in more stages with intermediate annealing, to a final thickness of between 0.15-1.0 mm.","The strip is then continuously annealed to carry out the primary recrystallization and then annealed to carry out the oriented secondary recrystallization.","The process further includes that after solidification of the strip, and before its coiling, a phase transformation from Ferrite to Austenite is induced into the metal matrix for a volume fraction between 25-60%, obtained by controlling the alloy composition so that the Austenite fraction is allowed within the stability equilibrium between the two phases.","The strip is then deformed by rolling in-line with the casting step to obtain a deformation higher than 20% in the temperature interval 1000-1300° C."],["1-8.","(canceled) 9.A process for the production of electrical grain oriented Fe—Si strips in which a Si-containing molten alloy composition is directly cast as continuous strips 2.5 to 5 mm thick, cold rolled in one step or more steps with intermediate annealing to a final thickness of between 1 and 0.15 mm, the strip being then continuously annealed to carry out oriented secondary recrystallisation, characterised in that after the strip solidification and before coiling of said strip in a coiling phase, a ferrite to austenite transformation is induced in the metal matrix via deformation, by rolling said strip between two cooled rolls to obtain a deformation over 20% in a temperature range of 1000-1300° C., thereby producing a volume fraction of austenite to be between 25 and 60%, said molten alloy composition being chosen such that said volume fraction of austenite is stable in a temperature interval of between 1100 and 1200° C. 10.The process according to claim 9, in which between the rolling phase and the coiling one, the strip is held between 1100 and 1200° C. for at least 5 s. 11.The process according to claim 9, in which the as-solidified strip thickness is comprised between 1.5 and 4.0 mm and after the rolling phase the strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.","12.The process according to claim 9, in which before cold rolling the strip is annealed at a maximum temperature of 1200° C. 13.The process according to claim 12, in which after said annealing the strip is continuously quenched from a temperature comprised between 750 and 950° C. down to 400° C. in less than 12 s. 14.The process according to claim 9, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5 wt % Cu, less than 2 wt % Cr+Ni+Mo.","Less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 15.The process according to claim 9, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 16.The process according to claim 9, in which in the alloy at least an element is added chosen between Sn, Sb, P, Bi.","17.A process for the production of grain oriented electrical Fe—Si strip, in which a Si-containing alloy is directly cast as continuous strip 2.5 to 5 mm thick, in-line hot-rolled and then cold-rolled in one step or more steps, with intermediate annealing, to a final thickness of between 1 and 0.15 mm, the cold-rolled strip being, then continuously annealed to carry out primary recrystallisation and subsequently again annealed to carry out secondary recrystallisation, characterized in that the Si-containing alloy composition is selected to induce in the alloy, during the hot-rolling step in which a deformation rate of at least 20% is utilized in a temperature interval of between 1000 and 1300° C., a ferrite to austenite phase transformation with an austenite volume fraction of between 25 to 60% stable in a temperature interval of between 1100 to 1200° C. 18.The process according to claim 17, in which between the hot-rolling and the coiling steps the strip is held in the temperature range of 1100 to 1200° C. for at least 5 s. 19.The process according to claim 17, in which an as-cast strip 2.5 to 4 mm thick is in-line hot-rolled and the thus obtained hot-rolled strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.","20.The process according to claim 17, in which before cold-rolling the strip is annealed at a maximum temperature of 1200° C. 21.The process according to claim 20, in which, after said annealing, the strip is continuously quenched from a temperature of between 750 and 950° C. down to 400° C. in less than 12 s. 22.The process according to claim 17, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5% Cu, less than 2.0% Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 23.The process according to claim 22, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 24.The process according to claim 22, in which in the allot at least an element is added chosen between Sn, Sb, P, Bi."],[" FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.","More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling."],[" SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.","Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.","Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.","Further objects of present invention will be evident from the following description of the invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.","More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.","STATE OF THE ART Grain oriented electrical steel strips (Fe—Si) are typically industrially produced as strips having a thickness comprised between 0,18 and 0,50 mm and are characterised by magnetic properties variable according to the specific product class.","Said classification substantially refers to the specific power losses of the strip subjected to given electromagnetic work conditions (e.g.","P50 Hz at 1,7 Tesla, in W/kg), evaluated along a specific reference direction (rolling direction).","The main utilisation of said strips is the production of transformer cores.","Good magnetic properties (strongly anisotropic) are obtained controlling the final crystalline structure of the strips to obtain all, or almost all, the grains oriented to have their easiest magnetization direction (the <001> axis) aligned in the most perfect way with the rolling direction.","In practice, final products are obtained having the grains mean diameter generally comprised between 1 and 20 mm having an orientation centred around the Goss orientation ({110} <001>).","The minor the angular dispersion around the Goss one, the better the product magnetic permeability and hence the lesser the magnetic losses.","The final products having low magnetic losses (core losses) and high permeability have interesting advantages in terms of design, dimensions and yield of the transformers.","The first industrial production of the above materials was described by the U.S. Firm ARMCO at the beginning of the thirties (U.S. Pat.","No.","1,956,559).","Many important improvements have been since introduced in the production technology of grain oriented electrical strips, in terms both of magnetic and physical quality of products and of transformation costs and cycles rationalisation.","All existing technologies exploit the same metallurgical strategy to obtain a very strong Goss structure in the final products, i.e.","the process of oriented secondary recrystallisation guided by uniformly distributed second phases and/or segregating elements.","The, non metallic, second phases and the segregating elements play a fundamental role in controlling (slowing down) the movement of grain boundaries doing the final annealing which actuates the selective secondary recrystallisation process.","In the original ARMCO technology, utilising MnS as inhibitor of the grain boundaries movement, and in the subsequent technology developed by NSC, in which the inhibitors are mainly aluminium nitrides (AIN+MnS) (EP 8.385, EP 17.830, EP 202.339), a very important binding step common to both production processes is the heating of the continuously cast slabs (ingots, in old times), immediately before the hot rolling, at very high temperatures (around 1400° C.) for a time sufficient to guarantee a complete dissolution of sulphides and/or nitrides coarsely precipitated during the slab cooling after casting, to re-precipitate them in a very fine and uniformly distributed form throughout the metallic matrix of the hot rolled strips.","Such a fine re-precipitation can be started and completed, as well as the precipitates dimensions adjusted, during the process, in any case, however, before the cold rolling.","The slab heating to said temperatures requires using special furnaces (pushing furnaces, liquid-slag walking-beam furnaces, induction furnaces) due to the ductility at high temperatures of the Fe-3% Si alloys and to formation of liquid slags.","New casting technologies of the liquid steel are intended to simplify the production processes to make them more compact and flexible and to reduce costs.","One of said technologies is the “thin slab” casting, consisting in the continuous casting of slabs having the typical thickness of conventional already roughened slabs, apt to a direct hot rolling, through a sequence of slabs continuous casting, treating in continuous tunnel-furnaces to rise/maintain the temperature of slabs and finishing-rolling, down to coiled strip.","The problems connected to the utilisation of said technique for grain oriented products mainly consist in the difficulty to maintain and control the high temperatures necessary to keep in solution the elements forming the second phases, which have to be finely precipitated at the beginning of the finishing hot-rolling step, if desired best micro-structural and magnetic characteristics are to be obtained in the end products.","Such problems were dealt with in different ways, for instance utilising the low thickness of the cast slabs in connection to specific concentration intervals of the micro-alloying elements to stably control the second phases precipitation (grain growth inhibitors) during hot rolling, or drastically modifying the strategy of the inhibitors formation in the metal matrix.","The casting technique potentially offering the highest rationalisation level of the processes and the higher production flexibility is the one consisting in the direct production of strips from the liquid steel (Strip Casting), totally eliminating the hot rolling step.","Such an exaordinary innovation was conceived and patented long time ago, and since long time were also devised and patented process conditions to produce electrical steel strips, and more particularly grain oriented ones.","However, up to now there is not an industrial production in the world of grain oriented electrical steel according to the above technique, though the state of the art relating to the casting machines is ready for industrial applications, as shown by existing plants, producing only carbon steels and stainless steels.","The present inventors believe that to industrially produce grain oriented electrical steel strips from direct solidification of a strip (Strip Casting) it is necessary to have a strip micro-structure before cold rolling significantly different from the one obtained during the casting stage.","The high solidification speed of the cast strip makes it difficult to have a homogeneous and reproducible grain structure throughout the strip and between different castings, due to the high sensitivity of the solidification structure to the fluctuations of the casting conditions and to the alloy composition.","The micro-structure of the intermediate products starting from strip casting is much more influenced by the solidification structure, with respect to the ones derived from conventional slab casting, because of the lack of deformation in the strip during the typical hot rolling.","SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.","Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.","Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.","Further objects of present invention will be evident from the following description of the invention.","DETAILED DESCRIPTION OF THE INVENTION A first important aspect of present invention resides in that a molten alloy containing silicon is directly solidified in the form of a strip, through the casting technology known as strip casting (casting between twin cooled and counter-rotating rolls), thus avoiding, with respect to currently utilised technologies, casting the alloy in slabs or ingots, subjecting said slabs to thermal treatment in special high-temperature furnaces for long times (to attain the necessary thermal homogeneity) and transforming said slabs into strips through hot rolling with total reductions which, according to the slab casting technologies, vary between 96 and 99%.","A second important aspect of present invention resides in that the chemical composition of the Silicon containing alloy is selected specifically to control the thermodynamic stability of the Austenite phase in the matrix (face-centered cubic lattice) in equilibrium with the Ferrite phase (body-centered cubic lattice).","More precisely, to obtain excellent final magnetic characteristics, it is convenient to adjust the chemistry of the alloy so that an Austenite fraction comprised between 25 and 60% is stable between 1100 and 1200° C. Consequently, to balance the strong tendency of silicon to stabilise the Ferrite phase, a number of elements are utilised, favouring the Austenite formation.","Aming those elements, Carbon is particularly important due to its intrinsic austenitising effect as well as to its particular mobility into the matrix, making it possible its easy elimination by means of solid-state decarburising processes which, in this field, are usually carried out by extraction from the strip surfaces utilising annealing atmospheres having a controlled oxidising potential.","The carbon is conveniently present in the steel composition in amount apt to control the desired Austenite fraction, in that in this way it is possible to rise again the stability of the Ferrite by means of a simple decarburisation process, and thus avoiding during the final secondary recrystallisation annealing important phase transition phenomena which would be detrimental for the final desired texture.","As known, however, in said materials itis necessary to reduce the carbon content in the final products at levels of under 50 ppm, to eliminate the adverse effect on the core losses due to formation of carbides.","The higher the carbon content of the alloy, the longer the time necessary to carry out the decarburisation.","For productivity reasons it is then convenient to keep the carbon content within a maximum of 0,1 wt %.","Present inventors eveluated the obtainable Austenite fractions according to different alloy compositions both experimentally and according to empirical relationships available in the literature.","A third aspect of the invention resides in that the Derrite to Austenite transformation in the metal matrix of the cast strip is induced, in a temperature interval centered around 1150° C., typically 1000-1300° C., by means of a sudden deformation higher than 20%, by rolling between cooled rolls, in-line with the continuous casting and before the coiling.","Said sudden and localised deformation imparts to the material the energy necessary to nucleation and formation of the Austenite phase in the matrix, which phase would not be obtained for kinetic reasons, though thermodynamically very stable.","In fact, to obtain equilibrium conditions between the two phases at the considered temperature very long times are necessary, while the working and cooling times are intrinsecally very short, particularly in the case of direct casting as strip (strip casting).","The phase transformation from Ferrite to Austenite is tunable, according to present invention, in quantity, according to selection of chemical composition, and consistently reproducible, as necessary in an industrial process.","As a consequence of the phase transformation induced in the temperature interval defined according to present invention, the grains distribution in the produced strip, in terms both of dimensions and of texture, is exrtremely homogeneous and reproducible through the whole geometrical profile of the strip.","This, in particular, solves the problem the drawback of microstructural etereogeneity, typical of the production of oriented grain steel strips, in that the selection process of the final texture is sensible even to small local differences in the structure and orientation of grains, and even more sensible in the case of strip-cast products.","In fact, in the traditional processes the strip structure before cold rolling is the result of a strong hot deformation of the cast slabs, which contributes to fragment, recrystallise and homogenise the solidification structure; on the contrary, in the strips obtained by direct solidification the structure directly depends on the solidification one, and due to the high solidification speed and to the strongly dynamic nature of the process any even small fluctuation of the casting conditions (such as strip thickness, casting speed, heat transfer to the casting rolls, etc.)","can induce local variations, periodic or random, in the solidification structure and therefore in the final strips micro-structure throughout its geometrical profile.","The process of the invention overcomes the drawbacks inherent in the directly cast steel strips, due to lack of high hot deformation levels refining and homogenising the micro-structure.","Said high deformation levels are typical of technologies based on conventional casting, and in present invention are very efficiently replaced by causing a controlled, as amount and distribution, phase transformation Ferrite to Austenute, able to refine and homogenise the micro-structure.","The high solification speeds proper of strip casting are also an important metallurgical opportunity to exploit in the best way the process according to present invention.","In fact, in the traditional technologies starting from slabs or ingots the Ferrite/Austenite transformation, if any, is localised in chemical segregation zones, in which austenitising elements are concentrated, particularly in the semi-products core.","Thus, in said zones the austenitic transformation can occur, due to local concentration of austenitising elements, even if the mean chemical composition of the steel would not consent it.","On the contrary, in the strip casting the high solidifation speeds strongly limit the segregating phenomena, thus making homogeneous in the matrix the distribution of austenitising elements.","In said conditions, by hot rolling in the prescribed temperature field, it is obtained in a stable and reproducible way the volumetric fraction of Austenite, defined by chosing the steel composition, throughout the whole geometrical profile of the strip.","A further element of present invention if the definition of a process utilising a controlled volumetric fraction of Austenite, induced within the strip as above defined, to obtain a controlled distribution of hard phases (Carbides, Cementite, Pearlite, Bainite) and to control the formation of some Martensite (tetragonal lattice) within the metal matrix, by quenching the strip between the in-line hot rolling and the coiling steps.","The presence of homogeneously distributed hard phases (quenching phases) permits the cold rolling to control the adequate deformation texture, clearly because of the different deformation models and of the higher hardening levels obtained by cold rolling when hard phases are present with reference to the case in which a quenching structure is not present.","This permits to reduce the thickness of the strip to be cold rolled (for the same final thickness) and consequently to reduce the thickness of the cast strip, with important advantages on the casting productivity.","In fact, the thinner the cast strip, the higher the casting productivity, in that the strip becomes longer in direct proportion to the thickness reduction, while the casting speed rises with the square of the thickness reduction.","A further element of present invention is a process in which the strip, after in-line deformation, is kept at a temperature around 1150° C., typically 1100-1200° C., for at least 5 s, utilising a continuous heating apparatus between the in-line rolling mill and the coiler.","This can be obtained for instance a heating chamber provided with burners, or with electric heating, or with infrared lamps, or with an induction-heating apparatus; however, any active or passive system apt to obtain the desired strip temperature in the prescribed interval and for at least 5 s. In this case, the optional quenching step will be carried out at the exit from said chamber.","Another aspect of present invention is a process in which the strip is annealed, before cold rolling, at temperature not exceeding 1200° C., preferably not exceeding 1170° C. Such an annealing can be advantegeous for the grain oriented electrical steel strip production process, for a number of reasons, particularly with respect to the magnetic characteristics control of the final products.","Some useful phenomena for the process are, for instance, the precipitation of non-metallic second phases, necessary in present products to the control of the oriented secondary recrystallisation, or the possibility to carry out a controlled surface decarburisation of the strips before the cold rolling, which can have positive effects on the texture of the cold rolled strip.","Moreover, this annealing can offer the possibility to shift to this process step the formation of quenching phases, instead of forming them before coiling the strip after the casting process.","In this case, at the end of the annealing furnace a suitable cooling device must be present able to reach the necessary cooling speed.","For instance, the strip cooling can be usefully obtained with respect to the teaching of present invention, by means of a group of lances provided with nuzzles to spray on the strip surface a mixture of water and steam, at a controlled pressure.","Typically, after the in-line rolling the strip is quanched to obtain a Martensite volume fraction comprised between 5 and 15%.","The quenching device operate starting from a temperature of between 750 and 950° C., to cool down the strip down to 400° C. in less than 12 s. A last element of present invention is a process in which the chemical composition requires the presence of elements chosen between two distinct classes: (i) elements useful to control the desired equilibtium bewteen Austenite anf Ferrite in the metal matrix and (ii) elements useful to control a second phases distribution, such as sulphides, selenides, nitrides, carbo-nitrides etc., necessary for the grain growth control and of grain orientation during the primary and secondary recrystallisation steps.","Typically, the cast steel composition comprises 2,5-5 wt % Si; 200-1000 ppm C, 0,05-0,5 wt % Mn, 0,07-0,5 wt % Cu, less than 2 wt % Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. To this composition at least an element can be added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V and Co, and at least an element chosen in the group consisting of Sn, Sb, P, Bi.","Many are the elements useful to the equilibrium control berween Austenite and Ferrite phases and there are no specific choice limitations, but cost and yield convenience.","However, and specifically in electric-furnace steel shops utilising steel scraps as raw material, it can be convenient to balance the content of silicon as well as of chromium, nickel, molybdenum, niobium, copper, manganese and tin.","Many are also the elements useful to control the distribution of second phases particles for the grain growth inhibition.","It is convenient to chose said elements among the ones able to form suphides, selenides, carbonitrides, nitrides, to obtain a mix of second phases having different composition in which co-exist compounds thermally stable, as solubility, at different temperatures.","As a consequence of this choice, the drag force of the grain boundaries movement due to second phases particles gradually diminishes as temperature rises, in that during the heat treatments the more soluble particles will dissolve and/or grow before the less soluble ones.","This permits a better control of grain growth, with respect to the utilisation of inhibitors of a single composition type chacarterised by a narrower solubilisation temperatures interval.","The following Examples are intended solely to illustration purposes not limiting the scope of present invention.","EXAMPLE 1 A number of steels having the compositions shown in Table 1 were cast as a strip 3,5 mm thick in a strip casting machine provided with twin counter-rotating rolls.","The cast strips were then in-line hot rolled at the temperature of 1150° C. to a 2,0 mm thickness.","During the casting operation of each steel composition and at about mid casting time, the cast strip thickness was reduced to 2,0 mm and the in-line rolling suspended.","The hot rolled strips were then annealed at 1100° C. and single-stage cold rolled to 0,30 mm.","TABLE 1 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 500 3.1 0.2 75 300 100 250 0.1 B 300 3.1 0.1 68 350 120 270 0.15 C 350 3.2 0.4 70 320 110 230 0.3 D 400 3.1 0.3 80 290 150 280 0.25 E 500 3.1 0.4 50 400 100 280 0.2 The cold rolled strips were then decarburised, coated with an MgO based annealing separator, box annealed with an heating rate of 15° C./h up to 1200° C., held at this temperature for 20 h, and then received an insulating and tensioning coating.","On the as-cast strips the austenite (γ phase) content at 1150° C. was calculated by means of dilatometric measures; data obtained are shown in Table 2.TABLE 2 Steel γ(1150) (%) A 27 B 11 C 15 D 19 E 25 The magnetic characteristics measured on the final product for the different steel composition are shown in Table 3.TABLE 3 In-line hot rolled Not in-line rolled Steel B800 (mT) B800 (Mt) A 1950 1700 B 1720 1650 C 1730 1630 D 1900 1680 E 1945 1710 EXAMPLE 2 A number of steels having different compositions as shown in Table 4 were directy cast as strips 2,1 mm thick in a strip-casting machine provided with twin counter-rotating rolls.","TABLE 4 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 550 3.3 0.3 80 450 200 280 0.15 B 300 3.1 0.2 68 350 120 270 0.2 C 350 3.2 0.4 70 320 130 230 0.3 D 400 3.0 0.3 80 290 180 280 0.25 E 400 3.1 0.4 75 250 200 290 0.25 The cast strips were then in-line hot rolled at 1170° C. to a thickness of 1,0 mm, quenched by means of water and steam at high pressure down to a temperature of 150° C. and then coiled.","After casting about half of the steel the quenching was stopped and the strips wound at 700° C. Table 5 shows the Martensite fractions metallographically measured on the strip after coiling.","TABLE 5 Quenched strip Not-quenched strip Steel Martensite (%) Martensite (%) A 19 0 B 3 0 C 5 0 D 13 0 E 15 0 The strips were then divided into lesser coils, part of which were cold rolled to 0,3 mm (the casting A did show fragility problems during cold rolling and was not transformed into finished product), decarburised, coated with an MgO based annealing separator, then box annealed with a heating rate of 20° C./h up to 1200° C. and then held at this temperature for 20 h. Table 6 shows the magnetic characteristics (induction at 800 A/m) measured on the finished product.","TABLE 6 Quenched strip Not-quenched strip Steel B800 (mT) B800 (mT) A — 1830 B 1790 1650 C 1890 1630 D 1920 1820 E 1950 1830 EXAMPLE 3 The other lesser rolls of Example 2 without quenching and coiled at 700° C. were annealed at 1150° C. for 60 s, quenchecd by means of water and steam at high pressure down to 150° C., pickled and colied at room temperature.","The strips were then transformed into finished product as in preceding Example.","Table 7 shows the Martensite fractions measured on the coiled strips and relevant magnetic characteristics.","TABLE 7 Martensite B800 Steel (%) (mT) A 12 1950 B 2 1700 C 5 1740 D 8 1920 E 9 1920 EXAMPLE 4 Five different alloys of composition (in ppm) shown in Table 8 were cast directly as strips 2,2-2,4 mm thick in a casting machine with twin counter-rotating rolls.","TABLE 8 Si C Mn Cu Sn Cr Mo Nb Ni P Al Ce N S A 3.2 0.07 0.40 0.25 0.1 0.03 0.1 0.03 0.02 — 0.030 0.01 0.01 0.010 B 3.3 0.06 0.06 0.07 0.09 0.03 — 0.03 — 0.004 — 0.007 0.025 C 3.0 0.03 0.95 0.40 0.06 0.30 0.02 0.02 0.20 0.02 0.015 — 0.007 0.015 D 3.1 0.05 0.15 0.25 — 0.02 0.03 — 0.02 — 0.028 — 0.008 0.007 E 3.4 0.07 0.40 0.35 — 0.03 0.05 0.01 0.03 0.01 0.030 — 0.008 0.006 TABLE 9 Decarburation.","T (° C.) A1 B1 C1 D1 E1 A2 B2 C2 D2 E2 830 1890 1800 1920 1930 1910 1690 1520 1730 1640 1580 850 1930 1750 1940 1910 1920 1730 1540 1780 1540 1630 870 1940 1590 1890 1900 1890 1780 1530 1690 1520 1540 The cast steels were in-line hot rolled at 1150° C. to a thickness of 1,2 mm.","From said coiled strips were obtained lesser coils.","For each condition a strip was then double-stage annealed with quick heating to 1170° C., cooling at 1100° C. and quenched to room temperature with water plus steam jets (strips A1, B1, C1, D1, E1).","A second group of strips, similar the the previous one was annealed with a similar thermal cycle, without however the quenching step (strips A2, B2, C2, D2, E2).","All the strips were then single-stage cold rolled to a final thickness of 0,29 mm.","The strips were then treated in a continuous pilot line for primary recrystallisation, nitriding, secondary recrystallisation.","Each strip was then treated as follows: in the first treating zone (primary recrystallisation) the temperatures of 830, 850 and 870° C. were adopted, in a wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,60 and for 180 s (50 of which for the heating at treating temperature) in the second treating zone nitriding was carried out at 890° C. in wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,09, with the addition of 30% vol of ammonia, for 50 s in the third zone, at 1100° C. in a wet Nitrogen/Hydrogen atmosphere with a pH2O/pH2 ratio of 0,01 for 50 s. After coating with an Mg/O based annealing separator the strips treated in the pilot line were then box annealed with a heating rate of about 60° C./h up to 1200° C. in a 50% Nitrogen-Hydrogen atmosphere, held at this temperature for 3 h in pure hydrogen and cooled down to 800° C. in hydrogen and subsequently to room temperature in nitrogen.","The magnetic characteristics measured on samples of each of said strips were measured as induction mean value B800 in mT, and are shown in Table 9."]],"string":"[\n [\n \"PROCESS FOR THE PRODUCTION OF GRAIN ORIENTED ELECTRICAL STEEL STRIPS\",\n \"A process for the production of grain oriented electrical Fe—Si strips in which a Si-containing alloy is directly cast as a strip between 2.5-5.0 mm thick and cold rolled in one stage, or in more stages with intermediate annealing, to a final thickness of between 0.15-1.0 mm.\",\n \"The strip is then continuously annealed to carry out the primary recrystallization and then annealed to carry out the oriented secondary recrystallization.\",\n \"The process further includes that after solidification of the strip, and before its coiling, a phase transformation from Ferrite to Austenite is induced into the metal matrix for a volume fraction between 25-60%, obtained by controlling the alloy composition so that the Austenite fraction is allowed within the stability equilibrium between the two phases.\",\n \"The strip is then deformed by rolling in-line with the casting step to obtain a deformation higher than 20% in the temperature interval 1000-1300° C.\"\n ],\n [\n \"1-8.\",\n \"(canceled) 9.A process for the production of electrical grain oriented Fe—Si strips in which a Si-containing molten alloy composition is directly cast as continuous strips 2.5 to 5 mm thick, cold rolled in one step or more steps with intermediate annealing to a final thickness of between 1 and 0.15 mm, the strip being then continuously annealed to carry out oriented secondary recrystallisation, characterised in that after the strip solidification and before coiling of said strip in a coiling phase, a ferrite to austenite transformation is induced in the metal matrix via deformation, by rolling said strip between two cooled rolls to obtain a deformation over 20% in a temperature range of 1000-1300° C., thereby producing a volume fraction of austenite to be between 25 and 60%, said molten alloy composition being chosen such that said volume fraction of austenite is stable in a temperature interval of between 1100 and 1200° C. 10.The process according to claim 9, in which between the rolling phase and the coiling one, the strip is held between 1100 and 1200° C. for at least 5 s. 11.The process according to claim 9, in which the as-solidified strip thickness is comprised between 1.5 and 4.0 mm and after the rolling phase the strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.\",\n \"12.The process according to claim 9, in which before cold rolling the strip is annealed at a maximum temperature of 1200° C. 13.The process according to claim 12, in which after said annealing the strip is continuously quenched from a temperature comprised between 750 and 950° C. down to 400° C. in less than 12 s. 14.The process according to claim 9, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5 wt % Cu, less than 2 wt % Cr+Ni+Mo.\",\n \"Less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 15.The process according to claim 9, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 16.The process according to claim 9, in which in the alloy at least an element is added chosen between Sn, Sb, P, Bi.\",\n \"17.A process for the production of grain oriented electrical Fe—Si strip, in which a Si-containing alloy is directly cast as continuous strip 2.5 to 5 mm thick, in-line hot-rolled and then cold-rolled in one step or more steps, with intermediate annealing, to a final thickness of between 1 and 0.15 mm, the cold-rolled strip being, then continuously annealed to carry out primary recrystallisation and subsequently again annealed to carry out secondary recrystallisation, characterized in that the Si-containing alloy composition is selected to induce in the alloy, during the hot-rolling step in which a deformation rate of at least 20% is utilized in a temperature interval of between 1000 and 1300° C., a ferrite to austenite phase transformation with an austenite volume fraction of between 25 to 60% stable in a temperature interval of between 1100 to 1200° C. 18.The process according to claim 17, in which between the hot-rolling and the coiling steps the strip is held in the temperature range of 1100 to 1200° C. for at least 5 s. 19.The process according to claim 17, in which an as-cast strip 2.5 to 4 mm thick is in-line hot-rolled and the thus obtained hot-rolled strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.\",\n \"20.The process according to claim 17, in which before cold-rolling the strip is annealed at a maximum temperature of 1200° C. 21.The process according to claim 20, in which, after said annealing, the strip is continuously quenched from a temperature of between 750 and 950° C. down to 400° C. in less than 12 s. 22.The process according to claim 17, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5% Cu, less than 2.0% Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 23.The process according to claim 22, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 24.The process according to claim 22, in which in the allot at least an element is added chosen between Sn, Sb, P, Bi.\"\n ],\n [\n \" FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.\",\n \"More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.\",\n \"Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.\",\n \"Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.\",\n \"Further objects of present invention will be evident from the following description of the invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.\",\n \"More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.\",\n \"STATE OF THE ART Grain oriented electrical steel strips (Fe—Si) are typically industrially produced as strips having a thickness comprised between 0,18 and 0,50 mm and are characterised by magnetic properties variable according to the specific product class.\",\n \"Said classification substantially refers to the specific power losses of the strip subjected to given electromagnetic work conditions (e.g.\",\n \"P50 Hz at 1,7 Tesla, in W/kg), evaluated along a specific reference direction (rolling direction).\",\n \"The main utilisation of said strips is the production of transformer cores.\",\n \"Good magnetic properties (strongly anisotropic) are obtained controlling the final crystalline structure of the strips to obtain all, or almost all, the grains oriented to have their easiest magnetization direction (the <001> axis) aligned in the most perfect way with the rolling direction.\",\n \"In practice, final products are obtained having the grains mean diameter generally comprised between 1 and 20 mm having an orientation centred around the Goss orientation ({110} <001>).\",\n \"The minor the angular dispersion around the Goss one, the better the product magnetic permeability and hence the lesser the magnetic losses.\",\n \"The final products having low magnetic losses (core losses) and high permeability have interesting advantages in terms of design, dimensions and yield of the transformers.\",\n \"The first industrial production of the above materials was described by the U.S. Firm ARMCO at the beginning of the thirties (U.S. Pat.\",\n \"No.\",\n \"1,956,559).\",\n \"Many important improvements have been since introduced in the production technology of grain oriented electrical strips, in terms both of magnetic and physical quality of products and of transformation costs and cycles rationalisation.\",\n \"All existing technologies exploit the same metallurgical strategy to obtain a very strong Goss structure in the final products, i.e.\",\n \"the process of oriented secondary recrystallisation guided by uniformly distributed second phases and/or segregating elements.\",\n \"The, non metallic, second phases and the segregating elements play a fundamental role in controlling (slowing down) the movement of grain boundaries doing the final annealing which actuates the selective secondary recrystallisation process.\",\n \"In the original ARMCO technology, utilising MnS as inhibitor of the grain boundaries movement, and in the subsequent technology developed by NSC, in which the inhibitors are mainly aluminium nitrides (AIN+MnS) (EP 8.385, EP 17.830, EP 202.339), a very important binding step common to both production processes is the heating of the continuously cast slabs (ingots, in old times), immediately before the hot rolling, at very high temperatures (around 1400° C.) for a time sufficient to guarantee a complete dissolution of sulphides and/or nitrides coarsely precipitated during the slab cooling after casting, to re-precipitate them in a very fine and uniformly distributed form throughout the metallic matrix of the hot rolled strips.\",\n \"Such a fine re-precipitation can be started and completed, as well as the precipitates dimensions adjusted, during the process, in any case, however, before the cold rolling.\",\n \"The slab heating to said temperatures requires using special furnaces (pushing furnaces, liquid-slag walking-beam furnaces, induction furnaces) due to the ductility at high temperatures of the Fe-3% Si alloys and to formation of liquid slags.\",\n \"New casting technologies of the liquid steel are intended to simplify the production processes to make them more compact and flexible and to reduce costs.\",\n \"One of said technologies is the “thin slab” casting, consisting in the continuous casting of slabs having the typical thickness of conventional already roughened slabs, apt to a direct hot rolling, through a sequence of slabs continuous casting, treating in continuous tunnel-furnaces to rise/maintain the temperature of slabs and finishing-rolling, down to coiled strip.\",\n \"The problems connected to the utilisation of said technique for grain oriented products mainly consist in the difficulty to maintain and control the high temperatures necessary to keep in solution the elements forming the second phases, which have to be finely precipitated at the beginning of the finishing hot-rolling step, if desired best micro-structural and magnetic characteristics are to be obtained in the end products.\",\n \"Such problems were dealt with in different ways, for instance utilising the low thickness of the cast slabs in connection to specific concentration intervals of the micro-alloying elements to stably control the second phases precipitation (grain growth inhibitors) during hot rolling, or drastically modifying the strategy of the inhibitors formation in the metal matrix.\",\n \"The casting technique potentially offering the highest rationalisation level of the processes and the higher production flexibility is the one consisting in the direct production of strips from the liquid steel (Strip Casting), totally eliminating the hot rolling step.\",\n \"Such an exaordinary innovation was conceived and patented long time ago, and since long time were also devised and patented process conditions to produce electrical steel strips, and more particularly grain oriented ones.\",\n \"However, up to now there is not an industrial production in the world of grain oriented electrical steel according to the above technique, though the state of the art relating to the casting machines is ready for industrial applications, as shown by existing plants, producing only carbon steels and stainless steels.\",\n \"The present inventors believe that to industrially produce grain oriented electrical steel strips from direct solidification of a strip (Strip Casting) it is necessary to have a strip micro-structure before cold rolling significantly different from the one obtained during the casting stage.\",\n \"The high solidification speed of the cast strip makes it difficult to have a homogeneous and reproducible grain structure throughout the strip and between different castings, due to the high sensitivity of the solidification structure to the fluctuations of the casting conditions and to the alloy composition.\",\n \"The micro-structure of the intermediate products starting from strip casting is much more influenced by the solidification structure, with respect to the ones derived from conventional slab casting, because of the lack of deformation in the strip during the typical hot rolling.\",\n \"SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.\",\n \"Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.\",\n \"Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.\",\n \"Further objects of present invention will be evident from the following description of the invention.\",\n \"DETAILED DESCRIPTION OF THE INVENTION A first important aspect of present invention resides in that a molten alloy containing silicon is directly solidified in the form of a strip, through the casting technology known as strip casting (casting between twin cooled and counter-rotating rolls), thus avoiding, with respect to currently utilised technologies, casting the alloy in slabs or ingots, subjecting said slabs to thermal treatment in special high-temperature furnaces for long times (to attain the necessary thermal homogeneity) and transforming said slabs into strips through hot rolling with total reductions which, according to the slab casting technologies, vary between 96 and 99%.\",\n \"A second important aspect of present invention resides in that the chemical composition of the Silicon containing alloy is selected specifically to control the thermodynamic stability of the Austenite phase in the matrix (face-centered cubic lattice) in equilibrium with the Ferrite phase (body-centered cubic lattice).\",\n \"More precisely, to obtain excellent final magnetic characteristics, it is convenient to adjust the chemistry of the alloy so that an Austenite fraction comprised between 25 and 60% is stable between 1100 and 1200° C. Consequently, to balance the strong tendency of silicon to stabilise the Ferrite phase, a number of elements are utilised, favouring the Austenite formation.\",\n \"Aming those elements, Carbon is particularly important due to its intrinsic austenitising effect as well as to its particular mobility into the matrix, making it possible its easy elimination by means of solid-state decarburising processes which, in this field, are usually carried out by extraction from the strip surfaces utilising annealing atmospheres having a controlled oxidising potential.\",\n \"The carbon is conveniently present in the steel composition in amount apt to control the desired Austenite fraction, in that in this way it is possible to rise again the stability of the Ferrite by means of a simple decarburisation process, and thus avoiding during the final secondary recrystallisation annealing important phase transition phenomena which would be detrimental for the final desired texture.\",\n \"As known, however, in said materials itis necessary to reduce the carbon content in the final products at levels of under 50 ppm, to eliminate the adverse effect on the core losses due to formation of carbides.\",\n \"The higher the carbon content of the alloy, the longer the time necessary to carry out the decarburisation.\",\n \"For productivity reasons it is then convenient to keep the carbon content within a maximum of 0,1 wt %.\",\n \"Present inventors eveluated the obtainable Austenite fractions according to different alloy compositions both experimentally and according to empirical relationships available in the literature.\",\n \"A third aspect of the invention resides in that the Derrite to Austenite transformation in the metal matrix of the cast strip is induced, in a temperature interval centered around 1150° C., typically 1000-1300° C., by means of a sudden deformation higher than 20%, by rolling between cooled rolls, in-line with the continuous casting and before the coiling.\",\n \"Said sudden and localised deformation imparts to the material the energy necessary to nucleation and formation of the Austenite phase in the matrix, which phase would not be obtained for kinetic reasons, though thermodynamically very stable.\",\n \"In fact, to obtain equilibrium conditions between the two phases at the considered temperature very long times are necessary, while the working and cooling times are intrinsecally very short, particularly in the case of direct casting as strip (strip casting).\",\n \"The phase transformation from Ferrite to Austenite is tunable, according to present invention, in quantity, according to selection of chemical composition, and consistently reproducible, as necessary in an industrial process.\",\n \"As a consequence of the phase transformation induced in the temperature interval defined according to present invention, the grains distribution in the produced strip, in terms both of dimensions and of texture, is exrtremely homogeneous and reproducible through the whole geometrical profile of the strip.\",\n \"This, in particular, solves the problem the drawback of microstructural etereogeneity, typical of the production of oriented grain steel strips, in that the selection process of the final texture is sensible even to small local differences in the structure and orientation of grains, and even more sensible in the case of strip-cast products.\",\n \"In fact, in the traditional processes the strip structure before cold rolling is the result of a strong hot deformation of the cast slabs, which contributes to fragment, recrystallise and homogenise the solidification structure; on the contrary, in the strips obtained by direct solidification the structure directly depends on the solidification one, and due to the high solidification speed and to the strongly dynamic nature of the process any even small fluctuation of the casting conditions (such as strip thickness, casting speed, heat transfer to the casting rolls, etc.)\",\n \"can induce local variations, periodic or random, in the solidification structure and therefore in the final strips micro-structure throughout its geometrical profile.\",\n \"The process of the invention overcomes the drawbacks inherent in the directly cast steel strips, due to lack of high hot deformation levels refining and homogenising the micro-structure.\",\n \"Said high deformation levels are typical of technologies based on conventional casting, and in present invention are very efficiently replaced by causing a controlled, as amount and distribution, phase transformation Ferrite to Austenute, able to refine and homogenise the micro-structure.\",\n \"The high solification speeds proper of strip casting are also an important metallurgical opportunity to exploit in the best way the process according to present invention.\",\n \"In fact, in the traditional technologies starting from slabs or ingots the Ferrite/Austenite transformation, if any, is localised in chemical segregation zones, in which austenitising elements are concentrated, particularly in the semi-products core.\",\n \"Thus, in said zones the austenitic transformation can occur, due to local concentration of austenitising elements, even if the mean chemical composition of the steel would not consent it.\",\n \"On the contrary, in the strip casting the high solidifation speeds strongly limit the segregating phenomena, thus making homogeneous in the matrix the distribution of austenitising elements.\",\n \"In said conditions, by hot rolling in the prescribed temperature field, it is obtained in a stable and reproducible way the volumetric fraction of Austenite, defined by chosing the steel composition, throughout the whole geometrical profile of the strip.\",\n \"A further element of present invention if the definition of a process utilising a controlled volumetric fraction of Austenite, induced within the strip as above defined, to obtain a controlled distribution of hard phases (Carbides, Cementite, Pearlite, Bainite) and to control the formation of some Martensite (tetragonal lattice) within the metal matrix, by quenching the strip between the in-line hot rolling and the coiling steps.\",\n \"The presence of homogeneously distributed hard phases (quenching phases) permits the cold rolling to control the adequate deformation texture, clearly because of the different deformation models and of the higher hardening levels obtained by cold rolling when hard phases are present with reference to the case in which a quenching structure is not present.\",\n \"This permits to reduce the thickness of the strip to be cold rolled (for the same final thickness) and consequently to reduce the thickness of the cast strip, with important advantages on the casting productivity.\",\n \"In fact, the thinner the cast strip, the higher the casting productivity, in that the strip becomes longer in direct proportion to the thickness reduction, while the casting speed rises with the square of the thickness reduction.\",\n \"A further element of present invention is a process in which the strip, after in-line deformation, is kept at a temperature around 1150° C., typically 1100-1200° C., for at least 5 s, utilising a continuous heating apparatus between the in-line rolling mill and the coiler.\",\n \"This can be obtained for instance a heating chamber provided with burners, or with electric heating, or with infrared lamps, or with an induction-heating apparatus; however, any active or passive system apt to obtain the desired strip temperature in the prescribed interval and for at least 5 s. In this case, the optional quenching step will be carried out at the exit from said chamber.\",\n \"Another aspect of present invention is a process in which the strip is annealed, before cold rolling, at temperature not exceeding 1200° C., preferably not exceeding 1170° C. Such an annealing can be advantegeous for the grain oriented electrical steel strip production process, for a number of reasons, particularly with respect to the magnetic characteristics control of the final products.\",\n \"Some useful phenomena for the process are, for instance, the precipitation of non-metallic second phases, necessary in present products to the control of the oriented secondary recrystallisation, or the possibility to carry out a controlled surface decarburisation of the strips before the cold rolling, which can have positive effects on the texture of the cold rolled strip.\",\n \"Moreover, this annealing can offer the possibility to shift to this process step the formation of quenching phases, instead of forming them before coiling the strip after the casting process.\",\n \"In this case, at the end of the annealing furnace a suitable cooling device must be present able to reach the necessary cooling speed.\",\n \"For instance, the strip cooling can be usefully obtained with respect to the teaching of present invention, by means of a group of lances provided with nuzzles to spray on the strip surface a mixture of water and steam, at a controlled pressure.\",\n \"Typically, after the in-line rolling the strip is quanched to obtain a Martensite volume fraction comprised between 5 and 15%.\",\n \"The quenching device operate starting from a temperature of between 750 and 950° C., to cool down the strip down to 400° C. in less than 12 s. A last element of present invention is a process in which the chemical composition requires the presence of elements chosen between two distinct classes: (i) elements useful to control the desired equilibtium bewteen Austenite anf Ferrite in the metal matrix and (ii) elements useful to control a second phases distribution, such as sulphides, selenides, nitrides, carbo-nitrides etc., necessary for the grain growth control and of grain orientation during the primary and secondary recrystallisation steps.\",\n \"Typically, the cast steel composition comprises 2,5-5 wt % Si; 200-1000 ppm C, 0,05-0,5 wt % Mn, 0,07-0,5 wt % Cu, less than 2 wt % Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. To this composition at least an element can be added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V and Co, and at least an element chosen in the group consisting of Sn, Sb, P, Bi.\",\n \"Many are the elements useful to the equilibrium control berween Austenite and Ferrite phases and there are no specific choice limitations, but cost and yield convenience.\",\n \"However, and specifically in electric-furnace steel shops utilising steel scraps as raw material, it can be convenient to balance the content of silicon as well as of chromium, nickel, molybdenum, niobium, copper, manganese and tin.\",\n \"Many are also the elements useful to control the distribution of second phases particles for the grain growth inhibition.\",\n \"It is convenient to chose said elements among the ones able to form suphides, selenides, carbonitrides, nitrides, to obtain a mix of second phases having different composition in which co-exist compounds thermally stable, as solubility, at different temperatures.\",\n \"As a consequence of this choice, the drag force of the grain boundaries movement due to second phases particles gradually diminishes as temperature rises, in that during the heat treatments the more soluble particles will dissolve and/or grow before the less soluble ones.\",\n \"This permits a better control of grain growth, with respect to the utilisation of inhibitors of a single composition type chacarterised by a narrower solubilisation temperatures interval.\",\n \"The following Examples are intended solely to illustration purposes not limiting the scope of present invention.\",\n \"EXAMPLE 1 A number of steels having the compositions shown in Table 1 were cast as a strip 3,5 mm thick in a strip casting machine provided with twin counter-rotating rolls.\",\n \"The cast strips were then in-line hot rolled at the temperature of 1150° C. to a 2,0 mm thickness.\",\n \"During the casting operation of each steel composition and at about mid casting time, the cast strip thickness was reduced to 2,0 mm and the in-line rolling suspended.\",\n \"The hot rolled strips were then annealed at 1100° C. and single-stage cold rolled to 0,30 mm.\",\n \"TABLE 1 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 500 3.1 0.2 75 300 100 250 0.1 B 300 3.1 0.1 68 350 120 270 0.15 C 350 3.2 0.4 70 320 110 230 0.3 D 400 3.1 0.3 80 290 150 280 0.25 E 500 3.1 0.4 50 400 100 280 0.2 The cold rolled strips were then decarburised, coated with an MgO based annealing separator, box annealed with an heating rate of 15° C./h up to 1200° C., held at this temperature for 20 h, and then received an insulating and tensioning coating.\",\n \"On the as-cast strips the austenite (γ phase) content at 1150° C. was calculated by means of dilatometric measures; data obtained are shown in Table 2.TABLE 2 Steel γ(1150) (%) A 27 B 11 C 15 D 19 E 25 The magnetic characteristics measured on the final product for the different steel composition are shown in Table 3.TABLE 3 In-line hot rolled Not in-line rolled Steel B800 (mT) B800 (Mt) A 1950 1700 B 1720 1650 C 1730 1630 D 1900 1680 E 1945 1710 EXAMPLE 2 A number of steels having different compositions as shown in Table 4 were directy cast as strips 2,1 mm thick in a strip-casting machine provided with twin counter-rotating rolls.\",\n \"TABLE 4 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 550 3.3 0.3 80 450 200 280 0.15 B 300 3.1 0.2 68 350 120 270 0.2 C 350 3.2 0.4 70 320 130 230 0.3 D 400 3.0 0.3 80 290 180 280 0.25 E 400 3.1 0.4 75 250 200 290 0.25 The cast strips were then in-line hot rolled at 1170° C. to a thickness of 1,0 mm, quenched by means of water and steam at high pressure down to a temperature of 150° C. and then coiled.\",\n \"After casting about half of the steel the quenching was stopped and the strips wound at 700° C. Table 5 shows the Martensite fractions metallographically measured on the strip after coiling.\",\n \"TABLE 5 Quenched strip Not-quenched strip Steel Martensite (%) Martensite (%) A 19 0 B 3 0 C 5 0 D 13 0 E 15 0 The strips were then divided into lesser coils, part of which were cold rolled to 0,3 mm (the casting A did show fragility problems during cold rolling and was not transformed into finished product), decarburised, coated with an MgO based annealing separator, then box annealed with a heating rate of 20° C./h up to 1200° C. and then held at this temperature for 20 h. Table 6 shows the magnetic characteristics (induction at 800 A/m) measured on the finished product.\",\n \"TABLE 6 Quenched strip Not-quenched strip Steel B800 (mT) B800 (mT) A — 1830 B 1790 1650 C 1890 1630 D 1920 1820 E 1950 1830 EXAMPLE 3 The other lesser rolls of Example 2 without quenching and coiled at 700° C. were annealed at 1150° C. for 60 s, quenchecd by means of water and steam at high pressure down to 150° C., pickled and colied at room temperature.\",\n \"The strips were then transformed into finished product as in preceding Example.\",\n \"Table 7 shows the Martensite fractions measured on the coiled strips and relevant magnetic characteristics.\",\n \"TABLE 7 Martensite B800 Steel (%) (mT) A 12 1950 B 2 1700 C 5 1740 D 8 1920 E 9 1920 EXAMPLE 4 Five different alloys of composition (in ppm) shown in Table 8 were cast directly as strips 2,2-2,4 mm thick in a casting machine with twin counter-rotating rolls.\",\n \"TABLE 8 Si C Mn Cu Sn Cr Mo Nb Ni P Al Ce N S A 3.2 0.07 0.40 0.25 0.1 0.03 0.1 0.03 0.02 — 0.030 0.01 0.01 0.010 B 3.3 0.06 0.06 0.07 0.09 0.03 — 0.03 — 0.004 — 0.007 0.025 C 3.0 0.03 0.95 0.40 0.06 0.30 0.02 0.02 0.20 0.02 0.015 — 0.007 0.015 D 3.1 0.05 0.15 0.25 — 0.02 0.03 — 0.02 — 0.028 — 0.008 0.007 E 3.4 0.07 0.40 0.35 — 0.03 0.05 0.01 0.03 0.01 0.030 — 0.008 0.006 TABLE 9 Decarburation.\",\n \"T (° C.) A1 B1 C1 D1 E1 A2 B2 C2 D2 E2 830 1890 1800 1920 1930 1910 1690 1520 1730 1640 1580 850 1930 1750 1940 1910 1920 1730 1540 1780 1540 1630 870 1940 1590 1890 1900 1890 1780 1530 1690 1520 1540 The cast steels were in-line hot rolled at 1150° C. to a thickness of 1,2 mm.\",\n \"From said coiled strips were obtained lesser coils.\",\n \"For each condition a strip was then double-stage annealed with quick heating to 1170° C., cooling at 1100° C. and quenched to room temperature with water plus steam jets (strips A1, B1, C1, D1, E1).\",\n \"A second group of strips, similar the the previous one was annealed with a similar thermal cycle, without however the quenching step (strips A2, B2, C2, D2, E2).\",\n \"All the strips were then single-stage cold rolled to a final thickness of 0,29 mm.\",\n \"The strips were then treated in a continuous pilot line for primary recrystallisation, nitriding, secondary recrystallisation.\",\n \"Each strip was then treated as follows: in the first treating zone (primary recrystallisation) the temperatures of 830, 850 and 870° C. were adopted, in a wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,60 and for 180 s (50 of which for the heating at treating temperature) in the second treating zone nitriding was carried out at 890° C. in wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,09, with the addition of 30% vol of ammonia, for 50 s in the third zone, at 1100° C. in a wet Nitrogen/Hydrogen atmosphere with a pH2O/pH2 ratio of 0,01 for 50 s. After coating with an Mg/O based annealing separator the strips treated in the pilot line were then box annealed with a heating rate of about 60° C./h up to 1200° C. in a 50% Nitrogen-Hydrogen atmosphere, held at this temperature for 3 h in pure hydrogen and cooled down to 800° C. in hydrogen and subsequently to room temperature in nitrogen.\",\n \"The magnetic characteristics measured on samples of each of said strips were measured as induction mean value B800 in mT, and are shown in Table 9.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450969"}}},{"rowIdx":290,"cells":{"text":{"kind":"list like","value":[["Generic architecture for adaptable software","This invention concerns adaptable software.","In particular it concerns a method for constructing an adaptable soft-ware application.","A first step involves instantiating a first set of knowledge elements (kitems) related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application and used to produce dynamic and flexible templates.","A second step involves instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","In a further aspect it concerns an adaptable software application comprising at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations."],["1.A method for constructing an adaptable software application, comprising the steps of: instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application; and, instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","2.A method according to claim 1, where the step of instantiating the second set of kitems occurs dynamically, during the use of the application.","3.A method according to claim 1, where further instantiating steps produce further sets of kitems from the immediately preceding set.","4.A method according to claim 1, comprising the further step of directing enquiries against kitems at any level of instantiation using a consultation process.","5.A method according to claim 1, comprising the further step of accessing the business objects using a consultation process during use of the application.","6.A method according to claim 1, comprising the further step of accessing kitems related to the type of business objects for the purposes of inspecting or managing the design of the application.","7.An adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.","8.An application comprising adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable system of claim 7.9.An application according to claim 8, further comprising adaptable meta-module to define and implement the logic between the other modules.","10.An application according to claim 9, where the meta-module monitors a part of, or the whole chain of modules."],[" BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.","These are often, but not exclusively, documents.","Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.","Such applications are normally hard-coded using standard programming techniques.","The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.","The consequence of this approach is that systems are difficult to design and develop.","They are also difficult to maintain and modify.","When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.","In many cases, the effort is so difficult and time consuming that it is not attempted.","Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.","This has the advantage that the business rules can, in principle, be changed without having to change the application.","In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.","Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.","The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.","Few applications use expert systems technology; it is a niche technology.","A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.","The contents of these three applications are incorporated into this specification by reference.","A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.","The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.","A knowledge application is typically characterised by a single instantiation process for all the kitems.","The kitems can also be used as contexts or templates from which further kitems can be instantiated.","Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.","This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.","The enquiry is itself a kitem instantiated from a kitem template.","Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems."],[" SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.","Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.","Alternatively, these templates can be activated manually.","The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.","The business objects are easily created, and can be easily manipulated.","Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.","Instantiating the second set of kitems may take place dynamically, during the use of the application.","Applications can be built with a greater number of cascades or instantiations larger than two.","Enquiries can be directed against kitems at any level of instantiation using a consultation process.","The business objects may be accessed using a consultation process during use of the application.","The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.","The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.","The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.","So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.","The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.","This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.","Each adaptable module implements the knowledge about the best way to run that module.","These modules are used to facilitate development and to increase the functionality and adaptability of the solution.","An adaptable meta-module may define and implement the logic between the other modules.","In addition the meta-module may monitor a part of, or the whole chain of modules.","This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.","It may also adapt dynamically to the needs of users as software is being used.","Adaptablability is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation, customization and flexibility.","In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.","Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.","Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.","Using adaptable software generic applications can be quickly produced.","The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.","An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.","Also client specific applications can be quickly produced.","Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements."],["TECHNICAL FIELD This invention concerns adaptable software.","In particular it concerns a method for constructing an adaptable software application.","In a further aspect it concerns an adaptable software application.","BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.","These are often, but not exclusively, documents.","Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.","Such applications are normally hard-coded using standard programming techniques.","The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.","The consequence of this approach is that systems are difficult to design and develop.","They are also difficult to maintain and modify.","When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.","In many cases, the effort is so difficult and time consuming that it is not attempted.","Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.","This has the advantage that the business rules can, in principle, be changed without having to change the application.","In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.","Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.","The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.","Few applications use expert systems technology; it is a niche technology.","A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.","The contents of these three applications are incorporated into this specification by reference.","A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.","The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.","A knowledge application is typically characterised by a single instantiation process for all the kitems.","The kitems can also be used as contexts or templates from which further kitems can be instantiated.","Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.","This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.","The enquiry is itself a kitem instantiated from a kitem template.","Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.","SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.","Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.","The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.","Alternatively, these templates can be activated manually.","The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.","The business objects are easily created, and can be easily manipulated.","Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.","Instantiating the second set of kitems may take place dynamically, during the use of the application.","Applications can be built with a greater number of cascades or instantiations larger than two.","Enquiries can be directed against kitems at any level of instantiation using a consultation process.","The business objects may be accessed using a consultation process during use of the application.","The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.","The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.","The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.","So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.","The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.","This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.","Each adaptable module implements the knowledge about the best way to run that module.","These modules are used to facilitate development and to increase the functionality and adaptability of the solution.","An adaptable meta-module may define and implement the logic between the other modules.","In addition the meta-module may monitor a part of, or the whole chain of modules.","This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.","It may also adapt dynamically to the needs of users as software is being used.","Adaptablability is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation, customization and flexibility.","In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.","Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.","Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.","Using adaptable software generic applications can be quickly produced.","The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.","An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.","Also client specific applications can be quickly produced.","Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.","BRIEF DESCRIPTION OF THE DRAWINGS An example of the invention will now be described with reference to the accompanying drawings, in which: FIG.","1 is a block diagram showing the steps in the construction and use of an adaptable software application.","FIG.","2 is a block diagram showing the architecture of an adaptable software application.","BEST MODES OF THE INVENTION Adaptable software differs from the knowledge applications described in the background art in that there are at least two levels of instantiation instead of one; and the templates for the second level instantiations are the first level instantiations (they can be objects and not classes in object oriented programming).","Adaptable software extends the architecture of known knowledge applications.","The use of a two level instantiation, or cascading, design enables the first level to define the application (without hard-coding) and the second to define the documents that are handled by the application.","Adaptable software transforms the definition of the application (typically hardcoded) into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.","The process for building an adaptable software application will now be described with reference to FIG.","1.The process starts in a knowledge application development environment 10 where the business needs of the application determine the knowledge to be instantiated, or created, from contexts and templates.","The first step 11 is the instantiation of this knowledge.","The outcome is an application module dynamic framework 12 in which the kitems relate to the type of business objects or operations which will be handled by the adaptable software application.","This first step is distinguished from the construction of an application knowledge base since it does not produce the documents or the knowledge to be accessed via a consultation.","Instead these kitems describe the application as a framework that will be used to generate the dynamic templates for the objects, documents, that relate to the application.","A second step 13 then involves populating the adaptable software application with business objects instantiated from contexts or templates produced in the first step.","The process for defining these business objects is similar to defining an enquiry in a knowledge application.","That is, parameters are specified that determine the features of the business object which is then created.","The outcome is an application module 14 populated with business objects.","The business objects so created can be managed 15, that is accessed, edited, displayed, distributed, etc.","These operations are simple to implement as they correspond to manipulating kitems, and they use standard kitem manipulation methods, and other specific methods if required.","For example, the business objects could be accessed using a question-answer session.","Specific applications (or modules) are implemented by customising the generic framework produced in the first step, and this corresponds to instantiating different kitems as business objects for each application or module.","This customizing can take place dynamically, during the use of the application.","This corresponds to dynamic adaptation of the application to the needs of users.","Enquiries can be directed against both levels of instantiated kitems via a consultation process.","Enquiries directed to the upper level of documents, produced by the first step, are typically used for inspecting and managing the design of the application.","To users, adaptable applications do not need to look different from normal applications.","For example, a dialogue could take place that enquires about the needs or intentions of a user.","Knowing the user's needs or intentions then enables the application to adapt or customize dynamically the template that is presented to the user; this templates defines the documents, or the type of documents, that the user enters into the application.","The two level instantiation process can also be described as cascading knowledge items.","That is, some knowledge items determine which other knowledge items will be activated or instantiated to define documents.","Applications can be built with a greater number of cascades or instantiations larger than two.","Most software applications are made of modules that communicate with each other.","For example, a typical business management application is made of a client module, a project module, a contact module, a resource module and an accounting module.","FIG.","2 shows adaptable, intelligent modules or agents.","In particular there are adaptable intelligent client 21, project 22, contact 23, resource 24 and accounting 25 modules.","Each module is adaptable.","For example, in the client module, a different set of questions could be asked based on the age or location or socio-economic profile.","Similarly for the other modules.","Each module has its business logic defined and implemented using an adaptable architecture.","This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.","Each adaptable module implements the knowledge about the best way to run that module.","These modules are used to facilitate development and to increase the functionality and adaptability of the solution.","Adaptable meta-module(s) 30 are used to define and implement the logic between the other modules.","This could include workflow or project costing for example, where a client would be treated differently based on the number of projects commissioned in the past eighteen months and the success of these projects.","In addition the meta-modules are adaptable.","In FIG.","2, the meta-module monitors a part of, or the whole chain of modules.","For example, the accounting module may detect that some projects from some clients are not profitable.","That means that the project module would need to be modified (using its adaptable feature) to ensure that these projects are run differently.","It could also mean that additional information should be asked of these clients to perhaps rejects them as clients for a certain type of project.","In this case the client module is modified using its adaptable feature.","A specific example is an access control database.","All the operations possible with a database that needs to be controlled are listed as the context in a GKMS type application.","Access is to be controlled for each group of users and for users in the groups (control is determined by the group and, in some cases, by the user).","In this case, the first level of instantiation corresponds to defining the template to be used to give rights to each group of users.","Each user, when linked to a group get an access rights document based on the template for the group it belongs to.","This is the second level of instantiation.","This document represents the right of that user.","The administrator can then modify each document if desired to take account of the characteristics of the user within the group.","It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described.","The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive."]],"string":"[\n [\n \"Generic architecture for adaptable software\",\n \"This invention concerns adaptable software.\",\n \"In particular it concerns a method for constructing an adaptable soft-ware application.\",\n \"A first step involves instantiating a first set of knowledge elements (kitems) related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application and used to produce dynamic and flexible templates.\",\n \"A second step involves instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"In a further aspect it concerns an adaptable software application comprising at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\"\n ],\n [\n \"1.A method for constructing an adaptable software application, comprising the steps of: instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application; and, instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"2.A method according to claim 1, where the step of instantiating the second set of kitems occurs dynamically, during the use of the application.\",\n \"3.A method according to claim 1, where further instantiating steps produce further sets of kitems from the immediately preceding set.\",\n \"4.A method according to claim 1, comprising the further step of directing enquiries against kitems at any level of instantiation using a consultation process.\",\n \"5.A method according to claim 1, comprising the further step of accessing the business objects using a consultation process during use of the application.\",\n \"6.A method according to claim 1, comprising the further step of accessing kitems related to the type of business objects for the purposes of inspecting or managing the design of the application.\",\n \"7.An adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\",\n \"8.An application comprising adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable system of claim 7.9.An application according to claim 8, further comprising adaptable meta-module to define and implement the logic between the other modules.\",\n \"10.An application according to claim 9, where the meta-module monitors a part of, or the whole chain of modules.\"\n ],\n [\n \" BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.\",\n \"These are often, but not exclusively, documents.\",\n \"Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.\",\n \"Such applications are normally hard-coded using standard programming techniques.\",\n \"The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.\",\n \"The consequence of this approach is that systems are difficult to design and develop.\",\n \"They are also difficult to maintain and modify.\",\n \"When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.\",\n \"In many cases, the effort is so difficult and time consuming that it is not attempted.\",\n \"Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.\",\n \"This has the advantage that the business rules can, in principle, be changed without having to change the application.\",\n \"In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.\",\n \"Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.\",\n \"The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.\",\n \"Few applications use expert systems technology; it is a niche technology.\",\n \"A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.\",\n \"The contents of these three applications are incorporated into this specification by reference.\",\n \"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.\",\n \"The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.\",\n \"A knowledge application is typically characterised by a single instantiation process for all the kitems.\",\n \"The kitems can also be used as contexts or templates from which further kitems can be instantiated.\",\n \"Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.\",\n \"This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.\",\n \"The enquiry is itself a kitem instantiated from a kitem template.\",\n \"Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.\",\n \"Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.\",\n \"Alternatively, these templates can be activated manually.\",\n \"The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.\",\n \"The business objects are easily created, and can be easily manipulated.\",\n \"Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.\",\n \"Instantiating the second set of kitems may take place dynamically, during the use of the application.\",\n \"Applications can be built with a greater number of cascades or instantiations larger than two.\",\n \"Enquiries can be directed against kitems at any level of instantiation using a consultation process.\",\n \"The business objects may be accessed using a consultation process during use of the application.\",\n \"The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.\",\n \"The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\",\n \"The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.\",\n \"So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.\",\n \"The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.\",\n \"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.\",\n \"Each adaptable module implements the knowledge about the best way to run that module.\",\n \"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.\",\n \"An adaptable meta-module may define and implement the logic between the other modules.\",\n \"In addition the meta-module may monitor a part of, or the whole chain of modules.\",\n \"This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.\",\n \"It may also adapt dynamically to the needs of users as software is being used.\",\n \"Adaptablability is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation, customization and flexibility.\",\n \"In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.\",\n \"Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.\",\n \"Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"Using adaptable software generic applications can be quickly produced.\",\n \"The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.\",\n \"An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.\",\n \"Also client specific applications can be quickly produced.\",\n \"Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.\"\n ],\n [\n \"TECHNICAL FIELD This invention concerns adaptable software.\",\n \"In particular it concerns a method for constructing an adaptable software application.\",\n \"In a further aspect it concerns an adaptable software application.\",\n \"BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.\",\n \"These are often, but not exclusively, documents.\",\n \"Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.\",\n \"Such applications are normally hard-coded using standard programming techniques.\",\n \"The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.\",\n \"The consequence of this approach is that systems are difficult to design and develop.\",\n \"They are also difficult to maintain and modify.\",\n \"When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.\",\n \"In many cases, the effort is so difficult and time consuming that it is not attempted.\",\n \"Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.\",\n \"This has the advantage that the business rules can, in principle, be changed without having to change the application.\",\n \"In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.\",\n \"Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.\",\n \"The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.\",\n \"Few applications use expert systems technology; it is a niche technology.\",\n \"A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.\",\n \"The contents of these three applications are incorporated into this specification by reference.\",\n \"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.\",\n \"The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.\",\n \"A knowledge application is typically characterised by a single instantiation process for all the kitems.\",\n \"The kitems can also be used as contexts or templates from which further kitems can be instantiated.\",\n \"Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.\",\n \"This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.\",\n \"The enquiry is itself a kitem instantiated from a kitem template.\",\n \"Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.\",\n \"SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.\",\n \"Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.\",\n \"The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.\",\n \"Alternatively, these templates can be activated manually.\",\n \"The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.\",\n \"The business objects are easily created, and can be easily manipulated.\",\n \"Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.\",\n \"Instantiating the second set of kitems may take place dynamically, during the use of the application.\",\n \"Applications can be built with a greater number of cascades or instantiations larger than two.\",\n \"Enquiries can be directed against kitems at any level of instantiation using a consultation process.\",\n \"The business objects may be accessed using a consultation process during use of the application.\",\n \"The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.\",\n \"The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.\",\n \"The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.\",\n \"So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.\",\n \"The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.\",\n \"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.\",\n \"Each adaptable module implements the knowledge about the best way to run that module.\",\n \"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.\",\n \"An adaptable meta-module may define and implement the logic between the other modules.\",\n \"In addition the meta-module may monitor a part of, or the whole chain of modules.\",\n \"This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.\",\n \"It may also adapt dynamically to the needs of users as software is being used.\",\n \"Adaptablability is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation, customization and flexibility.\",\n \"In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.\",\n \"Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.\",\n \"Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"Using adaptable software generic applications can be quickly produced.\",\n \"The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.\",\n \"An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.\",\n \"Also client specific applications can be quickly produced.\",\n \"Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS An example of the invention will now be described with reference to the accompanying drawings, in which: FIG.\",\n \"1 is a block diagram showing the steps in the construction and use of an adaptable software application.\",\n \"FIG.\",\n \"2 is a block diagram showing the architecture of an adaptable software application.\",\n \"BEST MODES OF THE INVENTION Adaptable software differs from the knowledge applications described in the background art in that there are at least two levels of instantiation instead of one; and the templates for the second level instantiations are the first level instantiations (they can be objects and not classes in object oriented programming).\",\n \"Adaptable software extends the architecture of known knowledge applications.\",\n \"The use of a two level instantiation, or cascading, design enables the first level to define the application (without hard-coding) and the second to define the documents that are handled by the application.\",\n \"Adaptable software transforms the definition of the application (typically hardcoded) into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.\",\n \"The process for building an adaptable software application will now be described with reference to FIG.\",\n \"1.The process starts in a knowledge application development environment 10 where the business needs of the application determine the knowledge to be instantiated, or created, from contexts and templates.\",\n \"The first step 11 is the instantiation of this knowledge.\",\n \"The outcome is an application module dynamic framework 12 in which the kitems relate to the type of business objects or operations which will be handled by the adaptable software application.\",\n \"This first step is distinguished from the construction of an application knowledge base since it does not produce the documents or the knowledge to be accessed via a consultation.\",\n \"Instead these kitems describe the application as a framework that will be used to generate the dynamic templates for the objects, documents, that relate to the application.\",\n \"A second step 13 then involves populating the adaptable software application with business objects instantiated from contexts or templates produced in the first step.\",\n \"The process for defining these business objects is similar to defining an enquiry in a knowledge application.\",\n \"That is, parameters are specified that determine the features of the business object which is then created.\",\n \"The outcome is an application module 14 populated with business objects.\",\n \"The business objects so created can be managed 15, that is accessed, edited, displayed, distributed, etc.\",\n \"These operations are simple to implement as they correspond to manipulating kitems, and they use standard kitem manipulation methods, and other specific methods if required.\",\n \"For example, the business objects could be accessed using a question-answer session.\",\n \"Specific applications (or modules) are implemented by customising the generic framework produced in the first step, and this corresponds to instantiating different kitems as business objects for each application or module.\",\n \"This customizing can take place dynamically, during the use of the application.\",\n \"This corresponds to dynamic adaptation of the application to the needs of users.\",\n \"Enquiries can be directed against both levels of instantiated kitems via a consultation process.\",\n \"Enquiries directed to the upper level of documents, produced by the first step, are typically used for inspecting and managing the design of the application.\",\n \"To users, adaptable applications do not need to look different from normal applications.\",\n \"For example, a dialogue could take place that enquires about the needs or intentions of a user.\",\n \"Knowing the user's needs or intentions then enables the application to adapt or customize dynamically the template that is presented to the user; this templates defines the documents, or the type of documents, that the user enters into the application.\",\n \"The two level instantiation process can also be described as cascading knowledge items.\",\n \"That is, some knowledge items determine which other knowledge items will be activated or instantiated to define documents.\",\n \"Applications can be built with a greater number of cascades or instantiations larger than two.\",\n \"Most software applications are made of modules that communicate with each other.\",\n \"For example, a typical business management application is made of a client module, a project module, a contact module, a resource module and an accounting module.\",\n \"FIG.\",\n \"2 shows adaptable, intelligent modules or agents.\",\n \"In particular there are adaptable intelligent client 21, project 22, contact 23, resource 24 and accounting 25 modules.\",\n \"Each module is adaptable.\",\n \"For example, in the client module, a different set of questions could be asked based on the age or location or socio-economic profile.\",\n \"Similarly for the other modules.\",\n \"Each module has its business logic defined and implemented using an adaptable architecture.\",\n \"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.\",\n \"Each adaptable module implements the knowledge about the best way to run that module.\",\n \"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.\",\n \"Adaptable meta-module(s) 30 are used to define and implement the logic between the other modules.\",\n \"This could include workflow or project costing for example, where a client would be treated differently based on the number of projects commissioned in the past eighteen months and the success of these projects.\",\n \"In addition the meta-modules are adaptable.\",\n \"In FIG.\",\n \"2, the meta-module monitors a part of, or the whole chain of modules.\",\n \"For example, the accounting module may detect that some projects from some clients are not profitable.\",\n \"That means that the project module would need to be modified (using its adaptable feature) to ensure that these projects are run differently.\",\n \"It could also mean that additional information should be asked of these clients to perhaps rejects them as clients for a certain type of project.\",\n \"In this case the client module is modified using its adaptable feature.\",\n \"A specific example is an access control database.\",\n \"All the operations possible with a database that needs to be controlled are listed as the context in a GKMS type application.\",\n \"Access is to be controlled for each group of users and for users in the groups (control is determined by the group and, in some cases, by the user).\",\n \"In this case, the first level of instantiation corresponds to defining the template to be used to give rights to each group of users.\",\n \"Each user, when linked to a group get an access rights document based on the template for the group it belongs to.\",\n \"This is the second level of instantiation.\",\n \"This document represents the right of that user.\",\n \"The administrator can then modify each document if desired to take account of the characteristics of the user within the group.\",\n \"It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described.\",\n \"The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450974"}}},{"rowIdx":291,"cells":{"text":{"kind":"list like","value":[["Generic knowledge agents","This invention concerns a generic knowledge software agent, or kuark, existing within a knowledge environment.","The kuark comprises: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark, and a destination context of information identifiable by the kuark.","The kuark has a defined source region, or pattern, within the and problems source context and a mapping between that source region and a defined destination region, or pattern, within the destination context.","The kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","It also concerns systems or kitems, made up of kuarks and a process for constructing them.","It also concerns mult-kitem systems and searching processes for those systems.","Finally it also concerns software agents and a knowledge management system comprising kitems."],["1.A generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","2.A software agent according to claim 1, and further comprising: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; or, links to other kuarks.","3.A software agent according to claim 2, where the links are: the hierarchical parent's universal id, and more than one parents could be allowed; the id of the previous version kuark; the ids of the kuarks that are part of this kuark; or, the ids of the kuarks this kuark belongs to or is associated with.","4.A software agent according to claim 1, operating to: create a daughter/parent kuark; adopt an existing kuark as daughter or parent; display an internal state (contexts, regions, mapping); or make a copy each time it fires.","5.A software agent according to claim 1 where, depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines the relevance of the environment.","6.A software agent according to claim 1 where, the mapping is a program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context, and.","the mapping executes when the environment is compatible with the source context and the region.","7.A software agent according to claim 1, implemented as classes and objects using an object oriented language.","8.A system, or kitem, comprising more than one software agent according to claim 1, where the kitem has its own properties and methods in addition to those of its components 9.A process for constructing a system, or kitem, according to claim 8 from its components, comprising the steps of: concatenating their source contexts; concatenating their destination contexts; concatenating their source regions; concatenating their destination regions; concatenating their mappings; concatenating their explanations.","10.A process according to claim 9, comprising the additional step of building a list of the component kuarks or kitems.","11.A system, or kitem, according to claim 8, where all its components (kuarks or kitems) keep their individual contexts.","12.A system, or kitem, according to claim 8, where all its components have their individual contexts concatenated.","13.A system, or kitem, according to claim 8, expressed as an object containing another object.","14.A multi-kitem system comprising two or more systems, or kitems, according to claim 8, where the kitems are linked to one another.","15.A multi-kitem system according to claim 14, comprising a serial arrangement of kitems in which the destination context of one kitem is used as at least part of the source context of another.","16.A multi-kitem system according to claim 14, comprising a parallel arrangement of kitems in which the kitems that have independent (or disjointed), but can also have overlapping, source contexts.","17.A kitem according to claim 8, including within itself a multi-kitem system and having access to all the properties and methods of kitems contained within it, in addition to the properties that belong only to that kitem.","18.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as its source context and region, and a has a mapping which is a program or procedure that produces material to be displayed as output when the relevance mapping fires.","19.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as part of its source context and its source region, and a has a mapping which is a program or procedure that produces a destination region as output when the relevance mapping fires.","20.A searching process for identifying those kitems in a multi-kitem system according to claim 14, that have source regions compatible with an enquiry, the process comprising the step of: determining the ‘relevance status’ of a kitem with respect to the enquiry or consultation so far, where an available status is the default status of a kitem before a search takes place, a candidate kitem is one that is not incompatible with the enquiry so far, and a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","21.A searching process according to claim 20, where the source context is organised as a tree structure, and each kitem is indexed against the context tree so that each object in the context possesses a list of the kitems it is associated with, and during the search questions are context objects and all the indexed kitems to the questions asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","22.A searching process according to claim 21, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","23.A searching process according to claim 20, where the source context is organised as a tree structure in which each element is a kitem and the search process is also a kitem which flags the kitems in the tree structurre that correspond to the questions answered so far.","24.A searching process according to claim 23, where context tree element determines whether all the indexed elements have taken into account the values of the context tree element, and if so, it passes the value(s) to the indexed kitems and waits until all the responses have been received before the next step in the enquiry can proceed.","25.A searching process according to claim 24, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","26.A searching process according to claim 20, where the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","27.A searching process according to claim 20, where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it also possess a set of applicability addresses that correspond to the kitems in its source region, and during processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","28.A searching process according to claim 27, where each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process; and the enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry, or enquiry step.","29.An agent comprising a kitem according to claim 15, and having the following structure: Beliefs, Desires and Intentions are parallel kitems and their respective components are also kitems; and the Beliefs, Desires and Intentions kitems are organised in a serial architecture, that is the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","30.A knowledge management system, comprising: a context element is a display kitem according to claim 18; a source or destination context kitem is a parallel display kitem, and its members comprise the list of all the context elements defined in the domain of discourse; a context executable kitem to edit a context element, edit a context space or display a context space as a tree; a source or destination context tree as an executable kitem, where the display kitem display the elements that belong to the source and destination kitems, its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context; a knowledge editing kitem is an executable kitem that has: as source space: the source and destination contexts, as destination space: the source and destination contexts, as source region a subset of the source context that defines the applicability of the knowledge kitem, as mapping a link to a subset of the destination context, or a procedure or executable (sub-)kitem, as destination region a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure, and an explanation; a search kitem is an executable kitem with: as source context the source and destination contexts, as source region the enquiry as defined so far, as destination context the kitems in the domain of discourse (if known), as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry, as destination region the candidate and definite kitems ‘retrieved’ by the mapping; an enquiry kitem is an executable kitem with: as source space the enquiry (the consultation process, showing each step in the question-answer session), as destination space the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems; a display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way."],[" BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.","PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.","A summary of these knowledge models will now be given with reference to FIGS.","1 to 4 .","A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.","1 .","The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.","2 .","FIGS.","3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.","FIG.","4 is an extension of FIG.","3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.","Once again it involves signal exchanges between servers and clients.","The accepted architecture for agents is summarized in FIGS.","5 and 6 .","FIG.","5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.","FIG.","5 shows that in each part of the BDI model, agents need knowledge to be able to operate.","They must have belief which is knowledge about what their role in existence is.","Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.","Similarly, each of the DBI boxes in FIG.","5 is made of components, each representing a kind of knowledge in its category.","All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.","In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.","This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.","It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.","5 , but to any knowledge that may be useful in agents.","FIG.","6 shows how an agent situated in an environment interacts with this environment.","Th interaction is very similar to FIGS.","3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.","Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process."],[" SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","So the kuark makes predetermined information automatically available to a user when input criteria are met.","The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.","The source region may be identicle to the source context, or it may be a subset within it.","A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.","The links implement relationships to other kuarks.","The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.","This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.","The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.","It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.","A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.","The relevance can be expressed as a probability.","The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.","For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.","Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.","This kuark executes its mapping when the environment is compatible with the source context and the region.","That is, the input to the procedure is the source region in the source context.","The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.","Kuarks may be implemented as classes and objects using an object oriented language such as Java.","A kuark is a class in OOP and some of its properties are static, final, etc.","A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.","A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.","In a kitem all the component kuark and kitems may keep their identities.","The new kitem may have its own properties and methods in addition to those of its component kitems.","The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.","A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.","Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.","The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.","Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.","Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.","A kuark or kitem can be represented as an object using an object oriented language such as Java.","A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.","This means that kitem objects can be generated without programming.","The other features disclosed in the GKMS patent also apply to kitems implemented as classes.","Kitems can be linked.","A link has a source or origin kitem and a destination kitem.","The links can be stored inside the kitem or kuark, as a property of the kitem.","An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.","A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.","More than two kitems can be chained.","Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.","Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.","An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.","Some of the kitems may be linked in series or in parallel.","A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.","Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.","A display kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the display kitem.","The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.","The material to be displayed comprises the destination region and the explanation.","An executable kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the executable kitem.","The second kuark is a procudure kuark.","It has the relevance (the output of the first kuark) as region and as part of its source context.","When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.","The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.","A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.","Searching involves identifying those kitems that have source regions compatible with the enquiry.","It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.","Searches may be synchronous or asynchronous.","An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","The source context may be organised as a tree structure.","Each kitem may be indexed against the context tree.","This means that each object in the context possesses a list of the kitems it is associated with.","During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","The search process updates the status of the kitems according to the enquiry.","Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.","Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.","If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.","The search process waits until all the tree elements corresponding to the enquiry so far have replied.","When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).","Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","Each kitem updates its status according to the enquiry and informs the searc process.","Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.","That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","The state of the relevance region determines what happens to the application region.","Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.","During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.","The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).","Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.","It is up to the enquiry process to manage all the replies it gets from the network.","The solution outlined here is applicable to distributed processing.","Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).","The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.","Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.","When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.","The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.","This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.","In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).","In a multi-kitem system, knowledge processing involves running a question-answer session.","The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.","The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.","Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.","As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.","Therefore the questions are selected dynamically.","Alternatively the search may have processing delegated to each kitem.","Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.","It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.","The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.","Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.","Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.","There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.","Therefore they are included in the compiled kitem.","This is faster than the partial compilation.","An agent may be represented as a kitem with the following structure: Beliefs 61 , Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61 , Desires 62 and Intentions 63 kitems are organised in a serial architecture.","That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).","A source or destination context kitem is a parallel display kitem.","Its members comprise the list of all the context elements defined in the domain of discourse.","Context executable kitems may edit a context element, edit a context space or display a context space as a tree.","A source or destination context tree is an executable kitem.","The display kitem may display the elements that belong to the source and destination kitems.","Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.","A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.","As destination space: the source and destination contexts.","As source region: a subset of the source context that defines the applicability of the knowledge kitem.","As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.","As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.","An explanation.","A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.","An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems.","A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.","This technology supports rapid application development (RAD).","RAD is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation and customization.","RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.","RAD is also a very serious technical challenge for designers.","Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.","In addition agents, with proper architecture, should scale up.","That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.","Currently agents are difficult to design and buid.","This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.","They are: control theory, cognitive psychology and artificial intelligence."],["TECHNICAL FIELD This invention concerns generic knowledge software agents, or kuarks, and their use as the building blocks for agent based applications.","It also concerns systems or kitems, made up of kuarks and a process for constructing them.","It also concerns mult-kitem systems and searching processes for those systems.","Finally it also cocerns software agents and a knowledge management system comprising kitems.","BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.","PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.","A summary of these knowledge models will now be given with reference to FIGS.","1 to 4.A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.","Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.","1.The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.","2.FIGS.","3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.","FIG.","4 is an extension of FIG.","3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.","Once again it involves signal exchanges between servers and clients.","The accepted architecture for agents is summarized in FIGS.","5 and 6.FIG.","5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.","FIG.","5 shows that in each part of the BDI model, agents need knowledge to be able to operate.","They must have belief which is knowledge about what their role in existence is.","Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.","Similarly, each of the DBI boxes in FIG.","5 is made of components, each representing a kind of knowledge in its category.","All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.","In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.","This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.","It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.","5, but to any knowledge that may be useful in agents.","FIG.","6 shows how an agent situated in an environment interacts with this environment.","Th interaction is very similar to FIGS.","3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.","Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.","SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.","So the kuark makes predetermined information automatically available to a user when input criteria are met.","The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.","The source region may be identicle to the source context, or it may be a subset within it.","A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.","The links implement relationships to other kuarks.","The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.","This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.","The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.","It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.","A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.","The relevance can be expressed as a probability.","The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.","For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.","Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.","This kuark executes its mapping when the environment is compatible with the source context and the region.","That is, the input to the procedure is the source region in the source context.","The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.","Kuarks may be implemented as classes and objects using an object oriented language such as Java.","A kuark is a class in OOP and some of its properties are static, final, etc.","A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.","A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.","In a kitem all the component kuark and kitems may keep their identities.","The new kitem may have its own properties and methods in addition to those of its component kitems.","The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.","A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.","Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.","The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.","Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.","Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.","A kuark or kitem can be represented as an object using an object oriented language such as Java.","A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.","This means that kitem objects can be generated without programming.","The other features disclosed in the GKMS patent also apply to kitems implemented as classes.","Kitems can be linked.","A link has a source or origin kitem and a destination kitem.","The links can be stored inside the kitem or kuark, as a property of the kitem.","An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.","A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.","More than two kitems can be chained.","Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.","Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.","An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.","Some of the kitems may be linked in series or in parallel.","A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.","Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.","A display kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the display kitem.","The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.","The material to be displayed comprises the destination region and the explanation.","An executable kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the executable kitem.","The second kuark is a procudure kuark.","It has the relevance (the output of the first kuark) as region and as part of its source context.","When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.","The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.","A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.","Searching involves identifying those kitems that have source regions compatible with the enquiry.","It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.","Searches may be synchronous or asynchronous.","An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","The source context may be organised as a tree structure.","Each kitem may be indexed against the context tree.","This means that each object in the context possesses a list of the kitems it is associated with.","During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","The search process updates the status of the kitems according to the enquiry.","Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.","Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.","If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.","The search process waits until all the tree elements corresponding to the enquiry so far have replied.","When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).","Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","Each kitem updates its status according to the enquiry and informs the searc process.","Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.","That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","The state of the relevance region determines what happens to the application region.","Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.","During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.","The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).","Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.","It is up to the enquiry process to manage all the replies it gets from the network.","The solution outlined here is applicable to distributed processing.","Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).","The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.","Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.","When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.","The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.","This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.","In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).","In a multi-kitem system, knowledge processing involves running a question-answer session.","The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.","The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.","Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.","As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.","Therefore the questions are selected dynamically.","Alternatively the search may have processing delegated to each kitem.","Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.","It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.","The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.","Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.","Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.","There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.","Therefore they are included in the compiled kitem.","This is faster than the partial compilation.","An agent may be represented as a kitem with the following structure: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.","That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).","A source or destination context kitem is a parallel display kitem.","Its members comprise the list of all the context elements defined in the domain of discourse.","Context executable kitems may edit a context element, edit a context space or display a context space as a tree.","A source or destination context tree is an executable kitem.","The display kitem may display the elements that belong to the source and destination kitems.","Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.","A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.","As destination space: the source and destination contexts.","As source region: a subset of the source context that defines the applicability of the knowledge kitem.","As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.","As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.","An explanation.","A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.","An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems.","A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.","This technology supports rapid application development (RAD).","RAD is a very important commercial feature for software.","It holds the promise of speeding up software development, implementation and customization.","RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.","RAD is also a very serious technical challenge for designers.","Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.","In addition agents, with proper architecture, should scale up.","That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.","Currently agents are difficult to design and buid.","This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.","They are: control theory, cognitive psychology and artificial intelligence.","BRIEF DESCRIPTION OF THE DRAWINGS The background to the invention has been described above with reference to the following drawings: FIG.","1 is a diagram illustrating knowledge as mappings between a problem space and a solution space.","FIG.","2 is a diagram illustrating a network of client-servers, each able to run agents.","FIG.","3 is a diagram illustrating a knowledge capture loop in the GKMS.","FIG.","4 is a diagram illustrating an expanded knowledge capture loop in an on-the-job problem-solving system.","FIG.","5 is a diagram illustrating the agent DBI model.","FIG.","6 is a diagram illustrating an agent situated in and interacting with its environment.","The invention will now be described with reference to the following drawings: FIG.","7 is a diagram illustrating a generic elementary cognitive element (kuark).","FIG.","8 is a diagram illustrating a relevance kuark.","FIG.","9 is a diagram illustrating a procedure kuark.","FIG.","10 is a diagram illustrating a serial architecture.","FIG.","11 is a diagram illustrating a parallel architecture.","FIG.","12 is a diagram illustrating the agent implementing the DBI model as a serial kitem.","BEST MODES OF THE INVENTION Generic Elementary Cognitive Agent (Kuark) Referring to FIG.","7, the generic elementary cognitivey agent (kuark) 10 is the building block of agent-based applications.","The kuark knowledge model implements and extends the GKMS model described in the GKMS patent.","The kuark 10 monitors the environment by detecting all environment parameters compatible with the source region 11 and source context 12.When the source region 11, which determines the applicability of the mapping 13, is satisfied, the mapping 13 fires.","The mapping determines the output region 14 in the destination context 15 and makes the region available to the output, and hence to the environment.","Regions are sometimes referred to as patterns.","EXAMPLE 1 The source context comprises two sets of integers and the destination context one set of integers.","The source region is the positive integers for the two sets, the mapping a multiplication of the two inputs accepted in the source region.","The destination region is the result of the multiplication of the two positive numbers.","EXAMPLE 2 The source context is the state of a logical variable and the region the value ‘true’ of that variable.","The destination context is the state of the same variable and the mapping the ‘negation’ of the original value, that is ‘false’.","EXAMPLE 3 The source context is the temperature and pressure inside a boiler and the destination context a list of commands triggering alarms and associated processes (that can be software based).","The source region is the range of pressures and temperatures that trigger a ‘code red’ alert and the destination region the command triggering the ‘code red’ alarm.","EXAMPLE 4 The source context and region is as in the example 3.The destination context is the list of the relevance status of the kuark and the region the actual relevance status of the kuark, based on the source region.","That is, the ‘code red’ alert is given a relevance status (perhaps expressed as a probability) based on the environment.","Kuark Properties The common properties of a kuark are: univeral id; title; explanation (multimedia); time and date created; author; time and date when last modified; person who last modified agent; version number; domain of discourse source context (what it knows about) 12; destination context 15; applicability source region or pattern 11 (can be identical to source context); relevance status to an enquiry (available, candidate, rejected, definite); mapping 13 (the process that produces the outcome based on the state of source context) outcome result of the mapping between source and destination context; the mapping determines the destination region or pattern; status (enabled/disabled/archived); state of source context at time of last firing (describes source context and region); state of destination context after last firing (describes destination region); links to other kuarks the hierarchical parent's universal id (more than one parents could be allowed); the id of the previous version kuark; the ids of the kuarks that are part of this kuark (see later for multi-kuark kuarks); the ids of the kuarks this kuark belongs to or is associated with (see later for multi-kuark kuarks).","The property ‘links to other kuarks’ is the mechanism used to implement relationships between kuarks.","The fourth link mentioned may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.","Kuark Methods The main methods of a kuark are: monitor applicability (detect only what its context allows); determines whether environment fits within source region, if yes triggers/fires mapping; execute mapping (modifies destination state) mapping determines the outcome which is a subset of the destination context; create daughter/parent kuark; adopt existing kuark as daughter or parent; display internal state (contexts, regions, mapping); make copy each time it fires.","It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.","Relevance Kuark Referring now to FIG.","8, a relevance kuark 20 is a kuark 10 (as described previously) that determines the relevance of the environment.","Depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping 23 that determines (calculates) the relevance of the environment.","The relevance can be expressed as a probability.","The destination context 15 is made of the different relevance values that the environment can take with respect to the source context and region.","For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.","Procedure Kuark Referring now to FIG.","9, a procedure kuark 30 is one that contains a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.","The kuark executes its procedure mapping 33, when the environment is compatible with the source context and the region.","That is, the input to the procedure is the source region in the source context.","The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.","The procedure mapping can be a trivial mapping.","It can simply display a fixed pattern and an explanation in a fixed destination context.","The context item, see later, is an implementation of the trivial procedure kuark.","Relevance kuarks tend to have the same destination context, whereas procedure kuarks typically have context that are strongly application dependent and that can vary within an application.","Kuarks and Object Oriented Programming Kuarks can be implemented as classes and objects using an object oriented language such as Java.","A kuark is a class in OOP and some of its properties are static, final, etc.","A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.","Multi-Kuark Systems or Applications This section describes how kuarks can be used together to increase the power of applications.","Multi-Kuark Kitems A system made of several kuarks is still a kuark with all the allowed properties and methods.","However, the term kuark is reserved for simple (or elementary) kuarks, as described above.","A multi-kuark kuark is named a kitem.","Kitems are constructed as follows: the designer identifies the kuarks (or kitems) to be included in the new kitem; the designer clicks ‘construct kitem’.","The construction process simply puts a wrapper around the kuarks and kitems to include in the new kitem.","All the component kuark and kitems keep their identities.","The new kitem can have its own properties and methods in addition to those of its component kitems.","The new kitem has its own title and explanation, and can also have its own source and destination contexts and regions, and its own mapping.","One can also view the new kuark as having undergone a construction process that carried the following operations: concatenates the source contexts; concatenates the destination contexts; concatenates the source regions; concatenates the destination regions; concatenates the mappings; concatenates the explanations.","Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.","The kitem construction process also builds the list of kuarks or kitems that are part of the new kitem.","Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.","Alternatively the kitem construction process can also be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.","Kitems have explicit domains of discourse, the source and destination contexts, and explicit regions; kitems are ‘aware’ of their domains of discourse and when their knowledge, expressed as their mappings and destination regions, can be applied.","Not all kuarks can be assembled in kitems as some concatenation may not be allowed; this issue is not treated further here.","Refer later for different architectures for multi-kitem systems.","Kitem Hierarchy When defining kitems for an application, it is often convenient to organise them as a hierarchy of objects, as in a file system for example.","In fact, all kitems are defined within a hierarchical structure similar to that of the domain names on the Internet.","Each kitem has a parent, except the root kitem which is the top of the tree.","The hierarchical organisation of the kitems imposes no restriction on how they can be grouped (in multi-kitem systems) to achieve some purposes in an application.","That is, composed kitems can be assembled out of any kitem from any location in the hierarchy.","Kitems and Object Oriented Programming A kitem as outlined above, together with its GKMS features, mean that kitems can be implemented using an object oriented language, as objects or instances of the kuark or kitem classes.","As indicated above, a kuark or kitem can be represented as an object using an object oriented language such as Java.","A multi-kuark kitem as outlined above, can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.","This means that kitem objects can be generated without programming.","The other features disclosed in the GKMS patent also apply to kitems implemented as classes.","Rationale for Multi-Kitem Systems The advantages of building applications using kitems are very significant: the methods and properties can be used across the hierarchy (for example, a method to display the components kitems of a kitem can be re-used at all levels in the hierarchy of kitems); kitems provide a unified way for thinking and implementing applications; kitems can be fashioned as simple building blocks that offer maximum re-use; kitems implement knowledge at every level (in every kitem) that make up the application; knowledge permeates the design and the use of the application; applications can be largely soft-coded, instead of hard-coded; applications can be developed rapidly (RAD: rapid application development).","Links Between Kitems Kitems can be linked.","A link has a source or origin kitem and a destination kitem.","The links can be stored inside the kitem or kuark, as a property of the kitem.","An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.","In a link kitem, the source context contain the ID of the source kitem and the type of link (see above for list of possible links), and the destination regions contains the IDs of the corresponding links (the destination context is the domain of discourse of the source kitem).","It is also possible, indeed often preferable, to have the source and destination contexts and regions for a kitem expressed via links.","That is, one or several link kitems contain the source and destination context, and the regions (their kitems and values).","Multi-Kitem Architectures This section discusses the architectures available for building multi-kuark or multi-kitem systems.","In particular, these architectures can be used to build the display and executable kitems.","Serial Architecture In the serial architecture 40, the destination context of one kitem 41 is used as the source context (or part of the source context) of another 42.More than two kitems can be chained, as illustrated in FIG.","10.The state of the environment is used as input to kitem 41 (and perhaps to other kitems).","Similarly, the serial kitem output is the output from kitem n 45 (and perhaps from other kitems).","In the most simple case, the source context is the source context of kitem 41 and the destination context the destination context of kitem n. In general, the source context of the serial kitem is a subset of the source contexts of all its ‘internal kitems’.","Similarly for the destination context and for the regions in these contexts.","The source context of an internal kitem can comprise some elements from the environment in addition to the output from the previous kitem.","The serial kitem is a kitem and has all the properties of kitems discussed above.","Parallel Architecture Referring now to FIG.","11, a parallel kitem 50 is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.","The power of parallel kitems derives from the links that can be established between its internal kitems.","The objective is to ensure that the information and knowledge contained in two kitems becomes linked.","For example, a kitem could describe clients and another projects; and a link would connect a client with a project.","A link has an origin and a destination.","Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.","When the class of a parallel kitem is defined, the operator has to specify which kitems are instantiated as objects when the parallel kitem object is created.","In linked kitems, it is convenient, but not mandatory, to instantiate the origin kitem but not the destination kitem.","Links can be unidirectional or bidirectional.","For example, from clients to projects, or from clients to projects and from projects to clients.","Hierarchical Kitems The kitems that are part of a parallel (or serial kitem) keep their properties, notably the one identifying their parents.","Therefore the parallel kitem can, in fact, contain a hierarchy of kitems even if they are organised in a flat hierarchical (that is, parallel) way in the parallel kitem.","A Knowledge Application as A Parallel Architecture Kitem An application or knowledge base is a set of knowledge items (kitems) defined with respect to a domain of discourse (source and destination contexts and mappings available).","These kitems are arranged in parallel and some of them can be linked.","For example, in an application all the knowledge items are typically linked (that is refer) to the same domain of discourse, as explained above.","Hybrid Architecture It is possible to define a hybrid architecture, with the hybrid kitem containing some kitems linked in a serial way and others in a parallel way.","Meta-Kitems and Meta-Knowledge In FIGS.","10 and 11, each kitem is a class that supports the creation of objects, or knowledge elements.","It is desirable to be able to define a class that can have attributes and methods that span more than one of the kitems contained in the parallel, serial or hybrid kitem.","This can be achieved by giving the outside kitem (eg: the serial, parallel or hybrid kitem) access to all the properties and methods of the contained kitems, in addition to the properties that belong only to the outside kitems.","This means that it is possible to define meta-knowledge about the kitems contained in the outside kitems.","The outside kitem, when instanced as an object, can observe the behaviour of the kitems in it and take actions when necessary.","The outside kitems are referred to as meta-kitems able to express meta-knowledge.","Display and Executable Kitems In this section we consider two different types of very useful kitems: the display kitem and executable kitem.","We show that each can be constructed using one relevance kuark and one procedure kuark.","Display Kitem The display kitem is a serial kitem comprising two kuarks.","The first kuark is a relevance kuark.","It uses its region and the state of the environment to determines the relevance of the display kitem.","The second kuark has the relevance (the output of the first kuark) as source context and region.","The material to be displayed comprises the destination region and the explanation (the destination region can be the explanation.","Context Kitem Contexts and regions are made of elements or objects that describe the contexts and regions.","Each such object can have values.","Here we show that kitems are suitable for describing and implementing these context objects.","A context object is a kitem as shown below (only some properties are mentioned): kitem tile → object title source context → object values source region → instantiated values destination context → same as source context destination region → same as source region mapping → maps source to destination (contexts and regions) explanation → optional.","Explanation Kitem An explanation object is a kitem as shown below (only some properties are mentioned): kitem title → explanation title (can be ‘explanation’) source context → not used source region → not used destination context → not used destination region → not used mapping → not used explanation → multimedia explanation.","Context Element A context element is a kitem made of, typically, a context kitem and an explanation kitem (some context items can have more than one explanation).","To create such a kitem, the designer defines a parallel kitem using the procedure described in the GKMS patent.","It is also possible to have a context item made of a single context kitem, by using its explanation instead of the explanation kitem.","This solution does not work if more than one explanation is required.","For example, an explanation relating to the meaning of the context item and another relating to its use in a knowledge item.","Context Space (Source Or Destination) The context space is a kitem made of context items (also kitems) using the parallel architecture.","Typically no links are used between context items.","Executable Kitem The executable kitem is a serial kitem comprising two kuarks.","The first kuark uses its region and the state of the environment to determines the relevance of the executable kitem.","The second kuark has the relevance (the output of the first kuark) as region and as part of its source context.","When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.","The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.","Knowledge Item and Knowledge Item Definition A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.","Defining a knowledge item, as described in the GKMS patent, involves defining a region in the source context (and other things).","This is done using the context space kitem, that is made of context objects (or elements).","The architecture of a knowledge item is normally that of a parallel kitem.","It is often desirable to explain why a certain context element is part of a knowledge item or how to use or interpret this context element when it is attached to the knowledge item.","This can be achieved simply by giving the user defining the knowledge item the facility to add an explantion kitem to the list of kitem that are part of the knowledge item, and linking this explanation kitem to the context element (kitem) in question.","Alternatively, the explanation can be part of the context element kitem.","That is, the explanation kitem is already linked to the context element (with reference to OOP, the context element class ‘contains’ the explanation).","A knowledge item is defined with respect to a domain of discourse, defined by the source and destination context and the mappings available at definition time.","It would not be efficient to include the contexts kitems in the knowledge item.","Instead, the knowledge item is linked to the context space kitems.","This is in agreement with the definition of links in the parallel architecture kitem.","Kitem Hardcoding We have discussed how kitems can be implemented using other kitems.","However it is always possible to implement a kitem as a single hardcoded entity.","For example, in the GKMS patent, the context elements and the knowledge items were implemented as single entities with their specific architectures.","When this is done, many of the modular and flexible features of the kuark-kitem model are lost.","When a similar effect is desired, it is preferable to implement the kitem as a permanently compiled multi-kitem kitem (see later).","Knowledge Processing in Multi-Kitem Systems A multi-kitem system is a system comprising a set of kitems representing the body of knowledge about a certain domain of discourse.","Typically the domain of discourse is identical for all the kitems in the system, although it is not essential.","Such a system is identified as a knowledge module or database.","Knowledge processing involves the system (or server) running a question-answer session.","The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.","The steps involved are: identify the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asks these questions; on the basis of the answers to the questions, determine a) whether any answers can be found, and b) the next best questions to ask.","The processing refers to: the identification of the correct answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most promising provisional answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most important questions to ask on the basis of the information available.","Searching for relevant or important knowledge Knowledge access in a multi-kitem system (a knowledge module) follows the same pattern as for a GKMS knowledge base; that is, it is a question-answer session or consultation.","The consultation process is managed with, and recorded in, a document.","This document can also be a kitem called the consultation kitem.","That is, the consultation kitem has all the properties and methods of the kitem class.","All the utilities for kuarks and kitems can be used with consultation kitems.","Searching involves identifying those kitems that have source regions compatible with the enquiry (see GKMS patent for meaning of compatible).","It means determining the ‘relevance status’ (available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.","Searches can be synchronous or asynchronous.","An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.","Synchronous Searches Database Search The database search is a SQL search or equivalent.","This means that the kitems are stored as records in a database.","This is not discussed further in this document Full Text Search The full text search has to process each relevant field in each record where each record describes a knowledge element.","This process can be slow; it is not discussed further in this document.","Context Tree Search with Processing Delegated to A Search Agent The source context is organised as a tree structure.","A source region, which determines whether a kitem will fire (firing depends on the region's compatibility with an enquiry), is a subset of the source context.","Each kitem (eg: knowledge element) can be indexed against the context tree.","This means that each object in the context possesses a list of the kitems it is associated with.","The context tree is a formalisation of one of the kuarks'properties (see above).","During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.","Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","The search process updates the status of the kitems according to the enquiry.","Context Tree Search with Processing Delegated to Each Kitem The search process (also a kitem) flags these elements in the context tree that correspond to the questions answered so far.","Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.","If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.","The search process waits until all the tree elements corresponding to the enquiry so far have replied.","When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).","Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.","At the implementation level, the context tree is a modified context space kitem.","The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.","These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.","Each kitem updates its status according to the enquiry and informs the searc process.","Asynchronous Searches Processing Delegated to Each Kitem In asynchronous searches, the processing is distributed and controlled by each item.","There is no central authority that decides what to do when.","Each kitem (or agent) monitors its environment and reacts appropriately.","In doing so, it may change the environment and trigger new actions by other kitems.","This is in contrast to synchronous searches in which it is the search process that determines whether a kitem is applicable or not.","In particular, in asynchronous searches it is each kitem that determines whether it is applicable to the environment as it is detected via the source context and the source region.","In asynchronous searches, the source region determines whether the outcome of the kitem becomes applicable.","That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.","The state of the relevance region determines what happens to the application region.","In the case of non-executable mappings (or display mappings) the process is as illustrated in FIG.","8; the relevance mapping ‘releases’ the destination region to the kuark output.","State of Environment (or Context) Communicated to the Items As indicated above, each kitem has a unique ID or address that determines its location in a distributed memory space or network (such as the Internet for example).","It also possesses a set of applicability addresses that correspond to the kitems in its source region.","During processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.","In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.","The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.","These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).","Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.","It is up to the enquiry process to manage all the replies it gets from the network.","The solution outlined here is applicable to distributed processing.","The Kitems Seek to Know the Status of their Source Context Another model for asynchronous searches is to give even more responsibility to the distributed kitems than in the previous description.","In this model, each kitem has the address of each of the context objects in its context and in its region (a subset of the context).","The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.","This model means that the kitem takes the intiative (this has some important applications not discussed here).","However during searching, the model imposes much traffic on the network that is of no benefit, unless the context object has changed since the last status check.","Another problem is that there may be an unacceptable delay between status checks, during which time, if the status has changed, the kitem doing the interrogating cannot determine its applicability.","Cascading Knowledge Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.","When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.","The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.","This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.","In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).","Validity of the Relevant Knowledge For simple knowledge bases on a single server it is a relatively simple matter to ensure that all kitems they contain have the same domain of discourse.","This, however, does not apply for large or distributed knowledge bases where the knowledge comes from different sources.","In general, therefore, there is no certainty that all the knowledge items (kitems) accessed during a search have the same source and destination contexts.","In fact it is unlikely.","This means that a user inspecting a definite kitem (with respect to an enquiry) may not know what the domain of discourse of that kitem might be.","This information is very important as it defines the context for the knowledge in the kitem in question.","A solution to this situation is to give the user the ability to inspect the source and destination context of every kitem that is provided by the search process either as a candidate or definite kitem.","Identifying the Most Promising Questions Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Context objects which index a large number of available or candidate kitems are potentially very important when used as questions.","This is because their answers have the potential to impact on the relevance status of a large number of kitems.","This indicates that the importance of these context objects as questions in an enquiry is determined by the number of kitems with status available or candidate they index.","As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.","Therefore the questions are selected dynamically.","Context Tree Search with Processing Delegated to Each Kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.","It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","Asynchronous Searches Processing Delegated to Each Kitem The enquiry process (that sends the enquiry over the network for kitems to process it) performs several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.","In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.","Search for Hidden Knowledge Hidden knowledge refers to knowledge that may be relevant to an enquiry but that is not yet linked to any question answered in the enquiry process so far.","The question is how to identify this hidden knowledge and how to extract its ‘best questions’.","In situations where the domain of discourse of a body of knowledge is known, one can simply identify the kitems with the largest number of indexed kitems with relevance status available or candidate (see context tree above).","There is no simple solutions when the domain of discourse of a body of knowledge is not known.","Processing Speed Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Synchronous searches can be very quick as they are under the control of one process that can be optimised, based on known conditions in the server.","Context Tree Search with Processing Delegated to Each Kitem This processing can be distributed across a network, but it is infrequent.","This processing is slower than the previous one as it imposes communications overheads between kitems (those doing some processing and the enquiry process).","Nevertheless, the enquiry process runs on a single server and can be optimised.","When the process is distributed, several kitems can process simultaneously; this can partially offset the communication overheads.","Asynchronous Searches Processing Delegated to Each Kitem This processing is slowest unless the number of kitems doing processing is large enough to offset the communication overhead between kitems and the network delays.","Compilation Compilation is the process that takes a collection of kitems and organises them for the purpose of speeding up processing.","The result of compilation is a kitem.","In general, compilation is applied to any kitem.","In practice it is often applied to all the kitems that belong to a domain of discourse (also a kitem) on a single server.","That is, all the kitems involved have domains of discourse that are subset of the compiled domain of discourse.","Compilation is also applied to a group of kitems contained in a serial, parallel or hybrid kitem.","The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.","Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.","Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.","The processing model for the multi-agent systems described in this document imposes, like the Java language, that kitems call other kitems that in turn may call other kitems, etc, until the processing can be completed.","Once this is done, the results of the processing need to travel up the hierarchy of kitems, back to the initiating process.","The compilation process is designed to speed up the ‘down-hierarchy’ and ‘up-hierarchy’ travelling described above.","The context tree compilation achieves this purpose by ‘flattening’ the hierarchy to a single branch tree, where each context element (or node) in the branch contains all the kitems that have that context element as part of their regions.","There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.","Therefore they are included in the compiled kitem.","This is faster than the partial compilation.","Compilation can be be carried out to different depths: 1.build context tree at runtime; 2.get regions of indexed kitems in the context tree at runtime; 3.include regions of indexed kitems in the context tree; Compilations of type 1 and 2 above are just-in-time compilations.","They updates the context tree each time the search process is triggered; permanent compilation produces the context tree and leaves it untouched until another compilation is carried out.","Agents as Kitem Systems An agent, as illustrated in FIG.","5, can be represented as a kitem 60 with the structure illustrated in FIG.","12.: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.","That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.","It is recommended but not essential that the kitems that comprise the agent operate in a synchronous mode.","Internal synchronous processing is possible, even if a multi-agent systems behaves asynchronously.","As an agent is a kitem, a multi-agent system is a multi-kitem system with a parallel architecture and continuous asynchronous processing.","Kitems Needed for A Workable Solutions The kitems needed to produce a workable implementation for knowledge management are shown below: Context Management Context Elements A context element is a display kitem (a serial kitem made of two kuarks).","Source and Destination Contexts A source or destination context kitem is a parallel display kitem.","Its members comprise the list of all the context elements defined in the domain of discourse.","Context Executable Kitems Edit a context element Edit a context space Display a context space as a tree A source or destination context tree is an executable kitem.","The display kitem can display the elements that belong to the source and destination kitems.","Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.","Knowledge Definition Knowledge Editing Kitem The knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.","As destination space: the source and destination contexts.","As source region: a subset of the source context that defines the applicability of the knowledge kitem.","As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.","As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.","An explanation.","Knowledge Access and Display Search Kitem The search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.","Enquiry or Consolation Kitem The enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.","), the ids of the definite kitems.","Display Kitem The display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.","Administration of Contexts and Other Kitems The hierarchical (tree) structure of the kitems gives a very convenient way of controlling user access to kitems: the administrator can decide which branch of the hierarchy users can have access to and the type of access for the branch and each kitem in the branch; access control can also be carried out at the level of the kitem.","Dynamic Kitem Management The objective of dynamic kitem management is to be able to manage the integrity of the knowledge base when a kitem is edited.","This issue is important as kitems (eg: a context kitem) can be used in many knowledge elements and a change in such a kitem can have impacts on the validity of these knowledge elements (from a knowledge viewpoint, not operational viewpoint).","It is essential that the users are not left in the dark about the potential ramification of their changes and that they are helped in checking the validity of some affected kitems.","As much as possible, the system should ensure the integrity of the knowledge itself.","The process for achieving the above is to: use the enquiry process (by the system or the user) to retrieve all kitems that could be affected (the system or the user can specify the profile of the kitems affected by the change); make the changes automatically if acceptable; or ask the user to review/recertify the kitems affected (the system needs to provide guidance in this process).","The profile mentioned above refers to the values given to kitems in the source and destination context.","The system treats these values as an enquiry and retrieves all the kitems that are compatible with the enquiry.","Change to properties All the properties of the kitems can be changed.","Potentially, all the kitems that are linked to the kitem being edited are affected.","Some properties and the way their editing can be handled is listed below.","Change to Source Context and Region If a new context object is added: the system asks the user to specify the profile of the kitems that may be affected, the system retrieves these kitems and the user is asked to recertify them.","If an existing context object is edited or deleted: the system retrieves all the kitems that are related to this kitem (that contain it as part of a link for example) and asks the user to recertify them and to edit them if necessary before doing so.","Change to Destination Context and Region As for a change to the source context and region.","Change to Mapping The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.","Change to Status (Enabled/Disabled/Archived) The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.","Change to the ‘Links to Other Kitems’ No checks required, the change is localised.","Consistency Issues Consistency issues may arise in a knowledge base when some kitems have the following features: kitems have overlapping (at least one common kitem with at least a common value) or identical destination regions with non-identical source regions; kitems have disjointed destination regions with overlapping or identical source regions.","The system can easily have an agent that checks all the kitems in a knowledge base and identifies these kitems that may be inconsistent.","The system presents the kitems to their authors for editing if necessary and recertification.","Missing Knowledge Missing knowledge refers to parts of a domain of discourse for which there is no knowledge or inadequate knowledge.","When the domain of discourse for a set of kitems is explicit, it is a simple matter to check that the union of the source regions in the kitems cover the entire source context.","If it is not the case, it means that the some additional knowledge is required.","The user can be informed and the location of this missing knowledge presented.","The location of the missing knowledge can be pointed out on each dimension (or context kitem) of the source context.","The user selects a context item and a value (or range of values) for which there is no region, and the system responds by showing the other context items and their values for which there is no region.","The user then selects a second context item and gives it some values (where there is no region), and so on for the other kitems if desired, until the user is confident of being able to add some missing knowledge.","New Kitems When a new knowledge element is being saved (the user has finished its definition), the system checks that it is consistent with the other knowledge elements in the knowledge base (see section 12.2).","The system also retrieves all kitems that overlap with the new kitem's source and/or destination regions.","The system informs the user that these kitems may be affected (their knowledge may be superseeded by the new kitem) and presents them to the user for checking.","The system can also ask the user to define the profile of the kitems that may be superseeded by the new kitem.","Terminology Term Explanation Applicability region The region in the source context that determines whether the kitem is applicable.","Application context The context for the application made of the source or space and destination contexts.","Available kitem The status of a kitem that is available for an enquiry (the other status are definite, candidate and rejected.","Candidate kitem A kitem that is compatible with the enquiry so far but for which the applicability conditions are not completely met.","Definite kitem A kitem that is compatible with the enquiry so far with its applicability conditions completely met.","Destination context The space of solutions considered by an application or a kitem.","Domain of discourse The space of problems and solutions (or situations and outcomes) considered by an application or a kitem; it is made of the source and destination contexts.","Enquriy process The agent or process that controls the question- answer session or consultation.","Executable mapping A mapping that takes an input from the source context and applies an algorithm to it to produce an outcome.","Kitem A object or element that comprises a source context and region, a destination context and region, and a mapping.","Knowledge item A kitem that is used to express knowledge.","Kuark An elementary kitem (the smallest building block) that can be used to build other kitems and/or complete applications.","Mapping The transformation that is applied to a source region to produce a destination region or outcome.","Outcome Another term for destionation region or solution.","Parallel kitem A kitem comprising other kitems organised in parallel.","Rejected kitem An kitem that is not compatible with an enquiry.","Relevance context The destination context that expresses the range of possible relevance for kitems in an application.","Relevance region A region in the relevance context.","Relevance status The relevance of a kitem with respect to an enquiry (see GKMS patent for details).","Serial kitem A kitem comprising other kitems organised in series.","Solution Another term for destination region or outcome.","Source context The space of outcomes considered by an application or a kitem."]],"string":"[\n [\n \"Generic knowledge agents\",\n \"This invention concerns a generic knowledge software agent, or kuark, existing within a knowledge environment.\",\n \"The kuark comprises: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark, and a destination context of information identifiable by the kuark.\",\n \"The kuark has a defined source region, or pattern, within the and problems source context and a mapping between that source region and a defined destination region, or pattern, within the destination context.\",\n \"The kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"It also concerns systems or kitems, made up of kuarks and a process for constructing them.\",\n \"It also concerns mult-kitem systems and searching processes for those systems.\",\n \"Finally it also concerns software agents and a knowledge management system comprising kitems.\"\n ],\n [\n \"1.A generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"2.A software agent according to claim 1, and further comprising: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; or, links to other kuarks.\",\n \"3.A software agent according to claim 2, where the links are: the hierarchical parent's universal id, and more than one parents could be allowed; the id of the previous version kuark; the ids of the kuarks that are part of this kuark; or, the ids of the kuarks this kuark belongs to or is associated with.\",\n \"4.A software agent according to claim 1, operating to: create a daughter/parent kuark; adopt an existing kuark as daughter or parent; display an internal state (contexts, regions, mapping); or make a copy each time it fires.\",\n \"5.A software agent according to claim 1 where, depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines the relevance of the environment.\",\n \"6.A software agent according to claim 1 where, the mapping is a program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context, and.\",\n \"the mapping executes when the environment is compatible with the source context and the region.\",\n \"7.A software agent according to claim 1, implemented as classes and objects using an object oriented language.\",\n \"8.A system, or kitem, comprising more than one software agent according to claim 1, where the kitem has its own properties and methods in addition to those of its components 9.A process for constructing a system, or kitem, according to claim 8 from its components, comprising the steps of: concatenating their source contexts; concatenating their destination contexts; concatenating their source regions; concatenating their destination regions; concatenating their mappings; concatenating their explanations.\",\n \"10.A process according to claim 9, comprising the additional step of building a list of the component kuarks or kitems.\",\n \"11.A system, or kitem, according to claim 8, where all its components (kuarks or kitems) keep their individual contexts.\",\n \"12.A system, or kitem, according to claim 8, where all its components have their individual contexts concatenated.\",\n \"13.A system, or kitem, according to claim 8, expressed as an object containing another object.\",\n \"14.A multi-kitem system comprising two or more systems, or kitems, according to claim 8, where the kitems are linked to one another.\",\n \"15.A multi-kitem system according to claim 14, comprising a serial arrangement of kitems in which the destination context of one kitem is used as at least part of the source context of another.\",\n \"16.A multi-kitem system according to claim 14, comprising a parallel arrangement of kitems in which the kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"17.A kitem according to claim 8, including within itself a multi-kitem system and having access to all the properties and methods of kitems contained within it, in addition to the properties that belong only to that kitem.\",\n \"18.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as its source context and region, and a has a mapping which is a program or procedure that produces material to be displayed as output when the relevance mapping fires.\",\n \"19.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as part of its source context and its source region, and a has a mapping which is a program or procedure that produces a destination region as output when the relevance mapping fires.\",\n \"20.A searching process for identifying those kitems in a multi-kitem system according to claim 14, that have source regions compatible with an enquiry, the process comprising the step of: determining the ‘relevance status’ of a kitem with respect to the enquiry or consultation so far, where an available status is the default status of a kitem before a search takes place, a candidate kitem is one that is not incompatible with the enquiry so far, and a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"21.A searching process according to claim 20, where the source context is organised as a tree structure, and each kitem is indexed against the context tree so that each object in the context possesses a list of the kitems it is associated with, and during the search questions are context objects and all the indexed kitems to the questions asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"22.A searching process according to claim 21, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"23.A searching process according to claim 20, where the source context is organised as a tree structure in which each element is a kitem and the search process is also a kitem which flags the kitems in the tree structurre that correspond to the questions answered so far.\",\n \"24.A searching process according to claim 23, where context tree element determines whether all the indexed elements have taken into account the values of the context tree element, and if so, it passes the value(s) to the indexed kitems and waits until all the responses have been received before the next step in the enquiry can proceed.\",\n \"25.A searching process according to claim 24, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"26.A searching process according to claim 20, where the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"27.A searching process according to claim 20, where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it also possess a set of applicability addresses that correspond to the kitems in its source region, and during processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"28.A searching process according to claim 27, where each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process; and the enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry, or enquiry step.\",\n \"29.An agent comprising a kitem according to claim 15, and having the following structure: Beliefs, Desires and Intentions are parallel kitems and their respective components are also kitems; and the Beliefs, Desires and Intentions kitems are organised in a serial architecture, that is the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"30.A knowledge management system, comprising: a context element is a display kitem according to claim 18; a source or destination context kitem is a parallel display kitem, and its members comprise the list of all the context elements defined in the domain of discourse; a context executable kitem to edit a context element, edit a context space or display a context space as a tree; a source or destination context tree as an executable kitem, where the display kitem display the elements that belong to the source and destination kitems, its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context; a knowledge editing kitem is an executable kitem that has: as source space: the source and destination contexts, as destination space: the source and destination contexts, as source region a subset of the source context that defines the applicability of the knowledge kitem, as mapping a link to a subset of the destination context, or a procedure or executable (sub-)kitem, as destination region a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure, and an explanation; a search kitem is an executable kitem with: as source context the source and destination contexts, as source region the enquiry as defined so far, as destination context the kitems in the domain of discourse (if known), as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry, as destination region the candidate and definite kitems ‘retrieved’ by the mapping; an enquiry kitem is an executable kitem with: as source space the enquiry (the consultation process, showing each step in the question-answer session), as destination space the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems; a display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\"\n ],\n [\n \" BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.\",\n \"PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.\",\n \"A summary of these knowledge models will now be given with reference to FIGS.\",\n \"1 to 4 .\",\n \"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.\",\n \"1 .\",\n \"The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.\",\n \"2 .\",\n \"FIGS.\",\n \"3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.\",\n \"FIG.\",\n \"4 is an extension of FIG.\",\n \"3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.\",\n \"Once again it involves signal exchanges between servers and clients.\",\n \"The accepted architecture for agents is summarized in FIGS.\",\n \"5 and 6 .\",\n \"FIG.\",\n \"5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.\",\n \"FIG.\",\n \"5 shows that in each part of the BDI model, agents need knowledge to be able to operate.\",\n \"They must have belief which is knowledge about what their role in existence is.\",\n \"Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.\",\n \"Similarly, each of the DBI boxes in FIG.\",\n \"5 is made of components, each representing a kind of knowledge in its category.\",\n \"All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.\",\n \"In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.\",\n \"This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.\",\n \"It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.\",\n \"5 , but to any knowledge that may be useful in agents.\",\n \"FIG.\",\n \"6 shows how an agent situated in an environment interacts with this environment.\",\n \"Th interaction is very similar to FIGS.\",\n \"3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.\",\n \"Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"So the kuark makes predetermined information automatically available to a user when input criteria are met.\",\n \"The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.\",\n \"The source region may be identicle to the source context, or it may be a subset within it.\",\n \"A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.\",\n \"The links implement relationships to other kuarks.\",\n \"The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.\",\n \"This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.\",\n \"The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.\",\n \"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.\",\n \"A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.\",\n \"The relevance can be expressed as a probability.\",\n \"The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.\",\n \"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.\",\n \"Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.\",\n \"This kuark executes its mapping when the environment is compatible with the source context and the region.\",\n \"That is, the input to the procedure is the source region in the source context.\",\n \"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.\",\n \"Kuarks may be implemented as classes and objects using an object oriented language such as Java.\",\n \"A kuark is a class in OOP and some of its properties are static, final, etc.\",\n \"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.\",\n \"A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.\",\n \"In a kitem all the component kuark and kitems may keep their identities.\",\n \"The new kitem may have its own properties and methods in addition to those of its component kitems.\",\n \"The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.\",\n \"A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.\",\n \"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.\",\n \"The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.\",\n \"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.\",\n \"Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.\",\n \"A kuark or kitem can be represented as an object using an object oriented language such as Java.\",\n \"A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.\",\n \"This means that kitem objects can be generated without programming.\",\n \"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.\",\n \"Kitems can be linked.\",\n \"A link has a source or origin kitem and a destination kitem.\",\n \"The links can be stored inside the kitem or kuark, as a property of the kitem.\",\n \"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.\",\n \"A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.\",\n \"More than two kitems can be chained.\",\n \"Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.\",\n \"An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.\",\n \"Some of the kitems may be linked in series or in parallel.\",\n \"A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.\",\n \"Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.\",\n \"A display kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the display kitem.\",\n \"The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.\",\n \"The material to be displayed comprises the destination region and the explanation.\",\n \"An executable kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the executable kitem.\",\n \"The second kuark is a procudure kuark.\",\n \"It has the relevance (the output of the first kuark) as region and as part of its source context.\",\n \"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.\",\n \"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.\",\n \"A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.\",\n \"Searching involves identifying those kitems that have source regions compatible with the enquiry.\",\n \"It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.\",\n \"Searches may be synchronous or asynchronous.\",\n \"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"The source context may be organised as a tree structure.\",\n \"Each kitem may be indexed against the context tree.\",\n \"This means that each object in the context possesses a list of the kitems it is associated with.\",\n \"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"The search process updates the status of the kitems according to the enquiry.\",\n \"Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.\",\n \"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.\",\n \"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.\",\n \"The search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).\",\n \"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"Each kitem updates its status according to the enquiry and informs the searc process.\",\n \"Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.\",\n \"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"The state of the relevance region determines what happens to the application region.\",\n \"Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.\",\n \"During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.\",\n \"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).\",\n \"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.\",\n \"It is up to the enquiry process to manage all the replies it gets from the network.\",\n \"The solution outlined here is applicable to distributed processing.\",\n \"Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).\",\n \"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.\",\n \"Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.\",\n \"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.\",\n \"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.\",\n \"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.\",\n \"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).\",\n \"In a multi-kitem system, knowledge processing involves running a question-answer session.\",\n \"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.\",\n \"The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.\",\n \"Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.\",\n \"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.\",\n \"Therefore the questions are selected dynamically.\",\n \"Alternatively the search may have processing delegated to each kitem.\",\n \"Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.\",\n \"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.\",\n \"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.\",\n \"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.\",\n \"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.\",\n \"Therefore they are included in the compiled kitem.\",\n \"This is faster than the partial compilation.\",\n \"An agent may be represented as a kitem with the following structure: Beliefs 61 , Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61 , Desires 62 and Intentions 63 kitems are organised in a serial architecture.\",\n \"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).\",\n \"A source or destination context kitem is a parallel display kitem.\",\n \"Its members comprise the list of all the context elements defined in the domain of discourse.\",\n \"Context executable kitems may edit a context element, edit a context space or display a context space as a tree.\",\n \"A source or destination context tree is an executable kitem.\",\n \"The display kitem may display the elements that belong to the source and destination kitems.\",\n \"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.\",\n \"A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.\",\n \"As destination space: the source and destination contexts.\",\n \"As source region: a subset of the source context that defines the applicability of the knowledge kitem.\",\n \"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.\",\n \"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.\",\n \"An explanation.\",\n \"A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.\",\n \"An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems.\",\n \"A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\",\n \"This technology supports rapid application development (RAD).\",\n \"RAD is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation and customization.\",\n \"RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"RAD is also a very serious technical challenge for designers.\",\n \"Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.\",\n \"In addition agents, with proper architecture, should scale up.\",\n \"That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.\",\n \"Currently agents are difficult to design and buid.\",\n \"This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.\",\n \"They are: control theory, cognitive psychology and artificial intelligence.\"\n ],\n [\n \"TECHNICAL FIELD This invention concerns generic knowledge software agents, or kuarks, and their use as the building blocks for agent based applications.\",\n \"It also concerns systems or kitems, made up of kuarks and a process for constructing them.\",\n \"It also concerns mult-kitem systems and searching processes for those systems.\",\n \"Finally it also cocerns software agents and a knowledge management system comprising kitems.\",\n \"BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.\",\n \"PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.\",\n \"A summary of these knowledge models will now be given with reference to FIGS.\",\n \"1 to 4.A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.\",\n \"Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.\",\n \"1.The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.\",\n \"2.FIGS.\",\n \"3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.\",\n \"FIG.\",\n \"4 is an extension of FIG.\",\n \"3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.\",\n \"Once again it involves signal exchanges between servers and clients.\",\n \"The accepted architecture for agents is summarized in FIGS.\",\n \"5 and 6.FIG.\",\n \"5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.\",\n \"FIG.\",\n \"5 shows that in each part of the BDI model, agents need knowledge to be able to operate.\",\n \"They must have belief which is knowledge about what their role in existence is.\",\n \"Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.\",\n \"Similarly, each of the DBI boxes in FIG.\",\n \"5 is made of components, each representing a kind of knowledge in its category.\",\n \"All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.\",\n \"In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.\",\n \"This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.\",\n \"It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.\",\n \"5, but to any knowledge that may be useful in agents.\",\n \"FIG.\",\n \"6 shows how an agent situated in an environment interacts with this environment.\",\n \"Th interaction is very similar to FIGS.\",\n \"3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.\",\n \"Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.\",\n \"SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.\",\n \"So the kuark makes predetermined information automatically available to a user when input criteria are met.\",\n \"The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.\",\n \"The source region may be identicle to the source context, or it may be a subset within it.\",\n \"A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.\",\n \"The links implement relationships to other kuarks.\",\n \"The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.\",\n \"This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.\",\n \"The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.\",\n \"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.\",\n \"A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.\",\n \"The relevance can be expressed as a probability.\",\n \"The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.\",\n \"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.\",\n \"Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.\",\n \"This kuark executes its mapping when the environment is compatible with the source context and the region.\",\n \"That is, the input to the procedure is the source region in the source context.\",\n \"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.\",\n \"Kuarks may be implemented as classes and objects using an object oriented language such as Java.\",\n \"A kuark is a class in OOP and some of its properties are static, final, etc.\",\n \"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.\",\n \"A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.\",\n \"In a kitem all the component kuark and kitems may keep their identities.\",\n \"The new kitem may have its own properties and methods in addition to those of its component kitems.\",\n \"The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.\",\n \"A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.\",\n \"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.\",\n \"The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.\",\n \"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.\",\n \"Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.\",\n \"A kuark or kitem can be represented as an object using an object oriented language such as Java.\",\n \"A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.\",\n \"This means that kitem objects can be generated without programming.\",\n \"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.\",\n \"Kitems can be linked.\",\n \"A link has a source or origin kitem and a destination kitem.\",\n \"The links can be stored inside the kitem or kuark, as a property of the kitem.\",\n \"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.\",\n \"A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.\",\n \"More than two kitems can be chained.\",\n \"Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.\",\n \"An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.\",\n \"Some of the kitems may be linked in series or in parallel.\",\n \"A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.\",\n \"Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.\",\n \"A display kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the display kitem.\",\n \"The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.\",\n \"The material to be displayed comprises the destination region and the explanation.\",\n \"An executable kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the executable kitem.\",\n \"The second kuark is a procudure kuark.\",\n \"It has the relevance (the output of the first kuark) as region and as part of its source context.\",\n \"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.\",\n \"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.\",\n \"A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.\",\n \"Searching involves identifying those kitems that have source regions compatible with the enquiry.\",\n \"It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.\",\n \"Searches may be synchronous or asynchronous.\",\n \"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"The source context may be organised as a tree structure.\",\n \"Each kitem may be indexed against the context tree.\",\n \"This means that each object in the context possesses a list of the kitems it is associated with.\",\n \"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"The search process updates the status of the kitems according to the enquiry.\",\n \"Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.\",\n \"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.\",\n \"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.\",\n \"The search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).\",\n \"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"Each kitem updates its status according to the enquiry and informs the searc process.\",\n \"Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.\",\n \"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"The state of the relevance region determines what happens to the application region.\",\n \"Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.\",\n \"During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.\",\n \"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).\",\n \"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.\",\n \"It is up to the enquiry process to manage all the replies it gets from the network.\",\n \"The solution outlined here is applicable to distributed processing.\",\n \"Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).\",\n \"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.\",\n \"Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.\",\n \"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.\",\n \"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.\",\n \"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.\",\n \"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).\",\n \"In a multi-kitem system, knowledge processing involves running a question-answer session.\",\n \"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.\",\n \"The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.\",\n \"Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.\",\n \"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.\",\n \"Therefore the questions are selected dynamically.\",\n \"Alternatively the search may have processing delegated to each kitem.\",\n \"Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.\",\n \"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.\",\n \"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.\",\n \"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.\",\n \"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.\",\n \"Therefore they are included in the compiled kitem.\",\n \"This is faster than the partial compilation.\",\n \"An agent may be represented as a kitem with the following structure: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.\",\n \"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).\",\n \"A source or destination context kitem is a parallel display kitem.\",\n \"Its members comprise the list of all the context elements defined in the domain of discourse.\",\n \"Context executable kitems may edit a context element, edit a context space or display a context space as a tree.\",\n \"A source or destination context tree is an executable kitem.\",\n \"The display kitem may display the elements that belong to the source and destination kitems.\",\n \"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.\",\n \"A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.\",\n \"As destination space: the source and destination contexts.\",\n \"As source region: a subset of the source context that defines the applicability of the knowledge kitem.\",\n \"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.\",\n \"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.\",\n \"An explanation.\",\n \"A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.\",\n \"An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems.\",\n \"A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\",\n \"This technology supports rapid application development (RAD).\",\n \"RAD is a very important commercial feature for software.\",\n \"It holds the promise of speeding up software development, implementation and customization.\",\n \"RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.\",\n \"RAD is also a very serious technical challenge for designers.\",\n \"Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.\",\n \"In addition agents, with proper architecture, should scale up.\",\n \"That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.\",\n \"Currently agents are difficult to design and buid.\",\n \"This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.\",\n \"They are: control theory, cognitive psychology and artificial intelligence.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The background to the invention has been described above with reference to the following drawings: FIG.\",\n \"1 is a diagram illustrating knowledge as mappings between a problem space and a solution space.\",\n \"FIG.\",\n \"2 is a diagram illustrating a network of client-servers, each able to run agents.\",\n \"FIG.\",\n \"3 is a diagram illustrating a knowledge capture loop in the GKMS.\",\n \"FIG.\",\n \"4 is a diagram illustrating an expanded knowledge capture loop in an on-the-job problem-solving system.\",\n \"FIG.\",\n \"5 is a diagram illustrating the agent DBI model.\",\n \"FIG.\",\n \"6 is a diagram illustrating an agent situated in and interacting with its environment.\",\n \"The invention will now be described with reference to the following drawings: FIG.\",\n \"7 is a diagram illustrating a generic elementary cognitive element (kuark).\",\n \"FIG.\",\n \"8 is a diagram illustrating a relevance kuark.\",\n \"FIG.\",\n \"9 is a diagram illustrating a procedure kuark.\",\n \"FIG.\",\n \"10 is a diagram illustrating a serial architecture.\",\n \"FIG.\",\n \"11 is a diagram illustrating a parallel architecture.\",\n \"FIG.\",\n \"12 is a diagram illustrating the agent implementing the DBI model as a serial kitem.\",\n \"BEST MODES OF THE INVENTION Generic Elementary Cognitive Agent (Kuark) Referring to FIG.\",\n \"7, the generic elementary cognitivey agent (kuark) 10 is the building block of agent-based applications.\",\n \"The kuark knowledge model implements and extends the GKMS model described in the GKMS patent.\",\n \"The kuark 10 monitors the environment by detecting all environment parameters compatible with the source region 11 and source context 12.When the source region 11, which determines the applicability of the mapping 13, is satisfied, the mapping 13 fires.\",\n \"The mapping determines the output region 14 in the destination context 15 and makes the region available to the output, and hence to the environment.\",\n \"Regions are sometimes referred to as patterns.\",\n \"EXAMPLE 1 The source context comprises two sets of integers and the destination context one set of integers.\",\n \"The source region is the positive integers for the two sets, the mapping a multiplication of the two inputs accepted in the source region.\",\n \"The destination region is the result of the multiplication of the two positive numbers.\",\n \"EXAMPLE 2 The source context is the state of a logical variable and the region the value ‘true’ of that variable.\",\n \"The destination context is the state of the same variable and the mapping the ‘negation’ of the original value, that is ‘false’.\",\n \"EXAMPLE 3 The source context is the temperature and pressure inside a boiler and the destination context a list of commands triggering alarms and associated processes (that can be software based).\",\n \"The source region is the range of pressures and temperatures that trigger a ‘code red’ alert and the destination region the command triggering the ‘code red’ alarm.\",\n \"EXAMPLE 4 The source context and region is as in the example 3.The destination context is the list of the relevance status of the kuark and the region the actual relevance status of the kuark, based on the source region.\",\n \"That is, the ‘code red’ alert is given a relevance status (perhaps expressed as a probability) based on the environment.\",\n \"Kuark Properties The common properties of a kuark are: univeral id; title; explanation (multimedia); time and date created; author; time and date when last modified; person who last modified agent; version number; domain of discourse source context (what it knows about) 12; destination context 15; applicability source region or pattern 11 (can be identical to source context); relevance status to an enquiry (available, candidate, rejected, definite); mapping 13 (the process that produces the outcome based on the state of source context) outcome result of the mapping between source and destination context; the mapping determines the destination region or pattern; status (enabled/disabled/archived); state of source context at time of last firing (describes source context and region); state of destination context after last firing (describes destination region); links to other kuarks the hierarchical parent's universal id (more than one parents could be allowed); the id of the previous version kuark; the ids of the kuarks that are part of this kuark (see later for multi-kuark kuarks); the ids of the kuarks this kuark belongs to or is associated with (see later for multi-kuark kuarks).\",\n \"The property ‘links to other kuarks’ is the mechanism used to implement relationships between kuarks.\",\n \"The fourth link mentioned may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.\",\n \"Kuark Methods The main methods of a kuark are: monitor applicability (detect only what its context allows); determines whether environment fits within source region, if yes triggers/fires mapping; execute mapping (modifies destination state) mapping determines the outcome which is a subset of the destination context; create daughter/parent kuark; adopt existing kuark as daughter or parent; display internal state (contexts, regions, mapping); make copy each time it fires.\",\n \"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.\",\n \"Relevance Kuark Referring now to FIG.\",\n \"8, a relevance kuark 20 is a kuark 10 (as described previously) that determines the relevance of the environment.\",\n \"Depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping 23 that determines (calculates) the relevance of the environment.\",\n \"The relevance can be expressed as a probability.\",\n \"The destination context 15 is made of the different relevance values that the environment can take with respect to the source context and region.\",\n \"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.\",\n \"Procedure Kuark Referring now to FIG.\",\n \"9, a procedure kuark 30 is one that contains a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.\",\n \"The kuark executes its procedure mapping 33, when the environment is compatible with the source context and the region.\",\n \"That is, the input to the procedure is the source region in the source context.\",\n \"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.\",\n \"The procedure mapping can be a trivial mapping.\",\n \"It can simply display a fixed pattern and an explanation in a fixed destination context.\",\n \"The context item, see later, is an implementation of the trivial procedure kuark.\",\n \"Relevance kuarks tend to have the same destination context, whereas procedure kuarks typically have context that are strongly application dependent and that can vary within an application.\",\n \"Kuarks and Object Oriented Programming Kuarks can be implemented as classes and objects using an object oriented language such as Java.\",\n \"A kuark is a class in OOP and some of its properties are static, final, etc.\",\n \"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.\",\n \"Multi-Kuark Systems or Applications This section describes how kuarks can be used together to increase the power of applications.\",\n \"Multi-Kuark Kitems A system made of several kuarks is still a kuark with all the allowed properties and methods.\",\n \"However, the term kuark is reserved for simple (or elementary) kuarks, as described above.\",\n \"A multi-kuark kuark is named a kitem.\",\n \"Kitems are constructed as follows: the designer identifies the kuarks (or kitems) to be included in the new kitem; the designer clicks ‘construct kitem’.\",\n \"The construction process simply puts a wrapper around the kuarks and kitems to include in the new kitem.\",\n \"All the component kuark and kitems keep their identities.\",\n \"The new kitem can have its own properties and methods in addition to those of its component kitems.\",\n \"The new kitem has its own title and explanation, and can also have its own source and destination contexts and regions, and its own mapping.\",\n \"One can also view the new kuark as having undergone a construction process that carried the following operations: concatenates the source contexts; concatenates the destination contexts; concatenates the source regions; concatenates the destination regions; concatenates the mappings; concatenates the explanations.\",\n \"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.\",\n \"The kitem construction process also builds the list of kuarks or kitems that are part of the new kitem.\",\n \"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.\",\n \"Alternatively the kitem construction process can also be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.\",\n \"Kitems have explicit domains of discourse, the source and destination contexts, and explicit regions; kitems are ‘aware’ of their domains of discourse and when their knowledge, expressed as their mappings and destination regions, can be applied.\",\n \"Not all kuarks can be assembled in kitems as some concatenation may not be allowed; this issue is not treated further here.\",\n \"Refer later for different architectures for multi-kitem systems.\",\n \"Kitem Hierarchy When defining kitems for an application, it is often convenient to organise them as a hierarchy of objects, as in a file system for example.\",\n \"In fact, all kitems are defined within a hierarchical structure similar to that of the domain names on the Internet.\",\n \"Each kitem has a parent, except the root kitem which is the top of the tree.\",\n \"The hierarchical organisation of the kitems imposes no restriction on how they can be grouped (in multi-kitem systems) to achieve some purposes in an application.\",\n \"That is, composed kitems can be assembled out of any kitem from any location in the hierarchy.\",\n \"Kitems and Object Oriented Programming A kitem as outlined above, together with its GKMS features, mean that kitems can be implemented using an object oriented language, as objects or instances of the kuark or kitem classes.\",\n \"As indicated above, a kuark or kitem can be represented as an object using an object oriented language such as Java.\",\n \"A multi-kuark kitem as outlined above, can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.\",\n \"This means that kitem objects can be generated without programming.\",\n \"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.\",\n \"Rationale for Multi-Kitem Systems The advantages of building applications using kitems are very significant: the methods and properties can be used across the hierarchy (for example, a method to display the components kitems of a kitem can be re-used at all levels in the hierarchy of kitems); kitems provide a unified way for thinking and implementing applications; kitems can be fashioned as simple building blocks that offer maximum re-use; kitems implement knowledge at every level (in every kitem) that make up the application; knowledge permeates the design and the use of the application; applications can be largely soft-coded, instead of hard-coded; applications can be developed rapidly (RAD: rapid application development).\",\n \"Links Between Kitems Kitems can be linked.\",\n \"A link has a source or origin kitem and a destination kitem.\",\n \"The links can be stored inside the kitem or kuark, as a property of the kitem.\",\n \"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.\",\n \"In a link kitem, the source context contain the ID of the source kitem and the type of link (see above for list of possible links), and the destination regions contains the IDs of the corresponding links (the destination context is the domain of discourse of the source kitem).\",\n \"It is also possible, indeed often preferable, to have the source and destination contexts and regions for a kitem expressed via links.\",\n \"That is, one or several link kitems contain the source and destination context, and the regions (their kitems and values).\",\n \"Multi-Kitem Architectures This section discusses the architectures available for building multi-kuark or multi-kitem systems.\",\n \"In particular, these architectures can be used to build the display and executable kitems.\",\n \"Serial Architecture In the serial architecture 40, the destination context of one kitem 41 is used as the source context (or part of the source context) of another 42.More than two kitems can be chained, as illustrated in FIG.\",\n \"10.The state of the environment is used as input to kitem 41 (and perhaps to other kitems).\",\n \"Similarly, the serial kitem output is the output from kitem n 45 (and perhaps from other kitems).\",\n \"In the most simple case, the source context is the source context of kitem 41 and the destination context the destination context of kitem n. In general, the source context of the serial kitem is a subset of the source contexts of all its ‘internal kitems’.\",\n \"Similarly for the destination context and for the regions in these contexts.\",\n \"The source context of an internal kitem can comprise some elements from the environment in addition to the output from the previous kitem.\",\n \"The serial kitem is a kitem and has all the properties of kitems discussed above.\",\n \"Parallel Architecture Referring now to FIG.\",\n \"11, a parallel kitem 50 is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.\",\n \"The power of parallel kitems derives from the links that can be established between its internal kitems.\",\n \"The objective is to ensure that the information and knowledge contained in two kitems becomes linked.\",\n \"For example, a kitem could describe clients and another projects; and a link would connect a client with a project.\",\n \"A link has an origin and a destination.\",\n \"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.\",\n \"When the class of a parallel kitem is defined, the operator has to specify which kitems are instantiated as objects when the parallel kitem object is created.\",\n \"In linked kitems, it is convenient, but not mandatory, to instantiate the origin kitem but not the destination kitem.\",\n \"Links can be unidirectional or bidirectional.\",\n \"For example, from clients to projects, or from clients to projects and from projects to clients.\",\n \"Hierarchical Kitems The kitems that are part of a parallel (or serial kitem) keep their properties, notably the one identifying their parents.\",\n \"Therefore the parallel kitem can, in fact, contain a hierarchy of kitems even if they are organised in a flat hierarchical (that is, parallel) way in the parallel kitem.\",\n \"A Knowledge Application as A Parallel Architecture Kitem An application or knowledge base is a set of knowledge items (kitems) defined with respect to a domain of discourse (source and destination contexts and mappings available).\",\n \"These kitems are arranged in parallel and some of them can be linked.\",\n \"For example, in an application all the knowledge items are typically linked (that is refer) to the same domain of discourse, as explained above.\",\n \"Hybrid Architecture It is possible to define a hybrid architecture, with the hybrid kitem containing some kitems linked in a serial way and others in a parallel way.\",\n \"Meta-Kitems and Meta-Knowledge In FIGS.\",\n \"10 and 11, each kitem is a class that supports the creation of objects, or knowledge elements.\",\n \"It is desirable to be able to define a class that can have attributes and methods that span more than one of the kitems contained in the parallel, serial or hybrid kitem.\",\n \"This can be achieved by giving the outside kitem (eg: the serial, parallel or hybrid kitem) access to all the properties and methods of the contained kitems, in addition to the properties that belong only to the outside kitems.\",\n \"This means that it is possible to define meta-knowledge about the kitems contained in the outside kitems.\",\n \"The outside kitem, when instanced as an object, can observe the behaviour of the kitems in it and take actions when necessary.\",\n \"The outside kitems are referred to as meta-kitems able to express meta-knowledge.\",\n \"Display and Executable Kitems In this section we consider two different types of very useful kitems: the display kitem and executable kitem.\",\n \"We show that each can be constructed using one relevance kuark and one procedure kuark.\",\n \"Display Kitem The display kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark is a relevance kuark.\",\n \"It uses its region and the state of the environment to determines the relevance of the display kitem.\",\n \"The second kuark has the relevance (the output of the first kuark) as source context and region.\",\n \"The material to be displayed comprises the destination region and the explanation (the destination region can be the explanation.\",\n \"Context Kitem Contexts and regions are made of elements or objects that describe the contexts and regions.\",\n \"Each such object can have values.\",\n \"Here we show that kitems are suitable for describing and implementing these context objects.\",\n \"A context object is a kitem as shown below (only some properties are mentioned): kitem tile → object title source context → object values source region → instantiated values destination context → same as source context destination region → same as source region mapping → maps source to destination (contexts and regions) explanation → optional.\",\n \"Explanation Kitem An explanation object is a kitem as shown below (only some properties are mentioned): kitem title → explanation title (can be ‘explanation’) source context → not used source region → not used destination context → not used destination region → not used mapping → not used explanation → multimedia explanation.\",\n \"Context Element A context element is a kitem made of, typically, a context kitem and an explanation kitem (some context items can have more than one explanation).\",\n \"To create such a kitem, the designer defines a parallel kitem using the procedure described in the GKMS patent.\",\n \"It is also possible to have a context item made of a single context kitem, by using its explanation instead of the explanation kitem.\",\n \"This solution does not work if more than one explanation is required.\",\n \"For example, an explanation relating to the meaning of the context item and another relating to its use in a knowledge item.\",\n \"Context Space (Source Or Destination) The context space is a kitem made of context items (also kitems) using the parallel architecture.\",\n \"Typically no links are used between context items.\",\n \"Executable Kitem The executable kitem is a serial kitem comprising two kuarks.\",\n \"The first kuark uses its region and the state of the environment to determines the relevance of the executable kitem.\",\n \"The second kuark has the relevance (the output of the first kuark) as region and as part of its source context.\",\n \"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.\",\n \"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.\",\n \"Knowledge Item and Knowledge Item Definition A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.\",\n \"Defining a knowledge item, as described in the GKMS patent, involves defining a region in the source context (and other things).\",\n \"This is done using the context space kitem, that is made of context objects (or elements).\",\n \"The architecture of a knowledge item is normally that of a parallel kitem.\",\n \"It is often desirable to explain why a certain context element is part of a knowledge item or how to use or interpret this context element when it is attached to the knowledge item.\",\n \"This can be achieved simply by giving the user defining the knowledge item the facility to add an explantion kitem to the list of kitem that are part of the knowledge item, and linking this explanation kitem to the context element (kitem) in question.\",\n \"Alternatively, the explanation can be part of the context element kitem.\",\n \"That is, the explanation kitem is already linked to the context element (with reference to OOP, the context element class ‘contains’ the explanation).\",\n \"A knowledge item is defined with respect to a domain of discourse, defined by the source and destination context and the mappings available at definition time.\",\n \"It would not be efficient to include the contexts kitems in the knowledge item.\",\n \"Instead, the knowledge item is linked to the context space kitems.\",\n \"This is in agreement with the definition of links in the parallel architecture kitem.\",\n \"Kitem Hardcoding We have discussed how kitems can be implemented using other kitems.\",\n \"However it is always possible to implement a kitem as a single hardcoded entity.\",\n \"For example, in the GKMS patent, the context elements and the knowledge items were implemented as single entities with their specific architectures.\",\n \"When this is done, many of the modular and flexible features of the kuark-kitem model are lost.\",\n \"When a similar effect is desired, it is preferable to implement the kitem as a permanently compiled multi-kitem kitem (see later).\",\n \"Knowledge Processing in Multi-Kitem Systems A multi-kitem system is a system comprising a set of kitems representing the body of knowledge about a certain domain of discourse.\",\n \"Typically the domain of discourse is identical for all the kitems in the system, although it is not essential.\",\n \"Such a system is identified as a knowledge module or database.\",\n \"Knowledge processing involves the system (or server) running a question-answer session.\",\n \"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.\",\n \"The steps involved are: identify the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asks these questions; on the basis of the answers to the questions, determine a) whether any answers can be found, and b) the next best questions to ask.\",\n \"The processing refers to: the identification of the correct answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most promising provisional answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most important questions to ask on the basis of the information available.\",\n \"Searching for relevant or important knowledge Knowledge access in a multi-kitem system (a knowledge module) follows the same pattern as for a GKMS knowledge base; that is, it is a question-answer session or consultation.\",\n \"The consultation process is managed with, and recorded in, a document.\",\n \"This document can also be a kitem called the consultation kitem.\",\n \"That is, the consultation kitem has all the properties and methods of the kitem class.\",\n \"All the utilities for kuarks and kitems can be used with consultation kitems.\",\n \"Searching involves identifying those kitems that have source regions compatible with the enquiry (see GKMS patent for meaning of compatible).\",\n \"It means determining the ‘relevance status’ (available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.\",\n \"Searches can be synchronous or asynchronous.\",\n \"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.\",\n \"Synchronous Searches Database Search The database search is a SQL search or equivalent.\",\n \"This means that the kitems are stored as records in a database.\",\n \"This is not discussed further in this document Full Text Search The full text search has to process each relevant field in each record where each record describes a knowledge element.\",\n \"This process can be slow; it is not discussed further in this document.\",\n \"Context Tree Search with Processing Delegated to A Search Agent The source context is organised as a tree structure.\",\n \"A source region, which determines whether a kitem will fire (firing depends on the region's compatibility with an enquiry), is a subset of the source context.\",\n \"Each kitem (eg: knowledge element) can be indexed against the context tree.\",\n \"This means that each object in the context possesses a list of the kitems it is associated with.\",\n \"The context tree is a formalisation of one of the kuarks'properties (see above).\",\n \"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.\",\n \"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"The search process updates the status of the kitems according to the enquiry.\",\n \"Context Tree Search with Processing Delegated to Each Kitem The search process (also a kitem) flags these elements in the context tree that correspond to the questions answered so far.\",\n \"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.\",\n \"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.\",\n \"The search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).\",\n \"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.\",\n \"At the implementation level, the context tree is a modified context space kitem.\",\n \"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.\",\n \"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.\",\n \"Each kitem updates its status according to the enquiry and informs the searc process.\",\n \"Asynchronous Searches Processing Delegated to Each Kitem In asynchronous searches, the processing is distributed and controlled by each item.\",\n \"There is no central authority that decides what to do when.\",\n \"Each kitem (or agent) monitors its environment and reacts appropriately.\",\n \"In doing so, it may change the environment and trigger new actions by other kitems.\",\n \"This is in contrast to synchronous searches in which it is the search process that determines whether a kitem is applicable or not.\",\n \"In particular, in asynchronous searches it is each kitem that determines whether it is applicable to the environment as it is detected via the source context and the source region.\",\n \"In asynchronous searches, the source region determines whether the outcome of the kitem becomes applicable.\",\n \"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.\",\n \"The state of the relevance region determines what happens to the application region.\",\n \"In the case of non-executable mappings (or display mappings) the process is as illustrated in FIG.\",\n \"8; the relevance mapping ‘releases’ the destination region to the kuark output.\",\n \"State of Environment (or Context) Communicated to the Items As indicated above, each kitem has a unique ID or address that determines its location in a distributed memory space or network (such as the Internet for example).\",\n \"It also possesses a set of applicability addresses that correspond to the kitems in its source region.\",\n \"During processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.\",\n \"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.\",\n \"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.\",\n \"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).\",\n \"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.\",\n \"It is up to the enquiry process to manage all the replies it gets from the network.\",\n \"The solution outlined here is applicable to distributed processing.\",\n \"The Kitems Seek to Know the Status of their Source Context Another model for asynchronous searches is to give even more responsibility to the distributed kitems than in the previous description.\",\n \"In this model, each kitem has the address of each of the context objects in its context and in its region (a subset of the context).\",\n \"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.\",\n \"This model means that the kitem takes the intiative (this has some important applications not discussed here).\",\n \"However during searching, the model imposes much traffic on the network that is of no benefit, unless the context object has changed since the last status check.\",\n \"Another problem is that there may be an unacceptable delay between status checks, during which time, if the status has changed, the kitem doing the interrogating cannot determine its applicability.\",\n \"Cascading Knowledge Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.\",\n \"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.\",\n \"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.\",\n \"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.\",\n \"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).\",\n \"Validity of the Relevant Knowledge For simple knowledge bases on a single server it is a relatively simple matter to ensure that all kitems they contain have the same domain of discourse.\",\n \"This, however, does not apply for large or distributed knowledge bases where the knowledge comes from different sources.\",\n \"In general, therefore, there is no certainty that all the knowledge items (kitems) accessed during a search have the same source and destination contexts.\",\n \"In fact it is unlikely.\",\n \"This means that a user inspecting a definite kitem (with respect to an enquiry) may not know what the domain of discourse of that kitem might be.\",\n \"This information is very important as it defines the context for the knowledge in the kitem in question.\",\n \"A solution to this situation is to give the user the ability to inspect the source and destination context of every kitem that is provided by the search process either as a candidate or definite kitem.\",\n \"Identifying the Most Promising Questions Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Context objects which index a large number of available or candidate kitems are potentially very important when used as questions.\",\n \"This is because their answers have the potential to impact on the relevance status of a large number of kitems.\",\n \"This indicates that the importance of these context objects as questions in an enquiry is determined by the number of kitems with status available or candidate they index.\",\n \"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.\",\n \"Therefore the questions are selected dynamically.\",\n \"Context Tree Search with Processing Delegated to Each Kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.\",\n \"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"Asynchronous Searches Processing Delegated to Each Kitem The enquiry process (that sends the enquiry over the network for kitems to process it) performs several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.\",\n \"In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.\",\n \"Search for Hidden Knowledge Hidden knowledge refers to knowledge that may be relevant to an enquiry but that is not yet linked to any question answered in the enquiry process so far.\",\n \"The question is how to identify this hidden knowledge and how to extract its ‘best questions’.\",\n \"In situations where the domain of discourse of a body of knowledge is known, one can simply identify the kitems with the largest number of indexed kitems with relevance status available or candidate (see context tree above).\",\n \"There is no simple solutions when the domain of discourse of a body of knowledge is not known.\",\n \"Processing Speed Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Synchronous searches can be very quick as they are under the control of one process that can be optimised, based on known conditions in the server.\",\n \"Context Tree Search with Processing Delegated to Each Kitem This processing can be distributed across a network, but it is infrequent.\",\n \"This processing is slower than the previous one as it imposes communications overheads between kitems (those doing some processing and the enquiry process).\",\n \"Nevertheless, the enquiry process runs on a single server and can be optimised.\",\n \"When the process is distributed, several kitems can process simultaneously; this can partially offset the communication overheads.\",\n \"Asynchronous Searches Processing Delegated to Each Kitem This processing is slowest unless the number of kitems doing processing is large enough to offset the communication overhead between kitems and the network delays.\",\n \"Compilation Compilation is the process that takes a collection of kitems and organises them for the purpose of speeding up processing.\",\n \"The result of compilation is a kitem.\",\n \"In general, compilation is applied to any kitem.\",\n \"In practice it is often applied to all the kitems that belong to a domain of discourse (also a kitem) on a single server.\",\n \"That is, all the kitems involved have domains of discourse that are subset of the compiled domain of discourse.\",\n \"Compilation is also applied to a group of kitems contained in a serial, parallel or hybrid kitem.\",\n \"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.\",\n \"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.\",\n \"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.\",\n \"The processing model for the multi-agent systems described in this document imposes, like the Java language, that kitems call other kitems that in turn may call other kitems, etc, until the processing can be completed.\",\n \"Once this is done, the results of the processing need to travel up the hierarchy of kitems, back to the initiating process.\",\n \"The compilation process is designed to speed up the ‘down-hierarchy’ and ‘up-hierarchy’ travelling described above.\",\n \"The context tree compilation achieves this purpose by ‘flattening’ the hierarchy to a single branch tree, where each context element (or node) in the branch contains all the kitems that have that context element as part of their regions.\",\n \"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.\",\n \"Therefore they are included in the compiled kitem.\",\n \"This is faster than the partial compilation.\",\n \"Compilation can be be carried out to different depths: 1.build context tree at runtime; 2.get regions of indexed kitems in the context tree at runtime; 3.include regions of indexed kitems in the context tree; Compilations of type 1 and 2 above are just-in-time compilations.\",\n \"They updates the context tree each time the search process is triggered; permanent compilation produces the context tree and leaves it untouched until another compilation is carried out.\",\n \"Agents as Kitem Systems An agent, as illustrated in FIG.\",\n \"5, can be represented as a kitem 60 with the structure illustrated in FIG.\",\n \"12.: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.\",\n \"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.\",\n \"It is recommended but not essential that the kitems that comprise the agent operate in a synchronous mode.\",\n \"Internal synchronous processing is possible, even if a multi-agent systems behaves asynchronously.\",\n \"As an agent is a kitem, a multi-agent system is a multi-kitem system with a parallel architecture and continuous asynchronous processing.\",\n \"Kitems Needed for A Workable Solutions The kitems needed to produce a workable implementation for knowledge management are shown below: Context Management Context Elements A context element is a display kitem (a serial kitem made of two kuarks).\",\n \"Source and Destination Contexts A source or destination context kitem is a parallel display kitem.\",\n \"Its members comprise the list of all the context elements defined in the domain of discourse.\",\n \"Context Executable Kitems Edit a context element Edit a context space Display a context space as a tree A source or destination context tree is an executable kitem.\",\n \"The display kitem can display the elements that belong to the source and destination kitems.\",\n \"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.\",\n \"Knowledge Definition Knowledge Editing Kitem The knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.\",\n \"As destination space: the source and destination contexts.\",\n \"As source region: a subset of the source context that defines the applicability of the knowledge kitem.\",\n \"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.\",\n \"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.\",\n \"An explanation.\",\n \"Knowledge Access and Display Search Kitem The search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.\",\n \"Enquiry or Consolation Kitem The enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.\",\n \"), the ids of the definite kitems.\",\n \"Display Kitem The display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.\",\n \"Administration of Contexts and Other Kitems The hierarchical (tree) structure of the kitems gives a very convenient way of controlling user access to kitems: the administrator can decide which branch of the hierarchy users can have access to and the type of access for the branch and each kitem in the branch; access control can also be carried out at the level of the kitem.\",\n \"Dynamic Kitem Management The objective of dynamic kitem management is to be able to manage the integrity of the knowledge base when a kitem is edited.\",\n \"This issue is important as kitems (eg: a context kitem) can be used in many knowledge elements and a change in such a kitem can have impacts on the validity of these knowledge elements (from a knowledge viewpoint, not operational viewpoint).\",\n \"It is essential that the users are not left in the dark about the potential ramification of their changes and that they are helped in checking the validity of some affected kitems.\",\n \"As much as possible, the system should ensure the integrity of the knowledge itself.\",\n \"The process for achieving the above is to: use the enquiry process (by the system or the user) to retrieve all kitems that could be affected (the system or the user can specify the profile of the kitems affected by the change); make the changes automatically if acceptable; or ask the user to review/recertify the kitems affected (the system needs to provide guidance in this process).\",\n \"The profile mentioned above refers to the values given to kitems in the source and destination context.\",\n \"The system treats these values as an enquiry and retrieves all the kitems that are compatible with the enquiry.\",\n \"Change to properties All the properties of the kitems can be changed.\",\n \"Potentially, all the kitems that are linked to the kitem being edited are affected.\",\n \"Some properties and the way their editing can be handled is listed below.\",\n \"Change to Source Context and Region If a new context object is added: the system asks the user to specify the profile of the kitems that may be affected, the system retrieves these kitems and the user is asked to recertify them.\",\n \"If an existing context object is edited or deleted: the system retrieves all the kitems that are related to this kitem (that contain it as part of a link for example) and asks the user to recertify them and to edit them if necessary before doing so.\",\n \"Change to Destination Context and Region As for a change to the source context and region.\",\n \"Change to Mapping The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.\",\n \"Change to Status (Enabled/Disabled/Archived) The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.\",\n \"Change to the ‘Links to Other Kitems’ No checks required, the change is localised.\",\n \"Consistency Issues Consistency issues may arise in a knowledge base when some kitems have the following features: kitems have overlapping (at least one common kitem with at least a common value) or identical destination regions with non-identical source regions; kitems have disjointed destination regions with overlapping or identical source regions.\",\n \"The system can easily have an agent that checks all the kitems in a knowledge base and identifies these kitems that may be inconsistent.\",\n \"The system presents the kitems to their authors for editing if necessary and recertification.\",\n \"Missing Knowledge Missing knowledge refers to parts of a domain of discourse for which there is no knowledge or inadequate knowledge.\",\n \"When the domain of discourse for a set of kitems is explicit, it is a simple matter to check that the union of the source regions in the kitems cover the entire source context.\",\n \"If it is not the case, it means that the some additional knowledge is required.\",\n \"The user can be informed and the location of this missing knowledge presented.\",\n \"The location of the missing knowledge can be pointed out on each dimension (or context kitem) of the source context.\",\n \"The user selects a context item and a value (or range of values) for which there is no region, and the system responds by showing the other context items and their values for which there is no region.\",\n \"The user then selects a second context item and gives it some values (where there is no region), and so on for the other kitems if desired, until the user is confident of being able to add some missing knowledge.\",\n \"New Kitems When a new knowledge element is being saved (the user has finished its definition), the system checks that it is consistent with the other knowledge elements in the knowledge base (see section 12.2).\",\n \"The system also retrieves all kitems that overlap with the new kitem's source and/or destination regions.\",\n \"The system informs the user that these kitems may be affected (their knowledge may be superseeded by the new kitem) and presents them to the user for checking.\",\n \"The system can also ask the user to define the profile of the kitems that may be superseeded by the new kitem.\",\n \"Terminology Term Explanation Applicability region The region in the source context that determines whether the kitem is applicable.\",\n \"Application context The context for the application made of the source or space and destination contexts.\",\n \"Available kitem The status of a kitem that is available for an enquiry (the other status are definite, candidate and rejected.\",\n \"Candidate kitem A kitem that is compatible with the enquiry so far but for which the applicability conditions are not completely met.\",\n \"Definite kitem A kitem that is compatible with the enquiry so far with its applicability conditions completely met.\",\n \"Destination context The space of solutions considered by an application or a kitem.\",\n \"Domain of discourse The space of problems and solutions (or situations and outcomes) considered by an application or a kitem; it is made of the source and destination contexts.\",\n \"Enquriy process The agent or process that controls the question- answer session or consultation.\",\n \"Executable mapping A mapping that takes an input from the source context and applies an algorithm to it to produce an outcome.\",\n \"Kitem A object or element that comprises a source context and region, a destination context and region, and a mapping.\",\n \"Knowledge item A kitem that is used to express knowledge.\",\n \"Kuark An elementary kitem (the smallest building block) that can be used to build other kitems and/or complete applications.\",\n \"Mapping The transformation that is applied to a source region to produce a destination region or outcome.\",\n \"Outcome Another term for destionation region or solution.\",\n \"Parallel kitem A kitem comprising other kitems organised in parallel.\",\n \"Rejected kitem An kitem that is not compatible with an enquiry.\",\n \"Relevance context The destination context that expresses the range of possible relevance for kitems in an application.\",\n \"Relevance region A region in the relevance context.\",\n \"Relevance status The relevance of a kitem with respect to an enquiry (see GKMS patent for details).\",\n \"Serial kitem A kitem comprising other kitems organised in series.\",\n \"Solution Another term for destination region or outcome.\",\n \"Source context The space of outcomes considered by an application or a kitem.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10450975"}}},{"rowIdx":292,"cells":{"text":{"kind":"list like","value":[["Support and guiding structure for friction element in disc brakes","Method and apparatus for friction element control in spot-type automotive disc brakes provides for use in a disc brake (10) of the kind comprising a fixed caliper (32) and at least one axially slidable disc (12, 14), a friction element mounting in which the friction clement (20) is supported on lengthwise guides (52) in the region of its circumferentially spaced ends (62, 64) and is further supported by an axially-extending abutment element (66) fixed to its upper edge and received in a channel (68) in the brake caliper (32).","The abutment element an its channel serve to provide improved support for the friction element in resisting torque applied to the friction element during braking."],["1-9.","(canceled) 10.A method of mounting a friction element in a spot-type automotive disc brake, the disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation means therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at least one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and said method comprising; g) providing abutment structure adapted to resist torque applied to said at least one friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and the method comprising causing same to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.","11.A method of mounting a friction element in a disc brake comprising an axially slidable disc, the method comprising providing guide structure adapted to act at two spaced locations on said friction element between said friction element and a non-rotatable mounting therefor, and said method comprising providing abutment structure located between said spaced locations on said friction element, and said method comprising causing said abutment structure to resist torque applied to said friction element during use of said brake.","12.A brake friction element comprising abutment structure located between guide structure at spaced locations of said friction element and adapted to resist torque applied to said friction element during use in a brake system.","13.A spot-type automotive disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation device therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at lest one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and g) abutment structure adapted to resist torque applied to said at least on friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and adapted to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.","14.A disc brake comprising an axially slidable disc, and guide structure adapted to act at two spaced locations on a friction element and between said friction element and a non-rotatable mounting therefor, and said brake comprising abutment structure located intermediate said spaced locations and adapted to resist torque applied to said friction element during use of said brake.","15.A brake friction element comprising abutment structure located between circumferentially spaced ends of said friction element and adapted to resist torque applied to said friction element during use in a brake system.","16.A spot-type automotive disc brake according to claim 13, wherein said abutment structure is located generally radially in-line with the line of action of said actuation device.","17.A spot-type automotive disc brake according to claim 13, wherein said abutment structure comprises an abutment element fixed at the upper periphery of said at least one friction element and extending lengthwise in the direction of sliding brake-actuating motion of said friction element and slidingly received in a complementary channel formed in said non-rotatable mounting or caliper.","18.A brake according to claim 17, wherein said abutment element having a length-to-width ratio of at least in the range of 1 to 1 to 3 to 3."],["This invention relates to friction element control in disc brakes.","An example of the application of the invention is to the control of friction elements in spot-type automotive disc brakes, particularly such brakes of the kind in which a fixed caliper and sliding friction elements are employed.","The invention may find an application outside the strict limits of the technical field of the disc brakes disclosed in the specific embodiments described below, but principally the invention is concerned with controlling the movement of friction elements in relation to the associated structures of a disc brake, particularly the disc or discs themselves (which rotate during use), and the relatively fixed or non-rotatable structures such as that of the caliper on which the friction elements are mounted.","Generally, the friction elements with which the invention is concerned are those which are not actually fixed to a position-defined structure such as the end plate of a fixed caliper and thus, the invention is particularly applicable to friction elements of the kind employed as the middle one of three friction elements interleaved with twin discs in a fixed caliper/sliding disc assembly as disclosed below.","In such a disc brake, a need arises for excellent dynamic control of the movement/position/attitude of the friction elements during brake application in order to maximise the effectiveness of the brake and minimise the extent of uneven wear during use.","In this connection, we have already disclosed in co-pending applications arrangements whereby the friction elements are provided with anti-tilt systems to maintain as far as possible the parallel and co-planar relationship of the friction-producing faces of the friction elements and of the brake discs themselves, and this has largely been achieved by means of spring arrangements exerting substantial resilient forces on the sliding disc assemblies and likewise on the sliding friction elements assemblies to achieve the necessary degree of control under the dynamic conditions of use.","Developments thus far have enabled us to provide a satisfactory degree of control in respect of those functions which may be conveniently grouped under the heading “anti-tilt control”, but we have discovered that there is a further area in relation to friction element control which requires attention in order to provide further advances in relation to the dynamic aspects of the performance of the disc brake itself.","These further factors which require attention concern principally the question of the response of the friction elements to the effect of the torque exerted thereon by the frictional forces during brake application.","Thus, it will be readily understood that upon brake application and frictional engagement of the friction pads with the rotating disc, the applied forces acting on the friction elements produce a torque due to the rotary motion of the brake discs and such torque or turning effect on the pad and thus on its backing plate and thus on the friction element as a whole can cause destabilisation of the friction elements leading to a reduction in brake efficiency, or possibly uneven wear of the friction pads, and an object of the present invention is to provide improvements in this regard in relation to friction elements for disc brakes, or indeed improvements generally.","According to the invention there is provided a method and apparatus as defined in the accompanying claims.","In embodiments of the invention described below the axially slidable friction elements are provided with the basic guide means and supporting means whereby they are able to execute supported axial movement for frictional engagement with the brake discs.","However, in addition, the slidable friction elements are provided with abutment or torque-transmitting means in the form of an abutment or torque-transmitting element and an associated abutment or torque-transmitting channel in which the abutment or torque-transmitting element is received.","These structures (provided respectively at the upper edge of the friction element and at the underside of the brake caliper) serve to provide an important abutment or torque-transmitting function for the friction elements whereby the potentially twisting effect of the braking torque applied by the rotating brake discs is effectively neutralised by the abutting engagement of the abutment or torque-transmitting element with its corresponding abutment or torque-transmitting channel.","Moreover, although it would be possible to provide the abutment or torque-transmitting element as merely an upstanding peg or the like structure having the same thickness as the remainder of the friction element backing plate (and some benefit in terms of torque neutralisation would undoubtably be available), in the embodiments the abutment or torque-transmitting element is constructed so as to have a length dimension (extending lengthwise in the direction of the thickness of the backing plate material) whereby its torque neutralisation effects are significantly enhanced.","It also potentially assists in guiding its friction element.","A particular feature of the embodiments is the location of the abutment or torque-transmitting element in relation to the line of action of the actuation means for the brake, such as the centre line of the actuating piston.","In the embodiments, the abutment or torque-transmitting element is positioned directly above the line of action of the piston, and this is the preferred position.","In the case where twin actuating pistons are provided, then a central position between the pistons (and thus effectively at the centre of pressure produced by the pistons) is preferred.","Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which: FIG.","1 shows, in block diagram format a spot-type automotive disc brake comprising a pair of axially slidable discs and associated friction elements, an actuating mechanism therefor and a fixed caliper or bridge structure overlying same; FIG.","2 shows on a somewhat larger scale an exploded perspective view of the caliper and an associated friction element with an abutment or torque-transmitting element shown at an upper edge of the friction element together with an associated abutment or torque-transmitting channel to receive same; FIG.","3 shows a side elevation view of the assembly of FIG.","2 in its assembled condition; and FIG.","4 shows a plan view, as seen in the direction of arrow IV in FIG.","3, of the assembly of FIGS.","2 and 3.As shown in FIG.","1 a spot-type automotive disc brake 10 comprises a pair of rotatable brake discs 12, 14, a rotatable mounting 16 for the brake discs to permit rotation of the discs and which is adapted to drive the brake discs and have exerted thereon the braking effect by the discs when the disc brake 10 is actuated.","Two pairs of friction elements 18, 20 and 22 are provided and are adapted to frictionally engage braking surfaces 24, 26 provided at opposite sides of brake discs 12, 14 to effect braking on actuating actuation means for the brake.","Central friction element 20 is double-sided for frictional engagement with the mutually-inwardly facing braking surfaces 24, 26 of brake discs 12, 14 and is provided with appropriately facing friction pad material accordingly.","Friction elements 18, 20, 22 comprise (as shown in FIGS.","3 and 4) in each case a generally flat metal backing plate 28 and secured thereto and standing proud thereof a body of friction material of known construction for high durability frictional engagement with the relevant braking surface of the relevant brake disc.","In the case of central friction element 20, the friction material is provided at both faces of the backing plate 28.Brake discs 12, 14 are axially slidable in use with respect to their rotatable mounting 16 under the action of friction elements 18, 20, 22 and the actuation means (to be described below) therefor during braking.","For example the brake discs may be keyed to the rotatable mounting or hub 16 at three or more locations and resilient means may act there between.","A non-rotatable mounting 32 for friction elements 18, 20, 22 is provided comprising a caliper or bridge structure 34 which is mounted on a fixed structure of the vehicle to be braked, for example on the wheel mounting and which straddles the brake discs 12, 14 and also provides a mounting for actuation means 36,38 (indicated diagrammatically) which applies inwardly directed braking forces to the outer friction elements 18, 22, thereby causing frictional engagement with the brake discs 12, 14, and slight sliding movement of those discs with respect to their rotatable mounting 16.In FIG.","1 of course it can be seen that the clearances between the structures have been greatly exaggerated for simplicity of diagrammatic illustration.","The actuation means 36, 38 could comprise a pair of piston and cylinder assemblies.","However only one such is strictly needed since the actuation means can be one-sided with a fixed structure at one side or the other of the assembly of discs and friction elements (which fixed structure could simply be a stop extending from caliper 34), and against which fixed structure the assembly is pushed by the single actuation means.","Further details in this regard may be found in our co-pending applications WO 98/26192 (docket 2558).","Non-rotatable mounting 32 for the friction elements 18 to 22 is adapted to permit sliding movement of the friction elements into and out of frictional engagement with the brake discs while resisting movement of the friction elements under the action of frictional forces generated by engagement of the friction elements with the discs 12, 14.Turning now to the details of the structures shown in FIGS.","2, 3 and 4, we mention first that the friction element illustrated is taken to be the central friction element 20 which (though not shown in FIG.","2) is provided as shown in FIGS.","3 and 4 with pads 40, 42 of friction material facing axially at its opposite sides.","Other structures seen in the drawings include the casting 44 to which caliper 34 is secured and from which the actuating piston 46 can extend under the action of the usual hydraulic and driver-controlled brake actuation system having a line of action 48 which of course intersects the friction elements, this location being shown in FIG.","2 at 50 which identifies the line of action of the centre of piston 46.As also shown in FIGS.","2 to 4 axially-extending primary abutment or torque-transmitting means 52 is provided in relation to caliper 34 and is adapted to act on the slidable friction elements 20, 22 (end friction element 18 being secured or fixed to an end plate (not shown) extending downwards from the outboard end of caliper 34).","Primary guide means 52 comprises axially-extending guide rails 54, 56, one at each side of caliper 34 and adapted to receive corresponding hook elements 58, 60 provided at spaced locations namely the opposite circumferentially-spaced ends 62, 64 of friction element 20.Thus, guide rails 54, 56 and hook elements 58, 60 serve to ensure that friction element 20 (and likewise sliding friction element 22 in a similar way) is able to hang from the caliper for sliding axial movement lengthwise thereof towards and away from the brake discs 12, 14.In addition to the guide rails 54, 56 and hook elements 58, 60 there is provided further abutment or torque-transmitting means comprising an abutment or torque-transmitting element 66 located intermediate spaced locations namely the opposite ends 62, 64 of the friction element and adapted to enable the friction element to resist torque from frictional forces applied to it during use of the brake, while permitting the friction element to move towards and away from the brake discs.","Abutment or torque-transmitting element 66 is received in an abutment or torque-transmitting channel 68 formed in the underside 70 of caliper 34.It will be noted from FIG.","2 that abutment or torqur-transmitting element 66 is generally radially in-line with the line of action 48 of actuating piston 46 and indeed is directly above the piston centre line point 50.FIG.","2 of the drawings shows the proportions of abutment or torque-transmitting element 66 in a clear manner with respect to the remainder of the friction element structure.","Thus, the abutment or torque-transmitting element has an axial length L and a traverse width W such that L/W lies in the range of 2 to 3.In general, the ratio L/W should preferably be at least 1 and more preferably still in the range of 1 to 3.Such a ratio has the effect of maximising the abutment or torque-transmitting influence of the abutment or torque-transmitting element in relation to frictionally-developed torque forces applied to the friction element during use, thereby minimising the tendency for taper wear to develop in the pads 40, 42 of friction material.","The significance of the aspect ratio L/W in terms of maximising the torque-transmitting effect of the abutment or torque-transmitting element is discussed above.","So far as the length aspects of the dimensions of the abutment element 66 are concerned, the following applies.","As shown in FIG.","3, there is shown at 72 an indication of the possibility of friction element tilt in an axial plane which can occur when the brake is in its off condition.","Such tilting is limited by engagement of abutment element 66 with the top of groove 68.The possibility of tilt in the mode indicated at 72 is decreased with increasing length of the abutment element 66.Likewise, a similar tilt-inhibiting effect is exerted by minimising clearance between the top of the abutment element 66 and the top of groove 68.In the on condition of the brake, as indicated in FIG.","3, the brake-applying force Fp is indicated at 74 and acts along line 48 on the friction element 20 and has a turning (or taper wear) moment Fp×xp at the element 66 where xp is the radial distance (identified at 76) of off-set of line 48 from the top of channel 68.This force is resisted by a force at the end of the abutment element 66 acting on the top of groove 68 accordingly.","This function likewise is enhanced by increasing the length of abutment or torque-transmitting element 66.In use, disc brake 10 operates in substantially the usual manner, which is implicit from the above description, so far as concerns the general mode of frictional engagement of the relevant surfaces.","So far as guidance of the friction elements during use is concerned, the friction elements are supported on guide rails 54, 56 through hook elements 58, 60 and frictionally-developed torque forces applied to the pads 40, 42 of friction material which tend to produce twisting forces arising from the rotation of the brake discs are largely taken out of the friction element structure by the abutment or torque-transmitting element 66 and the engagement of same with the side walls of abutment or torque-transmitting channel 68 in which the abutment or torque-transmitting element is a close sliding fit.","Indeed, in principle, the tolerances in relation to the sliding fit of abutment or torque-transmitting element 66 within abutment or torque-transmitting channel 68 are tighter (meaning a closer fit) than exists between hook elements 58, 60 and guide rails 54, 56.As a result, wear on the pads 40, 42 or friction material is greatly improved in terms of its uniformity throughout the total working area of the friction material.","Amongst other modifications which could be made in the above embodiment while remaining within the scope of the appended claims are changes to the shape and dimensions and exact location of the abutment or torque-transmitting element 66 in relation to caliper 34."]],"string":"[\n [\n \"Support and guiding structure for friction element in disc brakes\",\n \"Method and apparatus for friction element control in spot-type automotive disc brakes provides for use in a disc brake (10) of the kind comprising a fixed caliper (32) and at least one axially slidable disc (12, 14), a friction element mounting in which the friction clement (20) is supported on lengthwise guides (52) in the region of its circumferentially spaced ends (62, 64) and is further supported by an axially-extending abutment element (66) fixed to its upper edge and received in a channel (68) in the brake caliper (32).\",\n \"The abutment element an its channel serve to provide improved support for the friction element in resisting torque applied to the friction element during braking.\"\n ],\n [\n \"1-9.\",\n \"(canceled) 10.A method of mounting a friction element in a spot-type automotive disc brake, the disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation means therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at least one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and said method comprising; g) providing abutment structure adapted to resist torque applied to said at least one friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and the method comprising causing same to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.\",\n \"11.A method of mounting a friction element in a disc brake comprising an axially slidable disc, the method comprising providing guide structure adapted to act at two spaced locations on said friction element between said friction element and a non-rotatable mounting therefor, and said method comprising providing abutment structure located between said spaced locations on said friction element, and said method comprising causing said abutment structure to resist torque applied to said friction element during use of said brake.\",\n \"12.A brake friction element comprising abutment structure located between guide structure at spaced locations of said friction element and adapted to resist torque applied to said friction element during use in a brake system.\",\n \"13.A spot-type automotive disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation device therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at lest one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and g) abutment structure adapted to resist torque applied to said at least on friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and adapted to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.\",\n \"14.A disc brake comprising an axially slidable disc, and guide structure adapted to act at two spaced locations on a friction element and between said friction element and a non-rotatable mounting therefor, and said brake comprising abutment structure located intermediate said spaced locations and adapted to resist torque applied to said friction element during use of said brake.\",\n \"15.A brake friction element comprising abutment structure located between circumferentially spaced ends of said friction element and adapted to resist torque applied to said friction element during use in a brake system.\",\n \"16.A spot-type automotive disc brake according to claim 13, wherein said abutment structure is located generally radially in-line with the line of action of said actuation device.\",\n \"17.A spot-type automotive disc brake according to claim 13, wherein said abutment structure comprises an abutment element fixed at the upper periphery of said at least one friction element and extending lengthwise in the direction of sliding brake-actuating motion of said friction element and slidingly received in a complementary channel formed in said non-rotatable mounting or caliper.\",\n \"18.A brake according to claim 17, wherein said abutment element having a length-to-width ratio of at least in the range of 1 to 1 to 3 to 3.\"\n ],\n [\n \"This invention relates to friction element control in disc brakes.\",\n \"An example of the application of the invention is to the control of friction elements in spot-type automotive disc brakes, particularly such brakes of the kind in which a fixed caliper and sliding friction elements are employed.\",\n \"The invention may find an application outside the strict limits of the technical field of the disc brakes disclosed in the specific embodiments described below, but principally the invention is concerned with controlling the movement of friction elements in relation to the associated structures of a disc brake, particularly the disc or discs themselves (which rotate during use), and the relatively fixed or non-rotatable structures such as that of the caliper on which the friction elements are mounted.\",\n \"Generally, the friction elements with which the invention is concerned are those which are not actually fixed to a position-defined structure such as the end plate of a fixed caliper and thus, the invention is particularly applicable to friction elements of the kind employed as the middle one of three friction elements interleaved with twin discs in a fixed caliper/sliding disc assembly as disclosed below.\",\n \"In such a disc brake, a need arises for excellent dynamic control of the movement/position/attitude of the friction elements during brake application in order to maximise the effectiveness of the brake and minimise the extent of uneven wear during use.\",\n \"In this connection, we have already disclosed in co-pending applications arrangements whereby the friction elements are provided with anti-tilt systems to maintain as far as possible the parallel and co-planar relationship of the friction-producing faces of the friction elements and of the brake discs themselves, and this has largely been achieved by means of spring arrangements exerting substantial resilient forces on the sliding disc assemblies and likewise on the sliding friction elements assemblies to achieve the necessary degree of control under the dynamic conditions of use.\",\n \"Developments thus far have enabled us to provide a satisfactory degree of control in respect of those functions which may be conveniently grouped under the heading “anti-tilt control”, but we have discovered that there is a further area in relation to friction element control which requires attention in order to provide further advances in relation to the dynamic aspects of the performance of the disc brake itself.\",\n \"These further factors which require attention concern principally the question of the response of the friction elements to the effect of the torque exerted thereon by the frictional forces during brake application.\",\n \"Thus, it will be readily understood that upon brake application and frictional engagement of the friction pads with the rotating disc, the applied forces acting on the friction elements produce a torque due to the rotary motion of the brake discs and such torque or turning effect on the pad and thus on its backing plate and thus on the friction element as a whole can cause destabilisation of the friction elements leading to a reduction in brake efficiency, or possibly uneven wear of the friction pads, and an object of the present invention is to provide improvements in this regard in relation to friction elements for disc brakes, or indeed improvements generally.\",\n \"According to the invention there is provided a method and apparatus as defined in the accompanying claims.\",\n \"In embodiments of the invention described below the axially slidable friction elements are provided with the basic guide means and supporting means whereby they are able to execute supported axial movement for frictional engagement with the brake discs.\",\n \"However, in addition, the slidable friction elements are provided with abutment or torque-transmitting means in the form of an abutment or torque-transmitting element and an associated abutment or torque-transmitting channel in which the abutment or torque-transmitting element is received.\",\n \"These structures (provided respectively at the upper edge of the friction element and at the underside of the brake caliper) serve to provide an important abutment or torque-transmitting function for the friction elements whereby the potentially twisting effect of the braking torque applied by the rotating brake discs is effectively neutralised by the abutting engagement of the abutment or torque-transmitting element with its corresponding abutment or torque-transmitting channel.\",\n \"Moreover, although it would be possible to provide the abutment or torque-transmitting element as merely an upstanding peg or the like structure having the same thickness as the remainder of the friction element backing plate (and some benefit in terms of torque neutralisation would undoubtably be available), in the embodiments the abutment or torque-transmitting element is constructed so as to have a length dimension (extending lengthwise in the direction of the thickness of the backing plate material) whereby its torque neutralisation effects are significantly enhanced.\",\n \"It also potentially assists in guiding its friction element.\",\n \"A particular feature of the embodiments is the location of the abutment or torque-transmitting element in relation to the line of action of the actuation means for the brake, such as the centre line of the actuating piston.\",\n \"In the embodiments, the abutment or torque-transmitting element is positioned directly above the line of action of the piston, and this is the preferred position.\",\n \"In the case where twin actuating pistons are provided, then a central position between the pistons (and thus effectively at the centre of pressure produced by the pistons) is preferred.\",\n \"Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which: FIG.\",\n \"1 shows, in block diagram format a spot-type automotive disc brake comprising a pair of axially slidable discs and associated friction elements, an actuating mechanism therefor and a fixed caliper or bridge structure overlying same; FIG.\",\n \"2 shows on a somewhat larger scale an exploded perspective view of the caliper and an associated friction element with an abutment or torque-transmitting element shown at an upper edge of the friction element together with an associated abutment or torque-transmitting channel to receive same; FIG.\",\n \"3 shows a side elevation view of the assembly of FIG.\",\n \"2 in its assembled condition; and FIG.\",\n \"4 shows a plan view, as seen in the direction of arrow IV in FIG.\",\n \"3, of the assembly of FIGS.\",\n \"2 and 3.As shown in FIG.\",\n \"1 a spot-type automotive disc brake 10 comprises a pair of rotatable brake discs 12, 14, a rotatable mounting 16 for the brake discs to permit rotation of the discs and which is adapted to drive the brake discs and have exerted thereon the braking effect by the discs when the disc brake 10 is actuated.\",\n \"Two pairs of friction elements 18, 20 and 22 are provided and are adapted to frictionally engage braking surfaces 24, 26 provided at opposite sides of brake discs 12, 14 to effect braking on actuating actuation means for the brake.\",\n \"Central friction element 20 is double-sided for frictional engagement with the mutually-inwardly facing braking surfaces 24, 26 of brake discs 12, 14 and is provided with appropriately facing friction pad material accordingly.\",\n \"Friction elements 18, 20, 22 comprise (as shown in FIGS.\",\n \"3 and 4) in each case a generally flat metal backing plate 28 and secured thereto and standing proud thereof a body of friction material of known construction for high durability frictional engagement with the relevant braking surface of the relevant brake disc.\",\n \"In the case of central friction element 20, the friction material is provided at both faces of the backing plate 28.Brake discs 12, 14 are axially slidable in use with respect to their rotatable mounting 16 under the action of friction elements 18, 20, 22 and the actuation means (to be described below) therefor during braking.\",\n \"For example the brake discs may be keyed to the rotatable mounting or hub 16 at three or more locations and resilient means may act there between.\",\n \"A non-rotatable mounting 32 for friction elements 18, 20, 22 is provided comprising a caliper or bridge structure 34 which is mounted on a fixed structure of the vehicle to be braked, for example on the wheel mounting and which straddles the brake discs 12, 14 and also provides a mounting for actuation means 36,38 (indicated diagrammatically) which applies inwardly directed braking forces to the outer friction elements 18, 22, thereby causing frictional engagement with the brake discs 12, 14, and slight sliding movement of those discs with respect to their rotatable mounting 16.In FIG.\",\n \"1 of course it can be seen that the clearances between the structures have been greatly exaggerated for simplicity of diagrammatic illustration.\",\n \"The actuation means 36, 38 could comprise a pair of piston and cylinder assemblies.\",\n \"However only one such is strictly needed since the actuation means can be one-sided with a fixed structure at one side or the other of the assembly of discs and friction elements (which fixed structure could simply be a stop extending from caliper 34), and against which fixed structure the assembly is pushed by the single actuation means.\",\n \"Further details in this regard may be found in our co-pending applications WO 98/26192 (docket 2558).\",\n \"Non-rotatable mounting 32 for the friction elements 18 to 22 is adapted to permit sliding movement of the friction elements into and out of frictional engagement with the brake discs while resisting movement of the friction elements under the action of frictional forces generated by engagement of the friction elements with the discs 12, 14.Turning now to the details of the structures shown in FIGS.\",\n \"2, 3 and 4, we mention first that the friction element illustrated is taken to be the central friction element 20 which (though not shown in FIG.\",\n \"2) is provided as shown in FIGS.\",\n \"3 and 4 with pads 40, 42 of friction material facing axially at its opposite sides.\",\n \"Other structures seen in the drawings include the casting 44 to which caliper 34 is secured and from which the actuating piston 46 can extend under the action of the usual hydraulic and driver-controlled brake actuation system having a line of action 48 which of course intersects the friction elements, this location being shown in FIG.\",\n \"2 at 50 which identifies the line of action of the centre of piston 46.As also shown in FIGS.\",\n \"2 to 4 axially-extending primary abutment or torque-transmitting means 52 is provided in relation to caliper 34 and is adapted to act on the slidable friction elements 20, 22 (end friction element 18 being secured or fixed to an end plate (not shown) extending downwards from the outboard end of caliper 34).\",\n \"Primary guide means 52 comprises axially-extending guide rails 54, 56, one at each side of caliper 34 and adapted to receive corresponding hook elements 58, 60 provided at spaced locations namely the opposite circumferentially-spaced ends 62, 64 of friction element 20.Thus, guide rails 54, 56 and hook elements 58, 60 serve to ensure that friction element 20 (and likewise sliding friction element 22 in a similar way) is able to hang from the caliper for sliding axial movement lengthwise thereof towards and away from the brake discs 12, 14.In addition to the guide rails 54, 56 and hook elements 58, 60 there is provided further abutment or torque-transmitting means comprising an abutment or torque-transmitting element 66 located intermediate spaced locations namely the opposite ends 62, 64 of the friction element and adapted to enable the friction element to resist torque from frictional forces applied to it during use of the brake, while permitting the friction element to move towards and away from the brake discs.\",\n \"Abutment or torque-transmitting element 66 is received in an abutment or torque-transmitting channel 68 formed in the underside 70 of caliper 34.It will be noted from FIG.\",\n \"2 that abutment or torqur-transmitting element 66 is generally radially in-line with the line of action 48 of actuating piston 46 and indeed is directly above the piston centre line point 50.FIG.\",\n \"2 of the drawings shows the proportions of abutment or torque-transmitting element 66 in a clear manner with respect to the remainder of the friction element structure.\",\n \"Thus, the abutment or torque-transmitting element has an axial length L and a traverse width W such that L/W lies in the range of 2 to 3.In general, the ratio L/W should preferably be at least 1 and more preferably still in the range of 1 to 3.Such a ratio has the effect of maximising the abutment or torque-transmitting influence of the abutment or torque-transmitting element in relation to frictionally-developed torque forces applied to the friction element during use, thereby minimising the tendency for taper wear to develop in the pads 40, 42 of friction material.\",\n \"The significance of the aspect ratio L/W in terms of maximising the torque-transmitting effect of the abutment or torque-transmitting element is discussed above.\",\n \"So far as the length aspects of the dimensions of the abutment element 66 are concerned, the following applies.\",\n \"As shown in FIG.\",\n \"3, there is shown at 72 an indication of the possibility of friction element tilt in an axial plane which can occur when the brake is in its off condition.\",\n \"Such tilting is limited by engagement of abutment element 66 with the top of groove 68.The possibility of tilt in the mode indicated at 72 is decreased with increasing length of the abutment element 66.Likewise, a similar tilt-inhibiting effect is exerted by minimising clearance between the top of the abutment element 66 and the top of groove 68.In the on condition of the brake, as indicated in FIG.\",\n \"3, the brake-applying force Fp is indicated at 74 and acts along line 48 on the friction element 20 and has a turning (or taper wear) moment Fp×xp at the element 66 where xp is the radial distance (identified at 76) of off-set of line 48 from the top of channel 68.This force is resisted by a force at the end of the abutment element 66 acting on the top of groove 68 accordingly.\",\n \"This function likewise is enhanced by increasing the length of abutment or torque-transmitting element 66.In use, disc brake 10 operates in substantially the usual manner, which is implicit from the above description, so far as concerns the general mode of frictional engagement of the relevant surfaces.\",\n \"So far as guidance of the friction elements during use is concerned, the friction elements are supported on guide rails 54, 56 through hook elements 58, 60 and frictionally-developed torque forces applied to the pads 40, 42 of friction material which tend to produce twisting forces arising from the rotation of the brake discs are largely taken out of the friction element structure by the abutment or torque-transmitting element 66 and the engagement of same with the side walls of abutment or torque-transmitting channel 68 in which the abutment or torque-transmitting element is a close sliding fit.\",\n \"Indeed, in principle, the tolerances in relation to the sliding fit of abutment or torque-transmitting element 66 within abutment or torque-transmitting channel 68 are tighter (meaning a closer fit) than exists between hook elements 58, 60 and guide rails 54, 56.As a result, wear on the pads 40, 42 or friction material is greatly improved in terms of its uniformity throughout the total working area of the friction material.\",\n \"Amongst other modifications which could be made in the above embodiment while remaining within the scope of the appended claims are changes to the shape and dimensions and exact location of the abutment or torque-transmitting element 66 in relation to caliper 34.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10451031"}}},{"rowIdx":293,"cells":{"text":{"kind":"list like","value":[["Lugged cap forming system","Transfer apparatus and method are provided from first (15A, 16A) to second (15B, 16B) stations of tooling in a cap (11) making press.","This cap is biased against the first upper tools by a first airstream (50) introduced under the cap and moves upward with the first station punch (45).","As the punch approaches its top location, a transfer airstream (52) begins while the first airstream is still on, and moves the cap out through a transfer chute (18) to the second station.","The cap departs the chute and passes detents (67) on a pair of closed retention fingers (60) which define an extension of the transfer path from the chute into the open second station tools.","A vacuum (85) applied to a port in the second station punch then holds the cup against the rising upper tools.","When the punch clears the closing fingers and approaches its top location, an ejection airstream (87) commences to propel the finished cup via a discharge chute (19)."],["1.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed position in which said tools are closely spaced to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing further operations thereon; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station tools; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute.","2.A system as defined in claim 1, wherein said chute terminates adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being movably supported on opposite sides of the second station tools for movement perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open.","3.A system as defined in claim 2, wherein said fingers have complementary tracks formed thereon to receive and guide a part exiting said chute, said tracks having inwardly curved end sections which align with the center of the second station tools to provide a termination of the path of a cap part entering the second station tools, and cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening.","4.A system as defined in claim 3, wherein said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts.","5.A system as defined in claim 1 wherein said second station tools are constructed to form lugs on a rolled rim of the cap parts.","6.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed interface position in which said tools are closely spaced (a) to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and (b) then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel, said tools also form an outward rolled rim on the side wall; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing a lug shaping operation on the rolled rim; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station, said chute terminating adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being pivotally supported on opposite sides of the second station tools for opening and closing motion perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open; said fingers have complementary tracks formed thereon to receive and guide a part exiting said cute, said tracks having inwardly curved end sections which align with the center of the second station tools, said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts and to provide a termination of the path of a cap part entering the second station tools; cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute, said control means including an input pulse generator driven in synchronism with the cyclic operation of the press to generate a train of control pulses related to the angular position of the press crankshaft as it rotates to open and close the tools, whereby the initiation and termination of said airstream projecting means is synchronized with cyclic operation of the tools.","7.A method for discharging and transferring a cup shaped object from a work station, having upper and lower tooling which is opened and closed to form the object, along a transfer path, the open position of the upper tooling defining a ready position, comprising the steps of: (a) locating the object within the work station in the ready position by directing a first stream of pressurized gas against the object to cause the object to remain with the upper tooling upon opening of the tooling whereby the object is supported by the first stream against the upper tooling; (b) prior to locating the object in said ready position, initiating a second flow of pressurized gas through orifice means located adjacent to and directed across said ready position, thereby causing the transfer of said object from the work station when the object is released at the ready position; and (c) discontinuing the second flow of pressurized gas through said orifice means after the object has exited the open tooling.","8.The method of claim 7 including the additional steps of: (d) receiving the object in a chute extending to a second work station which defines the transfer path to adjacent second upper and lower tools of the second work station, (e) directing the object into an extension of the transfer path defined by pivoting fingers located at opposite sides of the second tools, and (f) opening the fingers when the second tools are closing, and closing the fingers when the second tools are opening such as to guide the object into a centered position between the second station tools."],[" BACKGROUND OF THE INVENTION U.S.","Pat.","Nos.","6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.","That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.","The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.","However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.","With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.","a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.","in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.","To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.","Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.","Nos.","4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.","Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.","This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.","Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station."],[" SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.","The caps are of a type having substantial height with respect to their diameter.","Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.","Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.","The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.","This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.","As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.","This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.","At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.","As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.","As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute."],["BACKGROUND OF THE INVENTION U.S. Pat.","Nos.","6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.","That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.","The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.","However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.","With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.","a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.","in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.","To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.","Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.","Nos.","4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.","Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.","This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.","Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.","SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.","The caps are of a type having substantial height with respect to their diameter.","Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.","Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.","The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.","This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.","As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.","This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.","At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.","As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.","As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a bottom view of a container cap as provided by the invention.","FIG.","2 is a transverse cross-sectional view of a typical completed cap, as shown in FIG.","1.FIG.","3 is an over all perspective view of the tooling for a four-out cap making system.","FIG.","4 is an enlarged perspective view, with the die shoe removed, showing details of the tooling, including four first station tools in the center and four related second station tools outwardly in opposite directions of the first stations.","FIG.","5 is a further enlarged perspective view showing details of one pair of first and second stations of the tooling.","FIG.","6 is a cross-section view of a first station punch or upper tool.","FIG.","7 is a cross-section view of a first station die or lower tool.","FIG.","8 is a cross-section view illustrating the first station punch and die in closed position, and showing a cap formed except for an outward curl at the lower rim of the cap.","FIG.","9 is a cross-section view showing first station and corresponding second station tools, a transfer chute between them, the fingers which provide an extension of the chute into the second stage tool, and cams which control movement of the fingers.","FIGS.","10 and 11 are detail views of the top of the second station die, showing the open and closed positions of the fingers.","FIG.","12 is an enlarged cross-section view of the second station tools, and FIG.","12A is a further enlargement of the circled area in the center of FIG.","12.FIGS.","13A and 13B1 & 13B2 (three sheets) together are a schematic diagram of the pneumatic supply & control system of the press and tooling.","FIG.","14 is a flow diagram of the electronic control system for the tooling package.","DESCRIPTION OF PREFERRED EMBODIMENT The present invention provides a transfer apparatus and method for two station progressive tooling in a cap making press and system.","The caps are of a type having substantial height with respect to their diameter.","A typical such cap is shown in FIGS.","1 and 2, which is also shown in FIGS.","2, 4A and 4B of U.S. Pat.","No.","6,015,062.The preferably integral cap 11, in the general form of an inverted cup, includes a top panel 12, a peripheral sidewall 13, and a curled rim 14.In the present drawings, FIGS.","3-12A depict multi-lane progressive tooling comprising four lanes for simultaneously forming four caps 11, each lane comprising pairs of first and second tooling stations 15A, 15B, 16A, 16B, 17A,17B, and 18A, 18B, with the first stations 15A-18A arranged centrally of the tooling (FIGS.","3, 4 & 5) and corresponding second stations 16A-16B arranged outward of the first stations toward opposite sides of the upper and lower die plates 20A, 20B which support the upper (punch) and lower (die) tooling, and mount between the bed and slide of a reciprocating press (not shown).","Except for their orientation in the overall tooling package, the respective first station and second station tools are alike, and the following detailed description applies to all.","Taking stations 15A and 15B as examples, each pair of corresponding first and second stations has an associated transfer chute 19 (FIGS.","4, 5 & 9) between them, and each second station has a discharge chute 19A, the four of which are directed out opposite sides of the tooling (FIGS.","3 & 4).","Sheets of metal, with an appropriate pattern of lithographed materials for each cap, are fed centrally into the first stations by sheet feeding mechanism of known construction (not shown) which moves the sheets one at a time in step wise fashion, synchronized to the press strokes, along the feed path indicated by arrow IN in FIGS.","3 & 4.First Station The first station tools comprise an upper or blank punch tool 45 and a compound lower die.","During the initial operation of the first station tools, with the lithographed patterns aligned with respect to the first station tools, a blank is cut from the material (typically aluminum or thin cold rolled steel) on the down stroke of the press by blank punch 45.On the continuation of the down stroke, the blank punch and lower die tool cooperate such that the blank is drawn into a cup shaped cap part 11P (FIG.","8).","At the bottom of the stroke the panel shape 12 is formed into the top of cap part lip by the punch 45 and cooperating die 46 (FIGS.","6, 7 & 8).","On the up stroke, the lower curl ring 48, which is under spring pressure, raises with the blank punch.","The bottom edge of the cap part 11P is curled outward into the cavity 49 formed by curl ring 48 and blank punch 45, completing a formed cap 11 with an outward curled rim 15.The formed cap in the first station, which is in the nature of an inverted cup, is biased against the upper forming die by a first airstream introduced through passage 50 (see FIGS.","8 & 9) into the cap as the first station tooling opens, and causes the cap to follow upward against the bottom of the punch 45.During the upward travel of the cap, a second airstream is initiated through a nozzle 52 directed across the upper fist station tooling toward chute 18, and is at its full power when punch 45 (with a cap 11 held thereto by the upward directed first airstream) traverses the space between nozzle 52 and the entry 18E to chute 18 (see FIG.","9).","By the time the first station tooling reaches full open at the top-dead-center of a press stroke, the cap has actually been transferred into the second station 15B; see the Press Rotation timing chart below (page 8).","Thus, the first and second airstreams, appropriately switched on and off, together with the associated chute, constitute a first part of an essentially passive press transfer system.","Second Station At the second station, a separated pair of fingers 60 reach around the sides of the second station tooling (FIGS.","9, 10 & 11), defining partial sides of a receiving space 62 when the second station tooling is open, and an extension of the transfer chute when closed.","The fingers are supported by pivots 63 inward of their rear ends 61, and are biased into their closed position (FIG.","10, forward ends parallel) by spring mechanism 64.Each finger has a narrow ledge-like track 65 extending part way along its forward upper edge (FIGS.","10-12), facing each other and ending in a curved section 66.Extending horizontally inward over tracks 65 are spring-loaded ball detents 67 which, together with the curved track sections 66, define the termination of the transfer path for the incoming caps.","A cap propelled from the first station traverses the adjacent transfer chute 19 and enters receiving space 62, passing across ball detents 67 and resting against the curved track sections 66 (see FIG.","11).","The fingers and their operating mechanism form the remainder of the unique transfer system.","As the tooling proceeds to close during the beginning of a next stroke, fingers 60 are swung outward by descending cams 48 pressing against followers 49 on the rear ends 61 of the fingers to move the followers 49 inward and the forward section of the fingers outward (FIGS.","10 & 12) before the second station tools close.","The elongated cams 48 are mounted on the upper (die) tooling base (FIG.","12), and this action centers a cap (FIG.","11) and then opens the fingers associated with the second station tooling, before that tooling closes to form lugs on rim 15 of a cap 11.This closing action of fingers 60 is momentary, and they are then opened substantially before the second station tooling closes; see Press Rotation timing chart below.","In the second station 15B in the press, the tooling includes an upper punch 70 and a lower die 72.The punch tool 70 includes an annular knock out ring 71 with an inner shape conforming to a cap exterior, and a headed knock out pin 73 which is spring loaded toward a position flush with the lower edge of ring 71 (FIGS.","12 & 12A) when the tooling is opened.","In the lower die 72 there is a vertically extending tapered cam 74 mounted onto a base plate 75 and surrounded by a plurality of die pins 76 also mounted onto plate 75.The cam 74 and pins 76 project through a vertically movable, upwardly biased ring 78 which contains a plurality of lug forming dies 80, equal in number to pins 76 and movable laterally outward through forces from cam 74 as it is made to enter ring 78.The top of ring 78 has an upper surface 82 contoured to fit within a cap 11.Thus, when the second station tooling closes a cap is positioned with the curled rim 15 between the upper ends of pins 76 and the radially outward moving forming dies 80, to for the predetermined number of lugs in the rim (FIG.","1).","Then, the completed cap is held to the knockout pin by means of a vacuum created in passage 85 though the head of that pin 73, such that the cap is carried upward past an air nozzle 87 directed across the path of the rising upper tool toward an associated discharge chute 19A.","The vacuum is created by air flow through a small venturi (not shown) so the vacuum is relieved immediately upon cessation of air flow through that venturi.","A discharge air stream is started from nozzle 87 and moves the cap from the upper knock out ring and pin, into and through the associated discharge chute 19.Press Rotation Relation to Tooling Function 1st Station 0° Top of the Stroke; Top Dead Center 140° Material is Blanked 180° Form & draw complete; Bottom Dead Center; Cap Overall Height ˜0.810 inch 190° Cap Curl complete; Overall Height is ˜0.625 inch 190° 1st Air turned on; Blows cap against punch tool 220° Blank punch exits Die tool, cap against its face 230° 2nd Transfer Air on; Blows cap into transfer chute 330° 1st Cap arrives into catch fingers at 2nd Station.","2nd Station 0° Top of a stroke; Top Dead Center 60° Upper vacuum is turned on 136° Catch fingers start open, upper vacuum holds 1st cap against upper knockout and tools close 180° Lugs formed in 1st cap; Bottom Dead Center 181° 1st cap (completed) and tools move up together 224° Catch fingers close completely after tooling passes 270° Upper vacuum to knockout turned off 280° Discharge air valve to nozzle is turned on 290° Knock out air to nozzle 87 is turned on 330° 2nd Cap arrives into catch fingers 336° 1st Cap actually moves to discharge position 335° Vacuum actually turns off on Cap #1 350° 1st Cap actually leaves press via chute 19 FIGS.","13A and 13B comprise a pneumatic diagram for the pneumatic portion of the press control.","A “shop air” source of compressed air is supplied to the various electrically controlled valves (which are all labeled) to direct air under pressure to the above mentioned parts of the tooling, under management control of the electronics control (FIG.","14) of the system.","The details of such controls are apparent to persons skilled in this art from these diagrams FIG.","14 is a block diagram of electrical/electronics controls for the system, which selectively turn on and off the compressed air to the nozzles providing lift or “blow up” air to hold the cap part against the rising upper tools in the first station, and providing a transfer air stream to quickly move a cap part into a transfer chute 19.The third (upper) control provides timed discharge airstreams through nozzles 87 to move the finished caps into the discharge chutes 19A.","A pulse generator PG is driven by the press crankshaft (not shown) in typical fashion to generate a train of pulses related to the angular position of the crankshaft as it rotates, and these pulses are directed to the system PLC (Programmable Logic Controller).","Since the diagram is divided into three functions which occur during a press cycle, the controller PLC is shown in each of the three diagram parts, but in fact one PLC is employed in the control system.","While the method herein described, and the form of apparatus for carrying this method into effect, constitute preferred embodiments of this invention, it is to be understood that the invention is not limited to this precise method and form of apparatus, and that changes may be made in either without departing from the scope of the invention, which is defined in the appended claims."]],"string":"[\n [\n \"Lugged cap forming system\",\n \"Transfer apparatus and method are provided from first (15A, 16A) to second (15B, 16B) stations of tooling in a cap (11) making press.\",\n \"This cap is biased against the first upper tools by a first airstream (50) introduced under the cap and moves upward with the first station punch (45).\",\n \"As the punch approaches its top location, a transfer airstream (52) begins while the first airstream is still on, and moves the cap out through a transfer chute (18) to the second station.\",\n \"The cap departs the chute and passes detents (67) on a pair of closed retention fingers (60) which define an extension of the transfer path from the chute into the open second station tools.\",\n \"A vacuum (85) applied to a port in the second station punch then holds the cup against the rising upper tools.\",\n \"When the punch clears the closing fingers and approaches its top location, an ejection airstream (87) commences to propel the finished cup via a discharge chute (19).\"\n ],\n [\n \"1.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed position in which said tools are closely spaced to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing further operations thereon; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station tools; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute.\",\n \"2.A system as defined in claim 1, wherein said chute terminates adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being movably supported on opposite sides of the second station tools for movement perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open.\",\n \"3.A system as defined in claim 2, wherein said fingers have complementary tracks formed thereon to receive and guide a part exiting said chute, said tracks having inwardly curved end sections which align with the center of the second station tools to provide a termination of the path of a cap part entering the second station tools, and cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening.\",\n \"4.A system as defined in claim 3, wherein said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts.\",\n \"5.A system as defined in claim 1 wherein said second station tools are constructed to form lugs on a rolled rim of the cap parts.\",\n \"6.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed interface position in which said tools are closely spaced (a) to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and (b) then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel, said tools also form an outward rolled rim on the side wall; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing a lug shaping operation on the rolled rim; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station, said chute terminating adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being pivotally supported on opposite sides of the second station tools for opening and closing motion perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open; said fingers have complementary tracks formed thereon to receive and guide a part exiting said cute, said tracks having inwardly curved end sections which align with the center of the second station tools, said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts and to provide a termination of the path of a cap part entering the second station tools; cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute, said control means including an input pulse generator driven in synchronism with the cyclic operation of the press to generate a train of control pulses related to the angular position of the press crankshaft as it rotates to open and close the tools, whereby the initiation and termination of said airstream projecting means is synchronized with cyclic operation of the tools.\",\n \"7.A method for discharging and transferring a cup shaped object from a work station, having upper and lower tooling which is opened and closed to form the object, along a transfer path, the open position of the upper tooling defining a ready position, comprising the steps of: (a) locating the object within the work station in the ready position by directing a first stream of pressurized gas against the object to cause the object to remain with the upper tooling upon opening of the tooling whereby the object is supported by the first stream against the upper tooling; (b) prior to locating the object in said ready position, initiating a second flow of pressurized gas through orifice means located adjacent to and directed across said ready position, thereby causing the transfer of said object from the work station when the object is released at the ready position; and (c) discontinuing the second flow of pressurized gas through said orifice means after the object has exited the open tooling.\",\n \"8.The method of claim 7 including the additional steps of: (d) receiving the object in a chute extending to a second work station which defines the transfer path to adjacent second upper and lower tools of the second work station, (e) directing the object into an extension of the transfer path defined by pivoting fingers located at opposite sides of the second tools, and (f) opening the fingers when the second tools are closing, and closing the fingers when the second tools are opening such as to guide the object into a centered position between the second station tools.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION U.S.\",\n \"Pat.\",\n \"Nos.\",\n \"6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.\",\n \"That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.\",\n \"The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.\",\n \"However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.\",\n \"With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.\",\n \"a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.\",\n \"in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.\",\n \"To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.\",\n \"Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.\",\n \"Nos.\",\n \"4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.\",\n \"Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.\",\n \"This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.\",\n \"Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.\",\n \"The caps are of a type having substantial height with respect to their diameter.\",\n \"Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.\",\n \"Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.\",\n \"The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.\",\n \"This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.\",\n \"As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.\",\n \"This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.\",\n \"At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.\",\n \"As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.\",\n \"As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.\"\n ],\n [\n \"BACKGROUND OF THE INVENTION U.S. Pat.\",\n \"Nos.\",\n \"6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.\",\n \"That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.\",\n \"The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.\",\n \"However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.\",\n \"With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.\",\n \"a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.\",\n \"in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.\",\n \"To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.\",\n \"Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.\",\n \"Nos.\",\n \"4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.\",\n \"Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.\",\n \"This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.\",\n \"Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.\",\n \"SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.\",\n \"The caps are of a type having substantial height with respect to their diameter.\",\n \"Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.\",\n \"Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.\",\n \"The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.\",\n \"This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.\",\n \"As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.\",\n \"This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.\",\n \"At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.\",\n \"As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.\",\n \"As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a bottom view of a container cap as provided by the invention.\",\n \"FIG.\",\n \"2 is a transverse cross-sectional view of a typical completed cap, as shown in FIG.\",\n \"1.FIG.\",\n \"3 is an over all perspective view of the tooling for a four-out cap making system.\",\n \"FIG.\",\n \"4 is an enlarged perspective view, with the die shoe removed, showing details of the tooling, including four first station tools in the center and four related second station tools outwardly in opposite directions of the first stations.\",\n \"FIG.\",\n \"5 is a further enlarged perspective view showing details of one pair of first and second stations of the tooling.\",\n \"FIG.\",\n \"6 is a cross-section view of a first station punch or upper tool.\",\n \"FIG.\",\n \"7 is a cross-section view of a first station die or lower tool.\",\n \"FIG.\",\n \"8 is a cross-section view illustrating the first station punch and die in closed position, and showing a cap formed except for an outward curl at the lower rim of the cap.\",\n \"FIG.\",\n \"9 is a cross-section view showing first station and corresponding second station tools, a transfer chute between them, the fingers which provide an extension of the chute into the second stage tool, and cams which control movement of the fingers.\",\n \"FIGS.\",\n \"10 and 11 are detail views of the top of the second station die, showing the open and closed positions of the fingers.\",\n \"FIG.\",\n \"12 is an enlarged cross-section view of the second station tools, and FIG.\",\n \"12A is a further enlargement of the circled area in the center of FIG.\",\n \"12.FIGS.\",\n \"13A and 13B1 & 13B2 (three sheets) together are a schematic diagram of the pneumatic supply & control system of the press and tooling.\",\n \"FIG.\",\n \"14 is a flow diagram of the electronic control system for the tooling package.\",\n \"DESCRIPTION OF PREFERRED EMBODIMENT The present invention provides a transfer apparatus and method for two station progressive tooling in a cap making press and system.\",\n \"The caps are of a type having substantial height with respect to their diameter.\",\n \"A typical such cap is shown in FIGS.\",\n \"1 and 2, which is also shown in FIGS.\",\n \"2, 4A and 4B of U.S. Pat.\",\n \"No.\",\n \"6,015,062.The preferably integral cap 11, in the general form of an inverted cup, includes a top panel 12, a peripheral sidewall 13, and a curled rim 14.In the present drawings, FIGS.\",\n \"3-12A depict multi-lane progressive tooling comprising four lanes for simultaneously forming four caps 11, each lane comprising pairs of first and second tooling stations 15A, 15B, 16A, 16B, 17A,17B, and 18A, 18B, with the first stations 15A-18A arranged centrally of the tooling (FIGS.\",\n \"3, 4 & 5) and corresponding second stations 16A-16B arranged outward of the first stations toward opposite sides of the upper and lower die plates 20A, 20B which support the upper (punch) and lower (die) tooling, and mount between the bed and slide of a reciprocating press (not shown).\",\n \"Except for their orientation in the overall tooling package, the respective first station and second station tools are alike, and the following detailed description applies to all.\",\n \"Taking stations 15A and 15B as examples, each pair of corresponding first and second stations has an associated transfer chute 19 (FIGS.\",\n \"4, 5 & 9) between them, and each second station has a discharge chute 19A, the four of which are directed out opposite sides of the tooling (FIGS.\",\n \"3 & 4).\",\n \"Sheets of metal, with an appropriate pattern of lithographed materials for each cap, are fed centrally into the first stations by sheet feeding mechanism of known construction (not shown) which moves the sheets one at a time in step wise fashion, synchronized to the press strokes, along the feed path indicated by arrow IN in FIGS.\",\n \"3 & 4.First Station The first station tools comprise an upper or blank punch tool 45 and a compound lower die.\",\n \"During the initial operation of the first station tools, with the lithographed patterns aligned with respect to the first station tools, a blank is cut from the material (typically aluminum or thin cold rolled steel) on the down stroke of the press by blank punch 45.On the continuation of the down stroke, the blank punch and lower die tool cooperate such that the blank is drawn into a cup shaped cap part 11P (FIG.\",\n \"8).\",\n \"At the bottom of the stroke the panel shape 12 is formed into the top of cap part lip by the punch 45 and cooperating die 46 (FIGS.\",\n \"6, 7 & 8).\",\n \"On the up stroke, the lower curl ring 48, which is under spring pressure, raises with the blank punch.\",\n \"The bottom edge of the cap part 11P is curled outward into the cavity 49 formed by curl ring 48 and blank punch 45, completing a formed cap 11 with an outward curled rim 15.The formed cap in the first station, which is in the nature of an inverted cup, is biased against the upper forming die by a first airstream introduced through passage 50 (see FIGS.\",\n \"8 & 9) into the cap as the first station tooling opens, and causes the cap to follow upward against the bottom of the punch 45.During the upward travel of the cap, a second airstream is initiated through a nozzle 52 directed across the upper fist station tooling toward chute 18, and is at its full power when punch 45 (with a cap 11 held thereto by the upward directed first airstream) traverses the space between nozzle 52 and the entry 18E to chute 18 (see FIG.\",\n \"9).\",\n \"By the time the first station tooling reaches full open at the top-dead-center of a press stroke, the cap has actually been transferred into the second station 15B; see the Press Rotation timing chart below (page 8).\",\n \"Thus, the first and second airstreams, appropriately switched on and off, together with the associated chute, constitute a first part of an essentially passive press transfer system.\",\n \"Second Station At the second station, a separated pair of fingers 60 reach around the sides of the second station tooling (FIGS.\",\n \"9, 10 & 11), defining partial sides of a receiving space 62 when the second station tooling is open, and an extension of the transfer chute when closed.\",\n \"The fingers are supported by pivots 63 inward of their rear ends 61, and are biased into their closed position (FIG.\",\n \"10, forward ends parallel) by spring mechanism 64.Each finger has a narrow ledge-like track 65 extending part way along its forward upper edge (FIGS.\",\n \"10-12), facing each other and ending in a curved section 66.Extending horizontally inward over tracks 65 are spring-loaded ball detents 67 which, together with the curved track sections 66, define the termination of the transfer path for the incoming caps.\",\n \"A cap propelled from the first station traverses the adjacent transfer chute 19 and enters receiving space 62, passing across ball detents 67 and resting against the curved track sections 66 (see FIG.\",\n \"11).\",\n \"The fingers and their operating mechanism form the remainder of the unique transfer system.\",\n \"As the tooling proceeds to close during the beginning of a next stroke, fingers 60 are swung outward by descending cams 48 pressing against followers 49 on the rear ends 61 of the fingers to move the followers 49 inward and the forward section of the fingers outward (FIGS.\",\n \"10 & 12) before the second station tools close.\",\n \"The elongated cams 48 are mounted on the upper (die) tooling base (FIG.\",\n \"12), and this action centers a cap (FIG.\",\n \"11) and then opens the fingers associated with the second station tooling, before that tooling closes to form lugs on rim 15 of a cap 11.This closing action of fingers 60 is momentary, and they are then opened substantially before the second station tooling closes; see Press Rotation timing chart below.\",\n \"In the second station 15B in the press, the tooling includes an upper punch 70 and a lower die 72.The punch tool 70 includes an annular knock out ring 71 with an inner shape conforming to a cap exterior, and a headed knock out pin 73 which is spring loaded toward a position flush with the lower edge of ring 71 (FIGS.\",\n \"12 & 12A) when the tooling is opened.\",\n \"In the lower die 72 there is a vertically extending tapered cam 74 mounted onto a base plate 75 and surrounded by a plurality of die pins 76 also mounted onto plate 75.The cam 74 and pins 76 project through a vertically movable, upwardly biased ring 78 which contains a plurality of lug forming dies 80, equal in number to pins 76 and movable laterally outward through forces from cam 74 as it is made to enter ring 78.The top of ring 78 has an upper surface 82 contoured to fit within a cap 11.Thus, when the second station tooling closes a cap is positioned with the curled rim 15 between the upper ends of pins 76 and the radially outward moving forming dies 80, to for the predetermined number of lugs in the rim (FIG.\",\n \"1).\",\n \"Then, the completed cap is held to the knockout pin by means of a vacuum created in passage 85 though the head of that pin 73, such that the cap is carried upward past an air nozzle 87 directed across the path of the rising upper tool toward an associated discharge chute 19A.\",\n \"The vacuum is created by air flow through a small venturi (not shown) so the vacuum is relieved immediately upon cessation of air flow through that venturi.\",\n \"A discharge air stream is started from nozzle 87 and moves the cap from the upper knock out ring and pin, into and through the associated discharge chute 19.Press Rotation Relation to Tooling Function 1st Station 0° Top of the Stroke; Top Dead Center 140° Material is Blanked 180° Form & draw complete; Bottom Dead Center; Cap Overall Height ˜0.810 inch 190° Cap Curl complete; Overall Height is ˜0.625 inch 190° 1st Air turned on; Blows cap against punch tool 220° Blank punch exits Die tool, cap against its face 230° 2nd Transfer Air on; Blows cap into transfer chute 330° 1st Cap arrives into catch fingers at 2nd Station.\",\n \"2nd Station 0° Top of a stroke; Top Dead Center 60° Upper vacuum is turned on 136° Catch fingers start open, upper vacuum holds 1st cap against upper knockout and tools close 180° Lugs formed in 1st cap; Bottom Dead Center 181° 1st cap (completed) and tools move up together 224° Catch fingers close completely after tooling passes 270° Upper vacuum to knockout turned off 280° Discharge air valve to nozzle is turned on 290° Knock out air to nozzle 87 is turned on 330° 2nd Cap arrives into catch fingers 336° 1st Cap actually moves to discharge position 335° Vacuum actually turns off on Cap #1 350° 1st Cap actually leaves press via chute 19 FIGS.\",\n \"13A and 13B comprise a pneumatic diagram for the pneumatic portion of the press control.\",\n \"A “shop air” source of compressed air is supplied to the various electrically controlled valves (which are all labeled) to direct air under pressure to the above mentioned parts of the tooling, under management control of the electronics control (FIG.\",\n \"14) of the system.\",\n \"The details of such controls are apparent to persons skilled in this art from these diagrams FIG.\",\n \"14 is a block diagram of electrical/electronics controls for the system, which selectively turn on and off the compressed air to the nozzles providing lift or “blow up” air to hold the cap part against the rising upper tools in the first station, and providing a transfer air stream to quickly move a cap part into a transfer chute 19.The third (upper) control provides timed discharge airstreams through nozzles 87 to move the finished caps into the discharge chutes 19A.\",\n \"A pulse generator PG is driven by the press crankshaft (not shown) in typical fashion to generate a train of pulses related to the angular position of the crankshaft as it rotates, and these pulses are directed to the system PLC (Programmable Logic Controller).\",\n \"Since the diagram is divided into three functions which occur during a press cycle, the controller PLC is shown in each of the three diagram parts, but in fact one PLC is employed in the control system.\",\n \"While the method herein described, and the form of apparatus for carrying this method into effect, constitute preferred embodiments of this invention, it is to be understood that the invention is not limited to this precise method and form of apparatus, and that changes may be made in either without departing from the scope of the invention, which is defined in the appended claims.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10451057"}}},{"rowIdx":294,"cells":{"text":{"kind":"list like","value":[["Diving aid","Diving aid for divers, at least comprising a power supply, alarm sound-generating means (3) and activating means (2) for activating the sound-generating means (3), which activating means (2) are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means (6) for attaching to the divers's body or the divers's equipment in an unmistakable orientation.","The diving aid according to the invention is preferably designed in the form to a wristwatch."],["1.Diving aid for reducing the dive stress of divers, in particular pre-dive stress, at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and which device also comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.","2.Diving aid according to claim 1, wherein the diving aid also comprises light-signal-generating means that are connected to the activating means.","3.Diving aid according to claim 1, wherein the activating means comprise a push-button.","4.Diving aid according to claim 1, wherein the activating means comprise an acceleration detector.","5.Diving aid according claim 1, wherein the diving aid comprises water detection means by means of which the device can be activated.","6.Diving aid according to claim 1, wherein the diving aid is designed in the form of a wristwatch.","7.Diving aid according to claim 1, wherein the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.","8.Diving aid according to claim 1, wherein the diving aid forms part of an ordinary diving computer."],["The present invention relates to a diving aid.","A major problem encountered by both experienced and inexperienced divers during diving is so-called dive stress, in particular pre-dive stress.","This dive stress increases the likelihood of mistakes being made prior to and during diving, mistakes which can have fatal consequences.","The chance of a panic situation under water is also increased considerably by this dive stress.","In practice, it is found that most divers dive only once or twice a year.","These are the divers at greatest risk.","It has been found that when such an inexperienced diver is in distress or in danger he has only three seconds in which it is possible to take action.","In the first second the distress or danger is perceived, in the second second the diver becomes aware of the problem and his position, and in the third second the diver is in a panic.","The most common reaction of such divers is to swim as fast as possible towards the surface, with all the risks this involves.","In a panic situation inexperienced divers are found to be incapable of any coordinated movement, for example in order to activate an alarm device.","The known alarm devices for use as diving aids are therefore found to be inadequate.","In this connection reference is made to FR-A-2,695,747.This French patent application discloses an alarm device for attaching to the wrist.","This device comprises two parts that are rotatable relative to each other and can be locked relative to each other by means of a locking pin.","This locking pin has to be removed in order to activate the alarm device.","FR-A-2,755,023 also discloses an alarm device, which comprises a housing with a battery and sound-generating means.","The housing comprises a push-button for activating the alarm device.","This alarm device is attached to an eye present on the housing.","It has been found that a diver who is in a panic is incapable of removing a locking pin in a controlled manner or locating and pressing a push-button on a trailing device.","Divers also very often wear gloves during diving, which makes such actions even more difficult.","Furthermore, a fully equipped diver has very limited freedom of movement.","There are also alarm devices that are operated by compressed air and are therefore connected to the compressed air system of the diver.","A common problem is that the diver is not getting any air and there is a fault in the compressed air system.","Such devices are therefore not sufficiently reliable, even if the diver were capable of activating the activating means.","In other words, all devices known in the prior art require coordinated movements from the diver.","These devices are therefore found to be inadequate in practice.","Knowledge of this fact increases both pre-dive stress and dive stress among divers.","There is consequently a great demand for a diving aid that can give the diver a relaxed and safe feeling.","The object of the present invention is to meet this demand, and to that end the invention provides a diving aid at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.","The diving aid according to the invention is found to be extremely satisfactory, even in a situation where an inexperienced diver panics.","It appears that an inexperienced diver in a panic is in fact only capable of making an uncoordinated striking movement with one hand in the direction of the alarm device.","For the device according to the invention, this is sufficient to activate the latter and make co-divers aware of the distress or danger.","It has been proved in practice that knowledge of the substantially increased safety under water through use of the diving aid according to the invention substantially reduces dive stress even in the case of very experienced divers.","It is pointed out that alarm devices that can be activated by an uncontrolled shock load are known for use on dry land.","These devices are not, however, suitable for use under water.","In this connection; reference is made to GB-A-2,267,373, GB-A-2,295,910 and GB-A-2,316,784.Apart from alarm sound-generating means, other means can, of course, also be used for alerting co-divers.","The diving aid also preferably comprises light-signal-generating means, which are connected to the activating means.","Under water the speed of sound is many times higher than it is in air.","The result of this is that under water the human ear is not capable of determining the direction of sound.","It is therefore preferable to use light-signal-generating means, such as, for example, a stroboscopic lamp or a stroboscopic LED.","Such lighting means are known in the prior art.","Another advantage of using light-generating means is that a diver in distress can be located easily even when diving at night or in murky water.","Of course, the advantages also apply to a victim who is unconscious, who can be found more easily in this way.","The activating means can be designed in many different ways according to the invention.","The activating means preferably comprise a relatively large push-button.","The push-button is so large that it can be pressed with certainty by an arbitrary uncontrolled striking movement.","The surface of the push-button is advantageously at least 2 cm2.More advantageously, the push-button also projects from the surface of the diving aid.","In the case of a large push-button it is advantageous if said button is provided with passages for water, so that the water pressure of the environment under the button can be evened out.","This ensures that during diving the diving aid is not activated undesirably by the pressure of the water.","In particular, a striking colour, for example red or yellow, is selected for such a push-button.","The activating means advantageously comprise an acceleration detector.","An example of this is a so-called tilt detector.","Such detectors are switches that are known in the prior art and can be activated by a shock load, without the direction of the shock load being important.","The diving aid can comprise an ON/OFF button, for switching on the diving aid just before diving, and for switching it off after diving.","The diving aid preferably comprises water detection means for switching on the diving aid as soon as it comes into contact with water.","This has the advantage that the diving aid is therefore always switched on under water, even if the diver forgets to switch on the diving aid.","Such water detection means are known in the prior art.","An example of these means is the detection electrodes that are also used on diving computers.","In this context the term switching on means putting the diving aid into a state ready for use.","In other words, when the diving aid is not switched on operation of the activating means will not result in the generation of an alarm sound.","In particular, the diving aid is designed in such a way that it can be worn on the wrist.","In practice, it has been found that the wrist is a suitable place for attachment of the diving aid, since in practice said diving aid can be reached easily with the other hand, so that in a panic situation the diving aid can be activated by an uncoordinated movement.","It will be clear that the diving aid can likewise be attached to the diver's equipment, provided that this attachment involves an unmistakable orientation, so that the diver can always reach the diving aid.","As a particular example of this, the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.","In a special embodiment the diving aid forms part of an ordinary diving computer.","An ordinary diving computer is a device from which, inter alia, the diving depth, the diving time that has elapsed and the like can be read.","The invention will be explained in greater detail below with reference to the appended drawing, in which: FIG.","1 shows a top view of an embodiment of a diving aid according to the invention; FIG.","2 shows a perspective view of the embodiment according to FIG.","1; FIG.","3 shows a top view of a different embodiment of a diving aid according to the invention; and FIG.","4 shows a perspective view of the embodiment according to FIG.","3.FIG.","1 shows an embodiment of a diving aid according to the invention, which is suitable for attachment around the wrist.","The diving aid comprises a housing 1 on which a large push-button 2 and alarm sound-generating means 3 are situated.","Reference numeral 5 indicates two water detection electrodes, which serve to activate the diving aid on contact with water.","A power supply is not shown in the figures, but it will be clear that in principle any power supply that is suitable for use under water can be used.","An example in this case is a power supply for a diving computer or the like.","Reference numeral 6 indicates a wristband for attachment of the diving aid to a diver's wrist.","Finally, reference numeral 7 indicates a reset button, by means of which the diving aid can be switched off after being activated, for example when the diver in distress or danger has been rescued.","For testing the functioning of the diving aid according to the invention above water, the diving aid can be switched on by touching the electrodes with moistened fingertips.","It will be clear that the diving aid is certain to be activated when in a panic situation a striking movement with the hand is made in an uncontrolled way in the direction of the diving aid according to the invention.","All this has been confirmed by experiments.","The alarm sound-generating means in this case are in the form of a piezoelectric sound-generating device.","FIG.","2 shows the diving aid according to FIG.","1 in perspective view.","It can be seen clearly in this figure that the large push-button 2 projects slightly from the surface of the device, so that said push-button is easy to press.","FIGS.","3 and 4 show a different preferred embodiment of a diving aid according to the invention, in which similar parts are indicated by the same reference numerals.","Apart from the alarm sound-generating means, this diving aid also comprises a stroboscopic xenon gas discharge lamp 8, for generating a light alarm.","When a diver in distress/danger uses this diving aid, he will be able to indicate his position not only by means of an audible sound signal, but also by means of light signals.","This means that the diver can be found even more quickly by co-divers."]],"string":"[\n [\n \"Diving aid\",\n \"Diving aid for divers, at least comprising a power supply, alarm sound-generating means (3) and activating means (2) for activating the sound-generating means (3), which activating means (2) are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means (6) for attaching to the divers's body or the divers's equipment in an unmistakable orientation.\",\n \"The diving aid according to the invention is preferably designed in the form to a wristwatch.\"\n ],\n [\n \"1.Diving aid for reducing the dive stress of divers, in particular pre-dive stress, at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and which device also comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.\",\n \"2.Diving aid according to claim 1, wherein the diving aid also comprises light-signal-generating means that are connected to the activating means.\",\n \"3.Diving aid according to claim 1, wherein the activating means comprise a push-button.\",\n \"4.Diving aid according to claim 1, wherein the activating means comprise an acceleration detector.\",\n \"5.Diving aid according claim 1, wherein the diving aid comprises water detection means by means of which the device can be activated.\",\n \"6.Diving aid according to claim 1, wherein the diving aid is designed in the form of a wristwatch.\",\n \"7.Diving aid according to claim 1, wherein the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.\",\n \"8.Diving aid according to claim 1, wherein the diving aid forms part of an ordinary diving computer.\"\n ],\n [\n \"The present invention relates to a diving aid.\",\n \"A major problem encountered by both experienced and inexperienced divers during diving is so-called dive stress, in particular pre-dive stress.\",\n \"This dive stress increases the likelihood of mistakes being made prior to and during diving, mistakes which can have fatal consequences.\",\n \"The chance of a panic situation under water is also increased considerably by this dive stress.\",\n \"In practice, it is found that most divers dive only once or twice a year.\",\n \"These are the divers at greatest risk.\",\n \"It has been found that when such an inexperienced diver is in distress or in danger he has only three seconds in which it is possible to take action.\",\n \"In the first second the distress or danger is perceived, in the second second the diver becomes aware of the problem and his position, and in the third second the diver is in a panic.\",\n \"The most common reaction of such divers is to swim as fast as possible towards the surface, with all the risks this involves.\",\n \"In a panic situation inexperienced divers are found to be incapable of any coordinated movement, for example in order to activate an alarm device.\",\n \"The known alarm devices for use as diving aids are therefore found to be inadequate.\",\n \"In this connection reference is made to FR-A-2,695,747.This French patent application discloses an alarm device for attaching to the wrist.\",\n \"This device comprises two parts that are rotatable relative to each other and can be locked relative to each other by means of a locking pin.\",\n \"This locking pin has to be removed in order to activate the alarm device.\",\n \"FR-A-2,755,023 also discloses an alarm device, which comprises a housing with a battery and sound-generating means.\",\n \"The housing comprises a push-button for activating the alarm device.\",\n \"This alarm device is attached to an eye present on the housing.\",\n \"It has been found that a diver who is in a panic is incapable of removing a locking pin in a controlled manner or locating and pressing a push-button on a trailing device.\",\n \"Divers also very often wear gloves during diving, which makes such actions even more difficult.\",\n \"Furthermore, a fully equipped diver has very limited freedom of movement.\",\n \"There are also alarm devices that are operated by compressed air and are therefore connected to the compressed air system of the diver.\",\n \"A common problem is that the diver is not getting any air and there is a fault in the compressed air system.\",\n \"Such devices are therefore not sufficiently reliable, even if the diver were capable of activating the activating means.\",\n \"In other words, all devices known in the prior art require coordinated movements from the diver.\",\n \"These devices are therefore found to be inadequate in practice.\",\n \"Knowledge of this fact increases both pre-dive stress and dive stress among divers.\",\n \"There is consequently a great demand for a diving aid that can give the diver a relaxed and safe feeling.\",\n \"The object of the present invention is to meet this demand, and to that end the invention provides a diving aid at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.\",\n \"The diving aid according to the invention is found to be extremely satisfactory, even in a situation where an inexperienced diver panics.\",\n \"It appears that an inexperienced diver in a panic is in fact only capable of making an uncoordinated striking movement with one hand in the direction of the alarm device.\",\n \"For the device according to the invention, this is sufficient to activate the latter and make co-divers aware of the distress or danger.\",\n \"It has been proved in practice that knowledge of the substantially increased safety under water through use of the diving aid according to the invention substantially reduces dive stress even in the case of very experienced divers.\",\n \"It is pointed out that alarm devices that can be activated by an uncontrolled shock load are known for use on dry land.\",\n \"These devices are not, however, suitable for use under water.\",\n \"In this connection; reference is made to GB-A-2,267,373, GB-A-2,295,910 and GB-A-2,316,784.Apart from alarm sound-generating means, other means can, of course, also be used for alerting co-divers.\",\n \"The diving aid also preferably comprises light-signal-generating means, which are connected to the activating means.\",\n \"Under water the speed of sound is many times higher than it is in air.\",\n \"The result of this is that under water the human ear is not capable of determining the direction of sound.\",\n \"It is therefore preferable to use light-signal-generating means, such as, for example, a stroboscopic lamp or a stroboscopic LED.\",\n \"Such lighting means are known in the prior art.\",\n \"Another advantage of using light-generating means is that a diver in distress can be located easily even when diving at night or in murky water.\",\n \"Of course, the advantages also apply to a victim who is unconscious, who can be found more easily in this way.\",\n \"The activating means can be designed in many different ways according to the invention.\",\n \"The activating means preferably comprise a relatively large push-button.\",\n \"The push-button is so large that it can be pressed with certainty by an arbitrary uncontrolled striking movement.\",\n \"The surface of the push-button is advantageously at least 2 cm2.More advantageously, the push-button also projects from the surface of the diving aid.\",\n \"In the case of a large push-button it is advantageous if said button is provided with passages for water, so that the water pressure of the environment under the button can be evened out.\",\n \"This ensures that during diving the diving aid is not activated undesirably by the pressure of the water.\",\n \"In particular, a striking colour, for example red or yellow, is selected for such a push-button.\",\n \"The activating means advantageously comprise an acceleration detector.\",\n \"An example of this is a so-called tilt detector.\",\n \"Such detectors are switches that are known in the prior art and can be activated by a shock load, without the direction of the shock load being important.\",\n \"The diving aid can comprise an ON/OFF button, for switching on the diving aid just before diving, and for switching it off after diving.\",\n \"The diving aid preferably comprises water detection means for switching on the diving aid as soon as it comes into contact with water.\",\n \"This has the advantage that the diving aid is therefore always switched on under water, even if the diver forgets to switch on the diving aid.\",\n \"Such water detection means are known in the prior art.\",\n \"An example of these means is the detection electrodes that are also used on diving computers.\",\n \"In this context the term switching on means putting the diving aid into a state ready for use.\",\n \"In other words, when the diving aid is not switched on operation of the activating means will not result in the generation of an alarm sound.\",\n \"In particular, the diving aid is designed in such a way that it can be worn on the wrist.\",\n \"In practice, it has been found that the wrist is a suitable place for attachment of the diving aid, since in practice said diving aid can be reached easily with the other hand, so that in a panic situation the diving aid can be activated by an uncoordinated movement.\",\n \"It will be clear that the diving aid can likewise be attached to the diver's equipment, provided that this attachment involves an unmistakable orientation, so that the diver can always reach the diving aid.\",\n \"As a particular example of this, the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.\",\n \"In a special embodiment the diving aid forms part of an ordinary diving computer.\",\n \"An ordinary diving computer is a device from which, inter alia, the diving depth, the diving time that has elapsed and the like can be read.\",\n \"The invention will be explained in greater detail below with reference to the appended drawing, in which: FIG.\",\n \"1 shows a top view of an embodiment of a diving aid according to the invention; FIG.\",\n \"2 shows a perspective view of the embodiment according to FIG.\",\n \"1; FIG.\",\n \"3 shows a top view of a different embodiment of a diving aid according to the invention; and FIG.\",\n \"4 shows a perspective view of the embodiment according to FIG.\",\n \"3.FIG.\",\n \"1 shows an embodiment of a diving aid according to the invention, which is suitable for attachment around the wrist.\",\n \"The diving aid comprises a housing 1 on which a large push-button 2 and alarm sound-generating means 3 are situated.\",\n \"Reference numeral 5 indicates two water detection electrodes, which serve to activate the diving aid on contact with water.\",\n \"A power supply is not shown in the figures, but it will be clear that in principle any power supply that is suitable for use under water can be used.\",\n \"An example in this case is a power supply for a diving computer or the like.\",\n \"Reference numeral 6 indicates a wristband for attachment of the diving aid to a diver's wrist.\",\n \"Finally, reference numeral 7 indicates a reset button, by means of which the diving aid can be switched off after being activated, for example when the diver in distress or danger has been rescued.\",\n \"For testing the functioning of the diving aid according to the invention above water, the diving aid can be switched on by touching the electrodes with moistened fingertips.\",\n \"It will be clear that the diving aid is certain to be activated when in a panic situation a striking movement with the hand is made in an uncontrolled way in the direction of the diving aid according to the invention.\",\n \"All this has been confirmed by experiments.\",\n \"The alarm sound-generating means in this case are in the form of a piezoelectric sound-generating device.\",\n \"FIG.\",\n \"2 shows the diving aid according to FIG.\",\n \"1 in perspective view.\",\n \"It can be seen clearly in this figure that the large push-button 2 projects slightly from the surface of the device, so that said push-button is easy to press.\",\n \"FIGS.\",\n \"3 and 4 show a different preferred embodiment of a diving aid according to the invention, in which similar parts are indicated by the same reference numerals.\",\n \"Apart from the alarm sound-generating means, this diving aid also comprises a stroboscopic xenon gas discharge lamp 8, for generating a light alarm.\",\n \"When a diver in distress/danger uses this diving aid, he will be able to indicate his position not only by means of an audible sound signal, but also by means of light signals.\",\n \"This means that the diver can be found even more quickly by co-divers.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10451113"}}},{"rowIdx":295,"cells":{"text":{"kind":"list like","value":[["Composition comprising an oxidizing and complexing compound","The present invention is related to a composition comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","The oxidizing compound can be in the form of an aqueous solution.","The complexing compound is for complexing metal ions.","Metal ions can be present in the solution or in an external medium being contacted with the solution.","The present invention can be used for cleaning a semiconductor substrate."],["1.A composition comprising an aqueous solution, said aqueous solution comprising an oxidizing compound and a complexing compound, said complexing compound comprising a chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.","2.The composition as recited in claim 1, further comprising an alkaline compound.","3.The composition as recited in claim 1, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.","4.The composition as recited in claim 1, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.","5.The composition as recited in claim 1, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4( 1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.","6.The composition as recited in claim 1, wherein said oxidizing compound comprises hydrogen peroxide.","7.The composition as recited in recited in claim 2, wherein said alkaline compound comprises an inorganic basic compound or an organic basic compound.","8.The composition as recited in claim 7, wherein said alkaline compound is selected from the group consisting of ammonia and organic amine compounds.","9.The composition as recited in claim 2, wherein an amount of oxidizing compound comprises from 0.001 to 30 weight %.","10.The composition as recited in claim 1, wherein an amount of the complexing agent comprises from 0.1 to 1000 ppm of said solution.","11.The composition as recited in claim 7, wherein an amount of the inorganic basic compound or the organic basic compound comprises from 0.001 to 30 weight %.","12.The composition as recited in claim 1, wherein an amount of said oxidizing compound comprises from 0.001 to 95 weight %.","13.A method of treating a semiconductor substrate, wherein said method comprises treating a semiconductor substrate with a composition comprising an aqueous solution, said aqueous solution comprising a complexing compound comprising the chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.","14.The method as recited in claim 13, wherein said composition further comprises an oxidizing compound.","15.The method as recited in claim 13, wherein said composition further comprises an alkaline compound.","16.The method as recited in claim 13, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.","17.The method as recited in claim 13, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.","18.The method as recited in claim 13, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4(1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.","19.The method as recited in claim 14, wherein said oxidizing compound comprises hydrogen peroxide.","20.The method as recited in claim 15, wherein the alkaline compound comprises an inorganic basic compound or an organic basic compound.","21.The method as recited in claim 20, wherein said inorganic basic compound or said organic basic compound is selected from the group consisting of ammonia and organic amine compounds.","22.The method as recited in claim 14, wherein an amount of the oxidizing compound comprises from 0.001 to 30 weight %.","23.The method as recited in claim 15, wherein an amount of the alkaline compound comprises from 0.001 to 30 weight %.","24.The method as recited in claim 13, wherein an amount of said complexing compound comprises from 0.1 ppm to 1000 ppm.","25.Use of a composition as recited in claim 1 in a field selected from the group consisting of soil remediation, metal etch bath, denture bleaching, bleaching of fats, bleaching of oils, bleaching of waxes, wastewater treatment, paper pulp bleaching, and textile bleaching.","26.The composition as recited in claim 8, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds.","27.The method as recited in claim 21, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds."],[" FIELD OF THE INVENTION This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound."],[" SUMMARY OF THE INVENTION In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","The oxidizing compound can be in the form of an aqueous solution.","The complexing compound is for complexing metal ions.","Metal ions can be present in the solution or in an external medium being contacted with the solution.","Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.","In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.","In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.","Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.","Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.","In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.","In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.","Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.","Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said composition can further comprise an oxidizing compound.","Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.","The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.","Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.","Said solution is particularly suitable for cleaning a semiconductor substrate.","Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.","In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.","In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.","In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.","Said alkaline compound can comprise an inorganic or organic basic compound.","Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound.","STATE OF THE ART Chemical solutions comprising an oxidizing compound such as hydrogen peroxide are used in a wide area of applications, for example, as bleaching agent for paper pulp applications, dental bleaching composition or as cleaning agents.","Chemical solutions comprising an oxidizing compound have often problems related to the stability of the solution.","In pure form, aqueous solution are stable over extended periods of time.","However, the presence of certain metal ions in the solution causes decomposition of the oxidizing compound.","Consequently, stabilizers to prevent such decomposition should be added.","Stabilizers can be e.g.","a complexing compound, such that the complexing compound will bind to the metal and consequently, the metal is not available for reaction with the oxidizing compound.","Thus, the decomposition of the oxidizing compound is substantially inhibited and the lifetime of the solution is increased.","Very stringent specifications must be met by oxidizing solutions for specialized applications such as semiconductor applications or reagent chemicals.","An overview of stabilizing oxidizing compound, and more specifically hydrogen peroxide solutions, is given in Kirk-Othmer Encyclopedia of Chemical Technology (4th edition), vol.","13 p. 965.U.S.","Pat.","No.","4,239,643 discloses an aqueous peroxide-containing solutions used in bleaching of cellulose fiber, wherein the stability of the peroxide is very greatly increased by inclusion of alkali metal polyphosphate and alkali metal diethylene triamine penta(methylene phosphonate).","U.S. Pat.","No.","3,903,244 describes a hydrogen peroxide concentrate containing up to 50% hydrogen peroxide, 1 to 3% of a soluble amino(methyl phosphonic acid) or a salt thereof, and 0.05 to 0.5% of phenol.","The concentrate is useful in the preparation of highly acid metal pickling baths.","Stabilizing compounds are useful for a wide area of application, such as soil remediation, metal etch bath, denture bleaching, bleaching of fats, oils, waxes,, wastewater treatment, paper pulp bleaching, textile bleaching, CMP applications or semiconductor applications.","In cleaning application, for example in semiconductor application, oxidizing solutions have been investigated extensively.","Since the invention of the RCA cleaning by Kern et al.","in 1965 (W. Kern and D. A. Pautinen, RCA Review 31, 187, 1970) this cleaning cycle has become the most used for semiconductors.","The conventional RCA cleaning consists of two steps: an alkaline solution, the so called SC1 solution and an acidic solution, SC2.The SC1 solution is composed of 1 part ammonia (NH4OH), 1 part hydrogen peroxide (H2O2) and 5 parts ultra pure water (H2O) and is often referred to as APM-cleaning (i.e.","Ammonia Peroxide Mixture).","Originally it was used to remove organic residues by oxidation.","Later it has been proven to be very efficient to remove particles.","A drawback of the SC1 solution is that metals precipitate on silicon surfaces; especially aluminum, iron and zinc have been shown to adsorb strongly on the wafer surface (Mertens et al., Proc.","of the 8th Internat.","Symp.","On Silicon Materials Science and Technology PV98-1 (1998)).","In addition, especially Fe and Cu are found to catalyze the decomposition reaction of the peroxide (Mertens et al., Proc.","of the 5th Internat.","Symp.","on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997)) leading to a decrease in the bath lifetime.","In order to remove the metallic surface contamination, the SC2 solution consisting of 1 part hydrochloric acid, 1 part hydrogen peroxide and 6 parts ultra-pure water is used.","However, it is expensive to get hydrochloric acid of sufficient quality for the usage in SC2 solution.","There is also a risk of re-contaminating the surface with particles.","Problems also occur in spray tools due the corrosive behavior of hydrochloric acid.","With the progress in semiconductor manufacturing the requirements concerning particle and metal contamination as well as roughness of the silicon surfaces became more stringent.","This led to a number of variations of the RCA clean.","The potential problems related to the SC2 and the consideration to reduce process time and equipment by leaving out this acidic step led to the development of single-stage cleaning procedures.","This can be done by using chemicals with reduced amount of metallic impurities.","For that purpose, advanced purification procedures are established for obtaining ultra-pure water, ammonia and hydrogen peroxide.","However, these chemicals are very expensive and the purity is not always assured when they are used in a cleaning bath.","Moreover, the cleaning solution is not very robust with respect to metal contamination from the semiconductor substrate and from the hardware.","Besides this, having an extra step in the cleaning cycle to remove residual metallic contamination implies extra hardware, e.g.","a SC2-tank and a rinse tank need to be used, and more chemicals.","Leaving out this extra step will results in a reduction of the hardware cost and a reduction of the amount of chemicals used in the cleaning cycle.","EP 528053 describes a method for treating a surface of a substrate with a surface treatment composition.","The surface treatment composition comprises a liquid medium containing a complexing agent as a metal deposition preventive.","The surface treatment composition is improved by incorporating at least two complexing agents.","A first complexing agent is preferably an aromatic hydrocarbon ring with at least an OH or O− group bonded to a carbon atom constituting the ring.","A second complexing agent is compound having a donor atom, such as heterocyclic amines.","Since this cleaning composition comprises two complexing agents, drawbacks such as higher cost and increased waste treatment are observed.","Moreover, aromatic hydrocarbons such as Tiron, Catechol derivatives are hazardous for the environment and for humans.","U.S. Pat.","No.","5,290,361 and U.S. Pat.","No.","5,302,311 describes an aqueous hydrogen peroxide solution further comprising a complexing compound containing phosphonic acid groups and showing complexing ability.","Cleaning solution comprising phosphonic acid goups are not effective in removing or suppressing Al from the substrate.","Moreover, enhanced deposition of Cu has been measured.","This makes the cleaning solutions less suitable.","In the present invention, the problems related to removal of metals as mentioned in the prior art, are avoided.","AIMS OF THE INVENTION It is an aim of the invention to provide a stable composition comprising an oxidizing compound and a complexing compound.","It is a further aim of the invention to provide a new composition for treating a surface which is stable and provokes less or no metal precipitation on the surface.","Another aim of the invention is to provide a new cleaning solution.","Another aim of the present invention is to provide an efficient APM-cleaning solution having a good robustness with respect to metal contamination.","A further aim is to provide a new single-step method for cleaning semiconductor surfaces.","FIGURE CAPTIONS FIG.","1 chemical structure of complexing molecules FIG.","2 bath age (minutes) as function of the normalized H2O2 concentration, 1=no complexing agent; 30 2=DEHP; 3=10×DEHP; 4=1-EMHP; 5=10×1-EMHP; 6-DMHP and 7=10×DMHP.","SUMMARY OF THE INVENTION In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","The oxidizing compound can be in the form of an aqueous solution.","The complexing compound is for complexing metal ions.","Metal ions can be present in the solution or in an external medium being contacted with the solution.","Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.","In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.","In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.","Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.","Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.","In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.","In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.","Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.","Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said composition can further comprise an oxidizing compound.","Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.","The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.","Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.","Said solution is particularly suitable for cleaning a semiconductor substrate.","Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.","In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.","In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.","In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.","In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.","In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.","Said alkaline compound can comprise an inorganic or organic basic compound.","Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.","In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.","DETAILED DESCRIPTION OF THE INVENTION In relation to the appended drawings the present invention is described in detail in the sequel.","It is apparent however that a person skilled in the art can imagine several other equivalent embodiments or other ways of executing the present invention.","In an aspect of this invention, said composition comprises an oxidizing compound and a complexing compound.","Said oxidizing compound can be in the form of an aqueous solution.","Said complexing agent can have a chemical formula as given in FIG.","1.R1, R2, R3 and R4 are independently selected from the group comprising hydrogen (H) or any organic group.","R1, R2, R3,or R4 can have a different chemical structure.","For the purpose of this invention, said complexing agent as mentioned above is generally referred to as pyridinone.","Said organic group can be every possible sequence of C, N, O or S atoms linked to each other by single, double or triple bonds such that the complexing properties of the final complexing agent are assured.","Said organic group can be selected from the group comprising aliphatic side chains, heterocycles and aromatic structures.","Said organic side chain is every possible sequence of carbon, atoms linked to each other by a single, double or triple bound and optionally characterised by the presence of functional groups linked to the carbon atoms.","Functional groups can be alcohol, carboxyl, carbonyl, aldehyde, keton, ether, ester, amine, amide, halogen containing groups.","Said heterocycle can be one of the group comprising a crown ether, a cryptant, a calixarene, .",".",".","In a preferred embodiment, R3 and R4 are H atoms, while R1 and R2 are a methyl, ethyl, (iso)propyl or butyl group.","Said methyl, ethyl, (iso) propyl or butyl group can functionalized, e.g.","with a carboxyl group (COOH or COO−).","Preferably, the complexing molecules as described in FIG.","2 are used.","DEHP=1,2-Diethyl-3-hydroxy-4(1H)-pyridinone 1-EMHP=1-Ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone 2-EMHP=1-Methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone DMHP=1,2-Dimethyl-3-hydroxy-4(1H)-pyridinone PEPH=1-Propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone PMHP=1-Propyl-2-methyl-3-hydroxy-4(1H)-pyridinone ECMHP=1- (2′Carboxyethyl)-methyl-3-hydroxy-4(1H)-pyridinone ECEHP=1-(2′Carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.","Although the addition of the amount of complexing agent in the composition in this invention is not particularly limited, it depends on the degree of metal contamination -and on the kind of other compounds being present in the solution.","Furtheron, the amount of complexing agent depends on the specific chemical structure of the complexing compound.","The amount of the complexing agent lies 0.01 and 10000 ppm, between 0.1 and 1000 ppm of said composition.","For this application, ppm should be understood as parts per million in the composition.","The amount of complexing compound depends on the specific complexing compound.","Since said complexing agents do not form a 1:1 metal/complexing agent complex, higher concentrations of complexing agents are required, compared to other complexing agents such as EDTA.","Typical values for the stoichiometry CA/metal for Fe, Al, Cu and Zn are given in the table: Complex Stability Constants: Fe1 Al2 Cu Zn Fe2 Al2 Cu Zn2 DMHP DMHP DMHP DMHP DEHP DEHP DEHP DEHP [ML]/[M] [L] 15.10 12.20 10.62 7.19 15.2 13.42 10.74 7.70 [ML2]/[ML] 11.51 11.05 8.99 6.34 11.76 11.64 9.07 6.09 [L] [ML3]/[ML2] 9.27 9.37 9.78 8.48 5.12 [L] [MLx]/[M] 35.88 32.62 19.61 13.53 36.8 33.54 19.81 18.91 [L]x 2 DMHP 1-EMHP DEHP [MLx]/[M] 37.2/36.4 37.7 36.8 [L]x PFe 19.4 19.7 1X = 3 2Values have been taken from different authors and determined by different techniques.","Said oxidizing compound can be every chemical compound having oxidizing properties.","E.g.","organic species, metallic compounds, inorganic particles, silicon, etc.","can be oxidized.","The oxidizing compound is a compound selected from the group comprising hydrogen peroxide or oxidizing anions.","The oxidizing anions can be e.g.","nitric acid and its salts, nitrate, persulfate, periodate, perbromate, perchlorate, iodate, bromate and chlorate salts of ammonium.","Preferably, the oxidizing compound is hydrogen peroxide.","The concentration of the oxidizing compound can be, but is not limited hereto, between 0.0001 and 99 weight %, between 0.001 and 90 weight % and preferebly between 0.001 to 30 weight %.","In this application, weight % should be understood as the percentage of weight of the specified compound in the composition.","Said composition can further comprise an alkaline compound.","The alkaline compound or base can be every chemical compound with a pH higher than 7.The alkaline compound can be an organic or inorganic compound.","The alkaline compound can be an organic base, ammonia, ammoniumhydroxide, or an alkaline solution containing metal ions such as potassium or sodium.","Said organic base can be a quaternary ammonium hydroxide such as tetraalkyl ammonium hydroxide in which the alkyl groups can contain hydroxy- and alkoxy-containing groups with 1 to 4 carbon atoms in the alkyl or alkoxy group.","Said organic base can further be an organic amine such as an alkanol amine.","Alkanol amines can be 2-aminoethanol, 1-amino 2-propanol, 1-amino 3-propanol.","Preferably, the alkaline compounds are tetramethyl ammonium hydroxide, and trimethyl 2-hydroxy ethyl ammonium hydroxide (choline) and ammonium hydroxide.","The amount of the alkaline compound lies between 0.0001 and 90 weight %, between 0.001 and 50 weight %, between 0.001 and 30 weight %.","Said composiiton can further comprise a surfactant.","In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.","Said semiconductor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.","Said composition can further comprise an oxidizing compound.","Said composition can be, but is not limited hereto, the composition described in the first aspect of this invention.","Said composition is particularly usefull for cleaning a substrate such that particles are oxidized and metallic contamination is removed.","The complexing compound is for complexing metals being present on the substrate and in the solution.","Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.","A substrate can be, but is not limited hereto, a substrate such as semiconducting material, glass, quartz, ceramics, metal, plastic, magnetic material, superconductor and the like.","Preferably, said substrate is a semiconductor substrate.","Semiconductor substrate can be every possible substrate used in semiconductor processing.","Said semiconductor substrate can be a substrate selected from the group, but not limited hereto, comprising a substrate made of silicon, germanium, gallium arsenide, indium phosphide, etc.","The semiconductor substrate be e.g.","the substrates as mentioned above covered entirely op partially with a thin film of e.g.","an oxide, a nitride, a metal, a polymeric insulating layer, an anti-reflecting coating, a barrier, a photoresist layer, etc.","The present invention is particularly relevant for cleaning or etching a semiconductor substrate of which the surface should be highly clean.","When the composition is used for treating a substrate, the weight concentration range of the alkaline compound in the-cleaning solution are typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.","For ammonium hydroxide, the weight concentration range of the alkaline compound in the cleaning solution are typically but not limited to 0.001-30%, 0.1-20% and preferably 0.1-5% by weight.","For other alkaline compounds, the weight concentration range is equivalent, and function of the strength of the alkaline compound.","For peroxide, the weight concentration the hydrogenperoxide is typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.","In the preferred embodiment of this invention, a composition for treating a semiconductor surface comprises ammonium hydroxide, hydrogen peroxide, water (hereafter called APM mictures) and additionally a complexing agent.","Said complexing agent is one of the group consisting of the molecules as described in FIG.","2.APM-cleaning mixtures comprising a complexing agent according to the present invention are robust with respect to metal contamination coming from the fresh chemicals as well as with respect to metal contamination introduced in the course of its use for cleaning.","The robustness of the basic APM process can be improved by the addition of complexing agents which keep the metals in solution and prevent the above mentioned catalysis of the peroxide decomposition.","The volume mixing ratio of NH4OH(296)/H2O2(30%)/H2O is typically, but not limited hereto, 0.25/1/5.The cleaning solution is prepared with the amounts as described above and afterwards the semiconductor substrate is treated with the cleaning solution.","In the best mode known to the applicant, molecule DMHP is selected and added in the amounts described above.","The complexing agent can be added as the pure compound to the cleaning solution.","Alternatively, the complexing agent can be dissolved in either water, ammonia or peroxide or a dilution of the two latter chemicals and added as such to the cleaning solution.","It is a further aim of the invention to disclose a process for treating a semiconductor substrate comprising the steps of treating said semiconductor substrate with the cleaning solution as described above and drying said semiconductor substrate.","An optional step: rinsing said semiconductor substrate can be performed after said treating said semiconductor substrate with the cleaning solution as described above.","In the step of treating said semiconductor substrate with said cleaning solution, the semiconductor substrate can be immersed in a bath containing the cleaning solution.","Alternatively, the cleaning solution can be dispensed or sprayed onto the semiconductor substrate for instance by using a spray processor.","In all cases, the cleaning performance of the solution can be enhanced by using a megasonic transducer.","The temperature range for treating the semiconductor substrate with the cleaning solution is typically but not limited to 0-95 degrees Celcius, 10-80 degrees Celcius and preferably between 20-70 degrees Celcius.","In the step of drying the semiconductor substrate, several techniques known in the art can be used, e.g.","spin-drying, Maragoni-drying, drying techniques using organic vapours.","The step of rinsing the semiconductor substrate comprises treating the semiconductor substrate with DI water or treating the semiconductor substrate with a diluted acidic solution or with DI water containing the complexing agent in an amount of 1 to 100000 ppm, 10 to 10000 ppm and by preference 100 to 1000 ppm.","It is a further aim of the invention to describe a process for treating a semiconductor substrate comprising the step of: treating said semiconductor substrate with any cleaning solution and/or treating said semiconductor substrate with any rinsing solution Said any cleaning solution can be any cleaning solution, not being limited to the compositions described in this application.","Said rinsing solution comprises said complexing agent and water.","Said complexing agent can be any complexing agent described in this application.","The amount of the complexing agent in the composition can be between 1 and 100000 ppm, 10 and 10000 ppm and by preference between 100 and 1000 ppm.","This rinsing solution can also comprise a surfactant in an amount of 0.1 ppm to 10 w %.","A surfactant is a surface-active agent comprising a lyophobic group and a lyophilic group.","The lyophobic group can be a straight-chain alkyl group or a branched-chain alkyl group (C8-C20), an long-chain (C8-C20) alkyl benzene residue, an alkylnaphtalene residue (C3 and greater-length alkyl groups), high-molecular-weight propylene oxide polymers (polyoxypropylene glycol derivatives), long-chain perfluoroalkyl or polysiloxane groups.","Depending upon the lyophilic group, the surfactant can be an anionic, cationic, nonionic or zwitterionic surfactant.","Anionic surfactants can be carboxylic acids or carboxylic acid salts (such as sodium and potassium salts of straight-chain fatty acids), sulfonic acids or sulfonic acid salts (such as linear alkylbenzenesulfonates, higher alkylbenzenesulfonates, benzene-, toluene-, xylene- and cumenesulfonates, ligninsulfonates, petroleum sulfonates, N-acyl-n-alkyltaureates, paraffin sulfonates, secondary n-alkanesulfonates, α-olefin sulfonates, sulfosuccinate esters, alkylnaphtalenesulfonates or isethionates), sulfuric acid ester salts (such as sulfated linear primary alcohols, sulfated polyoxyethylenated straight-chain alcohols or sulfated triglyceride oils), phosphoric and polyphosphoric acid esters.","Cationic surfactants can be primary amines and their salts, diamines and polyamines and their salts, quaternary ammonium salts (such as tetralkylammonium salts or imidazolinium salts), polyoxyethylenated long-chain amines (RN(CH2CH2O)xH]2), quaternized polyoxyethylenated long-chain amines or amine oxides (such as N-alkyldimethylamine oxides).","Nonionic surfactants can be polyoxyethylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long-chain carboxylic acid esters (such as glyceryl and polyglyceryl esters of natural fatty acids, propylene glycol, sorbitol or polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters and polyoxyethylenated fatty acids), alkanolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones or alkylpolyglycosides.","Zwitterionic surfactants have both anionic and cationic charges present in the lyophilic portion (such as β-N-alkylaminopropionic acids, N-alkyl-β-iminodipropionic acids, imidazoline carboxylates, N-alkylbetaines, amine oxides, sulfobetaines or sultaines) (M. J. Rosen, Surfactants and Interfacial phenomena, 2nd Edition, John Wiley and Sons, New York, 1989]) No additional alkaline compound should need to be added to the said rinsing solution.","The pH range of said rinsing solution can typically be, but not limited to, between 5 and 8.Said Rinse solution can be dispensed or sprayed onto the semiconductor surface as described above.","During rinsing the performance can also be enhanced by using a megasonic transducer.","The process of treating a semiconductor substrate with a cleaning solution comprising the above mentioned steps can be performed for a certain number of semiconductor substrates.","After treating at least one substrate, but preferably after treating more substrates, the composition of the cleaning solution can be modified by e.g.","adding extra alkaline compound, adding extra complexing compound, adding oxidizing compound such that the initial composition of the cleaning solution is kept constant as function of the process time.","COMPARATIVE EXAMPLES The present invention will be further described using non-limiting examples and drawings.","The effectiveness of the new class of complexing agents on the inhibition of the metal catalyzed decomposition of peroxide and the prevention of metal outplating on silicon wafers in metal contaminated APM cleaning solutions is described next.","For complexing agents as disclosed in this invention, different model compounds are selected (1-EMHP, 2-EMHP, DMHP, DEHP, PEHP, PMHP, ECMHP and ECEHP) and added to the bath at a concentration of 2.67×10−5 M and at a 10×, 40×, or 50× higher concentration (namely 2.65×10−4 10.6×10−4 and 13.25×10−4 M).","A comparison is made with other types of complexing agents containing as functional groups either phosphonic acids, such as diethylene (kleine letter) triamine penta-methylenephosphonic acid (DTPMP), carboxylic acids, such as ethylene diamino tetra acetic acid (EDTA).","An overview of the different chemicals used for the experiments is given in Table 1.All experiments were done in a class 1000 clean room environment or better.","TABLE 1 Chemicals used for preparation of APM baths.","Chemical Vendor Grade H2O2 30 (w/w) % Ashland TB(*) NH4OH 29 (w/w) % Ashland TB(*) EDTA Fluka DTPMP Monsanto Dequest 2060S DMHP Aldrich (*)TB-grade corresponds with a specification of maximal 100 ppt of metal ions in the chemical.","Example 1 Metal Outplating from APM Mixtures in Presence of Different Complexing Agents The efficiency of complexing agents to suppress the deposition of metallic contamination onto wafer surfaces was studied.","This was done through intentionally spiking well controlled trace amounts of metallic contamination to cleaning solutions.","For these metal deposition tests, p-type monitor wafers with a diameter of 150 mm and <100> orientation were used.","The wafers were pre-cleaned using IMEC Clean® in an automated Steag wet bench (i.e.","SOM+dHF+O3-rinse rendering a perfectly clean hydrophilic surface).","The metal deposition experiments were performed in a static quartz tank with a quartz cover plate.","This tank was not equipped with a megasonic transducer.","APM mixtures were prepared containing 1 w-ppb of different metals of interest with/without the complexing agent added.","The metals spiked to the APM bath were added from AAS-standard solutions (Merck).","After a bath age of 5 minutes, three wafers were immersed for 10 minutes, rinsed for 10 minutes in an overflow rinse tank and dried with a commercially available Marangoni drier (STEAG).","The resulting metal contamination was measured with straight TXRF or VPD-DSE-DC-TXRF (Vapor Phase Decomposition-Droplet Surface Etching-Droplet Collection Total X-Ray Fluorescence).","Determination of Al water surface concentration was done using VPD-DC GF-AAS (Graphite Furnace Atomic Absorption Spectroscopy).","An overview of the resulting metal surface contamination after dipping a clean wafer in an APM spiked with metals and different complexing agents is given in Table 2.TABLE 2 Measured metal surface concentrations after immersion in metal contaminated 0.25/1/5 APM solution at 20-50° C. with different complexing agents present.","Complexing Conc.","CA Conc.","Mt Al (1010 Fe agent (2.7 × 10−5 M) (w-ppb) at/cm2) (1010 at/cm2) Pre-clean only NA NA 2.34
- FIELD OF THE INVENTION
- BACKGROUND OF THE INVENTION
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"Storage medium convey apparatus and convey method and recording and/or reproduction apparatus",
"Apparatus that carries or transfers a memory medium having a solid-state memory over the inside and outside portions of a recording and/or reproducing apparatus is adapted so that, by selective engagement with respect to a chassis or a holder of a fluctuation lever the memory medium is inserted and held.",
"The memory medium is moved in a manner bridging between an eject position where insertion/withdrawal of the memory medium is permitted and a loading position.",
"A first biasing member moves and biases the holder in an eject direction opposite to a memory medium insertion direction is caused to store biasing force in the eject direction.",
"A second biasing member biases the holder in the memory medium insertion direction and is caused to be operative to carry out switching between movement in the memory medium insertion direction of the holder and movement in the eject direction."
],
[
"1.A carrying apparatus for a memory medium, comprising: a holder adapted to have the memory medium inserted therein including a connection terminal connected to a contact provided at a front end side of the inserted memory medium; a lock mechanism provided on the holder and locked with respect to a chassis of the holder so that the holder is movably supported, the lock mechanism serving to limit movement with respect to the chassis of the holder, so that when the contact of the memory medium inserted into the holder is connected to the connection terminal of the holder, the lock mechanism is pressed by the memory medium to thereby release a locking state with respect to the chassis; a fluctuation lever engaged with the holder and moved when the lock mechanism is released, the fluctuation lever moved in an insertion direction of the memory medium as one with the holder; first biasing means for biasing the fluctuation lever in an eject direction opposite to the insertion direction of the memory medium; and second biasing means for biasing the holder in the insertion direction of the memory medium, wherein the fluctuation lever is moved in the insertion direction of the memory medium as one with the holder to thereby allow the first biasing means to store a biasing force that moves the fluctuation lever in the eject direction, and engagement with respect to the holder is released by movement in the insertion direction of the memory medium of the fluctuation lever and only the holder into which the memory medium is inserted is drawn in the insertion direction of the memory medium by the second biasing means in accordance with engagement with respect to the chassis.",
"2.The carrying apparatus for a memory medium as set forth in claim 1, wherein the fluctuation lever is rotationally biased by the first biasing means in a direction where engagement with respect to the holder is released.",
"3.The carrying apparatus for memory medium as set forth in claim 2, wherein when the fluctuation lever is moved in the insertion direction of the memory medium as one with the holder, release of engagement with respect to the holder is limited by rotation limiting means provided at a side of the chassis that movably supports the holder.",
"4.The carrying apparatus for a memory medium as set forth in claim 1, wherein the lock mechanism includes a pressing operation portion caused to undergo a pressing operation by a lock piece engaged with the chassis and the memory medium inserted into the holder and a lock lever rotatably supported by the holder and rotationally biased by a biasing member in a direction to engage the lock piece with the chassis.",
"5.The carrying apparatus for a memory medium as set forth in claim 1, wherein a cover body rotatably attached to the chassis opens and closes a memory medium insertion/withdrawal opening adapted so that the memory medium to be inserted into the holder is inserted thereinto or is withdrawn therefrom, the cover body being rotationally biased in a direction to close the memory medium insertion/withdrawal opening by a biasing member at all times.",
"6.The carrying apparatus for a memory medium as set forth in claim 1, wherein the apparatus further comprises a slider movably supported by the chassis in the insertion direction with respect to the holder of the memory medium, and adapted so that the fluctuation lever is rotatably supported, wherein the first biasing means includes a first biasing member provided in a manner bridging between the slider and the chassis and biases the holder in the eject direction opposite to the insertion direction of the memory medium, wherein the second biasing means includes a second biasing member provided in a manner bridging between the slider and the holder and biases the holder in the insertion direction of the memory medium, and wherein the fluctuation lever is adapted so that the locking state with respect to the chassis by the lock mechanism is released and is moved in the insertion direction of the memory medium in one body with the holder moved in the insertion direction of the memory medium to allow the first biasing member to store a biasing force that moves the slider in the eject direction, whereby when a fluctuation lever lock mechanism provided at the fluctuation lever is moved to a position opposite to a cut portion provided at the chassis side, the fluctuation lever lock mechanism is inserted into the cut portion and engagement between the engagement portion and the holder is released by a rotational force produced as a result of a force in the insertion direction of the memory medium by the holder and a biasing force in the eject direction by the first biasing member with respect to the fluctuation lever act with respect to an engagement portion of the fluctuation lever formed at a position engaged with the holder, and the fluctuation lever and the slider are locked with respect to the chassis and only the holder into which the memory medium is inserted is drawn in the insertion direction of the memory medium by the biasing force of the second biasing member.",
"7.The carrying apparatus for a memory medium as set forth in claim 1, wherein the apparatus further comprises eject operation means for causing the holder moved in the insertion direction of the memory medium by the second biasing means to undergo movement operation in the eject direction against a biasing force of the second biasing means, and wherein when only the holder into which the memory medium is inserted is drawn in the insertion direction of the memory medium by the second biasing means so that it is held at a loading position of the memory medium, the holder is moved in the eject direction against the biasing force of the second biasing means through the eject operation means to thereby release engagement with respect to the chassis of the fluctuation lever to receive the biasing force of the first biasing means to move the holder in the eject direction as one with the fluctuation lever to move the holder to an initial position.",
"8.The carrying apparatus for a memory medium as set forth in claim 7, wherein the eject operation means comprises an eject operation piece moved as one with the holder.",
"9.The carrying apparatus for a memory medium as set forth in claim 7, wherein the apparatus further comprises a slider movably supported by the chassis in the insertion direction with respect to the holder of the memory medium and adapted so that the fluctuation lever is rotatably supported, wherein the first biasing means includes a first biasing member provided in a manner bridging between the slider and the chassis for biasing the slider in the eject direction opposite to the insertion direction of the memory medium, wherein the second biasing means includes a second biasing member provided in a manner bridging between the slider and the holder for biasing the holder in the insertion direction of the memory medium, wherein the fluctuation lever is adapted so that the locking state with respect to the chassis by the lock mechanism is released and the fluctuation lever is moved in the insertion direction of the memory medium as one with the holder moved in the insertion direction of the memory medium to thereby allow the first biasing member to store the biasing force that moves the slider in the eject direction, and when a fluctuation lever lock mechanism provided at the fluctuation lever is moved to a position opposite to a cut portion provided at the chassis side, the fluctuation lever lock mechanism is inserted into the cut portion and engagement between the engagement portion and the holder is released by a rotational force produced as a result of a force in the insertion direction of the memory medium by the holder and the biasing force in the eject direction by the first biasing member with respect to the fluctuation lever act with respect to engagement portion of the fluctuation lever formed ay a position engaged with the holder, and the fluctuation lever and the slider are locked with respect to the chassis and only the holder into which the memory medium is inserted is drawn in the insertion direction of the memory medium by a biasing force of the second biasing means, and wherein the holder is moved in the eject direction against the biasing force of the second biasing member through the eject operation means, and the fluctuation lever is rotated by the biasing force by the first biasing member to release the locking state with respect to the chassis by the fluctuation lever lock mechanism to receive the biasing force of the first biasing member to move the holder in the eject direction as one with the fluctuation lever to move the holder to an initial position.",
"10.A recording and/or reproducing apparatus, comprising: a holder adapted to have a memory medium inserted therein including a connection terminal connected to a contact provided at a front end side of the inserted memory medium; recording and/or reproducing means for carrying out recording and/or reproduction of data with respect to the memory medium having the contact connected to the connection terminal of the holder; a lock mechanism provided on the holder and locked by a chassis of the holder adapted so that the holder is movably supported, the lock mechanism serving to limit movement with respect to the chassis of the holder, so that when the contact of the memory medium inserted into the holder is connected to the connection terminal of the holder, the lock mechanism is pressed by the memory medium to thereby release a locking state with respect to the chassis; a fluctuation lever engaged with the holder and moved when the lock mechanism is released in an insertion direction of the memory medium as one with the holder; first biasing means for biasing the fluctuation lever in an eject direction opposite to the insertion direction of the memory medium; and second biasing means for biasing the holder in the insertion direction of the memory medium, wherein the fluctuation lever is moved in the insertion direction of the memory medium as one with the holder to thereby allow the first a biasing means to store biasing force that moves the fluctuation lever in the eject direction, and engagement with respect to the holder is released by movement in the insertion direction of the memory medium of the fluctuation lever and only the holder into which the memory medium is inserted is drawn in the insertion direction of the memory medium by the second biasing means in accordance with engagement with respect to the chassis.",
"11.The recording and/or reproducing apparatus as set forth in claim 10, wherein the recording and/or reproducing means carries out recording and/or reproduction of data with respect to the memory medium when the contact is connected to the connection terminal of the holder at a reproduction position where the memory medium is located as a result of only the holder into which the memory medium is inserted is drawn in the insertion direction of the memory medium by the second biasing means.",
"12.A carrying method for a memory medium comprising: a step of inserting a memory medium into a holder; a step of electrically connecting a contact of the memory medium and a connection terminal within the holder; a step of pressing lock means that locks the holder with respect to a chassis of the holder by the memory means after the contact of the memory medium and the connection terminal within the holder have been electrically connected to thereby release a locking state by the lock means; a step of further moving the holder in an insertion direction along with the memory medium; a step of engaging a lever moved in the insertion direction as one with the holder as a result of the lever being engaged with the holder, and of releasing engagement with respect to the holder; a step of storing a biasing force in an eject direction opposite to the insertion direction at first biasing means for a time period during which the lever is moved in the insertion direction where engagement with the holder is released; and a step of drawing only the holder in which engagement by the lever has been released in the insertion direction along with the memory medium by second biasing means.",
"13.The carrying method for memory medium as set forth in claim 12, wherein the method further comprises: a step of drawing only the holder in which engagement by the lever has been released in the insertion direction along with the memory medium by the second biasing means to hold the holder at a loading position; and a step of moving the holder held at the loading position in the eject direction against a biasing force of the second biasing means to thereby release engagement with respect to the chassis of the fluctuation lever to receive a biasing force of the first biasing means to move the holder in the eject direction as one with the fluctuation lever to move the holder to an initial position."
],
[
"<SOH> BACKGROUND ART <EOH>Hitherto, memory medium using a solid-state memory as a memory element is used as a memory medium for information signal.",
"Since the memory medium using the solid-state memory can be formed compact while ensuring large memory capacity, further miniaturization of the recording and/or reproducing apparatus using such memory medium can be realized."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a perspective view showing plane side of a memory medium used in the present invention.",
"FIG.",
"2 is a perspective view showing bottom face side of the memory medium shown in FIG.",
"1 .",
"FIG.",
"3 is a perspective view showing an example of a recording/reproducing apparatus to which the present invention is applied.",
"FIG.",
"4 is a perspective view showing another example of the recording/reproducing apparatus to which the present invention is applied.",
"FIG.",
"5 is an exploded perspective view showing loading unit to which carrying apparatus according to the present invention is applied.",
"FIG.",
"6 is a bottom view showing connector to which memory medium is connected along with the memory medium.",
"FIG.",
"7 is a bottom view showing the state where memory medium is connected to connector.",
"FIG.",
"8 is a plan view showing the state where holder is moved to initial position.",
"FIG.",
"9 is a side view showing the side where loading control mechanism including fluctuation lever is provided.",
"FIG.",
"10 is a side view showing the side where lock mechanism which locks holder at chassis is provided.",
"FIG.",
"11 is a side view showing loading control mechanism when holder is moved to initial position.",
"FIG.",
"12 is a plan view showing the state where memory medium is inserted into holder so that contact portion of the memory medium is connected to connector.",
"FIG.",
"13 is a side view showing the state where memory medium is inserted into holder so that contact portion of the memory medium is connected to connector.",
"FIG.",
"14 is a plan view showing the state where fluctuation lever is moved by holder in memory medium insertion direction and is then fluctuated so that it is engaged with chassis.",
"FIG.",
"15 is a side view showing the state where engagement between fluctuation lever and holder is released so that the fluctuation lever is engaged with chassis.",
"FIG.",
"16 is a side view of loading control mechanism showing the state where engagement between fluctuation lever and holder is released so that the fluctuation lever is engaged with chassis.",
"FIG.",
"17 is a plan view showing the state where memory medium inserted into holder is caused to undergo loading at loading position.",
"FIG.",
"18 is a side view showing the state where holder is caused to undergo biasing force of second extension coil spring so that it is moved to memory medium loading position.",
"FIG.",
"19 is a side view showing loading control mechanism when holder is caused to undergo biasing force of second extension coil spring so that the holder is placed in the state where it is moved to memory medium loading portion.",
"FIG.",
"20 is a plan view showing the state where memory medium loaded into holder is ejected.",
"FIG.",
"21 is a side view showing the state where memory medium loaded into holder is ejected.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to a carrying apparatus and a carrying method for a memory medium which are adapted for carrying a memory medium using a solid-state memory as a memory element to, e.g., loading position within a recording and/or reproducing apparatus, and further relates to a recording and/or reproducing apparatus using such a carrying apparatus.",
"BACKGROUND ART Hitherto, memory medium using a solid-state memory as a memory element is used as a memory medium for information signal.",
"Since the memory medium using the solid-state memory can be formed compact while ensuring large memory capacity, further miniaturization of the recording and/or reproducing apparatus using such memory medium can be realized.",
"DISCLOSURE OF THE INVENTION An object of the present invention is to realize further miniaturization of such a carrying apparatus for a memory medium using a solid-state memory as a memory element which can be formed compact while ensuring large memory capacity, and to realize further miniaturization of a recording and/or reproducing apparatus using such a carrying apparatus.",
"Another object of the present invention is to provide a carrying apparatus and a carrying method which can securely carry a memory medium using a solid-state memory as a memory element to the inside and outside of the apparatus.",
"A further object of the present invention is to provide a carrying apparatus and a recording and/or reproducing apparatus provided with eject mechanism which can securely eject a memory medium which has been carried to a predetermined loading position.",
"A carrying apparatus for a memory medium according to the present invention proposed in order to attain objects as described above comprises: a holder adapted so that the memory medium is inserted and provided with a connection terminal connected to a contact provided at the front side of the inserted memory medium; a lock mechanism provided at the holder and locked with respect to a chassis adapted so that the holder is movably supported, the lock mechanism serving to limit movement with respect to the chassis of the holder, and being such that when the contact of the memory medium inserted into the holder is connected to the connection terminal of the holder, the lock mechanism is pressed by the memory medium to thereby release lock with respect to the chassis; a fluctuation lever engaged with the holder moved as the result of the fact that lock with respect to the chassis by the lock mechanism is released, and moved in an insertion direction of the memory medium in one body with the holder; first biasing-means for biasing the fluctuation lever in an eject direction opposite to the insertion direction of the memory medium; and second biasing-means for biasing the holder in the insertion direction of the memory medium of the holder.",
"In this carrying apparatus, the fluctuation lever is moved in theinsertion direction of the memory medium in one body with the holder to thereby allow the first biasing means to store biasing force which moves the fluctuation lever in the ejection direction, and engagement with respect to the holder is released by movement in the insertion direction of the memory medium of the fluctuation lever and only the holder into which the memory medium is inserted is drawn or pulled in the insertion direction of the memory medium by the second biasing means in accordance with engagement with respect to the chassis.",
"Here, the fluctuation lever is rotationally biased by the first biasing means in the direction where engagement with respect to the holder is released.",
"Moreover, the fluctuation lever is adapted so that when it is moved in the insertion direction of the memory medium in one body with the holder, release of engagement with respect to the holder is limited by a rotation limiting portion provided at the -side of a chassis which movably supports the holder.",
"The lock mechanism used in the carrying apparatus according to the present invention includes a pressing operation portion which is caused to undergo pressing operation by a lock piece engaged with the chassis and the memory medium inserted into the holder, and comprises a lock lever rotatably supported by the holder and rotationally biased by a biasing member in a direction to engage the lock piece with the chassis.",
"At the chassis of the carrying apparatus, a cover body which opens or closes a: memory medium insertion/withdrawal opening adapted so that the memory medium to be inserted into the holder is inserted thereinto or withdrawn therefrom is rotatably attached.",
"The cover body is rotationally biased by the biasing member in-a direction to close the memory medium insertion/withdrawal opening at all times.",
"The carrying apparatus according to the present invention further comprises a slider movably supported by the chassis in the insertion direction with respect to the holder of the memory medium, and adapted so that the fluctuation lever is rotatably supported.",
"Here, the first biasing means comprises a first biasing member which is provided in a manner bridging between the slider and the chassis, and biases the slider in the eject direction opposite to the insertion direction of the memory medium.",
"The second biasing means comprises a second biasing member which is provided in a manner bridging between the slider and the holder, and biases the holder in the insertion direction of the memory medium.",
"The fluctuation lever is adapted so that lock with respect to the chassis by the lock mechanism is released so that it is moved in the insertion direction of the memory medium in one body with the holder moved in the insertion direction of the memory medium to thereby allow the first biasing member to store biasing force which moves the slider in the eject direction, and is adapted so that when a fluctuation lever lock mechanism provided at the fluctuation lever is moved up to the position opposite to a cut portion provided at the chassis side, the fluctuation lever lock mechanism is inserted into the cut portion and engagement between the engagement portion and the holder is released by rotational force produced as the result of the fact that force in the insertion direction of the memory medium by the holder and biasing force in the eject direction by the first biasing member with respect to the fluctuation lever act with respect to an engagement portion of the fluctuation lever formed at the position engaged with the holder, and the fluctuation lever and the slider are locked with respect to the chassis and only the holder inserted into the memory medium is drawn or pulled in the insertion direction of the memory medium by biasing force of the second biasing member.",
"The carrying apparatus according to the present invention further comprises eject operation means for allowing the holder moved in the insertion direction of the memory medium by the second biasing means to undergo -movement operation in the eject direction against biasing force of the second biasing means, whereby when only the holder to be inserted into the memory medium is drawn or pulled in the insertion direction of the memory medium by the second biasing means and is held at a loading position of the memory medium, the holder is moved in the eject direction against biasing force of the second biasing means through the eject operation means to thereby release engagement with respect to the chassis of the fluctuation lever to receive biasing force of the first biasing means to move the holder in the eject direction in one body with the fluctuation lever to move the holder up to the initial position.",
"Here, the eject operation means comprises an eject operation piece moved in one body with the holder.",
"The carrying apparatus according to the present invention further comprises a slider movably supported by the chassis in the insertion direction with respect to the holder of the memory median, and adapted so that the fluctuation lever is rotatably supported.",
"The first biasing means constituting this apparatus includes a first biasing member which is provided in a manner bridging between the slider and the chassis, and biases the slider in an eject direction opposite to the insertion direction of the memory medium, and the second biasing means comprises a second biasing member which is provided in a manner bridging between-the slider and the holder, and biases the-holder in the insertion direction of the memory medium.",
"The fluctuation lever is adapted so that lock with respect to the chassis by the lock mechanism is released so that it is moved into the insertion direction of the memory medium in one body with the holder moved in the insertion direction of the memory medium to thereby allow the first biasing member to store biasing force which moves the slider in the eject direction, whereby when a fluctuation lever lock mechanism provided at the fluctuation lever is moved up to the position opposite to a cut portion provided at the chassis side, the fluctuation lever lock mechanism is inserted into the cut portion and engagement between the engagement portion and the holder is released by rotational force produced as the result of the fact that force in the insertion direction of the memory medium by the holder and biasing force in the eject direction by the first biasing member with respect to the fluctuation lever act with respect to engagement portion of the fluctuation lever formed at the position engaged with the holder, and the fluctuation lever and the slider are locked with respect to the chassis and only the holder into which the memory medium is inserted is drawn or pulled in the insertion direction of the memory-medium by biasing force of the second biasing member so that it is held at loading position of the memory medium to move the holder in eject direction against biasing force of the second biasing member through eject operation means, and to rotate the fluctuation lever by biasing force by the first biasing means to release lock with respect to the chassis by the fluctuation lever lock mechanism to receive biasing force of the first biasing member to move the holder in the eject direction in one-body with the fluctuation lever to move the holder to initial position.",
"A recording and/or reproducing apparatus according to the present invention comprises: a holder adapted so that a memory medium is inserted, and provided with a connection terminal connected to a contact provided at the front side of the inserted memory medium; recording and/or reproducing means for carrying out recording and/or reproduction of data with respect to the memory medium of which contact is connected to the connection terminal of the holder; a lock mechanism provided at the holder and locked by a chassis adapted so that the holder is movably supported, and the lock mechanism serving to limit movement with respect to the chassis of the holder and being such that when the contact of the memory medium inserted into the holder is connected to the connection terminal of the holder, the lock mechanism is pressed by the memory medium to thereby release lock with respect to the chassis; a fluctuation lever engaged with the holder moved as the result of the fact that lock with respect to the chassis by the lock mechanism is released and moved in insertion direction of the memory medium in one body with the holder; first biasing means for biasing the fluctuation lever in an eject direction opposite to the insertion direction of the memory medium, and second biasing means for biasing the holder in the insertion direction of the memory medium.",
"In this recording and/or reproducing apparatus, the fluctuation lever is moved in the insertion direction of the memory medium in one body with the holder to thereby allow the first biasing means to store biasing force which moves the fluctuation lever in the eject direction, and engagement with respect to the holder is released by movement in the insertion direction of the memory medium of the fluctuation lever and only the holder into which the memory medium is inserted is drawn or pulled in the insertion direction of the memory medium by the second biasing means in accordance with engagement with respect to the chassis.",
"The present invention is directed to a method of carrying a memory medium, comprising: a step of inserting a memory medium into a holder; a step of electrically connecting a contact of the memory medium and a connection terminal within the holder; a step of pressing, by the memory medium, lock means which locks the holder at a chassis after the contact of the memory medium and the connection terminal within the holder are electrically connected to thereby release lock by the lock means; a step of further moving the holder along with the memory medium in an insertion direction; a step of engaging a lever moved in the insertion direction in one body with the holder as the result of the fact that the lever is engaged with the holder with the chassis and of releasing engagement with the holder; a step of storing biasing force in an eject direction opposite to the insertion direction at first biasing means for a time period during which the lever is moved with the holder is moved in the insertion direction where engagement with the holder is released; and a step of drawing or pulling only the holder in which engagement by the lever has been released in the insertion direction along with the memory medium by second biasing means.",
"This method of carrying out memory medium further comprises a step of drawing or pulling only the holder in which engagement by the lever has been released in the insertion direction along with the memory medium by the second biasing means to hold it at a loading position, and a step of moving the holder held at the loading position to move it in the eject direction against biasing force of the second biasing means to thereby release engagement with respect to the chassis of a fluctuation lever to receive biasing force of the first biasing means to move the holder in one body with the fluctuation lever in the eject direction to move the holder to initial position.",
"Still further objects of the present invention and practical merits obtained by the present invention will become more apparent from the description of the embodiments which will be given below with reference to the attached drawings.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a perspective view showing plane side of a memory medium used in the present invention.",
"FIG.",
"2 is a perspective view showing bottom face side of the memory medium shown in FIG.",
"1.FIG.",
"3 is a perspective view showing an example of a recording/reproducing apparatus to which the present invention is applied.",
"FIG.",
"4 is a perspective view showing another example of the recording/reproducing apparatus to which the present invention is applied.",
"FIG.",
"5 is an exploded perspective view showing loading unit to which carrying apparatus according to the present invention is applied.",
"FIG.",
"6 is a bottom view showing connector to which memory medium is connected along with the memory medium.",
"FIG.",
"7 is a bottom view showing the state where memory medium is connected to connector.",
"FIG.",
"8 is a plan view showing the state where holder is moved to initial position.",
"FIG.",
"9 is a side view showing the side where loading control mechanism including fluctuation lever is provided.",
"FIG.",
"10 is a side view showing the side where lock mechanism which locks holder at chassis is provided.",
"FIG.",
"11 is a side view showing loading control mechanism when holder is moved to initial position.",
"FIG.",
"12 is a plan view showing the state where memory medium is inserted into holder so that contact portion of the memory medium is connected to connector.",
"FIG.",
"13 is a side view showing the state where memory medium is inserted into holder so that contact portion of the memory medium is connected to connector.",
"FIG.",
"14 is a plan view showing the state where fluctuation lever is moved by holder in memory medium insertion direction and is then fluctuated so that it is engaged with chassis.",
"FIG.",
"15 is a side view showing the state where engagement between fluctuation lever and holder is released so that the fluctuation lever is engaged with chassis.",
"FIG.",
"16 is a side view of loading control mechanism showing the state where engagement between fluctuation lever and holder is released so that the fluctuation lever is engaged with chassis.",
"FIG.",
"17 is a plan view showing the state where memory medium inserted into holder is caused to undergo loading at loading position.",
"FIG.",
"18 is a side view showing the state where holder is caused to undergo biasing force of second extension coil spring so that it is moved to memory medium loading position.",
"FIG.",
"19 is a side view showing loading control mechanism when holder is caused to undergo biasing force of second extension coil spring so that the holder is placed in the state where it is moved to memory medium loading portion.",
"FIG.",
"20 is a plan view showing the state where memory medium loaded into holder is ejected.",
"FIG.",
"21 is a side view showing the state where memory medium loaded into holder is ejected.",
"BEST MODE FOR CARRYING OUT THE INVENTION First, a memory medium used in a recording and/or reproducing apparatus to which a carrying apparatus for memory medium according to the present invention is applied will be explained.",
"This memory medium 1 uses solid-state memory as a memory element, and comprises a casing 2 constituting the memory body obtained by molding synthetic resin.",
"Within this casing 2, memory element such as flash memory, etc.",
"having, e.g., large memory capacity of 32 mega byte or more is provided.",
"As shown in FIG.",
"1, the memory medium 1 is formed so as to take substantially rectangular shape in which, e.g., length W, of the short side is caused to be substantially 21.45 mm, length L, of the long side is caused to be substantially 50 mm, and thickness D, is caused to be substantially 2.8 mm.",
"As shown in FIGS.",
"1 and 2, at one end side serving as the side of one short side of the casing 2 constituting the memory medium 1, there is formed a contact portion 3 in a manner bridging over the bottom face from one end surface.",
"At this contact portion 3, there are provided plural contacts 3b separated by partitioning walls 3a from each other.",
"Read or write operation of information with respect to the memory element provided within the casing 2 is carried out through the contacts 3b provided at the contact portion 3.As shown in FIGS.",
"1 and 2, at one corner portion of one end surface side where the contact portion 3 is formed of the casing 2, there is provided a cut portion 4, which is cut so as to take circular arc shape, indicating insertion direction into recording and/or reproducing apparatus.",
"As shown in FIG.",
"2, at one side surface of the side where the cut portion 4 is formed of the casing 2, an erroneous insertion preventing groove 5 where the bottom face side of the casing 2 is opened is formed in a manner continuous to the cut portion 4.The cut portion 4 and the erroneous insertion preventing groove 5 serve to limit insertion direction with respect to the recording and/or reproducing apparatus to prevent erroneous insertion when the plate-shaped memory 1 is loaded with respect to the recording and/or reproducing apparatus.",
"At the bottom face side of the casing 2, an erroneous recording prevention switch 6 which prevents that information signal is erroneously recorded with respect to semiconductor memory is provided in the state positioned in the vicinity of the contact portion 3.The erroneous recording prevention switch 6 is connected to operation element within the casing 2 and is adapted so that when it is slid to one side, recording of information signal is permitted, while when it is slid to the other side, it is inhibited that new information signal is overwritten.",
"At one side surface and the other side surface of the casing 2, there are provided a first engagement recessed portion 7 which is engaged with engagement holding portion provided at the recording and/or reproducing apparatus side and a second engagement recessed portion 8 with which detecting portion which detects presence or absence of loading is engaged when the memory medium 1 is inserted into the recording and or reproducing apparatus.",
"As a recording/reproducing apparatus to which the present invention is applied using the memory medium 1 constituted as described above, there is a recording/reproducing apparatus constituted as shown in FIG.",
"3.The recording/reproducing apparatus shown in FIG.",
"3 serves to record information signal such as audio data, etc.",
"With respect to the recording medium 1, or to carry out reproduction of information signal such as audio data, etc.",
"Stored in the memory medium 1.This recording/reproducing apparatus 11 is caused to be of configuration of a size to such a degree that it is used in the state grasped by hand, and comprises, as shown in FIG.",
"3, a housing 12 constituting the apparatus body formed so as to take elongated rectangular parallelepiped shape.",
"Within the housing 12, there is included a loading unit for the memory medium 1 to which carrying apparatus according to the present invention constituted as described later is applied.",
"Loading of the memory medium 1 is carried out through this loading unit.",
"At the bottom face of the housing 12, there is provided a memory median insertion/withdrawal opening 13 for inserting/withdrawing the memory medium 1 with respect to the loading unit included within the housing 12.The memory medium insertion/withdrawal opening 13 is formed so as to take rectangular shape having dimensions sufficient so that the memory medium 1 is inserted with one end side where the contact portion 3 is provided being as insertion end.",
"The memory medium insertion/withdrawal opening 13 is closed by cover body rotatably supported at the loading unit side.",
"Within the housing 12, there are provided a recording/reproducing circuit for carrying out recording of information signal such as audio data, etc.",
"With respect to the loaded memory medium 1, or carrying out reproduction of information signal such as audio data, etc.",
"Stored with respect to the memory medium 1, and other signal processing units.",
"As shown in FIG.",
"3, at one side surface of the housing 12, there are provided a recording mode select key 19 for selecting recording mode for carrying out recording of information signal such as audio data, etc.",
"With respect to the recording medium 1, a reproduction (playback) mode select key 14, a stop key 15 for stopping operation mode such as recording mode, etc., and a various kind operation key 16 for carrying out selection of other operation modes.",
"Moreover, at the position of the upper side of the front side of the housing 12, there is provided a display unit 17 comprised of liquid crystal device, etc.",
"On this display unit 17, current operation mode and/or index of information recorded with respect to the recording medium, etc.",
"are displayed.",
"At the corner portion of the upper side of the front side where this display unit 17 is provided, there is provided a microphone 18 for carrying out collection of external sound.",
"Moreover, although not shown, at the housing 12, there is provided a connection unit for realizing connection to personal computer or disc drive unit through connection cord.",
"This connection unit is constituted as unit caused to be in conformity with USB (Universal Serial Bus).",
"In the recording/reproducing apparatus having such a configuration, the memory medium 1 as shown in FIGS.",
"1 and 2 is loaded at loading position within the apparatus body through the memory medium insertion/withdrawal opening 13 to operate the recording mode select key 19 so that there results recording operation mode, whereby recording of audio data inputted from external equipment such as personal computer, etc.",
"through the connection unit and or sound (voice) collected through the microphone 18 is carried out.",
"In addition, the reproduction (playback) mode select key 14 is operated so that there results reproduction (playback) operation mode, whereby reproduction of information signal such as audio data, etc.",
"recorded with respect to the loaded memory medium 1 is carried out.",
"As another example of the recording/reproducing apparatus to which the present invention is applied, there is a recording/reproducing apparatus 21 within which optical disc drive using optical disc such as Compact Disc (CD), etc.",
"as memory medium or hard disc drive using magnetic disc as recording medium is integrally included.",
"In this recording/reproducing apparatus 21, as shown in FIG.",
"4, at the.",
"upper portion side of a housing 22 constituting the equipment body, there is provided an optical disc drive 23 using optical disc as recording medium.",
"The optical disc used as a recording medium of the optical disc drive 23 is detachably loaded to the rotational drive mechanism of the optical disc drive.",
"Attachment/detachment of optical disc with respect to the optical disc drive 23 is carried out by rotationally operating a cover body 24 provided at the upper portion of the housing 22 to thereby open the upper portion of the housing 22 to allow the disc rotational drive mechanism of the optical disc drive 23 to be faced to the external.",
"The opening operation of the cover body 24 is carried out by carrying out slide operation of an opening operation button 25 provided at the central portion of the front side of the housing 22.Since the hard disc drive in which magnetic disc is integrally attached does not require exchange of magnetic disc, it is included within the housing 22.In the recording/reproducing apparatus 21 shown in FIG.",
"4, a memory medium loading portion 26 adapted so that the memory medium 1 shown in FIGS.",
"1 and 2 is detachably loaded is further provided within the housing 22.Loading operation of the memory medium 1 with respect to the memory medium loading portion 26 is carried out by using loading unit constituted as described later to which the present invention is applied, which is included within the housing 22.As shown in FIG.",
"4, insertion/withdrawal of the memory medium 1 with respect to the loading unit is carried out through a memory medium insertion/withdrawal opening 27 provided at the front central portion of the housing 22.This memory medium insertion/withdrawal opening 27 is also formed so as to take rectangular shape having dimensions sufficient so that the memory medium 1 is inserted with one end side where the contact portion 3 is provided being as insertion end as in the case of the previously described portable recording/reproducing apparatus 11, and is closed by cover body rotatably supported at the loading unit side.",
"Also in this recording/reproducing apparatus 21, within the housing 22, there are provided a recording/reproducing circuit for carrying out recording of information signal such as audio data, etc.",
"With respect to the loaded memory medium 1, or for carrying out reproduction of information signal such as audio data, etc stored in the memory medium 1, and other signal processing units.",
"As shown in FIG.",
"4, at the upper surface of the cover body 24 of the apparatus body side provided at the upper portion of the housing 22, there are provided plural mode select keys 28 for selecting operation modes of the optical disc drive 23 and the hard disc drive.",
"The compound type recording/reproducing apparatus 21 in which the memory medium loading portion 26 is provided along with the optical disc drive 23 and the hard disc drive to permit recording/reproduction of information signal with respect to the memory medium 1 is caused to be of the configuration so as to mutually permit transmission of information signals between the optical disc drive 23, the hard disc drive and the memory medium recording/reproducing unit, and to record, e.g., audio data stored on optical disc with respect to hard disc or the memory medium 1, and has function to record audio data, etc.",
"recorded on optical disc with respect to the memory medium 1, or to record audio data, etc.",
"recorded on hard disc with respect to the memory medium 1, and to record audio data, etc.",
"recorded at the memory medium 1 onto hard disc.",
"Transmission of information signals is mutually carried out between the optical disc, the hard disc and the memory medium 1 as stated above, and recording of information signals such as audio data, etc.",
"between respective recording media is executed by..operation of a recording mode selecting unit 29 comprised of plural operation keys provided at the upper surface of the cover body 24.At the upper surface of the cover body 24, a display unit 30 comprised of liquid crystal device, etc.",
"is provided similarly to the portable recording/reproducing apparatus 11.On this display unit 30, current operation mode and/or index of information recorded with respect to the recording medium 1, etc.",
"are displayed.",
"As shown in FIG.",
"5, a loading unit 51 used for loading the memory medium 1 with respect to the memory medium loading portion within the recording/reproducing apparatus 11 or 12 as described above comprises a holder 52 adapted so that the memory medium 1 loaded at the recording/reproducing apparatus 11 or 21 is inserted and held.",
"The holder 52 is formed by punching and bending thin metallic plate.",
"The holder 52 has a width sufficient so that the memory medium 1 is inserted with one end side where the contact portion 3 is provided being as insertion end, and is adapted so that a pair of side walls 53, 54 which support both sides of the memory medium 1 inserted into both sides opposite to each other are formed in a projected manner, and one end side is caused to be insertion side of the memory medium 1.At one side wall 53, there is provided an engagement holding piece 55 engaged with the first engagement recessed portion 7 provided at the memory medium 1 inserted into the holder 52.The engagement holding piece 55 is formed by cutting and raising a portion of one side wall 53, and is adapted so that the base end portion side is caused to be connecting portion to the side wall 53, the engagement holding piece 55 is extended toward insertion direction of the memory medium 1, and an engagement portion 56 engaged with the first engagement recessed portion 7 of the memory medium 1 is provided at the front portion.",
"The engagement portion 56 is formed by bending the front end portion of the engagement holding piece 55 so as to take circular arc shape, and is swelled toward the insertion side of the memory medium 1 of the holder 52.At the other end side of the holder 52, there is disposed a connector 58 provided with plural connection terminals 57 to which respective contacts 3b of the contact portion 3 provided at one end side of the memory medium 1 inserted into the holder 52 are respectively electrically and mechanically connected.",
"As shown in FIG.",
"6, the connector 58 is adapted so that plural connection terminals 57 are supported in parallel at a terminal supporting body 59 consisting of synthetic resin serving as insulating body, and a flexible printed board 60 for external connection is connected to the other end side of the terminal supporting body 59.The connector 58 is attached to a connector case 61 formed so as to take channel shape in cross section by punching and bending thin metallic plate, and is attached to the other side of the holder 52 through the connector case 61.The connector case 61 is adapted so that a pair of side walls 62,63 which realize supporting of the connector 58 and serve as attachment piece with respect to the holder 52 are formed in a projected manner at both sides opposite to each other, and a rising wall 64 which limits attachment position of the connector 58 is formed at the rear side.",
"The connector 58 is adapted so that both ends of the terminal supporting body 59 are put by the pair of side walls 62, 63 therebetween to collide the rear side of the terminal supporting body 59 with the rising wall 64 so that attachment position is caused to undergo positional limitation.",
"Thus, the connector 58 is supported by the connector case 61.At this time, in order to prevent falling off from the connector case 61, the connector 58 is attached in the state where engagement recessed portions provided at both ends of the terminal supporting body 59 are engaged with engagement projections formed by swelling a portion of the side walls 62, 63.As shown in FIG.",
"5, the connector case 61 which has attached the connector 58 is attached in such a manner that the connector 58 side is opposed to the holder 52, and the pair of side walls 62,63 are fitted into the portion between the pair of side walls 53, 54.At this time, the connector case 61 is integrally attached to the holder 52 by respectively fitting plural engagement pieces 65 formed by cutting and raising a portion of the side walls 62, 63 into plural engagement holes 66 bored at the side walls 53, 54 of the holder 52 side.",
"The connector case 61 is attached to the holder 52 to realize function to hold the memory medium 1 along with the holder 52.Namely, at the front end side of the side walls 62, 63, L-shaped memory medium supporting pieces 67, 68 which support the bottom face side of the memory medium 1 inserted into the holder 52 are formed in such a manner that they are bent.",
"As described above, by combining the connector case 61 to which the connector 58 has been attached with the holder 52, a storage holding unit for the memory medium 1 is constituted between the holder 52 and the connector case 61.The memory medium 1 is inserted into the storage holding unit between the holder 52 and the connector case 61 from opened one end side opposite to the side where the connector 58 is disposed of the holder 52 with the contact portion 3 side being as insertion end to connect the contact portion 3 to the connector 58.Meanwhile, as shown in FIG.",
"6, length L1 of two connection terminals 571 and 5710 serving as ground terminal of power supply positioned at the outermost side among plural connection terminals 57 provided at the connector 58 is formed so that it is longer than length L2 of other connection terminals 57.This realizes the fact that when the memory medium 1 is inserted into the holder 52, contacts 3a of both sides are connected, first of all, to two connection terminals 571, 5710 serving as ground terminal so that secure connection between the apparatus body side and power supply is established.",
"Moreover, connection terminal 575 which detects insertion/withdrawal of the memory medium 1 provided at the central portion of plural connection terminals 57 is formed so as to have the shortest length L3.By forming the connection terminal 575 for detection of insertion/withdrawal in this way, as shown in FIG.",
"7, when the memory medium 1 is inserted into the holder 52, contacts 3a are connected to other connection terminals 57, and are then connected to the connection terminal 57, for detection of insertion/withdrawal, and when eject operation to pull the memory medium 1 from the holder 52 is carried out, connection to the contact 3a is released first of all.",
"Accordingly, it is possible to detect precise connecting state with respect to the connector 58 of the memory medium 1.Thus, it is possible to realize reliable transmission/reception of signals between the memory medium 1 and the -apparatus body side.",
"In this example, at the other side wall 54 side of the holder 52, there is provided an eject operation piece 69 operated by eject operation mechanism in ejecting the memory medium 1 which has been caused to undergo loading at the loading position.",
"As described above, the holder 52 to which the connector.58 where the memory medium 1 is electrically connected is attached is movably attached to a chassis 71 fixedly disposed within the houding 12 or 22 constituting the apparatus body in order to permit the memory medium 1 inserted and held at the holder 52 to undergo loading at a predetermined loading position within the apparatus body.",
"The chassis 71 is formed by punching and bending thin metallic plate, and is formed so as to constitute rectangular frame body as shown in FIG.",
"5, wherein first and second supporting walls 72, 73 which support the holder 52 are formed in a projected manner at both sides opposite to each other.",
"At the first and second supporting walls 72, 73, there are bored slide guide holes 76, 77 formed as elongated hole adapted so that spindles 74, 75 which project portions of the first and second side walls 53, 54 of the holder 52 and are integrally formed with the first and second side walls 53, 54 are engaged, and there are provided holder supporting pieces 78, 79 which supports the bottom face side of the holder 52.The slide guide holes 76, 77 are provided at the side positioned inwardly of the equipment body when the chassis 71 is attached within the equipment body, and the holder supporting pieces 78, 79 are formed so as to take L-shape in cross section in the state positioned at the side where the memory medium 1 is inserted.",
"As shown in FIGS.",
"8 and 9, the holder 52 is disposed in such a manner that it is fitted between the first and second supporting walls 72, 73 of the chassis 71, and is adapted so that the spindles 74, 75 are inserted and engaged with respect to the slide guide holes 76, 77, and both sides of the bottom face side thereof are supported by the holder supporting pieces 78, 79, whereby the holder 52 is supported so that it can be moved in a manner bridging between eject position where insertion/withdrawal of the memory medium 1 is permitted and loading position where the memory medium 1 is moved to loading position.",
"In this example, at the first and second side walls 53, 54 of the holder 52, there are formed projection portions 53a, 54a for reducing contact resistance with respect to the first-and second supporting walls 72, 73 of the chassis 71 side.",
"As the result of the fact that such projection portions 53a, 54a are formed, smooth movement with respect to the chassis 71 of the holder 52 is guaranteed.",
"As shown in FIG.",
"8, at the holder 52 movably supported by the chassis 71 as described above, there is provided a lock mechanism 81 adapted to lock the holder 52 until the holder 52 is located at initial position where insertion/withdrawal of the memory medium 1 is permitted, and the contact portion 3 of the memory medium 1 inserted into this holder 52 is connected to the connector 58 provided at the holder 52 side-thereafter to release lock to permit movement in the memory medium loading position direction of the holder 52.The lock mechanism 81 comprises a lock lever 82 rotatably supported at one corner portion of the internal side of the holder 52.As shown in FIGS.5, the lock lever 82 is adapted so that a lock piece 83 engaged with a portion of the chassis 71 is provided at the front end side, and a pivotal hole 84 is bored at the base end portion side.",
"As shown in FIG.",
"8, this lock lever 82 is rotatably supported with a spindle 85 formed by projecting a portion of plane surface portion of the holder 52 being as center by pivoting the pivotal hole 84 with respect to the spindle 85.The lock lever 82 is rotatably biased in the direction indicated by arrow A, in FIG.",
"8 where the lock piece 83 of the front end side is projected toward the chassis 71 side by a compression coil spring 88 serving as a biasing member disposed between a memory medium pressing piece 86 provided at the base end side and a spring engagement recessed portion 87 provided at the terminal supporting body 59 of the connector 58 attached to the holder 52.As shown in FIG.",
"8, the lock lever 82 is biased by the compression coil spring 88 when the holder 52 is located at the initial position where insertion/withdrawal of the memory medium 1 is permitted, and is projected toward the chassis 71 side through an insertion hole 89 bored by forming the engagement holding piece 55 at the first side wall 53 of the holder 52.As shown in FIG.",
"10, the lock piece 83 projected toward the chassis 71 side is engaged with an engagement recessed portion 90 formed by cutting a portion of the first supporting wall 72 of the chassis 71 to limit movement in the direction indicated by arrow X1 in FIG.",
"8 of memory medium insertion direction of the holder 52.In the loading unit to which the carrying apparatus according to the present invention is applied, there is provided a loading control mechanism 91 which controls loading operation and eject operation of the holder 52 into which the memory medium 1 has been inserted.",
"As shown in FIGS.",
"8 and 9, the loading control mechanism 91 is provided at the second supporting wall 73 side of the chassis 71 which movably supports the holder 52 in the insertion/withdrawal direction of the memory medium 1.This loading control mechanism 91 comprises a slider 92 movably supported in the direction indicated by arrow X1 or in the direction indicated by arrow.",
"X2 in FIG.",
"9 on the second supporting wall 73, a fluctuation lever 93 rotatably supported by the slider 92 and moved along with the slider 92, a first extension coil spring 94 serving as a first biasing member which is stretched between the slider 92 and the chassis 71 and movably biases the slider 92 in the eject direction of the direction indicated by arrow X2 in FIG.",
"9, and a second extension coil spring 95 serving as a second biasing member which is streatched in a manner bridging between the slider 92 and the holder 52 and movably biases the holder 52 in the insertion direction of the memory medium 1 of the direction indicated by arrow X1 in FIG.",
"9 As shown in FIG.",
"5, the slider 92 is formed so as to take elongated thin plate shape, and is adapted so that both sides in length direction are held by plural holding pieces 96, 97 formed at both sides of the second supporting wall 73 of the chassis 71 so that it is movably supported on the second supporting wall 73.The fluctuation lever 93 is rotatably supported in the direction indicated by arrow C and in the direction indicated by arrow D in FIGS.",
"9 and 11 with a spindle 98 provided at the base end portion side being as center by pivoting the spindle 98 with respect to a pivotal hole 103 bored at one end side of the slider 92.The rotational range with the spindle 98 being as center of this fluctuation lever 93 is limited by a portion of the chassis 71 and the holder 52 movably supported by this chassis 71.Namely, as shown in FIGS.",
"9 and 11, the rotational range of the fluctuation lever 93 is limited so that the fluctuation lever 93 is rotated between a rotational limiting piece 99 formed by bending one side edge of the second supporting wall 73 and a rotation limiting piece 100 projected from one side edge of the holder 52.The fluctuation lever 93 is disposed in such a manner that it is put between the side wall 73 of the chassis 71 and the slider 92 so that falling off from the slider 92 is prevented.",
"The spindle 98 is formed by projecting a portion of the fluctuation lever 93 by striking out.",
"In this example, at the slider 92, as shown in FIG.",
"8, there are provided bent portions 92a, 92b for ensuring space for disposing the fluctuation lever 93 between the slider 92 and the side wall 73.At one side of the front end side of the fluctuation lever 93, there is provided an engagement portion 101 formed as inclined surface portion engaged with a portion of the holder 52.As shown in FIGS.",
"8 and I1, this engagement portion 101 is provided in the state inclined with respect to the holder 52 movement direction, and is engaged with a portion of the holder 52 when the holder 52 is located at the initial position shown in FIG.",
"8.The portion with which the engagement portion 101 is engaged of the holder 52 side is a portion formed by cutting and raising a portion of the front end side of a rotation limiting piece 100 which limits rotation of the fluctuation lever 93, and is a pressing operation piece 102 of the fluctuation lever 93.At the middle portion of the side edge opposite to the side where the engagement portion 101 is provided of the fluctuation lever 93, there is projected an engagement piece 106 in contact with the rotation limiting piece 99 of the chassis 71 side, and engaged with an engagement hold 105 formed by cutting a portion of the rotation limiting piece 99 of the chassis 71 side when the holder 52 is moved in memory medium insertion direction, i.e., in the direction indicated by arrow X1 in FIG.",
"8 by a predetermined quantity from the initial position.",
"In this example, at the side edge of the side where the engagement piece 106 is engaged of the engagement hole 105, there is formed a rising piece 107 in order to realize reliable engagement with the engagement piece.",
"The slider 92 which has supported the fluctuation lever 93 constituted as described above is biased in the direction indicated by arrow X2 in FIGS.",
"9 and 11 by the first extension coil spring 94 stretched between a spring holding piece 108 formed at the middle portion in length direction and a spring holding piece 109 provided at the chassis 71.Namely, the slider 92 is biased in the eject direction opposite to the direction where the memory medium 1 is inserted into the holder 52 by the first extension coil spring 94.Moreover, the second extension coil spring 95 stretched between the slider 92 and the holder 52 is stretched between a spring holding piece 110 formed at the front end side of the slider 92 and a spring holding piece 111 formed at the middle portion of one side of the holder 52 to move and bias the holder 52 in the insertion direction of the memory medium 1 of the direction indicated by arrow X1 in FIG.",
"9.At one end side of the chassis 71, i.e., the side where a memory medium insertion/withdrawal portion 112 for carrying out insertion/withdrawal of the memory medium 1 is provided with respect to the holder 52, there is attached an opening/closing cover 115 which opens or closes the memory insertion/withdrawal portion 112, and is opposed to the memory medium insertion/withdrawal opening 13 or 27 provided at the recording/reproducing apparatus 11 or 21 when this loading unit 51 is assembled into the previously described recording/reproducing apparatus 11 or 21 as shown in FIG.",
"3 or 4 to open or close the insertion/withdrawal opening 13 or 27.As shown in FIGS.",
"8 and 9, the opening/closing cover 115 is rotatably attached by supporting a pair of rotation arms 117, 118 provided at both sides of a cover portion 116 which opens or closes the memory medium insertion/withdrawal portion 112 at both sides of the side where the memory medium insertion/withdrawal portion 112 is formed of the chassis 71 to thereby open or close the memory medium insertion/withdrawal portion 112.Namely, the opening/closing cover 115 is rotatably attached with these spindles 119, 120 bored at the first and second supporting wall side walls 72, 73 of the chassis 71 being as center in the state where pivotal holes 117a, 118a bored at the front end portions of rotation axis 117, 118 are pivoted with respect to the spindles 119, 120.The opening/closing cover 115 is rotationally biased in the direction indicated by arrow E in FIG.",
"9 of the direction to always close the memory medium insertion/withdrawal portion 112 or the memory medium insertion/withdrawal opening 13 or 27 by a torsion coil spring 121 serving as a rotation biasing member.",
"The torsion coil spring 121 rotationally biases the opening/closing cover 115 in the direction indicated by arrow E in FIG.",
"9 by inserting coil portion into spindle 118b bored at the other rotation arm 118 to hold one arm portion 121a at the spring holding piece 109 provided at the chassis 71 side to form the other arm portion 121b at a spring holding portion 123 provided at the opening/closing cover 115 side.",
"The opening/closing cover 115 is adapted to allow the rotation arms 117, 118 to be in contact with rotation limiting pieces 124, 125 projected at the both sides of the chassis 71 so that rotation biasing position by the torsion coil spring 121 is limited, and is held at the position where the memory medium insertion/withdrawal portion 112 is closed.",
"As shown in FIGS.",
"5 and 8, the opening/closing cover 115 is attached in such a manner to project the cover portion 116 from the front end of the chassis 71.The upper surface of the cover portion 116 projected from the chassis 71 is caused to be a pressing operation portion 116b which is caused to undergo pressing operation by the memory medium 1 inserted into the holder 52.Namely, the opening/closing-cover 115 is caused to undergo pressing operation from the pressing operation portion 116b side of the upper surface of the cover portion 116, whereby it is rotationally operated in the direction indicated by arrow F in FIG.",
"9 of the direction to open the memory medium insertion/withdrawal portion 112 against biasing force of the torsion coil spring 121.As shown in FIG.",
"9, the inner surface of the cover portion 116, i.e., the surface opposite to the rear surface of the memory medium 1 inserted into the holder 52 is caused to be curved surface 116a or inclined surface.",
"By providing the curved surface 116a or the inclined surface at the inner surface of the cover portion 116, the curved surface 116a or the inclined surface is caused to undergo pressing operation by the memory medium 1 ejected from the holder 52, thereby making it possible to rotate the opening/closing cover 115 against biasing force of the torsion coil spring 121.The opening/closing cover 115 is rotated in a direction to open the memory medium insertion/withdrawal portion 112 or the memory medium insertion/withdrawal opening 13 or 27 in relation to the eject operation of the memory medium 1.In the loading apparatus 51 according to the present invention constituted as described above, when the holder 52 is located at the initial position serving as eject position shown in FIGS.",
"8 and 9 where insertion/withdrawal of the memory medium 1 is permitted, the holder 52 is placed in the state where the insertion/withdrawal side of the memory medium 1 is caused to be located at the position closest to the memory medium insertion/withdrawal portion 112 of the chassis 71.At this time, the slider 92 which has supported the fluctuation lever 93 undergoes biasing force of the first extension coil spring 94 so that it is moved and biased in the direction indicated by arrow X2 in FIGS.",
"9 and 11, and the fluctuation lever 93 supported by this slider 92 is also moved and biased in the same direction.",
"When the holder 52 is placed at the initial position, the fluctuation lever 93 is adapted to allow the engagement piece 106 to be in contact with the rotational limiting piece 99 provided at the chassis 71 to allow the engagement portion 101 to be in contact with the pressing operation piece 102 provided at the holder 52 so that it is engaged therewith in the state where movement in the direction indicated by arrow C in FIG.",
"11 is limited.",
"At this time, since the fluctuation lever 93 supported by the slider 92 moved and biased in the direction indicated by arrow X2 in FIGS.",
"9 and 11 by the first extension coil spring 94 allows the engagement piece 106 to be in contact with the rotation limiting piece 99 while being moved and biased in the direction indicated by arrow X2 in FIGS.",
"9 and 11, the fluctuation lever 93 is placed in the state where it is rotated and biased in the direction indicated by arrow D in FIGS.",
"9 and 11 with the spindle 98 in a direction opposite to the direction in contact with the rotation limiting piece 99 being as center to press the engagement portion 101 toward the pressing operation piece 102 of the holder 52.When the holder 52 is located at the initial position shown in FIGS.",
"8 and 9, the engagement portion 101 of the fluctuation lever 93 is in contact with the pressing operation piece 102.Accordingly, there results the state where movement in the memory medium insertion direction of the direction indicated by arrow X1 in FIGS.",
"9 and 11 by the second extension coil spring 95 stretched between the holder 52 and the slider 92 is limited.",
"It is to be noted that the initial position of the holder 52 is limited by allowing spindles 74, 75 to be in contact with the end portions of slide guide holes 76, 77 of the chassis 71 side.",
"Further, when the holder 52 is located at the initial position, the lock lever 82 constituting the lock mechanism 81 undergoes biasing force of the compression coil spring 88 so that it is rotationally biased in the direction indicated by arrow A in FIG.",
"8.Thus, the lock piece 83 is engaged with the engagement recessed portion 90 of the chassis 71 to place the holder 52 in the state where movement in the memory medium insertion direction of the direction indicated by arrow X1 in FIG.",
"8 thereof is limited.",
"Furthermore, the opening/closing cover 115 is also caused to undergo biasing force of the torsion coil spring 121 so that it is rotationally biased at the position where the memory medium insertion/withdrawal portion 112 is closed.",
"In order to load the memory medium 1 at the memory medium loading portion within the recording/reproducing apparatus by using the loading unit 51 to which the carrying apparatus according to the present invention has been applied, the memory medium 1 is inserted into the holder 52 through the memory medium insertion/withdrawal portion 112 with one end side where the contact portion 3 is provided being as insertion end as shown in FIGS.",
"12 and 13.When the memory medium 1 is inserted into the holder 52, a pressure operation portion 116b is pressed by the plane surface of the insertion end side of the memory medium 1 to rotate the opening/closing cover 115 against biasing force of the torsion coil spring 121 to open the memory medium insertion/withdrawal portion 112.At this time, the memory medium 1 is inserted in the state where the side in which the erroneous insertion preventing groove 5 is provided is positioned at the side where the lock lever 82 within the holder 52 is provided.",
"At the side where the contact portion 3 serving as insertion end of the memory medium 1, partitioning walls which partition plural contacts 3 a are projected.",
"Accordingly, even if the contact portion 3 side is caused to be in contact with the opening/closing cover 115, it is prevented that the opening/closing cover 115, etc.",
"is directly in contact with the contact 3a.",
"When the opening/closing cover 115 is rotated to face the side where the contact portion 3 is provided to the holder 52 to further insert the memory medium 1 into the holder 52, respective contacts 3a of the contact portion 3 are electrically and mechanically connected to respective connection terminals 57 of the connector 58 provided at the holder 52 side as shown in FIG.",
"7.When the contact portion 3 is connected to the connector 58, contacts 3a of both sides are connected, first of all, to two connection terminals 571, 5710 serving as the longest ground terminal as previously described, and central contact 3a is last connected to the shortest connection terminal 575.Accordingly, as the result of the fact that all contacts 3a are electrically connected to connection terminals 57 of the connector 58 side, it is detected that the memory medium 1 is inserted into the holder 52.By employing such configuration, reliable insertion into the holder 52 of the memory medium can be realized.",
"When the memory medium 1 is inserted into the holder 52 until the position where connection between the contact portion 3 and the connector 58 is carried out, the engagement portion 56 of the engagement holding piece 55 is engaged with the first engagement recessed portion 7 to realize reliable holding with respect to the holder 52 of the memory medium 1.The holder 52 is placed in the state where movement thereof is limited by the lock mechanism 81 until the contact portion 3 of the memory medium 1 is completely connected to the connector 58 attached to the holder 52.Namely, until the contact portion 3 of the memory medium 1 is completely connected to the connector 58, the lock lever 82 is engaged with the engagement recessed portion 87 of the chassis 71 as shown in FIG.",
"12 to limit movement of the holder 52.Until connection between the contact portion 3 and the connector 58 is carried out, movement of the holder 52 is limited.",
"Accordingly, connection between the contact portion 3 and the connector 58 can be carried out only by insertion operation into the holder 52 of the memory medium 1.Thus, insertion operation into the holder 52 of the memory medium 1, and electric and mechanical connecting operation thereof extremely become easy.",
"After connection between the contact portion 3 and the connector 5 8 is carried out, when the memory medium 1 is further inserted in the memory medium insertion direction of the direction indicated by arrow X1 in FIG.",
"12, the memory medium pressing piece 86 is pressed by insertion end of the memory medium 1 so that the lock lever 82 is rotated in the direction indicated by arrow A2 in FIG.",
"12 against biasing force of the compression coil spring 88.Thus, as shown in FIG.",
"14, engagement with respect to the engagement recessed portion 90 of the lock piece 83 is released.",
"As a result, there results the state where further insertion in the memory medium insertion direction of the direction indicated by arrow X1 in FIG.",
"14 of the holder 52 is permitted.",
"Since the holder 52 is located at the initial position until the time point when the memory medium 1 is inserted into the holder 52 so that lock of the lock lever 82 is released, there results the state where the engagement portion 101 of the fluctuation lever 93 constituting the loading control mechanism 91 is in contact with the pressing operation piece 102 as shown in FIG.",
"11.Namely, the holder 52 is adapted so that lock by the lock mechanism 81 is released so that there results the state where movement in the memory medium insertion direction is permitted, and there results the state where the pressing operation piece 102 is caused to be in contact with the engagement portion 101 of the fluctuation lever 93.Here, when the memory medium 1 is caused to undergo pressing operation to further insert the holder 52 in the memory medium insertion direction of the direction indicated by arrow X1 in FIG.",
"14, the engagement portion 101 is pressed by the pressing operation piece 102.As a result, the fluctuation lever 93 is moved in the direction indicated by arrow X1 in FIG.",
"13.Thus, the slider 92.where this fluctuation lever 93 is supported is moved in the same direction so as to expand the first extension coil spring 94.The first extension coil spring 94 is expanded as the result of the fact that the slider 92 is moved, whereby biasing force which moves the holder 52 in the eject direction of the initial position direction is stored.",
"At this time, the fluctuation lever 93 is in the state where the engagement piece 106 is in contact with the rotation limiting piece 99 of the chassis 7 1 so that rotation in the direction indicated by arrow C in FIG.",
"13 is limited to maintain the state where the engagement portion 101 is caused to be in contact with the pressing operation piece 102 of the holder 52 side.",
"Namely, as shown in FIG.",
"11, when the engagement piece 101 provided at the position deviating toward one side from center line P1 passing through the spindle 89 serving as fulcrum of rotation is pressed in the line direction of the direction indicated by arrow G in FIG.",
"11 by the pressing operation piece 102 of the holder 52 side, the fluctuation lever 93 is rotationally biased in the direction indicated by arrow C in FIG.",
"11 with the spindle 89 being as center.",
"Accordingly, when the fluctuation lever 93 is caused to undergo pressing operation by the holder 52, it is rotationally biased in a direction to allow the engagement portion I 01 to be away from the pressing operation piece 102.Here, as shown in FIG.",
"11, the engagement portion 101 is formed as inclined surface portion inclined by a predetermined angle θ with respect to the pressing direction by the pressing operation piece 102.Namely, since the engagement portion 101 is inclined in the direction where it is rotationally biased by the pressing operation piece 102, it can receive pressing force by the pressing operation piece 102 from the direction substantially perpendicular thereto.",
"Thus, it is possible to efficiently press the fluctuation lever 93 by the holder 52.When the memory medium 1 inserted into the holder 52 is pressed by finger in the state where the pressing operation piece 102 is engaged with the engagement portion 101 to further insert the holder 52 in the memory medium insertion direction of the direction indicated by arrow X1 in FIG.",
"14, the fluctuation lever 93 is moved in the direction indicated by arrow XI in FIG.",
"14 in one body with the slider 92 while expanding the first extension coil spring 94.When the engagement piece 106 reaches the position opposite to the engagement hole 105 formed by cutting a portion of the rotation limiting piece 99 provided at the chassis 71 side, rotation limitation of the fluctuation lever 93 by the rotation limiting piece 99 is released so that the fluctuation lever 93 is rotated in the direction indicated by arrow C in FIGS.",
"15 and 16 to release engagement with the pressing operation piece 102 of the engagement portion 101 to engage the engagement piece 106 with the engagement hole 105.When the engagement piece 106 of the fluctuation lever 93 is engaged with the engagement hole 105 of the chassis 71 side, the slider 92 is caused to undergo movement operation by the holder 52 so that it is held at the position where the first extension coil spring 94 is expanded as shown in FIG.",
"15.At this time, the holder 52 is placed in the state movable with respect to the slider 92 held at the position where the first extension coil spring 94 has been expanded as the result of the fact that engagement with respect to the engagement portion 101 of the fluctuation lever 93 of the pressing operation piece 102 is released.",
"The holder 52 undergoes biasing force of the second extension coil spring 95 placed in the state expanded between this holder 52 and the slider 92 so that it is moved in the memory medium insertion direction of the direction indicated by arrow X1 in FIGS.",
"14 and 15.Thus, as shown in FIGS.",
"17 and 18, the holder 52 is moved up to the memory medium loading position to allow the held memory medium 1 to undergo loading at the loading position.",
"In this example, when the holder 52 is moved in the memory medium insertion direction, the fluctuation lever 93 is placed in the state where one side of the front end side where the engagement portion 101 is provided is in contact with the rotation limiting piece 100 of the holder 52 side so that rotation in the direction indicated by arrow D in FIG.",
"19 is limited.",
"As the result of the fact that rotation in the direction indicated by arrow D in FIG.",
"19 of the fluctuation lever 93 is limited, engagement state with respect to the engagement hole 105 of the chassis 71 side of the engagement piece 106 is securely maintained to maintain the state where the first extension coil spring 94 is expanded.",
"When the holder 52 which has held the memory medium 1 is drawn or pulled up to the memory medium loading position, the memory medium 1 is completely drawn or pulled into the apparatus.",
"As a result, the opening/closing cover 115 which has been rotated by the memory medium 1 inserted into the holder 52 undergoes biasing force of the torsion coil spring 121 so that it is rotated to close the memory medium insertion/withdrawal portion 112 as shown in FIG.",
"18 to complete loading operation of the memory medium 1.It is to be noted that a drawing force which draws or pulls the holder 52 into which the memory medium 1 is inserted can be arbitrarily set by changing force value of the second extension coil spring 95.As described above, the loading unit 51 according to the present invention is adapted to insert the memory medium 1 into the holder 52 until the contact portion 3 is connected to the connector 58, whereby when the memory medium 1 is further pressed by finger, the memory medium 1 can be automatically loaded with respect to a predetermined memory medium loading portion by utilizing biasing force of biasing member such as spring, etc.",
"As shown in FIGS.",
"17 and 18, in order to eject the memory medium 1 loaded at the memory medium loading position, there is employed an approach to operate the eject operation piece 69 provided at the holder 52 to move the holder 52 in a direction to eject the memory medium 1 in the direction indicated by arrow X2 in FIGS.",
"17 and 18 against biasing force of the second extension coil spring 95.In the case where the loading unit 51 to which the carrying apparatus according to the present invention is applied is applied to the previously described recording/reproducing apparatus 11 or 21 as shown in FIG.",
"3 or FIG.",
"4, the eject operation mechanism comprised of eject lever, etc.",
"provided at the recording/reproducing apparatus 11, or 21 side is caused to be related to eject operation piece 69 to allow eject operation buttons 41, 42 constituting the eject operation mechanism to undergo pressing operation to thereby allow the eject operation piece 69 to undergo movement operation to thereby move the holder 52.It is to be noted that, as the eject operation mechanism used here, there may be employed a mechanism operated by manual operation or electric operation using electromagnetic drive means, etc.",
"When the holder 52 is moved in the eject operation of the direction indicated by arrow X2 in FIGS.",
"17 and 18, it is relatively moved with respect to the fluctuation lever 93 placed in the state where the engagement piece 106 is engaged with the engagement hole 105 of the chassis 71 side.",
"When the holder 52 is further moved in the direction indicated by arrow X2 in FIGS.",
"17 and 18, one side of the front end side thereof is caused to be in contact with the portion on the rotation limiting piece 100 so that the fluctuation lever placed in the state where rotation in the direction indicated by arrow D in FIG.",
"18 has been limited is rotated in the direction indicated by arrow D in FIG.",
"18.The fluctuation lever 93 which is held at the position where the first extension coil spring 94 has been expanded and is supported by the slider 92 which has been moved and biased in the direction indicated by arrow X2 in FIG.",
"21 engages the engagement piece 106 with the engagement hole 105 of the chassis 71 sidewhile being moved and biased in the direction indicated by arrow X2 in FIG.",
"21, whereby the fluctuation lever 93 is placed in the state where it is rotationally biased in the direction indicated by arrow D in FIG.",
"21.When the holder 52 is further moved in the direction indicated by arrow X2 in FIG.",
"21 to reach the position where contact between one side of the front end side of the fluctuation lever 93 and the rotation limiting piece 100 is released, the fluctuation lever 93 is rotated in the direction indicated by arrow D in FIG.",
"21 with the spindle 98 being as center to engage the engagement portion 101 provided at one side of the front end side with the pressing operation piece 102 of the holder 52 side.",
"Simultaneously with this engagement, the fluctuation lever 93 releases engagement of the engagement piece 106 with respect to the engagement hole 105 of the chassis 71 side.",
"The fluctuation lever 93 releases engagement with respect to the chassis 71 side, whereby the fluctuation lever 93 undergoes biasing force of the first extension coil spring 94 so that it is placed in movable state.",
"At this time, since the fluctuation lever 93 engages the engagement portion 101 with the pressing operation piece 102 of the holder 52, the fluctuation lever 93 is placed in the state where it can be moved in one body with the holder 52.At this time, since biasing force which moves the slider 92 in the eject direction of the direction indicated by arrow X2 in FIGS.",
"20 and 21 is stored at the first extension coil spring 94, the holder 52 is moved in the eject direction of the direction indicated by arrow X2 in FIGS.",
"20 and 21 through the fluctuation lever 93 moved in one body with the slider 92.When the holder 52 is moved in the direction indicated by arrow X2 in FIGS.",
"20 and 21, curved surface 116a of the opening/closing cover 115 is caused to-undergo pressing operation by the memory medium 1 held by the holder 52.As a result, the opening/closing cover 115 is rotated against biasing force of the torsion coil spring 121 so that the memory medium insertion/withdrawal portion 112 is opened.",
"Thus, a portion of the memory medium 1 is projected toward the external of the apparatus through the memory medium insertion/withdrawal portion 112.By grasping the portion projected from the memory medium insertion/withdrawal portion 112 to pull the memory medium 1, the contact portion 3 is caused to be away from the connector 58.Thus, eject operation is completed.",
"The opening/closing cover 115 undergoes biasing force of the torsion coil spring 121 along with the eject operation of the memory medium 1 so that it is rotated to close the memory medium insertion/withdrawal portion.",
"At this time, the holder 52 is moved to the initial position shown in FIG.",
"8.Thus, the lock lever 82 of the lock mechanism 81 undergoes biasing force of the compression coil spring 88 so that it is moved in the direction indicated by arrow A1 in FIG.",
"8.As a result, the holder 52 is engaged with the chassis 71 so that it is placed in locked state.",
"Meanwhile, the engagement piece 106 engaged with the engagement hole 105 of the chassis 71 side provided at the fluctuation lever 93 is provided in the state positioned at the side edge of the front end side in length direction of the fluctuation lever 93.Namely, the engagement piece 106 is provided at the position deviating with respect to center line P1 passing through the spindle 89 serving as fulcrum of rotation of the fluctuation lever 93 and at the position spaced from the spindle 89.Accordingly, when the fluctuation lever 93 is moved and biased in a direction in parallel to center line P1 by the first extension coil spring 94, it comes into contact with the engagement hole 105 from a direction having a predetermined angle.",
"At this time, the fluctuation lever 93 is placed in the state where it is rotationally biased in the direction indicated by arrow D in FIG.",
"19.Further, contact surface to the engagement hole 105 of the engagement piece 106 is formed in a manner substantially perpendicular to the center line P1, thereby making it possible to easily carry out hoding/withdrawal with respect to the engagement hole 105.Namely, this is because force applied when the fluctuation lever 93 is biased so that the engagement piece 106 comes into contact with the engagement hole 105 is directed to the direction where the fluctuation lever 93 is rotationally biased.",
"Accordingly, it is possible to easily carry out, by small force, eject operation of the memory medium 1 loaded with respect to the holder 52.While the fluctuation lever 93 which constitutes the loading control mechanism 91 is caused to be of a configuration such that it is supported by the slider 92 and is moved in movement direction of the holder 52 along with the slider 92 in the above-described example, it is not necessarily required that the slider 92 is used.",
"There may be employed a configuration such that the fluctuation lever 93 is moved in relation to movement of the holder 52, and fluctuates with the spindle 98 being as center in accordance with movement position of the holder 52 so that it is engaged with the chassis 71 or the holder 52 to thereby give biasing forces of first and second extension coil springs 94,.95 which produce biasing forces opposite to each other to the holder 52.Namely, the fluctuation lever 93 may be directly supported at the second supporting wall 73 of the chassis 71 so that it can be rotated and can be moved in the movement direction of the holder 52.Industrial Applicability As described above, in the present invention, by selective engagement with respect to the chassis or the holder of the fluctuation lever which is moved and fluctuated in relation to movement of the holder adapted so that the memory medium is inserted and held and moved in a manner bridging between the eject position where insertion/withdrawal of the memory medium is permitted and the loading position for the memory medium, the first biasing member which moves and biases the holder in the eject direction opposite to the memory medium insertion direction is caused to store biasing force in the eject direction, and the second biasing member which biases the holder in the memory medium insertion direction is caused to be operative.",
"Accordingly, it is possible to carry out switching between movement in the memory medium insertion direction of the holder and movement in the eject direction only by movement and fluctuation of one fluctuation lever.",
"Thus, the mechanism for controlling loading of the memory medium can become simple, and can become compact.",
"Further, in the present invention, since the memory medium is caused to undergo insertion operation on one direction, connection to the connector within the recording/reproducing apparatus is realized, and loading with respect to the memory medium loading position can be carried out.",
"Accordingly, it is possible to easily carry out carrying operation of the memory medium.",
"In addition, in the present invention, movement in the memory medium insertion direction or in the eject direction of the holder is carried out by using biasing member such as extension coil spring, etc.",
"Accordingly, by suitably selecting strength of the biasing member, it is possible to arbitrarily set action force and it is possible to realize stable loading operation and eject operation of the memory medium."
]
] | Patent_10399003 |
[
[
"Apparatus and method for hydroforming a tubular part",
"A method and apparatus for shaping a raw tube into a formed part.",
"The part can be configured within a die assembly including a hydroforming die structure and a pair of tube-engaging punches.",
"The punches are inserted into the ends of the raw tube to shape the ends into the desired configuration.",
"The middle portion of the raw tube is shaped into the desired configuration by hydroforming.",
"Thus, the method and apparatus can shape the raw tube along its entire length, leaving no remnants of the raw tube that must be trimmed away."
],
[
"1.A hydroforming die assembly 10 for hydroforming a part from a tubular blank 40, said part having a desired configuration different from a configuration of said blank and including a desired cross section at one end thereof, said die assembly 10 including a die structure having interior surfaces defining a die cavity 52, said die cavity 52 having a cross sectional configuration conforming generally to the desired exterior shape of said part, said die assembly 10 further comprising: a pair of tube-end engaging structures 81 disposed at opposite ends of said die cavity 52 and constructed and arranged to engage opposite ends of said tubular blank 40, said tube-end engaging structures 81 being constructed and arranged to seal said opposite ends of said tubular blank 40 and to pressurize hydroforming fluid within said tubular blank 40 for expanding said tubular blank 40 into conformity with said interior surfaces of said die cavity 52, a first of said tube-end engaging structures 81 having an outer cross-sectional configuration corresponding to said desired cross section at said one end of said part, said first of said tube-end engaging structures 81 being movable into forced engagement with one end of said tubular blank 40 to conform said one end of said tubular blank 40 to said outer cross-sectional configuration of said first of said tube-engaging structures 81 and hence said predetermined cross section at said one end of said part.",
"2.A hydroforming die assembly 10 according to claim 1, wherein said first tube-end engaging structure 81 has an exterior surface including a beveled portion 82 which is partially inserted into said one end of said tubular blank 40, said first tube-end engaging structure 81 being moved further into said one end of said tubular blank 40 so as to cause inner surface portions of said one end of said tubular blank 40 to slide in forced relation along said beveled portion 82 and cause said one end of said tubular blank 40 to be deformed i) over said first tube-end engaging structure 81 and ii) into conformity with said exterior surface of said first tube-end engaging structure 81, and wherein one end of said die cavity 52 receives said first tube-engaging structure 81, said interior surfaces of said die cavity 52 at said one end of said die cavity 52 having a configuration conforming to an exterior surface configuration of said part at said one end of said part.",
"3.A method of producing a hydroformed part using a hydroforming die assembly 10 for hydroforming a part from a tubular blank 40, the part having a desired configuration different from a configuration of the blank and including a desired cross section at one end of the part, the die assembly 10 including a die structure having interior surfaces defining a die cavity 52, the die cavity 52 having a cross sectional configuration conforming to the desired cross section of the part, the method comprising the steps of: providing and a pair of tube-end engaging structures 81 disposed at opposite ends of the die cavity 52 and constructed and arranged to engage opposite ends of the tubular blank 40, the tube-end engaging structures 81 being constructed and arranged to seal the opposite ends of the tubular blank 40 and to pressurize hydroforming fluid within the tubular blank 40 for expanding the tubular blank 40 into conformity with the interior surfaces of the die cavity 52, a first of the tube-end engaging structures 81 having an outer cross-sectional configuration corresponding to the desired cross section at one end of the part; moving the first of the tube-engaging structures 81 into forced engagement with one end of the tubular blank 40 to conform the one end of said tubular blank 40 to the outer cross-sectional configuration of the first of the tube-engaging structures 81 and hence the predetermined cross section at the one end of the part; and applying pressure within the tubular blank 40 to form the tubular blank 40 in to the desired configuration of the part.",
"4.A method according to claim 3, further comprising the step of: incorporating the part into a product without cutting off the one end of the part formed by the forced engagement of the first of the tube-engaging structures 81.5.A assembly according to claim 1, wherein a first of said tube-engaging structures 81 has a forward end 33 and a lateral shoulder 88 protruding from a multifaceted portion 84 such that the first of said tube engaging structures 81 can be inserted into said tubular blank 40 continuously from the forward end 33 of said tube-engaging structure to said lateral shoulder 88 along said multifaceted portion 84 until said lateral shoulder 88 abuts said tube-engaging structure 81 and halts further insertion of said first of said tube-engaging structures 81 relative to said tubular blank 40.6.A method according to claim 3, wherein the moving of the first of the tube-engaging structures 81 includes inserting the first of the tube-engaging structures 81 into the tubular blank 40 continuously from a forward end 33 of the first of the tube-engaging structures 81, along a multifaceted portion 84 of the first of the tube-engaging structures 81, and to a lateral shoulder 88 of the first of the tube-engaging structures 81 that protrudes from the multifaceted portion 84, until the lateral shoulder 88 abuts the end of tubular blank 40 and halts further insertion of the tube-engaging structure 81 relative to the tubular blank 40.7.A method according to claim 3, wherein the moving of the first of the tube-engaging structures 81 into forced engagement with one end of the tubular blank 40 is a complete insertion of the first of the tube-engaging structures 81 into the tubular blank 40 and a complete reconfiguration of the one end of the tubular blank 40 into the final, desired configuration of the part by fully forcing the one end of the tubular blank 40 to conform to the outer surface of the first of the tube-engaging structures 81 and to the inner surface of the die cavity 52."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Typically, to form a tubular part by hydroforming, a raw tube is positioned within a hydroforming tool and the tube is secured at its ends.",
"The middle portion of the raw tube is then subjected to hydroforming, leaving a transitional zone between the ends of the raw tube and the hydroformed middle portion.",
"The hydroformed part is then finished by having the two transition zones removed from the tube, leaving only the fully hydroformed middle portion.",
"The ends of the tube can be secured by tip portions being generally wedge-shaped as disclosed in EP 1022073A1.Hydroforming is also disclosed in the U.S. Pat.",
"Nos.",
"5,987,950 to Horton and U.S. Pat.",
"No.",
"6,014,950 to Jaekel et al.",
"Removing the ends of the hydroformed part creates inefficiencies.",
"For example, the cut away ends become wasted raw material.",
"Also, cutting away ends requires additional cutting tools, which complicates the apparatus needed to create the finished part.",
"Further, time is wasted performing the added step of cutting off the transitional zones at each end."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>One object of the present invention is to provide an improved apparatus and method for forming a hollow part.",
"Another object of the present invention is to provide an improved apparatus and method for efficiently and cost effectively shaping a hollow part by mechanically shaping at least one end of the part and by hydroforming a portion of the part.",
"Still another object of the invention is to provide an apparatus and method for forming a part that uses a punch to secure each end of the part while the punch shapes the end so that each end has the same configuration as a hydroformed, middle portion.",
"The forgoing objects are basically attained by providing a hydroforming die assembly for hydroforming a part from a tubular blank, the part having a desired configuration different from a configuration of the blank and including a desired cross section at one end thereof, the die assembly comprising: a die structure having interior surfaces defining a die cavity, the die cavity having a cross sectional configuration conforming to the desired cross section of the part; and a pair of tube-end engaging structures disposed at opposite ends of the die cavity and constructed and arranged to engage opposite ends of the tubular blank, the tube-end engaging structures being constructed and arranged to seal the opposite ends of the tubular blank and to pressurize hydroforming fluid within the tubular blank for expanding the tubular blank into conformity with the interior surfaces of the die cavity, a first of the tube-end engaging structures having an outer cross-sectional configuration corresponding to the desired cross section at one end of the part, the first of the tube-engaging structures being movable into forced engagement with one end of the tubular blank to conform the one end of the tubular blank to the outer cross-sectional configuration of the first of the tube-engaging structures and hence the predetermined cross section at the one end of the part.",
"The forgoing objects are also attained by providing a method of forming a hydroformed part comprising the steps of: providing a hydroforming die assembly for hydroforming a part from a tubular blank, the part having a desired configuration different from a configuration of the blank and including a desired cross section at one end of the part, the die assembly including a die structure having interior surfaces defining a die cavity, the die cavity having a cross sectional configuration conforming to the desired cross section of the part, and a pair of tube-end engaging structures disposed at opposite ends of the die cavity and constructed and arranged to engage opposite ends of the tubular blank, the tube-end engaging structures being constructed and arranged to seal the opposite ends of the tubular blank and to pressurize hydroforming fluid within the tubular blank for expanding the tubular blank into conformity with the interior surfaces of the die cavity, a first of the tube-end engaging structures having an outer cross-sectional configuration corresponding to the desired cross section at one end of the part; moving the first of the tube-engaging structures into forced engagement with one end of the tubular blank to conform the one end of the tubular blank to the outer cross-sectional configuration of the first of the tube-engaging structures and hence the predetermined cross section at the one end of the part; and applying pressure within the tubular blank to form the tubular blank in to the desired configuration of the part.",
"Other objects, advantages, and features of the invention will become apparent from the following detailed description, appended drawings, and claims."
],
[
"This application claims the benefit of U.S.",
"Provisional Application Ser.",
"No.",
"60/241,337, filed on Oct. 19, 2000, the entire contents of which are incorporated herein by reference thereto.",
"FIELD OF THE INVENTION The invention relates generally to an improved apparatus and method for more efficiently hydroforming a tubular part.",
"More specifically, the invention relates to an apparatus and method that uses a punch to shape each end of the part into the desired configuration and hold the part during hydroforming.",
"BACKGROUND OF THE INVENTION Typically, to form a tubular part by hydroforming, a raw tube is positioned within a hydroforming tool and the tube is secured at its ends.",
"The middle portion of the raw tube is then subjected to hydroforming, leaving a transitional zone between the ends of the raw tube and the hydroformed middle portion.",
"The hydroformed part is then finished by having the two transition zones removed from the tube, leaving only the fully hydroformed middle portion.",
"The ends of the tube can be secured by tip portions being generally wedge-shaped as disclosed in EP 1022073A1.Hydroforming is also disclosed in the U.S. Pat.",
"Nos.",
"5,987,950 to Horton and U.S. Pat.",
"No.",
"6,014,950 to Jaekel et al.",
"Removing the ends of the hydroformed part creates inefficiencies.",
"For example, the cut away ends become wasted raw material.",
"Also, cutting away ends requires additional cutting tools, which complicates the apparatus needed to create the finished part.",
"Further, time is wasted performing the added step of cutting off the transitional zones at each end.",
"SUMMARY OF THE INVENTION One object of the present invention is to provide an improved apparatus and method for forming a hollow part.",
"Another object of the present invention is to provide an improved apparatus and method for efficiently and cost effectively shaping a hollow part by mechanically shaping at least one end of the part and by hydroforming a portion of the part.",
"Still another object of the invention is to provide an apparatus and method for forming a part that uses a punch to secure each end of the part while the punch shapes the end so that each end has the same configuration as a hydroformed, middle portion.",
"The forgoing objects are basically attained by providing a hydroforming die assembly for hydroforming a part from a tubular blank, the part having a desired configuration different from a configuration of the blank and including a desired cross section at one end thereof, the die assembly comprising: a die structure having interior surfaces defining a die cavity, the die cavity having a cross sectional configuration conforming to the desired cross section of the part; and a pair of tube-end engaging structures disposed at opposite ends of the die cavity and constructed and arranged to engage opposite ends of the tubular blank, the tube-end engaging structures being constructed and arranged to seal the opposite ends of the tubular blank and to pressurize hydroforming fluid within the tubular blank for expanding the tubular blank into conformity with the interior surfaces of the die cavity, a first of the tube-end engaging structures having an outer cross-sectional configuration corresponding to the desired cross section at one end of the part, the first of the tube-engaging structures being movable into forced engagement with one end of the tubular blank to conform the one end of the tubular blank to the outer cross-sectional configuration of the first of the tube-engaging structures and hence the predetermined cross section at the one end of the part.",
"The forgoing objects are also attained by providing a method of forming a hydroformed part comprising the steps of: providing a hydroforming die assembly for hydroforming a part from a tubular blank, the part having a desired configuration different from a configuration of the blank and including a desired cross section at one end of the part, the die assembly including a die structure having interior surfaces defining a die cavity, the die cavity having a cross sectional configuration conforming to the desired cross section of the part, and a pair of tube-end engaging structures disposed at opposite ends of the die cavity and constructed and arranged to engage opposite ends of the tubular blank, the tube-end engaging structures being constructed and arranged to seal the opposite ends of the tubular blank and to pressurize hydroforming fluid within the tubular blank for expanding the tubular blank into conformity with the interior surfaces of the die cavity, a first of the tube-end engaging structures having an outer cross-sectional configuration corresponding to the desired cross section at one end of the part; moving the first of the tube-engaging structures into forced engagement with one end of the tubular blank to conform the one end of the tubular blank to the outer cross-sectional configuration of the first of the tube-engaging structures and hence the predetermined cross section at the one end of the part; and applying pressure within the tubular blank to form the tubular blank in to the desired configuration of the part.",
"Other objects, advantages, and features of the invention will become apparent from the following detailed description, appended drawings, and claims.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is an exploded perspective view showing upper and lower die structures and box-shaped punches of a hydroforming die assembly in accordance with the principles of the present invention; FIG.",
"2 is a longitudinal section view of the hydroforming die assembly along section line 1-1 in FIG.",
"1, and including a tubular blank positioned within the lower die structure, the upper die structure shown in the raised or fully open position, and box-shaped punches engaging the ends of the tubular blank; FIG.",
"3 is a longitudinal sectional view of the hydroforming die assembly, similar to FIG.",
"2, but showing the upper die structure in a fully lowered position, with the tubular blank positioned within the lower die structure, the box-shaped punches inserted into the ends of the tubular blank, and fluid injected into the tubular blank; FIG.",
"4 is a cross-section taken through section line 4-4 in FIG.",
"3 and showing an unexpanded oval tubular blank disposed within the hydroforming assembly and filled with hydroforming fluid; FIG.",
"5 is a cross-section taken through line 5-5 in FIG.",
"9 showing the tubular blank expanded by fluid under pressure within the expansion region of the cooperating upper and lower hydroforming dies; FIG.",
"6 is a partial perspective view, partially in cross-section, of a box-shaped punch engaging a tubular blank; FIG.",
"7 is a longitudinal sectional view taken through line 7-7 in FIG.",
"6 showing a box-shaped punch; FIG.",
"8 is a cross-sectional view along section line 8-8 in FIG.",
"9 showing the end of the tubular blank between upper and lower clamping structures with a box-shaped punch inserted therein; FIG.",
"9 is a longitudinal section view showing a hydroforming step wherein the upper die structure is in the fully lowered position and a tubular blank has been hydroformed into an expanded configuration by fluid under pressure; FIG.",
"10 is a longitudinal section view showing the use of hydroforming ram extenders attached to punches to permit a relatively short blank to be hydroformed in a relatively long hydroforming die assembly; and FIG.",
"11 is a longitudinal section view of a hydroforming die assembly with a box-shaped punch and a round punch engaging opposite ends of a tubular blank.",
"DETAILED DESCRIPTION OF THE DRAWINGS Shown generally in FIG.",
"1 is an exploded perspective view of a hydroforming die assembly generally indicated at 10 in accordance with the present invention.",
"The hydroforming die assembly 10 includes a movable upper die structure 12, a movable lower die structure 14, a fixed die structure 16, and a fixed base 18 on which the fixed die structure 16 is mounted.",
"A plurality of pneumatic or nitrogen spring cylinders 20 mount the lower die structure 14 for movement on the fixed base 18.The upper die structure 12, lower die structure 14, and fixed die structure 16 cooperate to define a longitudinal die cavity therebetween, having a substantially boxed-shaped or multifaceted cross section as will be described herein.",
"Preferably, the upper die structure 12, lower die structure 14, fixed die structure 16, and fixed base 18 are each made of an appropriate steel material such as P-20 steel and/or 2714 steel.",
"As shown in FIG.",
"1, the upper die structure 12 defines a pair of cradle areas 22 at opposite longitudinal ends thereof.",
"The cradle areas 22 are shaped and arranged to receive and accommodate upper clamping structures 26, at opposite longitudinal ends of the upper die structure 12.Particularly, the clamping structures 26 are each connected to the upper die structure 12 at the respective cradle areas 22, by a plurality of pneumatic or nitrogen spring cylinders 24 which permit relative vertical movement between the clamping structures 26 and the upper die structure 12.The lower die structure 14 has similar cradle areas 30 at opposite longitudinal ends thereof which are constructed and arranged to accommodate lower clamping structures 28 in a similar fashion.",
"As shown, the longitudinal ends, indicated at 15, forming cradle area 30 of the lower die structure 14 have a generally U-shaped configuration.",
"The lower clamping structures 28 each have an upwardly facing surface 34 having a cross-sectional configuration that defines one-half of a multifaceted surface configuration.",
"In the context of the present invention, the term multifaceted means square, rectangular, parallelepiped, polygonal, or any other closed, non-circular or oval configuration.",
"In the illustrated embodiment, surface 34 defines one half of a rectangle.",
"In the embodiment shown, the upper two clamping structures 26 are substantially identical to the lower clamping structures 28 but are inverted with respect thereto.",
"More particularly, each upper clamping structure 26, has a downwardly facing surface 36 having a cross-sectional configuration that defines a second half of the multifaceted (i.e., rectangular) surface configuration.",
"The surface 36, of each clamping structure 26, cooperates with surface 34, of the respective lower clamping structures 28, to form a multifaceted clamping surface that captures end portions of a tubular blank 40 when the upper die structure 12 is lowered.",
"As can be appreciated from, for example, FIGS.",
"2, 3, and 4, the upper die structure 12 defines a longitudinal channel 38 having a substantially inverted U-shaped cross section.",
"The channel 38 is defined by a downwardly facing, generally horizontal longitudinally extending surface 44, and a pair of spaced, longitudinally extending vertical side surfaces 43, which extend parallel to one another from opposite sides of surface 44.The lower die structure 14 has a central opening 42 extending vertically therethrough, between the U-shaped longitudinal ends 15.The opening 42 receives fixed die structure 16.Interior vertical surfaces 41 in the lower die structure 14 define the aforementioned central opening 42.More particularly, a pair of longitudinally extending side surfaces 41, define the lateral extremities of the opening 42.The surfaces are vertically disposed in parallel facing relationship with one another.",
"The U-shaped end portions 15 of the lower die structure 14 define the longitudinal extremities of the opening 42, and have interior surfaces (not shown) vertically disposed in parallel facing relation to one another.",
"The fixed base 18 is in the form of a substantially rectangular metal slab.",
"The fixed die structure 16 is affixed to an upper surface 46 of the fixed base 18.The fixed die structure 16 is an elongate structure which extends along a major portion of the length of the upper surface 46 of the fixed base 18, generally along the center of the fixed base 18.The fixed die structure 16 projects upwardly from the fixed base 18 and has substantially vertical side surfaces 48 on opposite longitudinal sides thereof.",
"The fixed die structure 16 is constructed and arranged to extend within the opening 42 in the lower die structure 14, with minimal clearance between the generally vertical side surfaces 48 of the fixed die structure and vertical surfaces 41 of the lower die structure 16.Similarly, there is minimal clearance between the interior transverse side surfaces (not shown) of end portions 15 of the lower die structure 14 and the vertical end surfaces 49 of the fixed die structure 16.The fixed die structure 16, further includes an upwardly facing, generally horizontal and longitudinally extending die surface 50, which is constructed and arranged to extend in spaced facing relation to the longitudinally extending, downwardly facing die surface 44 of the upper die structure 12.As can best be seen in FIGS.",
"4 and 5, the aforementioned side surfaces 41, the upwardly facing surface 50, the side surfaces 43 and downwardly facing surface 44 cooperate to define a die cavity 52, having a multifaceted cross sectional configuration substantially throughout its longitudinal extent.",
"The die cavity surfaces define the desired shape of a part to be hydroformed from a circular or oval blank tube.",
"FIG.",
"2 shows the upper die structure 12 in an opened or raised position.",
"In this position the hydroforming die assembly 10 enables the tubular blank 40 to be placed within the lower die structure 14.After the blank 40 is placed in the lower die structure 14, the upper die structure 12 is lowered to form the die cavity 52.The die cavity may be ultimately smaller than what is illustrated in FIG.",
"4 to effect a slight crushing of the tubular blank 40 before the blank 40 is expanded in the hydroforming operation, as disclosed in U.S. Pat.",
"No.",
"5,987,950 to Horton, which is incorporated herein by reference.",
"With the tubular blank 40 positioned between the closed upper die structure 12 and lower die structure 14, hydroforming rams 80 having punches 81 attached to mounting structures 90 are advanced from opposite sides of the hydroforming die assembly 10 to engage opposite ends of the tubular blank 40.As shown most clearly in FIGS.",
"6 and 7, each punch 81 includes an initial beveled portion 82 which transitions into a multifaceted, here rectangular, portion 84.A base 86 forming a lateral shoulder 88 is formed at one end of the multifaceted portion 84 opposite the initial beveled portion 82.The punch 81 is secured to the end of the mounting structure 90 by means of mechanical fasteners 92, such as bolts, extending through counter-bored apertures 94 formed in the punch 81 and into the holder 92.Base 86 preferably has a size and shape that is complementary to the size and shape of the mounting structure 90 so as to form a smooth, uniform transition between the punch 81 and the mounting structure 90.In the embodiment shown, the beveled portion 82 is preferably formed at an angle θ (see FIG.",
"7) of between about 13-17°, and most preferably, about 15° with respect to the sides of the box-shaped portion 84.The multifaceted portion 84 preferably has straight sides so as to have a perimeter that defines a multifaceted shape, such as a polygon, square rectangle, skewed parallelogram, etc.",
"The perimeter shape of the box-shaped portion 84 corresponds substantially to the shape of the clamping surface formed by the upwardly facing surface 34 of the lower clamping structure 28 and the downwardly facing surface 36 of the upper clamping structure 26.Also, the size of the multifaceted portion 84 is defined so as to provide a sealing interference fit with the wall of the tubular blank 40, with the clamping surfaces providing external support for blank 40.The forward end 83 of the punch 81 at the free end of the beveled portion 82 has dimensions that are smaller than the multifaceted portion 84, thus permitting the forward end 83 to be inserted into the unexpanded end of the tubular blank 40 as shown in FIG.",
"2.With the forward end 83 of the punch 81 engaged with the end of the blank 40, the hydroforming ram can be further advanced under the force of hydraulic pressure, thus forcing the punch 81 into the end of the tubular blank 40 after the upper die structure 12 is lowered, as shown in FIG.",
"3.The beveled portion 82 of the punch 81 gradually forms the end of the blank 40 until the multifaceted portion 84 is fully inserted into the end of the blank 40.During this process, the end portions of blank 40 may be stretched outwardly as they are conformed to the multifaceted portion 84 and hence, the adjacent clamping surfaces 34, 36, as best shown in FIGS.",
"3 and 8.The width of the lateral shoulder 88 is preferably substantially the same as the thickness of the tubular blank 40 so that the outer surface of the tubular blank 40 transitions smoothly with the outer surfaces of the base 86 and the holder 90.Thus, when the tube is formed over the punch to fit the finished tube shape it will not be necessary to remove the scrap portion of the blank, this eliminates the need for cut-off tooling, which saves money and time.",
"While the above description refers to only one punch, it should be appreciated that this discussion may apply to both punches 81 at opposite ends of the tube 40.The tubular blank 40 may be round (circular cross section).",
"Punches 81 have a similar height and width dimensions as the blank.",
"The blank may be oval for punches that are rectangular or otherwise elongated along a height or width dimension.",
"Hydroforming processes using oval tubular blanks are disclosed in U.S. Pat.",
"No.",
"5,987,950, the disclosure of which is hereby incorporated by reference, as stated above.",
"Providing a tubular blank having an oval cross-section is advantageous in comparison with the conventional circular cross-section because it provides a circumference that conforms more closely to the final cross sectional perimeter of the generally box-shaped (not square) cross-sectional shaped die cavity 52.Thus, less expansion of the blank 40 is required when expanding the blank into conformity with the surfaces forming cavity 52.In addition, the closer conformity of blank 40 and cavity surfaces allows the blank to be more easily expanded into the corners of the cavity 52, where expansion becomes most difficult due to the increasing frictional surface contact between the exterior surface of the blank and cavity surfaces during expansion of the blank 40.As can be seen in FIG.",
"3, when the blank 40 is substantially rigidly held in place between die structures 12 and 14 and the hydroforming cylinders or rams 80 are telescopically and sealingly inserted into both opposite ends of the blank 40, so that beveled surfaces 82 engage the opposite edges of blank 40.The rams 80 are forced inwardly to cause the opposing edges of blank 40 to ride down surface 82 until it engages surface 88 and thus converts the end portions of the blank into the exterior shape of portion 84 of punch 81.The hydroforming cylinders then preferably pre-fill, but do not pressurize to any large extent, the tubular blank 40 with hydroforming fluid (preferably water) as indicated by reference character F. The hydraulic fluid is injected through a channel 87 formed in one or both punches 81 which communicates with a channel 97 formed in the corresponding mounting structure 90.Although the pre-filling operation is preferred to reduce cycle times, and to achieve a more smoothly contoured part, for some applications the upper die structure 12, may be fully lowered before any fluid is provided internally to tubular blank 40.End portions of the sealed die cavity 52 are generally rectangular in shape as defined by surface portions 54 having generally the same size and shape as the clamping surfaces 34, 36 of the clamping structures 28, 26, respectively.",
"Thus, the portions 54 define areas of the die cavity which have a cross-sectional area that is the same as or only slightly larger than the area defined by the cross-sectional shape of the end portions of the tubular member 40 after the punches 81 have been forced into the ends of the tubular member as illustrated in FIG.",
"6.Otherwise stated, the portions 54 of the die cavity 52 define areas of the die cavity which are used to expand the tubular blank 40 during the hydroforming process only to the extent required to convert the shape of the blank from a round or oval cross section to a multifaceted (here rectangular) cross sectional configuration.",
"Because the end portions of the blank 40 fitted with punches 81 (as illustrated in FIG.",
"6) will form the shape desired for the final part to be hydroformed, and will not constitute an unexpanded portion that must be cut off after the hydroforming process so that the remaining uncut portions of the tube correspond to the desired part, substantial scrap material is saved.",
"Each unexpanded surface portion 54 of the die structure defines a shape consistent with the shape of the portions 84 and 86 of the punch 81.The cavity 52 may also include an enlarged portion 56 towards the longitudinally central portions thereof.",
"With the upper die structure 12 closed with respect to the lower die structure 14 and with the punches 81 sealingly inserted into the ends of the blank 40, the fluid F can be pressurized to expand the tubular blank 40 into conformity with the surfaces defining die cavity 52 (see FIGS.",
"5 and 9).",
"The tubular blank 40 is expanded into the non-round, multifaceted (e.g., rectangular) of the die cavity 52.If the hydroforming assembly includes a die cavity with an enlarged portion 56, the blank 40 will be enlarged in that area.",
"Because the portions 54 of die cavity 52 define shapes that are consistent with the shape of the multifaceted portion 84 of the punches 81, the hydroformed member has a consistent shape out to its ends, and it is not necessary to cut the end portions off.",
"If a portion of the blank is to be significantly enlarged in its cross-sectional perimeter (e.g., greater than 5% relative to the original blank perimeter), it may be preferred that the longitudinal ends of the blank be pushed inwardly toward one another to replenish wall thickness as the blank is expanded.",
"If the blank is not to be enlarged, but is only expanded into conformity with a multifaceted die cavity, longitudinal movement of the ends during expansion of the blank may not be necessary.",
"More particulars on the preferred hydroforming process are disclosed in U.S. Pat.",
"No.",
"6,014,879 to Jaekel et al., the disclosure of which is hereby incorporated by reference.",
"In accordance with another embodiment, if a significant amount of perimeter expansion is required at one end of the tube part so that substantial wall thickness replenishment is required thereat, it is generally preferred to employ a circular or oval punch, as opposed to a multifaceted punch.",
"This is because material flows more effectively and evenly toward the enlargement area from a rounded end than from a box-shaped end.",
"Such a hydroforming configuration is shown in FIG.",
"11, in which the surrounding die structures are not shown for clarity of the illustration.",
"The arrangement shown includes one hydroforming ram 80 having a beveled, rectangular-shaped punch 81 and a rectangular-shaped mounting structure 90, as shown and described previously.",
"The arrangement also includes a second hydroforming ram 100 that includes a cylindrical base portion 112 and a smaller cylindrical portion 102 with an insertion bevel 116 formed at the end.",
"A circular, annular sealing shoulder 114 that engages the end of the tubular blank 40′ is defined between base portion 112 and cylindrical portion 102.At the end of the blank 40′ engaged by the multifaceted punch 81, the die structure (not shown) presents a surface configuration that forms the blank 40′ such that the cross sectional configuration at portion 110 of the blank is expanded only to the extent that the rounded cross section of the blank is converted to a multifaceted cross section.",
"The portion 110 is joined by a gradually tapered segment 108 which extends to an enlarged rectangular-shaped cross sectional portion 106.Conversely, at the end of the blank 40′ engaged by the cylindrical punch 100, the die structure presents a surface configuration that forms a relatively short, non-enlarged cylindrical portion 105 of the blank.",
"The blank then transitions from the rounded perimeter shape at 105 to the rectangular cross section at area 106.The cylindrical punch 100 allows for the relatively large expansion of enlarged area 106 and the abrupt transition region 104 because longitudinal pushing at the end of the tubular member 40′ is more effective for replenishing wall thickness if the punch is round.",
"The cylindrical end portion of the formed member shaped by the cylindrical punch 100 would typically be cut off during a subsequent finishing operation.",
"The box-shaped end formed by the rectangular-shaped punch 81, on the other hand, can be tailored to the desired final member shape so the end need not be cut off.",
"As shown in FIG.",
"10, one or more ram extenders 120 can be installed between the punch 81 and the holder 90.The extenders 120 have a rectangular-shaped cross sectional configuration that conforms with the rectangular-shaped cross sections of the mounting structure 90 and the base 86 of the punch 81.Accordingly, the hydroforming rams 80′ can extend further into the relatively an enlarged portions 54 of the die cavity 52 to accommodate hydroforming of shorter tubular blanks 40′ within the same die cavity 52.Of course, the extended hydroforming blanks 80′ cannot extend to an enlarged area 56 of the hydroforming cavity 52 or the seal between the end of the tubular blank 40′ and the shoulder 88 of the punch 81 will be lost as the tubular blank expands into the enlarged area 56.Thus, the present invention includes a hydroforming die assembly for hydroforming a part from a tubular blank comprising a die structure having interior surfaces defining a die cavity, the die cavity having a cross sectional configuration conforming to the predetermined cross section of the part, the part having a predetermined configuration different from a configuration of the blank and including a predetermined cross section at one end thereof.",
"It should be appreciated that the foregoing detailed description and accompanying drawings of the preferred embodiments are merely illustrative in nature, and that the present invention includes all other embodiments that are within the spirit and scope of the described embodiments and appended claims."
]
] | Patent_10399192 |
[
[
"Method and system for online sales and purchase",
"Internet-based commercial network connects multiple qualified participant buyers and sellers.",
"Items are made available for defined periods of time, referred to as an event (139).",
"Event terms and conditions include item types, price, quantity, volume or units, discount, shipping terms (138).",
"Reiterative offer and counteroffers permit negotiation of terms for an event.",
"Participants may view anonymous competitive quotes for comparison and analysis.",
"Pooled purchases and auctions are envisioned.",
"Participants may create web presences and are subject to restrictions on access (141) and purchase (142) authority.",
"Central system maintains participant data."
],
[
"1.A method of computer assisted procurement of products comprising: (a) providing an internet website for listing products sought to be purchased by a purchaser, (b) establishing an event at which qualified sellers may visit the website to offer terms for the sale of the products to the purchaser by: (i) making the website available for the posting of quotes during an identified time period, and (ii) inviting qualified sellers to offer terms of sale by posting those terms of sale on the website during the identified time period.",
"2.The method of computer assisted procurement of products according to claim 1, further comprising making available on the website during the identified time period to qualified sellers participating in the event at least one of the terms offered by the other qualified seller or sellers participating in the event.",
"3.The method of computer assisted procurement of products according to claim 2, wherein the at least one of the terms is price.",
"4.The method of computer assisted procurement of products according to claim 1, wherein step (a) comprises: providing on the website a location for indicating an offer of price for each listed product, and providing on the website a further location for indicating an offer of at least one term other than price by any qualified seller participating in the event.",
"5.The method of computer assisted procurement of products according to claim 4, wherein the at least one other term includes at least one of advertising contribution, shipping terms, payment terms, schedule of delivery, quality, and quantity discount.",
"6.The method of computer assisted procurement of products according to claim 1, further comprising revealing to each qualified seller participating in the event the terms offered by each other qualified seller participating in the event, and concealing from each qualified seller participating in the event the identity of each other qualified seller participating in the event.",
"7.The method of computer assisted procurement of products according to claim 6, further comprising: (c) affording the purchaser the ability to designate accepted quotes for a period of time after the close of the event, including: (i) affording the purchaser the ability to designate selected winning quotes item by item, and (ii) affording the purchaser the ability to designate winning quotes by designating acceptance of a totality of quotes from a particular seller.",
"8.The method of computer assisted procurement of products according to claim 1, further comprising establishing the event as a market price event in which each item listed for purchase has a set market price, and each quote received is required to be in terms of a variance from the market price.",
"9.The method of computer assisted procurement of products according to claim 8, further comprising automatically by computer converting each quoted variance into a full price for each listed item based on the sum of the market price and the variance quoted.",
"10.The method of computer assisted procurement of products according to claim 1, further comprising establishing the event as an event in which the listed items do not have a set market price, and requiring quotes to be the actual price intended on an item by item basis.",
"11.The method of computer assisted procurement of products according to claim 1, further comprising the step of providing the purchaser information concerning the cost of money over time in reference to non-price terms of a particular sellers quote.",
"12.The method of computer assisted procurement of products according to claim 1, further comprising providing the purchaser with information assisting the purchaser to compare quotes of branded products differing in brand.",
"13.The method of computer assisted procurement of products according to claim 1, further comprising accepting quotes in differing units of measure, and converting at least one of the quotes in differing units of measure to quotes based on a single unit of measure, whereby the purchaser can readily compare competitive quotes.",
"14.The method of computer assisted procurement of products according to claim 1, wherein the quotes of each seller are made known to the other sellers, and the identity of each seller is not made known to the other sellers.",
"15.The method of computer assisted procurement of products according to claim 1, further comprising constraining an event to goods of a particular kind.",
"16.The method of computer assisted procurement of products according to claim 1, further comprising enabling the purchaser to alter the volume of goods being sought during an event.",
"17.The method of computer assisted procurement of products according to claim 16, wherein enabling the purchaser to alter the volume of goods comprises, presetting price points at which automatically the volume of goods being sought will be reset.",
"18.The method of computer assisted procurement of products according to claim 17, further comprising automatically extending the time period of the event where the volume of goods being sought is reset to give sellers an opportunity to respond.",
"19.The method of computer assisted procurement of products according to claim 1, further comprising setting a period of time during which sellers may change their prior quotes.",
"20.The method of computer assisted procurement according to claim 1, further comprising step of recognizing quotes in a quoter's quantity unit of measure other than a quantity unit of measure specified by an event administrator, and automatically converting the quoter's quantity unit of measure and a quoter's quote to the specified quantity unit of measure, whereby quotes from all quoters are readily compared.",
"21.The method of computer assisted procurement according to claim 20, further comprising the step of calculating total quote for each quoter for comparison based on quotes from quoters in disparate quantity units of measure.",
"22.The method of computer assisted procurement according to claim 1, further comprising the step of receiving as a part of a quote an additional funds or terms portion of a quote in addition to a price.",
"23.The method of computer assisted procurement according to claim 22 further comprising calculating the additional value of the additional funds or terms portion of a quote and summing the additional value with a price portion of the quote.",
"24.The method of computer assisted procurement according to claim 22, wherein the step of receiving as part of a quote an additional funds or terms portions comprises receiving at least one of a quantity based discount, contributed advertising dollars, shipping terms, payment terms, quality of goods, and scheduling of deliveries.",
"25.The method according to claim 1, further comprising the step of user authentication comprising: (i) determining the existence of a cookie on a user's computer, (ii) determining from the user's cookie the user's permitted level of access; and (iii) permitting the user to an event at the level of access determined.",
"26.The method according to claim 25, further comprising determining whether an event to which the user seeks access is still open, before proceeding with steps (i) and (ii).",
"27.The method according to claim 26, further comprising, before proceeding with steps (iii), determining whether the event has been disabled, and if the event has not been disabled, determining whether the user has been disabled.",
"28.The method according to claim 25, wherein prior to steps (ii) and (iii), the method further comprises the steps of determining whether the user has responded to an invitation to an event in which the user seeks participation.",
"29.The method according to claim 1, wherein step (b) further comprises setting a recurrent period during which quotes may be posted by qualified sellers.",
"30.The method according to claim 29, wherein step (b) comprises establishing a list of items regularly needing replenishing and permitting qualified sellers to quote each recurrent period without repeated invitation.",
"31.A method of computer assisted procurement of products sought to be purchased by a purchaser comprising: (a) providing an internet website, (b) establishing classifications of users entitling users to varying degrees of access to information and activities available through the website, (c) providing a publicly available registration page at the website, (d) issuing at least one of a login number, login word, and password at a user request on the registration page, (e) using the at least one number or word and password of a user at a login by the user to verify the user's identity and status, (f) upon successful verification of the user's identity and status, placing a cookie on the user's computer, the cookie comprising an identification of the user, and (g) thereafter based on the information in the user's cookie, (i) determining the user's classification, and (ii) affording the user access to information and activities on the website to a degree consistent with that user's classification.",
"32.The method of computer assisted procurement of products according to claim 31, wherein each cookie identifies the particular user's time zone, the method further comprising identifying timed events to a user in the user's local time by converting from the time of an event occurring outside the user's time zone.",
"33.The method of computer assisted procurement of products according to claim 31, further comprising, providing at the website a plurality of different home pages, each home page being associated with a particular user classification and each home page being accessible to a user having the associated classification.",
"34.The method of computer assisted procurement of products according to claim 33, wherein one of said classifications is administrator, and further comprising providing each user classified as administrator access to all of said home pages.",
"35.The method of computer assisted procurement of products according to claim 33, wherein one of said classifications is that of a responding buyer or seller entitled to quote at an event, the method further comprising restricting access of a user classified as a responding buyer or seller to event information, including providing a page on the website with competitive quote information without identification of competitive responding buyers or sellers making competitive quotes.",
"36.The method of computer assisted procurement of products according to claim 34, wherein another of said classifications is that of a responding buyer or seller entitled to quote at an event, the method further comprising: (i) providing a homepage at the website for each user classified as a responding buyer or seller and limiting access to that homepage to that responding buyer or seller and to users classified as administrators, (i) providing a program details page at the website with competitive quote information from each user classified as a responding buyer or seller and without identification of the users classified as responding buyer or seller, and permitting access to the program details page by users classified as responding buyer or seller and users classified as administrator.",
"37.The method of computer assisted procurement of products according to claim 36, wherein the program details page provides for the entry of information about a scheduled event including: identification of all items being offered for purchase, item quantity, item descriptions, item price quotes, and shipping information.",
"38.The method of computer assisted procurement of products according to claim 37, wherein the details page further provides for the entry of a market price.",
"39.The method of computer assisted procurement of products according to claim 38, wherein the details page further provides for a quote increment, said quote increment setting increments in price above or below the market price by which quotes must be made.",
"40.The method of computer assisted procurement of products according to claim 31, wherein providing an internet website comprises providing a plurality of pages at said website, and affording user access to information and activity on the website to a degree consistent with that user's classification comprises, as a user seeks to move from page to page among said pages, authenticating the user by determining whether the user is authorized to have access to each page sought by the user.",
"41.The method of computer assisted procurement of products according to claim 40, wherein authenticating includes determining whether the user is logged in and validated, and determining whether the user is authorized includes event participation authentication including determining whether the user is a valid event participant and that the user has permission to use event related pages.",
"42.The method of computer assisted procurement of products according to claim 41, wherein the step of authenticating includes inspecting the content of the cookie on the user's computer.",
"43.The method of computer assisted procurement of products according to claim 40, wherein affording the user access includes providing event authorization comprising: retrieving information concerning an event and determining whether the event has been disabled, if the event has not been disabled, determining whether a user seeking access to the event has been disabled, and if the user has not been disabled, determining if the user has responded to an invitation to the event, and if the user has responded to an invitation to the event, affording the user access to a page at which the user can enter a quote.",
"44.The method of computer assisted procurement of products according to claim 31, wherein the step of affording a user access further comprises identifying a user as qualified to organize an event.",
"45.The method of computer assisted procurement of products according to claim 44, further comprising providing event organization web page for use by a user qualified to organize an event.",
"46.The method of computer assisted procurement of products according to claim 44, further comprising providing a user qualified to organize an event with the authority to select event respondents.",
"47.The method of computer assisted procurement of products according to claim 45, further comprising enabling a user qualified to organize an event access to quotes and quoter identities during the course of an event.",
"48.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to organize an event to set an event market price with respect to which price quotations must be made by respondent.",
"49.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to organize an event to set a price quote increment of which all price quotations must be a multiple.",
"50.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to organize an event to determine that the event is either an auction or a reverse auction.",
"51.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to organize an event to set a minimum or a maximum price quote.",
"52.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to organize an event to establish the time and duration of an event.",
"53.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to establish an event to identify quoters whose quotes have been accepted.",
"54.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to establish an event to alter terms of an event during the course of an event.",
"55.The method of computer assisted procurement of products according to claim 44, wherein enabling a user qualified to establish an event to alter terms of an event during the course of an event includes enabling the setting of price points at which quantities of items sought to be purchased change automatically.",
"56.The method of computer assisted procurement of products according to claim 44, further comprising enabling a user qualified to establish an event to negotiate terms of a quote subsequent to the close of an event.",
"57.The method of computer assisted procurement of products according to claim 49, further comprising automatically converting an incremental bid to market price at a web page accessible to a user qualified to establish an event.",
"58.The method of computer assisted procurement of products according to claim 52, further comprising setting an extension of time of an event to occur upon receipt of a quote within a predetermined time of the scheduled close of an event.",
"59.The method of computer assisted procurement of products according to claim 52, further comprising disabling any quoter that has not submitted a quote during a predetermined time period in the course of the running of an event.",
"60.The method of computer assisted procurement of products according to claim 52, further providing a participant award website page to which a quoter whose quote has succeeded in the award of a sale is given access for completion of details of the award.",
"61.The method of computer assisted procurement of products according to claim 60, further comprising the step of communicating to a successful quoter a link to the participant award website page.",
"62.The method according to claim 31, further comprising affording a buyer a web page for the listing of a list of items regularly needing replenishment, affording participating sellers the ability to quote on items on the list at any time, affording the buyer the ability to award a purchase to a participating seller on the basis of a current quote.",
"63.The method according to claim 62 wherein affording participating sellers the ability to quote comprises, designating participating sellers and permitting designated participating sellers the ability to quote repeatedly without redesignation.",
"64.A method of computer assisted procurement of products sought to be purchased comprising: (a) providing an internet website, (b) affording a participating buyer the ability to list on a page at the website a list of items requiring recurrent replenishment, (c) qualifying participating sellers to quote on items in the list of items, (d) affording qualified participating sellers the ability to award one or more sales to one of said qualified participating sellers based on that seller's quote on an item or items on the list, and (e) enabling the qualified participating sellers to quote on a recurring basis on items on the list absent the need for requalification.",
"65.The method according to claim 64, wherein step (e) includes scheduling recurrent events during which quotes on listed items can be received.",
"66.The method according to claim 64 wherein step (e) includes maintaining a price list of items on the list continuously open for receipt of quotes on listed items.",
"67.A method of computer assisted sales or procurement comprising: (a) providing an internet website for listing products to be sold or to be purchased, (b) establishing an event at which qualified buyers or sellers may visit the website to quote terms for the purchase or sale of the products listed, by: (i) malting the website available for the posting of quotes during an identified time period, and (ii) inviting qualified buyers or sellers to offer terms of purchase or sale by posting those terms on the website during the identified time period.",
"68.A method of negotiated contract implementation comprising: (a) providing an internet website for facilitating negotiation between a first party seeking a contract proposal and at least one second party responding with a proposal, (b) providing at least one page at the website adapted to permit the first party to create an event by placing a contract out for bid, (c) providing at least one page at the website adapted to permit the at least one second party to submit a proposal, and (d) providing at least one further page facilitating analysis by the first party of each proposal from the at least one second party.",
"69.The method of negotiated contract implementation according to claim 68, further comprising requiring the at least one second party to accept at least one of event details and event terms and conditions by the first party before permitting the at least one second party to submit a proposal.",
"70.The method of negotiated contract implementation according to claim 68, further comprising enabling the first party to further negotiate terms with the at least one second party, including permitting the at least one second party to revive a previously submitted term in response to negotiation with the first party.",
"71.The method of negotiated contract implementation according to claim 70, wherein enabling the first party to further negotiate includes enabling the first party to reveal to the at least one second party one or more terms of competitive proposals of other second parties for comparison by the at least one second party of the at least one second party's proposals for said one or more terms with the one or more terms proposed by the other second parties.",
"72.The method of negotiated contract implementation according to claim 70, further comprising, after a revision of a previously submitted term by the at least one second party in response to negotiation with the first party, refreshing at least a portion of the analysis facilitated at step (d) for indicating to the first party the effect of the revision of the at least one term by the at least one second party.",
"73.The method of negotiated contract implementation according to claim 68, wherein the first party is a buyer and step (b) comprises: (i) enabling the buyer to identify items to be purchased, (ii) setting an event time and duration at the buyer's instructions, and (iii) identifying at least one of contract begin date, contract duration, estimated contract volume, and discount and future rates of money at the buyer's request.",
"74.The method of negotiated contract implementation according to claim 73, wherein step (b) further comprises, identifying participating sellers at the buyer's request.",
"75.The method of negotiated contract implementation according to claim 73, further comprising requiring, at the buyer's request, sample submission.",
"76.The method of negotiated contract implementation according to claim 75, further comprising conveying by e-mail the buyer's invitations to sellers identified by the buyer.",
"77.The method of negotiated contract implementation comprising claim 68, wherein step (c) includes, at the least one second party's instructions: (i) proposing cost, and (ii) proposing at least one of annual dollar volume, proposal duration, payment terms, annual seasonal distribution and movement.",
"78.The method of negotiated contract implementation according to claim 77, wherein step (c) further comprises, at the least one second party's instructions: (iii) offering additional funding.",
"79.The method of negotiated contract implementation according to claim 68, wherein step (d) further comprise making available to the first party a side-by-side comparison of proposals from all participating second parties.",
"80.The method of negotiated contract implementation according to claim 68, wherein step (d) further comprises, at the instructions of the first party, categorizing each additional funds offering by a participating second party, and providing category-by-category comparison to the first party.",
"81.The method of negotiated contract implementation according to claim 68, wherein step (d) further comprises, providing the first party an analysis of the relationship between time and money involved in a proposal by the at least one second party.",
"82.The method of negotiated contract implementation according to claim 68, wherein step (d) further comprises, at the instructions of the first party, providing a contingency analysis.",
"83.The method of negotiated contract implementation according to claim 70, wherein step (d) further comprises affording the first party at least one of calculation of net present value, calculation of item list cost differential, and calculation of additional profitability.",
"84.A method of computer assisted competitive product procurement comprising: (a) providing an internet website for listing branded products for which proposals for sales are sought by a buyer, (b) listing for the buyer characteristics of a proposed program for each branded product, (c) affording suppliers of the branded products access to the website to post terms of a proposed sale to the buyer of each branded product in response to the buyer's proposed programs, and (d) displaying for the buyer a comparison of the suppliers' responsive proposed sale terms.",
"85.A method of computer assisted competitive product procurement according to claim 84, wherein step (d) further comprises displaying the comparison on the bases of how nearly each supplier's posted terms of a proposed sale approach the buyer's proposed program characteristics.",
"86.A method of computer assisted competitive product procurement according to claim 85, wherein the characteristics of the proposed program for each branded product includes the buyer's proposed gross margin on retail sales of the products, and the method further comprising: (e) displaying a gross margin calculated on the basis of the terms of said each proposed sale by a supplier to the buyer of each branded product for comparison with said buyer's proposed gross margin on retail sales.",
"87.A method of computer assisted competitive product procurement according to claim 85, wherein displaying the comparison on the basis of how nearly each supplier's posted terms of a proposed sale approached the buyer's proposed program characteristics includes calculating and displaying the difference in program cost between the program having the buyer's proposed program's characteristics and a program based on the supplier's terms of a proposed sale.",
"88.A method of computer assisted competitive product procurement according to claim 87, wherein calculating and displaying the difference in program costs comprises calculating and displaying said difference in at least one of total monetary difference and percent difference.",
"89.A method of computer assisted competitive product procurement according to claim 85, wherein displaying for the buyer a comparison includes displaying a normalized competitive comparison that compare variations of several supplier-proposed sales from the buyer's proposed program.",
"90.A system for computer assisted procurement of products including: (a) a web server, (b) programming to: (i) produce an internet website for listing products sought to be purchased by a purchaser; (iii) establish an event at which qualified sellers may visit the website to offer terms for the sale of the products to the purchaser, and (iv) make the website available for the posting of offers during an identified time period for the event.",
"91.The Computer programming for computer assisted sales or procurement including: (a) website programming establishing an interactive website having: (ii) first web page programming responsive to a first user establishing at least one event for buying and selling a plurality of items, (iii) second web page programming responsive to input by a plurality of second users quoting proposed terms of purchase and sale of one or more of the identified items to present said terms on the one or more pages of interactive website; and (iv) third web page programming presenting said second users' proposed terms for review by the first user and responsive to inputs from the first user to communicate selection and award to at least one of the second users.",
"92.The programming according claim 91, wherein the first web page programming includes event scheduling programming operative to open response, the second web page programming during a period of time defined by inputs from the first user.",
"93.The programming according claim 92, wherein said website programming further comprises: (iv) fourth web page programming responsive to the selection and award, by the first user to enable the at least one second user to accept the selection and award."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>For any product that can be described and priced per item, per weight or by how it is packaged, procurement has often been inefficient and time consuming for purchasers and sellers alike.",
"This applies to retail grocery and healthcare businesses and any that regularly purchase a variety of items from various sources.",
"Additionally, past procurement methods have not been certain to bring purchasers the best purchase terms available, or to assure sellers a reasonable opportunity to fairly compete.",
"Typically, in a retail business, sellers would need to schedule a meeting with a purchasing agent of a large retailer, travel to the scheduled meeting, and make offers on products with little or no knowledge of what competitive offer had been or would be made by other sellers.",
"This practice has been inefficient, time consuming and not competitive.",
"Sellers have been unable to react to competitive offers and purchasers have been denied the benefit of such reactive pricing.",
"A number of websites on the worldwide web offer auctions and reverse auctions.",
"These permit buyers or sellers to make offers or “quote” on various posted items.",
"The quoting often is set to close at a particular time, and a feature has been offered whereby quoting may be extended if a quote is received within an extension threshold time approaching the scheduled close.",
"These sites are not tailored to a particular business's procurement practices, however.",
"They do not afford the opportunity for a business purchaser to schedule an event among recognized, qualified business suppliers, during which many items required by the business purchaser are posted by that purchaser, and suppliers are unable to quote against one another, not just on price, but on other terms important to both the selling and purchasing parties.",
"Neither do prior sites allow for confirming a purchasing or selling activity to the business practices of the relevant business.",
"In addition prior sites have not allowed for actual negotiation of contract terms of a negotiated contract."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with this invention, a system, software and method for computer-assisted procurement or sales of products are provided in which an internet website allows for the listing of a multiplicity of products to be sold or purchased by a seller or a purchaser.",
"An “event” is established at which qualified buyers or sellers can visit the website to offer terms for the sale or purchase of the products listed there.",
"The establishment of such an event includes making the website available for the posting of offers (“quotes”) during an identified time period.",
"In a purchasing event, qualified sellers are invited to offer terms of sale by posting those terms at the website in association with the items sought to be purchased, and doing so during the identified time period for the event.",
"In a selling event buyers are similarly invited to the website to offer to purchase items listed there.",
"In a negotiated event, negotiable purchase contract terms are posted for negotiation between buyer and seller.",
"In a competitive branded product event, similar products of differing brands may be sought by a buyer, and sellers' proposals in response are objectively compared despite differing proposed terms.",
"Buying and Selling Events The system by which the invention operates includes one or more servers, the internet-connected computers of buyers and sellers, the internet equipped computer of the company that supports the system and maintains the website and the programming of each that establishes the interactions of the buyers, sellers and company with the webiste and each other.",
"During the course of the purchasing event, software by which the system operates provides that offers by each qualified seller participating in the event are made known on the website to the other participating qualified sellers.",
"The terms offered by the qualified sellers may be not just price, but such further important considerations as quantity discounts, contributed advertising dollars, shipping terms, payment terms, quality, scheduling of deliveries, and such other terms and conditions as may be commonly a part of a purchase contract in the particular business or industry.",
"The website can offer the qualified sellers the opportunity to quote on each item listed.",
"It is able to provide for each seller to convey the additional, non-price terms with respect to each product listed or with respect to the entirety of the products sought to be purchased.",
"In addition, the website shows a total of the prices offered by each seller and can indicate by a reduction the value of the non-price terms offer by a seller.",
"The purchaser can buy from a qualified seller with respect to individual items or can purchase from a single seller the entirety of the items posted.",
"Typically, the event is scheduled in advance for a fixed period of time, but if a quote by a seller is made within the extension threshold near to the close of the event, the event is extended to allow active participants placing quotes to respond.",
"Although the examples given here relate primarily to a purchasing event (e.g.",
"a reverse auction), the event may also be a selling event (a traditional auction).",
"Purchasing events may be replenishment events.",
"These are regularly scheduled events at which previously approved sellers quote on items designated by the purchaser from a list of regularly purchased items.",
"Purchasing events may also be price list events.",
"These are replenishment events of a different kind.",
"A list of regularly purchased products is maintained open for quotes by approved sellers.",
"Buyers, then, have the opportunity to purchase at what they perceive as the best quote for a particular item, as needed.",
"Further, purchasing events may be spot buys.",
"This event is opened to cover a pressing need for a particular item or items.",
"That may arise from a shortage occurring because, for example, a retailer-buyer has run a sale on the particular item, because the retailer—seller wishes to extend a particularly successful promotion, or because a retailer-buyer has scheduled an ad for a particular item.",
"The immediate need may be heightened by the buyer's inability to purchase the desired item from the buyer's usual suppliers.",
"The spot buy event enables a spur of the moment buy to cover a real or anticipated shortage.",
"Where the event is a purchasing event, the event may be of several kinds.",
"One is a market price event where the products sought to be purchased have a “going price” against which the sellers quote.",
"In this kind of event, the quote may be plus or minus some variance from the market price.",
"In that case, the system is able to convert this to a total price mathematically and display that to the purchaser.",
"The second kind of event does not have a market price with respect to the items sought to be purchased, but rather the sellers quote the actual price intended on an item by item basis.",
"In a preferred embodiment the system also provides certain improvements over the reverse auction that assists a buyer in the procurement of general merchandise items, large capital items or branded products.",
"One such improvement aids the purchaser in evaluating the cost of money over time.",
"Another improvement helps level the decision making process when purchasing similar products that are different brands.",
"The system is robust in the sense that it can accommodate variance in the unit of measure posted by a purchaser and the unit of measure on which a quote is made.",
"For example, the desired purchase may be posted in cases and the responsive quote may be in pounds.",
"The system can be equipped with conversion factors that will accommodate this, making the calculation of the total value of the quote so that the purchaser can readily compare quotes.",
"In the purchasing event, the quotes of each seller may be made known to the other sellers participating in the event, while the identities are not.",
"Often, as with retail groceries, the events may be constrained to related foodstuffs or goods, e.g.",
"meat, produce, paper products.",
"In a particular embodiment, the purchaser is able to increase or decrease the volume of goods being sought manually or have the system raise the volume base upon certain price points that are configured before the event is run by the purchaser.",
"The automated feature is called proxy volumes.",
"This may result, for example, from a seller having offered an attractive volume discount as a non-price term.",
"In response to an increase or decrease in volume being sought by the purchaser, an extension of the event is provided to give sellers an opportunity to respond.",
"Participating sellers may increase their price offers or quote within a specified period of time as well as decrease them.",
"This is to give inexperienced sellers the reassurance that it is possible to correct an error in pricing.",
"The integrity of the scheduled event is maintained by validation processes.",
"Buyers and sellers must register and login at the website.",
"A publicly available registration page or pages is afforded.",
"There the registrant acquires the login number (or word) and password.",
"At the login, the registrant enters his or her login number and password at a login page.",
"These are checked for validity, and if valid, the registrant's account is cross-checked against a list of disabled accounts.",
"If the account has not been disabled, a cookie is placed on the registrant's machine.",
"With the cookie a file is provided on the registrant's machine.",
"The file provided contains the classification of the registrant, i.e.",
"buyer, seller or power buyer, qualifications for particular events and other information particular to the registrant.",
"The cookie identifies the registrant by a code unique to that registrant, and it also identifies the time zone of the registrant.",
"Thereafter, all times will be shown to the registrant in the applicable time for that registrant's time zone.",
"From this point, the registrant can move to the registrant's home page.",
"Based upon the role of the registrant, there are three different home pages.",
"If the registrant is an “administrator” of the system (e.g.",
"one employed by the company operating the system) then that registrant gets a listing of events that the registrant is administering.",
"If the registrant is a “power buyer” of the system then that registrant is empowered to administer (or “host”) his or her own events and gets a listing of events that they are hosting.",
"If the registrant is a buyer or seller in the system (i.e.",
"participants that do not administer events) then that registrant gets a listing of events that they may participate in.",
"Only an administrator from the administrator's home page can update any user or company profile, add or update products or product categories.",
"Administrators can also update or create events, see events that have not yet run, see events that are pending award, see events that have not yet been scheduled, see events for contracts that are coming due and see a full calendar of events to be run on the system.",
"From their home pages power buyers have only the ability to update events that they created or create events for their companies, see their events that have not yet run, see their events that are pending award, see their events that have not yet been scheduled, see their events for contracts that are coming due, and see a full calendar of events that are associated with their company.",
"From their home page buyers and sellers have access to events in which they have been invited to participate or which are being administered for them by an administrator.",
"The participation in an event by a buyer or seller is initiated by either an administrator or power buyer when creating an event from an event setup page.",
"From this page the user can send invitations to prospective participants, send “thank you for participation” messages or “thank you and award notification” via email or fax.",
"Once an invitation is sent to prospective participants, they can either click on a link in the email or logon to the website and from their home pages enter the program details page for that event.",
"The program details page summarizes all details related to the event and allows a participant to accept or decline participation in the event.",
"The details will include: all items and item descriptions, additional funds and/or terms, samples, and all event options chosen by the host purchaser of the event.",
"If one accepts, then one will continue to have access to the event.",
"If one declines the event, then the event will be removed from one's home page.",
"With each new page that a registrant moves to, two types of authentication take place.",
"First, user authentication verifies that the current user is logged in and validated.",
"Second, event participant authentication verifies that the current user is a valid event participant and that he or she has the necessary permissions to the various event pages such as “quote view page,” “quote/note pages,” or “funds/terms pages.” These verifications are made checking the content of the cookie left during login.",
"If no cookie exists, the user is entitled only to publicly accessible pages of the website.",
"These include the homepage, registration page and login page.",
"There are several varying degrees of access login depending on the role(s) associated with the user.",
"In addition to the buyers, sellers, power buyers and administrators employed by the company that maintains the system and provides the website, these include shareholders of the company and brokers empowered to submit quotes for others.",
"During user authentication, if the user has logged in previously and has a cookie, the users id (GUID) and time zone is determined.",
"If the session is still open, i.e.",
"timeout has not occurred, then the user is allowed access to any requested page for which that user is qualified based upon the user's GUID.",
"Event authentication ensues if the user requests access to an event.",
"Information concerning the event is retrieved, and it is determined whether the event has been disabled, i.e.",
"cancelled or postponed.",
"If not, it is determined whether the user is authorized for the event.",
"If the user has been authorized, it is determined whether the user has been disabled, and if that is not the case, whether the user has accepted an invitation to the event (RSVP'd).",
"User disablement may occur by virtue of a user failing to offer a quote over a determined period or if the user's quote is not within a specified amount above or below the then-best quote (“a tolerance”).",
"If a user has RSVP'd, the quote view page is displayed to the user who may then submit a quote.",
"The quote view page shown to a qualified user lists the items that the buyer seeks to purchase.",
"Terms required by the buyer may be posted.",
"In the case of a market price type of event, the market price is given.",
"Shipping locations may also appear if the buyer desires quotes to be delivered pricing The internal administrator, mentioned above, is permitted access to all pages and all events.",
"On the other hand, a power buyer may change any detail of the particular event for which he or she is responsible.",
"By comparison, buyers for whom a reverse auction event is administered by an administrator are permitted only to see events for which they are registered.",
"Like power buyers, these buyers have the ability to determine which participants will be allowed to make offers.",
"They can bring up a list of sellers who have RSVP'd and signal their acceptance of particular sellers.",
"A quote view page is regularly updated as new quotes are made, the lowest quote in each category may be highlighted.",
"With each quote, authentication again occurs.",
"It is determined whether the participant is qualified or has been disabled.",
"It is further determined whether the event is still open.",
"A check is made to see if the item has associated with it a reserve price, which is to say, a minimum quote that will be accepted in the case of an auction and a maximum quote in the case of a reverse auction.",
"The system also determines whether the event has been established as a regular or a reverse auction.",
"It is further determined whether the particular item being quote upon has a quote increment, an amount by which a quote must vary from a previous low or high quote depending on the type of auction.",
"In the case where there is a quote increment, it is determined whether the quote is an even multiple of that increment.",
"If so, in the case of a market priced item, conversion is made to indicate the market price plus the quote (the total price).",
"Other features of the system include determination of the extended price at a particular quote, quantity or “unit of measure” conversions as previously mentioned, an indication of the time remaining to the close of an event, five different levels of transparency and many other features described below.",
"By “levels of transparency” is meant the set up of a page to show more or less information such as “full view” of quotes and quoters, low quote without color ranking, low quote only, “blind” (without competitive information), etc.",
"When the system and method is used for traditional auctions, rather than reverse auctions, a seller is the event originator and administrator.",
"A participant can be a seller in one instance and a buyer in another.",
"This is useful when a participant who is ordinarily a purchaser has overstocked an item.",
"The system and method is conducive to the establishment of such business rules as are already in place in a particular trade, or that may desirably be implemented for better proceedings.",
"For example, a quote within a predetermined “threshold” time before the time set for the close of quoting may result in an extension to give active participants placing quotes a chance to respond.",
"In this situation, further rules can be implemented that participants placing quotes who have not placed a quote or have not placed a quote within a specified tolerance for an item a prior to a configurable time period are disabled from further participation in the event.",
"Upon the closing of an event a host buyer or administrator can choose which sellers will be chosen to provide the specified item from the event from the view award page.",
"Subsequent to the awarding business the host buyer or administrator can adjust item volumes or pricing and add in additional purchase information (i.e.",
"PO number, comments, etc .",
".",
".",
").",
"Once the award information is completed the host buyer or administrator has the ability to send system generated notification to the sellers that were awarded business from the event.",
"The notification can be sent via email or fax.",
"If it is sent via email the seller can then click on a link in the email which will allow the seller to logon and go to the participant award page.",
"On the participant award page the seller can view the buyer awarded information, enter their invoice number, sales order number, update volumes and accept the award online.",
"The system will then send a message to the host buyer and administrator notifying them of the completion of the award process.",
"Negotiated Events In a negotiated event the user places a contract out for responsive quotes by setting out proposed terms or “parameters.” Items to be bought or sold are identified and invitations are sent to desired participants.",
"An invited participant gains admittance to the process by accepting, first, the event details and then accepting the initiating user's terms and conditions.",
"Using the website page provided, an invited participant then submits his or her proposed terms, pricing, and contributed funds such as advertising contribution or volume discounts.",
"The initiating user then is provided an analysis web page at which the various proposals he or she has received can be viewed side-by-side for comparison.",
"Proposed contributed funds can be categorized and compared by category.",
"Various contingencies (“what if scenarios”) may be run.",
"Negotiation with a responding user is enabled.",
"If desired that user can be shown the proposals of others or just the contributed funds of others.",
"The initiating user allows the responding user to open its proposal for changes, and a contract may then be awarded.",
"Competitive Branded Products Events In one preferred embodiment, the system and method of the invention facilitates a buyer's choice between competitive branded products.",
"This is done by affording a basis for comparing supplier's proposals.",
"An objective comparison is afforded even though such terms as price, quantity, and additional funds may differ among the proposals.",
"The system compares the gross margin that would result from a seller's proposal with the gross margin that would be achieved under the buyer's proposed terms.",
"Dollar and percentage differences are shown.",
"A comparison between sellers proposals is made based on how each proposal differs from each of the buyer's proposals.",
"The systems and methods provided in each of the above-described events benefit the buyer by: a.",
"Enhancing buyer-supplier communication; b.",
"Providing a more time-efficient negotiation process; c. Assisting in making quicker, better-informed decisions; and d. Showing real-time market information.",
"The same systems and methods benefit the supplier by: a.",
"Providing low cost, high volume sales opportunities; b.",
"Providing an opportunity for a broader customer sales base; c. Through transparency, exposing partial or full visibility of competitors' responses, providing important market information; and d. Shortening negotiation processes and quicker purchasing decisions.",
"The above and further objects and advantages of the invention will be better understood from the following detailed description of a preferred embodiment taken in consideration with the accompanying drawings."
],
[
"CROSS REFERENCE TO RELATED APPLICATION Priority is claimed from U.S. provisional patent application Ser.",
"No.",
"60/239,141, for subject matter common to that contained herein.",
"FIELD OF THE INVENTION This invention relates to a system, software and method for computerized procurement, sales, or contract formation using a computer network, and more particularly to a system, software and method using the internet to establish multi-item procurement, sale or contracting events.",
"BACKGROUND OF THE INVENTION For any product that can be described and priced per item, per weight or by how it is packaged, procurement has often been inefficient and time consuming for purchasers and sellers alike.",
"This applies to retail grocery and healthcare businesses and any that regularly purchase a variety of items from various sources.",
"Additionally, past procurement methods have not been certain to bring purchasers the best purchase terms available, or to assure sellers a reasonable opportunity to fairly compete.",
"Typically, in a retail business, sellers would need to schedule a meeting with a purchasing agent of a large retailer, travel to the scheduled meeting, and make offers on products with little or no knowledge of what competitive offer had been or would be made by other sellers.",
"This practice has been inefficient, time consuming and not competitive.",
"Sellers have been unable to react to competitive offers and purchasers have been denied the benefit of such reactive pricing.",
"A number of websites on the worldwide web offer auctions and reverse auctions.",
"These permit buyers or sellers to make offers or “quote” on various posted items.",
"The quoting often is set to close at a particular time, and a feature has been offered whereby quoting may be extended if a quote is received within an extension threshold time approaching the scheduled close.",
"These sites are not tailored to a particular business's procurement practices, however.",
"They do not afford the opportunity for a business purchaser to schedule an event among recognized, qualified business suppliers, during which many items required by the business purchaser are posted by that purchaser, and suppliers are unable to quote against one another, not just on price, but on other terms important to both the selling and purchasing parties.",
"Neither do prior sites allow for confirming a purchasing or selling activity to the business practices of the relevant business.",
"In addition prior sites have not allowed for actual negotiation of contract terms of a negotiated contract.",
"SUMMARY OF THE INVENTION In accordance with this invention, a system, software and method for computer-assisted procurement or sales of products are provided in which an internet website allows for the listing of a multiplicity of products to be sold or purchased by a seller or a purchaser.",
"An “event” is established at which qualified buyers or sellers can visit the website to offer terms for the sale or purchase of the products listed there.",
"The establishment of such an event includes making the website available for the posting of offers (“quotes”) during an identified time period.",
"In a purchasing event, qualified sellers are invited to offer terms of sale by posting those terms at the website in association with the items sought to be purchased, and doing so during the identified time period for the event.",
"In a selling event buyers are similarly invited to the website to offer to purchase items listed there.",
"In a negotiated event, negotiable purchase contract terms are posted for negotiation between buyer and seller.",
"In a competitive branded product event, similar products of differing brands may be sought by a buyer, and sellers' proposals in response are objectively compared despite differing proposed terms.",
"Buying and Selling Events The system by which the invention operates includes one or more servers, the internet-connected computers of buyers and sellers, the internet equipped computer of the company that supports the system and maintains the website and the programming of each that establishes the interactions of the buyers, sellers and company with the webiste and each other.",
"During the course of the purchasing event, software by which the system operates provides that offers by each qualified seller participating in the event are made known on the website to the other participating qualified sellers.",
"The terms offered by the qualified sellers may be not just price, but such further important considerations as quantity discounts, contributed advertising dollars, shipping terms, payment terms, quality, scheduling of deliveries, and such other terms and conditions as may be commonly a part of a purchase contract in the particular business or industry.",
"The website can offer the qualified sellers the opportunity to quote on each item listed.",
"It is able to provide for each seller to convey the additional, non-price terms with respect to each product listed or with respect to the entirety of the products sought to be purchased.",
"In addition, the website shows a total of the prices offered by each seller and can indicate by a reduction the value of the non-price terms offer by a seller.",
"The purchaser can buy from a qualified seller with respect to individual items or can purchase from a single seller the entirety of the items posted.",
"Typically, the event is scheduled in advance for a fixed period of time, but if a quote by a seller is made within the extension threshold near to the close of the event, the event is extended to allow active participants placing quotes to respond.",
"Although the examples given here relate primarily to a purchasing event (e.g.",
"a reverse auction), the event may also be a selling event (a traditional auction).",
"Purchasing events may be replenishment events.",
"These are regularly scheduled events at which previously approved sellers quote on items designated by the purchaser from a list of regularly purchased items.",
"Purchasing events may also be price list events.",
"These are replenishment events of a different kind.",
"A list of regularly purchased products is maintained open for quotes by approved sellers.",
"Buyers, then, have the opportunity to purchase at what they perceive as the best quote for a particular item, as needed.",
"Further, purchasing events may be spot buys.",
"This event is opened to cover a pressing need for a particular item or items.",
"That may arise from a shortage occurring because, for example, a retailer-buyer has run a sale on the particular item, because the retailer—seller wishes to extend a particularly successful promotion, or because a retailer-buyer has scheduled an ad for a particular item.",
"The immediate need may be heightened by the buyer's inability to purchase the desired item from the buyer's usual suppliers.",
"The spot buy event enables a spur of the moment buy to cover a real or anticipated shortage.",
"Where the event is a purchasing event, the event may be of several kinds.",
"One is a market price event where the products sought to be purchased have a “going price” against which the sellers quote.",
"In this kind of event, the quote may be plus or minus some variance from the market price.",
"In that case, the system is able to convert this to a total price mathematically and display that to the purchaser.",
"The second kind of event does not have a market price with respect to the items sought to be purchased, but rather the sellers quote the actual price intended on an item by item basis.",
"In a preferred embodiment the system also provides certain improvements over the reverse auction that assists a buyer in the procurement of general merchandise items, large capital items or branded products.",
"One such improvement aids the purchaser in evaluating the cost of money over time.",
"Another improvement helps level the decision making process when purchasing similar products that are different brands.",
"The system is robust in the sense that it can accommodate variance in the unit of measure posted by a purchaser and the unit of measure on which a quote is made.",
"For example, the desired purchase may be posted in cases and the responsive quote may be in pounds.",
"The system can be equipped with conversion factors that will accommodate this, making the calculation of the total value of the quote so that the purchaser can readily compare quotes.",
"In the purchasing event, the quotes of each seller may be made known to the other sellers participating in the event, while the identities are not.",
"Often, as with retail groceries, the events may be constrained to related foodstuffs or goods, e.g.",
"meat, produce, paper products.",
"In a particular embodiment, the purchaser is able to increase or decrease the volume of goods being sought manually or have the system raise the volume base upon certain price points that are configured before the event is run by the purchaser.",
"The automated feature is called proxy volumes.",
"This may result, for example, from a seller having offered an attractive volume discount as a non-price term.",
"In response to an increase or decrease in volume being sought by the purchaser, an extension of the event is provided to give sellers an opportunity to respond.",
"Participating sellers may increase their price offers or quote within a specified period of time as well as decrease them.",
"This is to give inexperienced sellers the reassurance that it is possible to correct an error in pricing.",
"The integrity of the scheduled event is maintained by validation processes.",
"Buyers and sellers must register and login at the website.",
"A publicly available registration page or pages is afforded.",
"There the registrant acquires the login number (or word) and password.",
"At the login, the registrant enters his or her login number and password at a login page.",
"These are checked for validity, and if valid, the registrant's account is cross-checked against a list of disabled accounts.",
"If the account has not been disabled, a cookie is placed on the registrant's machine.",
"With the cookie a file is provided on the registrant's machine.",
"The file provided contains the classification of the registrant, i.e.",
"buyer, seller or power buyer, qualifications for particular events and other information particular to the registrant.",
"The cookie identifies the registrant by a code unique to that registrant, and it also identifies the time zone of the registrant.",
"Thereafter, all times will be shown to the registrant in the applicable time for that registrant's time zone.",
"From this point, the registrant can move to the registrant's home page.",
"Based upon the role of the registrant, there are three different home pages.",
"If the registrant is an “administrator” of the system (e.g.",
"one employed by the company operating the system) then that registrant gets a listing of events that the registrant is administering.",
"If the registrant is a “power buyer” of the system then that registrant is empowered to administer (or “host”) his or her own events and gets a listing of events that they are hosting.",
"If the registrant is a buyer or seller in the system (i.e.",
"participants that do not administer events) then that registrant gets a listing of events that they may participate in.",
"Only an administrator from the administrator's home page can update any user or company profile, add or update products or product categories.",
"Administrators can also update or create events, see events that have not yet run, see events that are pending award, see events that have not yet been scheduled, see events for contracts that are coming due and see a full calendar of events to be run on the system.",
"From their home pages power buyers have only the ability to update events that they created or create events for their companies, see their events that have not yet run, see their events that are pending award, see their events that have not yet been scheduled, see their events for contracts that are coming due, and see a full calendar of events that are associated with their company.",
"From their home page buyers and sellers have access to events in which they have been invited to participate or which are being administered for them by an administrator.",
"The participation in an event by a buyer or seller is initiated by either an administrator or power buyer when creating an event from an event setup page.",
"From this page the user can send invitations to prospective participants, send “thank you for participation” messages or “thank you and award notification” via email or fax.",
"Once an invitation is sent to prospective participants, they can either click on a link in the email or logon to the website and from their home pages enter the program details page for that event.",
"The program details page summarizes all details related to the event and allows a participant to accept or decline participation in the event.",
"The details will include: all items and item descriptions, additional funds and/or terms, samples, and all event options chosen by the host purchaser of the event.",
"If one accepts, then one will continue to have access to the event.",
"If one declines the event, then the event will be removed from one's home page.",
"With each new page that a registrant moves to, two types of authentication take place.",
"First, user authentication verifies that the current user is logged in and validated.",
"Second, event participant authentication verifies that the current user is a valid event participant and that he or she has the necessary permissions to the various event pages such as “quote view page,” “quote/note pages,” or “funds/terms pages.” These verifications are made checking the content of the cookie left during login.",
"If no cookie exists, the user is entitled only to publicly accessible pages of the website.",
"These include the homepage, registration page and login page.",
"There are several varying degrees of access login depending on the role(s) associated with the user.",
"In addition to the buyers, sellers, power buyers and administrators employed by the company that maintains the system and provides the website, these include shareholders of the company and brokers empowered to submit quotes for others.",
"During user authentication, if the user has logged in previously and has a cookie, the users id (GUID) and time zone is determined.",
"If the session is still open, i.e.",
"timeout has not occurred, then the user is allowed access to any requested page for which that user is qualified based upon the user's GUID.",
"Event authentication ensues if the user requests access to an event.",
"Information concerning the event is retrieved, and it is determined whether the event has been disabled, i.e.",
"cancelled or postponed.",
"If not, it is determined whether the user is authorized for the event.",
"If the user has been authorized, it is determined whether the user has been disabled, and if that is not the case, whether the user has accepted an invitation to the event (RSVP'd).",
"User disablement may occur by virtue of a user failing to offer a quote over a determined period or if the user's quote is not within a specified amount above or below the then-best quote (“a tolerance”).",
"If a user has RSVP'd, the quote view page is displayed to the user who may then submit a quote.",
"The quote view page shown to a qualified user lists the items that the buyer seeks to purchase.",
"Terms required by the buyer may be posted.",
"In the case of a market price type of event, the market price is given.",
"Shipping locations may also appear if the buyer desires quotes to be delivered pricing The internal administrator, mentioned above, is permitted access to all pages and all events.",
"On the other hand, a power buyer may change any detail of the particular event for which he or she is responsible.",
"By comparison, buyers for whom a reverse auction event is administered by an administrator are permitted only to see events for which they are registered.",
"Like power buyers, these buyers have the ability to determine which participants will be allowed to make offers.",
"They can bring up a list of sellers who have RSVP'd and signal their acceptance of particular sellers.",
"A quote view page is regularly updated as new quotes are made, the lowest quote in each category may be highlighted.",
"With each quote, authentication again occurs.",
"It is determined whether the participant is qualified or has been disabled.",
"It is further determined whether the event is still open.",
"A check is made to see if the item has associated with it a reserve price, which is to say, a minimum quote that will be accepted in the case of an auction and a maximum quote in the case of a reverse auction.",
"The system also determines whether the event has been established as a regular or a reverse auction.",
"It is further determined whether the particular item being quote upon has a quote increment, an amount by which a quote must vary from a previous low or high quote depending on the type of auction.",
"In the case where there is a quote increment, it is determined whether the quote is an even multiple of that increment.",
"If so, in the case of a market priced item, conversion is made to indicate the market price plus the quote (the total price).",
"Other features of the system include determination of the extended price at a particular quote, quantity or “unit of measure” conversions as previously mentioned, an indication of the time remaining to the close of an event, five different levels of transparency and many other features described below.",
"By “levels of transparency” is meant the set up of a page to show more or less information such as “full view” of quotes and quoters, low quote without color ranking, low quote only, “blind” (without competitive information), etc.",
"When the system and method is used for traditional auctions, rather than reverse auctions, a seller is the event originator and administrator.",
"A participant can be a seller in one instance and a buyer in another.",
"This is useful when a participant who is ordinarily a purchaser has overstocked an item.",
"The system and method is conducive to the establishment of such business rules as are already in place in a particular trade, or that may desirably be implemented for better proceedings.",
"For example, a quote within a predetermined “threshold” time before the time set for the close of quoting may result in an extension to give active participants placing quotes a chance to respond.",
"In this situation, further rules can be implemented that participants placing quotes who have not placed a quote or have not placed a quote within a specified tolerance for an item a prior to a configurable time period are disabled from further participation in the event.",
"Upon the closing of an event a host buyer or administrator can choose which sellers will be chosen to provide the specified item from the event from the view award page.",
"Subsequent to the awarding business the host buyer or administrator can adjust item volumes or pricing and add in additional purchase information (i.e.",
"PO number, comments, etc .",
".",
".",
").",
"Once the award information is completed the host buyer or administrator has the ability to send system generated notification to the sellers that were awarded business from the event.",
"The notification can be sent via email or fax.",
"If it is sent via email the seller can then click on a link in the email which will allow the seller to logon and go to the participant award page.",
"On the participant award page the seller can view the buyer awarded information, enter their invoice number, sales order number, update volumes and accept the award online.",
"The system will then send a message to the host buyer and administrator notifying them of the completion of the award process.",
"Negotiated Events In a negotiated event the user places a contract out for responsive quotes by setting out proposed terms or “parameters.” Items to be bought or sold are identified and invitations are sent to desired participants.",
"An invited participant gains admittance to the process by accepting, first, the event details and then accepting the initiating user's terms and conditions.",
"Using the website page provided, an invited participant then submits his or her proposed terms, pricing, and contributed funds such as advertising contribution or volume discounts.",
"The initiating user then is provided an analysis web page at which the various proposals he or she has received can be viewed side-by-side for comparison.",
"Proposed contributed funds can be categorized and compared by category.",
"Various contingencies (“what if scenarios”) may be run.",
"Negotiation with a responding user is enabled.",
"If desired that user can be shown the proposals of others or just the contributed funds of others.",
"The initiating user allows the responding user to open its proposal for changes, and a contract may then be awarded.",
"Competitive Branded Products Events In one preferred embodiment, the system and method of the invention facilitates a buyer's choice between competitive branded products.",
"This is done by affording a basis for comparing supplier's proposals.",
"An objective comparison is afforded even though such terms as price, quantity, and additional funds may differ among the proposals.",
"The system compares the gross margin that would result from a seller's proposal with the gross margin that would be achieved under the buyer's proposed terms.",
"Dollar and percentage differences are shown.",
"A comparison between sellers proposals is made based on how each proposal differs from each of the buyer's proposals.",
"The systems and methods provided in each of the above-described events benefit the buyer by: a.",
"Enhancing buyer-supplier communication; b.",
"Providing a more time-efficient negotiation process; c. Assisting in making quicker, better-informed decisions; and d. Showing real-time market information.",
"The same systems and methods benefit the supplier by: a.",
"Providing low cost, high volume sales opportunities; b.",
"Providing an opportunity for a broader customer sales base; c. Through transparency, exposing partial or full visibility of competitors' responses, providing important market information; and d. Shortening negotiation processes and quicker purchasing decisions.",
"The above and further objects and advantages of the invention will be better understood from the following detailed description of a preferred embodiment taken in consideration with the accompanying drawings.",
"DESCRIPTION OF DRAWINGS FIG.",
"1 is a flowchart representing a login validation routine; FIG.",
"2A is an illustration of an event listings page for a participating seller; FIG.",
"2B is an illustration of an event listings page for an administrator; FIG.",
"2C is an illustration of an event listings page for a power buyer; FIG.",
"3 is an illustration of a program details page for a participating seller; FIG.",
"4 is a flowchart representing a user authentication routine; FIG.",
"5 is a flowchart representing an event authentication routine; FIG.",
"6A is an illustration of an administrator, buyer or power buyer's quote view page before an event begins; FIG.",
"6B is an illustration of a participating seller's quote view page, before an event begins; FIG.",
"6C is an illustration of a quote/note page in full view; FIG.",
"6D is an illustration of a further participating seller's subsequent quote view page for the same event as FIGS.",
"6A-6C after the event has begun with a low quote only view; FIG.",
"6E is an illustration of a further participating seller's quote view page for the same event as FIGS.",
"6A-6D after the event has begun, and with a blind with low quote identifier view; FIG.",
"6F is an illustration of a participating seller's quote view page for the same event as FIG.",
"6A-6E after the event has begun, and with a blind w/out low quote identifier view; FIG.",
"6G is an illustration of a participating seller's quote view page for the same event as FIG.",
"6A-6F after the event has begun, and with a ranking view; FIG.",
"6H is an illustration of a quote funds/terms page for the event of FIGS.",
"6A-6G; FIG.",
"7 is an illustration of an administrator, buyer or power buyer's quote view page for the same event as FIGS.",
"6A-6H after the event has begun; FIG.",
"5A is an illustration of a further, administrator, buyer or power buyer's quote view page for the same event as FIGS.",
"6A-7, during an extension; FIG.",
"8B is an illustration of a further participating seller's quote view page for the same event as FIGS.",
"6A-8A, during an extension; FIG.",
"9A is an illustration of an administrator, buyer or power buyer's quote view page for the same event as FIGS.",
"6A-8 during the review period; FIG.",
"9B is an illustration of a further responding seller's quote view page for the same event as FIGS.",
"6A-9A, during the review period; FIG.",
"10 is an illustration of a buyer's final quote view page for the same event of FIGS.",
"6-9 after the close of the event, FIG.",
"10A is an illustration the view award page for the event of FIGS.",
"6-10; FIG.",
"10B is an illustration the award default page for the event of FIGS.",
"6-10A; FIG.",
"10C is an illustration the seller award page for the event of FIGS.",
"6-10B; FIG.",
"11A is a flowchart of the award of business process; FIGS.",
"11B & 11C are flowcharts representing an item quoting routine; FIG.",
"12 is a flowchart representing an item quotes trigger routine; FIG.",
"13 is a flowchart representing an event items trigger routine; FIG.",
"14 is a flowchart representing an event extension routine; FIG.",
"15 is a flowchart representing a funds/terms quoting routine; FIG.",
"16 is an illustration of an administrator, buyer or power buyer's quote view page for a market price event similar to the event of FIGS.",
"6A-10B; FIG.",
"17 is an illustration of a participating seller's quote view page for the market price event of FIG.",
"16; FIG.",
"18 is a guide to the relationship of FIGS.",
"18A-F; FIGS.",
"18A-F are a diagrammatic illustration of a data model of a system according to the invention; FIG.",
"19 is a flowchart of a first, Initializing Event, component of a negotiated event; FIG.",
"19A is a flowchart of a second, Create Proposal, component of the negotiated event of FIG.",
"19; FIG.",
"19B is a flowchart of a third, Analyze/Negotiate Proposals, component of the negotiated event of FIGS.",
"19 and 19A; FIG.",
"20 is an illustration of the quote view page for a negotiated event; FIG.",
"21 is an illustration of the quote view page for a branded product event; FIG.",
"21A is an illustration of a administrator, buyer or power buyer's quote view page for a competitive brand event.",
"FIG.",
"21B is an illustration of a seller's quote view page for a competitive brand event.",
"FIG.",
"22 is an illustration of the event setup page; and FIG.",
"23 is an illustration of the event setup page with negotiated event options.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENT Initially, one desiring to participate in an event according to the present invention is asked to complete a registration page available from the systems website.",
"Typical information requested may include a login id (this may be assigned), a desired password, name, title, organization, address (actual and email), time zone, billing address, phone number(s), fax number(s), and categories.",
"A listing of categories may characteristically be made available for the user's assistance.",
"Default settings are made, unless overridden Following registration, and after a participant has been given a login id and password, the user is shown to a simple login page (not shown) and proceeds as indicated at 101 in FIG.",
"1.Once the user's id and password have been entered at the login page, the validity of these is checked at 103 and at 104 (FIG.",
"1), the determination is made whether the user's account has been disabled.",
"If either the id or password is invalid, or if in fact, the account has been disabled, then an error is displayed to the user as indicated at 105.On the other hand, if the id and password have been determined to be valid and the account is not disabled, then the server with which the user is in communication generates a cookie which is placed on the user's machine.",
"This constitutes a global unique identifier (GUID), a 128-bit number unique to this particular user, and which identifies the user as well as the user's time zone offset and time zone abbreviation.",
"The time zone offset and time zone abbreviation are used subsequently, so that in all further communications the user's time zone time is that which appears on all subsequent pages.",
"At this time a file is established in the user's machine.",
"Here the user's classification as administrator, buyer, seller or power buyer is recorded along with other authorizations of the user as may pertain.",
"Cookie placement and file establishment of this kind are well understood procedures.",
"The user then proceeds to their home page 108.This page, as illustrated in FIG.",
"3, lists at 109 all of the upcoming events for which this user is qualified.",
"(In the exemplary FIG.",
"3, only one event, a training event, appears.)",
"The beginning and ending dates and times for each event are shown and an event status column is entitled “Status” in FIG.",
"3.Only upcoming events and events that have an end date within the past 14 days are displayed on the buyer/seller home page 108 of FIG.",
"3.Internal administrators can view all events on their home pages as shown in FIG.",
"3A at 108′.",
"Event power buyers see on their home pages events they are hosting or events for their company as illustrated at 108″ of FIG.",
"3B.",
"If the event is not a regularly occurring replenishment event, prior to an event starting, the event organizer, whether a power buyer or an administrator, can invite sellers to participate in an event.",
"Event participants, i.e.",
"responding buyers or sellers, will receive a notification to respond to the invite by reviewing the program details of the event shown in FIG.",
"3D.",
"They are not allowed into the actual event until they have responded to the invite and accepted it on the program details page, FIG.",
"3D.",
"If the event is a replenishment event that is repeated at regularly scheduled times, then the responding sellers will be pre selected and will be able to participate in each replenishment event as it arises.",
"As seen in FIG.",
"3D, the exemplary event here is an olive-purchasing event.",
"The program details of the page shown in FIG.",
"3D include descriptions and packaging sizes of the items sought, event start and stop times, and a listing of event details set by a power buyer or by an administrator for a buyer as described more fully below in connection with FIGS.",
"22 and 23.Returning to the flowchart of FIG.",
"1, when a supplier chooses an event from the events listing page at 108 the determination is made at 110 whether they have accepted the invitation to participate.",
"If the answer is yes, the user is permitted access to the event at 114.Likewise, if at 110, it is determined that the user does not need to accept the invitation, for example because such a response has already been made in the case of replenishments event types, then the user is moved at 114 to the event.",
"At every new page, authentication takes place.",
"Additionally, event level authentication takes place for pages that display event information.",
"User authentication is a general authentication, whereby the user is recognized as a qualified user of the website.",
"Event participation is more specific and determines that the user is qualified to participate in the particular event.",
"As flowcharted at FIG.",
"4, user authentication proceeds as follows.",
"From the user's homepage 108, the user requests access to an event.",
"At 118 it is determined that this is a valid user.",
"At 119 the user's profile is checked for completeness.",
"If not complete, the user is directed to a page 120 where the profile can be completed.",
"At 121 the cookie on the user's computer is checked, the GUID is retrieved, as are timezone details.",
"A determination is made at 122 whether the requested event is still open, and if not, the user is returned to the login page at 123.If the event is open, the user's classification is determined using the GUID at 124.The role of the user is determined, and if appropriate, the user is afforded access to the event by providing the user with the appropriate page at 125.If initially, the user is not valid at 118, then it is determined whether the user is seeking access to the server homepage or the registration or login pages, each readily available to the public.",
"If that is the case, then the user is passed to that page that the user has requested.",
"However, if the user is seeking another page, then the user is returned to the login page as indicated at 123.Event participant authentication verifies that the current user is a valid event participant, and that the user has the necessary permissions to read various event pages.",
"This authentication proceeds as indicated in FIG.",
"5.Where an event page request by the user is detected at 135, but there is no event id or an erroneous id is the request detected at 135, an error display is made at 137.These can typically include the starting time, the ending time, the allowable terms, etc.",
"If at 135, a valid event id is detected, at 138 event information is retrieved.",
"Next, a determination is made at 139 whether the event has been disabled.",
"If it has been, again, an error is displayed to the user as indicated at 137.If the event has not been disabled, a determination is made at 141 whether the user is authorized for this event.",
"If the user is not authorized, an error is displayed at 137, but if the user is authorized, then at 142 the determination is made whether the user has been disabled.",
"Again, if this is the case, an error is displayed at 137, but if the user has not been disabled, a further determination is made at 143 whether the user has responded to an invitation to participate.",
"If the user has not so responded the user is denied access and may be returned to his or her homepage.",
"If at 143, it is determined that the user has accepted the invitation, then the event page being requested is provided to the user as indicated at 147.FIG.",
"6A is a typical participating sellers quote view page.",
"This example is an olive event such as might be established by a retail grocer.",
"This event is not yet open.",
"The header frame for the quote view page provides navigation and general event information like the time remaining in the event.",
"The header automatically refreshes every ten seconds.",
"Each time it refreshes, it checks to see if the quote view page has been refreshed since the last quote was submitted.",
"If it has not, then the page is automatically refreshed when the header is done loading.",
"This way the user will only, at worst, be given data that is ten seconds old.",
"At 150 is seen the date and time of opening of the event, shown in the user's local time.",
"At 152, the time remaining until opening of the event is seen.",
"At 153 are the items that the grocer (buyer) wants to purchase.",
"Each description line 153 includes a description of the product and a description of the number and size of containers per case, e.g., “DW Spanish Olives Thrown Stuffed Manz,” packed 12 jars of 5.75 ounces each per case.",
"At 154 appears the number of cases sought.",
"Because this is not a market price event, no market prices appear.",
"However, at 155 “Current Price” appears.",
"This is the price at which the item s are available on the market at the time for the purchaser's comparison with the quoted prices during the event.",
"Five sellers, identified by name, are participating as seen by the columns 158 headed with those designations.",
"Turning to FIG.",
"6B, the participating seller's quote view page of a not yet open event is shown.",
"Again at 150, date and time of event opening is shown.",
"Competing sellers are identified only as S1, S2, S3 and S4.Shown in the columns having the headings S1, S2, S3 and S4 will appear the quotes submitted by those four sellers.",
"FIG.",
"6C is a seller's quote view page for an open event.",
"The lowest quote for each item is listed and clearly highlighted (in yellow, for example) at 159 and in the fields of the columns 158.Two quotes by the present user are lowest in this example.",
"The sellers S2, S3 and S4 each have one low quote indicated at 161.The total of all quotes appears at the line entitled “Gross” at 163.The additional funds/terms appear at the line 164 entitled “Additional Funds/Terms”.",
"The overall total, taking into account both the total quotes and any additional funds/terms, appears in the line 165 entitled “Net Total.” Additional funds/terms may be give-backs of the nature previously discussed, such as freight, free advertising, quantity discount, etc.",
"FIG.",
"6D is a further quote view page.",
"This page has a different level of transparency from FIG.",
"6C.",
"Here only the user's quotes, at 160 and the low quotes at 159 are shown to the user.",
"FIG.",
"6E has yet another level of transparency available according to this preferred embodiment.",
"Here the page is “blind,” providing the user only the user's own quote at 160 and an indication when the user's quotes are the low quotes by the highlighting in color at 160′.",
"FIG.",
"6F illustrates an alternative page.",
"Again the view is blind and here only the user's quotes appear at 160.No low quotes are indicated.",
"FIG.",
"6G illustrates still another alternative page.",
"Again the view is blind, but here user's quotes are shown at 160, with their ranking among competitive sellers at 160″.",
"An additional funds/terms page of a seller is shown in FIG.",
"6H.",
"Minimum funds to be contributed by the seller as set by the purchaser are shown at 1161.The user's quoted fund contribution of $220 is shown at 1162 for the upper item.",
"The user has not quoted a fund for the lower item or the funded quoted was below the minimum and is not shown.",
"Competitive seller's fund contributions may be shown at column 1163.Quoted terms appear at 1164 for the upper item.",
"In FIG.",
"7, a refreshed administrator, buyer or power buyer's quote view page for the same olive purchase event as is the subject of the page shown in FIGS.",
"6A-6H.",
"It reflects, at 150, that the event is open and running and at 152 time remaining in the event.",
"Here the identity of the sellers is visible at column 165, so this page is typical of that accessible to an administrator, a buyer or a power buyer.",
"Low bids are highlighted in color at 161.Indicia 162 in the Tee Pee Olives, Inc. column show that the quoting seller has attached a note.",
"In FIG.",
"8A an administrator, buyer, power buyer quote view page is shown during an extension of the same olive purchase event as FIGS.",
"6A-7.That the event has been extended is indicated at 150.The time remaining in the extension is shown at 152.In FIG.",
"8B shows a seller quote page during an extension as indicated at 150.At 152 the time remaining in the extension is shown.",
"Low quotes are highlighted in color.",
"In FIG.",
"9A, the final administrator, buyer, power buyer quote view page for the olive purchase event appears.",
"Again, unlike the pages available to the seller, the actual bidder's names are available.",
"At 152, it can be seen that the event is closed and time remains in the review period, and to the right of that in the header frame, the fact that the event has ended and that the event is under review now appears at 150.The seller Tee Pee Olives has the lowest gross line item total and net total at lines 163 and 165.The business rules provide that the buyer has until a certain time as shown at 150 to choose among the quotes.",
"FIG.",
"9B is the final seller quote view page, similar to the page at FIG.",
"9A.",
"FIG.",
"10 is the buyer's quote view web page with award indicating boxes 166 for the buyer to “click” on.",
"The buyer may choose to award the sellers based on individual low quotes for individual items as indicated by the check marks made at 166.Alternatively, the buyer may award the quoting participant having the lowest net total, or may make a selection based on other factors such as reputation, past experience, etc.",
"FIG.",
"10A is a view award page.",
"It shows the awards from the olive buying event to which FIGS.",
"6-9 relate.",
"The awarded sellers are shown at 167, delivery date at 168, price at 169 and volume at 170.Purchase Order numbers appear at 171.In the award default page of FIG.",
"1B, the term of the olive sale contract that must be accepted by the seller are shown at 172, along with the purchaser's contract at 173.The time period for acceptance is set at 174.FIG.",
"10C illustrates a seller award page.",
"It lists the terms, delivery date, price, etc.",
"as established during the event and by the purchaser's award.",
"At the buttons 174 and 175, the seller may decline or accept.",
"FIG.",
"11A flowcharts the award process.",
"The purchaser begins the award process 177 at the close of an event.",
"The purchaser evaluates the quotes at 178.The purchaser must identify the awarded seller at 179, or an error is displayed at 180.At 182, the purchaser completes the award information.",
"At 184, the purchaser indicates that the awards are complete; otherwise, the purchaser returns to 179 to complete the awards.",
"At 185, the seller reviews the award or awards to him or her.",
"Acceptance or decline is made at 186.The purchaser is notified at 187 if the award is declined, at which time purchaser again identifies an award to another seller.",
"At 188, the seller also notifies the purchaser if the award is accepted, and this completes this award process at 190.The flowchart of FIGS.",
"11B and 11C illustrates how the system deals with a quote.",
"When a quote is submitted via the quote/note page, at 195, it is checked at 196 to determine if it is in the correct form, i.e.",
"numeric.",
"If it is not, an error is displayed as indicated at 198, but if it is, the quoting party at 200 is checked to see if that he or she is a participant.",
"If the quoting party is not a participant, again an error is displayed as indicated.",
"If the quoting party is a participant, the next determination that is made at 201 is whether the event remains open.",
"If not, again an error is displayed.",
"If the event is open, information concerning the event is checked at 203 to see if there is a reserve price in place.",
"If there is not, the routine proceeds to FIG.",
"11B, where at 204 it is determined whether there is a quote increment in place.",
"However, if at 203 (FIG.",
"11B), it is determined that there is a reserve price, then a determination is made at 206 whether this is a regular or reverse auction.",
"If it is a regular auction, the quote is compared to the reserve price at 207, and if it does not exceed the reserve price, an error is shown again as indicated at 198.However, if the quote does exceed the reserve price, then the routine moves to the quote increment determination 204 of FIG.",
"11C.",
"Similarly, if at 203 it is determined that there is a reserve price and at 206 that this is a reverse auction, then at 209, the determination is made whether the quote is more than the reserve price, in which case, an error is displayed at 198.If the quote is less than the reserve price, the routine progresses to the quote increment determination at 204 in FIG.",
"11C.",
"If a quote increment is in place, as determined at 204, then the routine determines at 210 whether the quote is an event multiple of the quote increment.",
"If not, an error is displayed as indicated at 212.If the quote is a multiple of the quote increment as required, then a determination is made whether the particular item is market price based at 213.Also, at 204, if it is determined that there is no quote increment, the program steps directly to the determination of whether the item is market price based.",
"If this is a market price based item, then the quote will have been made relative to the market price.",
"In other words, a quote of plus five cents would mean a quote five cents above the market price.",
"When it is determined that the item is market price based at 213, then the quote is converted to actual price, by addition of the quote to the market price at 215.At this point, the quote table is updated at 216 and the buyer's quote view page illustrates the full price as calculated at 215.If the event is determined not to be market price based at 213, then a determination is made whether the quote is greater than zero at 218.In other words, a quote less than zero is appropriate in a market price based event because that quote less than zero can be subtracted from the market price to arrive at a positive number, but in other auctions, a quote less than zero is nonsensical, and again, the error is displayed at 212.Having determined that the quote is appropriate at 216, and that update of the item quote table, i.e.",
"the quote view page, is appropriate, the routine then proceeds to the sub-routine identified as item quotes trigger 219.This sub-routine is shown in the flow chart of FIG.",
"12.In FIG.",
"12, the routine SP sub-add item quote is the routine described just above with respect of FIGS.",
"11A and 11B.",
"The item quotes trigger routine of FIG.",
"12 determines at 220 whether the amount field is changing.",
"If not, the routine of FIG.",
"12 is done.",
"If at 220 it is determined that the amount field is being changed, then the particular seller's item low quote is modified at 222.At 224, it is determined whether the new quote is less than the previous quote.",
"If it is, then a determination is made at 226 whether an extension of the event is to be made, because, for example, the new low quote is being made at a time close to the close of the event.",
"Whether the new quote is higher or lower, the quote total is updated at 229.Next, data for the chart of FIG.",
"17 is updated at 230 and the routine is complete.",
"In FIG.",
"13, a further routine is shown triggered by an update of the low quote field at 235.At the decision block 237, a determination is made whether the new low quote value exceeds the old low quote value or if the new low quote value equals zero (“NULL”).",
"One of these two cases could occur where the previous low quote contributor has been disabled.",
"If either of these is the case, then it is determined, based on the time of occurrence, whether the event needs to be extended to give participants an opportunity to respond as indicated at 238.In any event, at 240 a determination is made if the unit of measure (ounces, pounds, cases, quarts, etc.)",
"quantity or if the quantities has been updated, both occurrences that require new totals to be calculated.",
"If not, the routine is done, but if so, on the basis of previous quotes, the item quote totals for the changed event item is recalculated at 241.In FIG.",
"14, an event extension routine is flowcharted.",
"Before an event is extended for any of the reasons mentioned above or other reason, it is verified at 244 that the event is open, and enabled.",
"It is determined at 245 whether the event can be extended, which is to say that the allowable extensions have not been used up.",
"If it cannot be extended, the event is done.",
"If the event can be extended, the time remaining is determined at 246, and if this is determined, at 247 to be within the time-to-closing threshold for extension, then the event is extended by the extend time at 249, and any participants who have not quoted yet, i.e.",
"prior to the first extension, are disabled at 251.If however, the time remaining is not yet within the extension threshold determined at 247 then the routine is done.",
"FIG.",
"15 illustrates a routine that may be called upon by a buyer by which a quote of additional funds or terms is submitted via the webpage at 255.These may be of the nature previously discussed, i.e.",
"quantity discount or for advertising.",
"The quote is determined to be numeric at 257 or otherwise the error is displayed at 258.At 259, it is determined whether the quoter is a participant, otherwise again an error is displayed as indicated at 258.If the quoter is a participant, then at 261, it is determined whether the event is still opened.",
"If it is not, error is displayed.",
"If it is open, it is determined whether quoting is allowed for this funds/terms at 262.If the answer is no, then the error is displayed, but if the answer is yes, then it is determined whether the quote is above a minimum amount at 263.Though a minimum may be set as indicated at 263, still quoting may be allowed on this particular funds/terms, which is why the decision block 262 is provided.",
"If the quote is above the minimum amount as determined at 263, then it is accepted, and the funds/terms quote table is updated at 264.FIGS.",
"16 & 17 are quote view pages of a market price event.",
"The market price is displayed with respect to the first two items at 270 in FIG.",
"16 and at 271 in FIG.",
"17.The participating sellers are clearly visible to the buyer in FIG.",
"16, but not to the seller in FIG.",
"17.TABLE A Business rules for calculating the total quote for an event item: 1.If the “Quote Unit of Measure” equals the “Event Item Unit of Measure” “Item Total” = “Event Item Quantity”/“Quote Quantity” * “Item Quote Amount” 2.If the “Quote Unit of Measure” does not equal the ″Event Item Unit of Measure” a.",
"If a “Event Item Unit of Measure to Quote Unit of Measure Cross Reference” exists “Item Total” = ((”Event Item Quantity” * “Event Item Unit of Measure to Quote Unit of Measure Cross Reference Ratio”)/“Quote Quantity”) * “Item Quote Amount” b.",
"If a “Unit of Measure Cross Reference” exists from “Event Item Unit of Measure” to “Pack Unit of Measure” and a “Unit of Measure Cross Reference” exists from “Pack Unit of Measure” to “Quote Unit of Measure” Item Total ((“Event Item Quantity” * “Event Item Unit of Measure to Pack Unit of Measure Cross Reference Ratio” * “Pack Unit of Measure to Quote Unit of Measure Cross Reference.",
"Ratio”)/”Quote Quantity”) * “Item Quote Amount” c. If the “Event Item Unit of Measure” equals the “Package” and the “Quote Unit of Measure” equals the ‘Tack Unit of Measure” “Item Total” = ((“Event Item Quantity” * “Pack Quantity”)/“Quote Quantity”) * “Item Quote Amount” d. If the “Event Item Unit of Measure” equals the “Package” and “Pack Unit of Measure to Quote Unit of Measure Cross Reference” Exists “Item Total” = (((“Event Item Quantity” * “Pack Quantity) * “Event Item Unit of Measure to Pack Unit of Measure Cross Reference Ratio”) “Quote Quantity”) * “Item Quote Amount” “Gross Event Value” = Sum of the “Item Totals” for an event.",
"Business rules for calculating the event funds/terms item totals: 1.If the “Event Offering Type” is “Dollar” represented by the integer value “0” in the table.",
"a.",
"If the “Event Offering Quote Amount” is Null and the “Event Offering Minimum Amount” is not null “Offering Item Total” = “Event Offering Minimum Amount” b.",
"If the “Event Offering Quote Amount” is greater that the “Event Offering Minimum Amount” “Offering Item Total” = “Event Offering Quote Amount” 2.If the “Event Offering Type” is “Percent” represented by the integer value “1” in the table a.",
"If the “Event Offering Quote Amount” is Null and the “Event Offering Minimum Amount” is not null “Offering Item Total” = “Gross Event Value”/“Event Offering Minimum Amount” b.",
"If the “Event Offering Quote Amount” is greater that the “Event Offering Minimum Amount” “Offering Item Total” = “Gross Event Value”/“Event Offering Quote Amount” “Total Funds/Term” = Sum of the “Offering Item Totals” for an event “Net Event Value” = “Gross Event Value” - “Total Offering” In Table A, there are listed in pseudo code business rules for an event for arriving at “net event value” such as appears at line 165 (there called “net total”) of FIG.",
"6.First the items totals as appear at the lines 154 of FIG.",
"6 are calculated using the rule 1 or 2 a, b, c or d under “Business rules for calculating the total quote for an event item.” Under rule 1 if the quote is made in the same units of measure as the event item is listed, the calculation is straightforward.",
"The first line 154 in FIG.",
"6 illustrates this.",
"The event item unit of measure there is the case.",
"A quantity of 1,050 cases of jars of a given size of particular olives is listed.",
"The quote, by the seller S1 for example, is $7.92 per case.",
"In this case, the item total is 1,050 cases/1 case multiplied by $7.92 to equal $3,316, which is the “Item Total.” Where the quote is made in a unit of measure (“quote Unit of Measure”) other than the unit of measure listed in the event (“Event Item Unit of Measure”), if a cross reference or conversion figure exists between the two units of measure, then that is used in rule 2a to arrive at the item total.",
"An example would be where the quote is in dollars per ounce and the event quantity is listed in pounds the “Event Item Unit of Measure to Quote Unit of Measure Cross Reference Ratio” would be 16.Similarly, rule 2b applies where the quote is made in a unit of measure other than the event unit of measure and, instead of a direct conversion or cross reference as in rule 2a, there exists a conversion figure from event item unit of measure to a pack unit of measure and another conversion figure for the pack unit of measure to the quote unit of measure.",
"For example, if the event quantity were listed in pounds, but the quote was in ounces, and pounds per pack as well as ounces per pack were known, then the total item quote could be calculated.",
"Rule 2c applies when the event lists the quantity of product by the package, but the quote unit of measure by which the seller quotes is the pack unit of measure or in other words, the units packaged together in a single package.",
"For example, if the event item quantity is 100 cases and each case contains 12 cans, then the pack quantity is 12.The quote quantity, which is the quote unit of measure, is one can and the item quote amount is the amount quote per can so for 100 cases times 12 cans where the quote is $1.00 per can, the total quote for the item is $1,200.Rule 2d, immediately following, takes into account a further removed quote unit of measure.",
"If the event item quantity is 100 cases of beef, and the pack quantity is 100 pounds per case, if the bidder were to quote in price per ounce, then an event item unit of measure to pack unit of measure cross reference ratio of 16 would be necessary to arrive at the item total.",
"In this example, if the quote were of $1.00 per ounce, the total quote or item total would be $160,000 or 100 cases times 100 pounds per case times 16 ounces per pound times $1.00 per ounce.",
"The “Business rules for calculating the event funds/terms item totals” relate to the non-price funds/terms listed at line 164 of FIG.",
"6 as previously discussed.",
"The funds/terms may be in a dollar amount, such as dollars in freight allowance or advertising, or the funds/terms may be a percentage such as a percentage discount based on volume purchased.",
"For a dollar funds/terms, an integer is set to zero in a field 387 in a table 385 in FIG.",
"18B of the data model of FIG.",
"18, discussed below.",
"If the event funds/terms quote amount is null, which is to say no quoting of additional funds has been made, and if there is an event funds/terms minimum amount, then the funds/terms item total is the event funds/terms minimum amount.",
"What this means is that if there is an event funds/terms minimum amount that has been set by the purchaser as a requirement, the participating seller has accepted this at the outset and consequently, this is a default value.",
"If the event funds/terms quote amount is greater than the event finds/terms minimum amount, however, the event funds/terms quote amount is the funds/terms item total rather than the event funds/terms minimum amount.",
"If the event funds/terms type is of a percentage kind, then the field 387 in the table 385 has an integer set to 1.Again, as in rule 2a, if the event funds/terms quote amount is null and the event funds/terms minimum amount is not null, then the funds/terms item total is the gross event value divided by the event funds/terms minimum amount (a percentage).",
"Similarly, if the event funds/terms quote amount is greater than the event funds/terms minimum amount, then the funds/terms item total is the gross event value divided by the percentage which is the event funds/terms quote amount.",
"The “gross” is the sum of the “Item Totals” for the event, each as determined in the Business Rules for Calculating the Total quote for an Event Item described above.",
"“Total Funds/Terms” is the sum of the “Offering Item Totals” for an event under Business Rules for Calculating the Event Funds/Terms Item Totals.",
"Finally the Net Event Value is the Gross Event Value minus the Total Funds/Terms.",
"The data structure of the system is as schematically illustrated in the data model of FIGS.",
"18 A-F.",
"These figures join as indicated in FIG.",
"18.Table 350 defines the events.",
"An integer 351 identifies an event.",
"The event id serves as a primary key as indicated by the entry <pk>.",
"By this key, the event may be called up and characteristics of the event set forth in the remainder of the fields 352 of the table 350 are retrieved.",
"The entry “not null” indicates that the value in this field may not be blank, whereas the entry “null”, where present, indicates that the entry may be blank.",
"Other tables in the data model take the event id from the table 350 as indicated by the unnumbered arrows labeled “event id=event id.” Information for the chart of FIG.",
"17 is located in the table 353.The chart id is provided by an integer 354.The chart id is the primary key for this chart table 353.In the “event id” field, the entry <fk> indicates that the field “eventid” is a foreign key, i.e., the key to another table.",
"In fact, it is the primary key of the table 350 as just discussed.",
"Event items, many of which have been discussed above, are set forth in the table 356.An event identifier in the form of an integer 358 is the primary key for this table.",
"The entries <i1>, <i2> reference indices available as a quick way to find associated data.",
"Other event related data and cross references are found in tables 361-364.In table 390, FIG.",
"18D, user's identifications are located.",
"“Varchar” refers to variable characters of 10 to 255 characters in length used to identify the user name, company, email address, etc.",
"As described above, disablement, shown in field 392, is determined by the setting of a single bit.",
"Participants in an event are identified in the table 395 and cross referenced in table 396 of FIG.",
"18D.",
"In table 400 in FIG.",
"18E, product identity is contained.",
"In table 405, units of measure (UOM) are kept and UOM cross references are kept in table 401.Various categories assigned to e.g.",
"events, users and products are kept in table 440, FIG.",
"18E, and are retrieved using tables 441-446.Time zone information is in table 447.Phone information and cross references are in the tables 410, 411 and 412 of FIG.",
"18F.",
"Addresses, address cross references and other address related information are contained in the tables 420-428 of FIG.",
"18C.",
"Company identification is found in the table 430 of FIG.",
"118C.",
"User id and company id are brought together in a table 431 of FIG.",
"18D.",
"The user id or GUID is in a sessions table 435 of FIG.",
"18D.",
"The non-price items, called here “additional funds,” are found in table 385 of FIG.",
"18B.",
"The additional non-price funds that have been quote are in table 386, the history of additional fund quotes is found in the table 387, and an additional fund award table is table 388.Item quotes appear in table 450, the history of item quotes in table 452, and the winning quote award in table 455.Negotiated Event The Negotiated Event is an electronic process for gathering and analyzing proposals for a contract that has been sent out for bid.",
"The process is broken down into three components: Initializing Event, Create Proposal, and Analyze/Negotiate Proposals.",
"The first component, flowcharted at FIG.",
"19, is Initializing Event.",
"It is the process that starts the whole event cycle.",
"Once the buyer has decided to place a contract out for bid, he or she logs on to the website and creates an event as indicated at 601.In order to create this event a buyer must fill out and select information about the event.",
"At 602 the buyer begins by answering some parameter questions on the set-up page.",
"These include event time duration, contract begin date, contract duration, estimated contract volume, discount & future rates of money and item list indicator.",
"The buyer proceeds by selecting items for the event at 603 and if desired requests a sample to be delivered for qualification at 604.The buyer continues by selecting participants who he or she would like to be involved in the proposal process at 605.Once selected the last step is to click on a button to send out e-mail invitations to the selected participants notifying them of this contract that is up for bid at 606.As shown in the flowchart in FIG.",
"19A, the second component, Create Proposal, is the supplier's process for responding to the contract that is up for bid.",
"If a supplier decides to participate that supplier clicks on a link established on the e-mail he or she receives from the buyer, at 607.This automatically takes the supplier to the website login screen.",
"Once logged in, the supplier has the ability to review the details about the event for this contract at 608.Upon this review, the supplier must decide to accept or decline the event based on these details.",
"If at 609 the supplier declines, the supplier is not allowed to continue on at 610, but if at 608 he or she accepts, he or she is then sent on to the user terms and condition-screen, at 611.The supplier again has the decision to accept 611 or decline 612 with the same results as the previous decision, but this time if the supplier accepts he or she is taken into the proposal process.",
"The proposal begins at 613 by outlining the instructions on how to proceed through this process.",
"Once this has been reviewed, the supplier enters into the proposal set-up screen at 614.Here the foundation is built for the supplier's whole proposal.",
"Required information about the foundation is entered and submitted.",
"This may include Proposed Annual Dollar Volume, Proposal Duration, Payment Terms and Annual Seasonal Distribution.",
"The supplier is then faced with entering in cost and movement for each individual item within the event at 615.",
"(“Movement” is a term understood in the grocery industry to mean volume moved over time, e.g.",
"case per week or trucks per month.)",
"Once completed, the supplier has the option to add at 616 any additional funding (by event level or item level) to round out their offering.",
"The supplier has the ability to change any aspect of this proposal until the deadline that the buyer has determined in the set up process, but once the deadline has passed changes only are allowed at the buyer's discretion.",
"As shown in the flowchart at FIG.",
"19B, the third component, Analyze/Negotiate Proposals, is an analytical review process for the buyer that takes into account the relationship between time and money.",
"From the buyer's perspective the buyer is able to see each proposal in a side by side comparison at 621.He or she is able to categorize any additional funds from the proposals at 622.This permits the buyer to compare additional funds categories such as advertising contributions, volume discounts, etc.",
"The buyer is able to run a “what if” scenario, or contingency, analysis on any selected proposal at 623.The “what if” scenario is run by permitting the buyer to change one or more terms of a quote and then based upon the same analytical review, observing how the buyer's results are effected.",
"This flexibility for looking at the proposal along with the comparative analytics such as the calculation of the net present value, the item list cost differential, and additional profitability between the different proposals assists the buyer in his or her decision making process on which proposal is a better option for the buyer.",
"Net Present Value is determined using the formula: PV=CF/((1+i)t) The net present value for a given cash flow is stated as the cash flow divided by 1+interest rate (i) taken to the power of the number of periods being calculated (t).",
"The inputs for these various elements in the application are as follows: (a) Cash Flow (CF)—this amount derives from the quotes being entered and the shipment distribution filled out by the supplier.",
"(b) Interest Rate (i)—this is the “Net Present Value Discount Rate” entered into the Setup Page.",
"Typically, this will be the return on investment or the interest rate on cash investments for the client.",
"(c) Number of Periods (t)—this is the number of periods for which the payment will be made.",
"In the present case, compounding occurs on a monthly basis, so this is the number of months the payment will be made from the beginning of the contract.",
"Item List Cost Differential is a means of weighting the extended amount to take into account the different volume of product the totals cannot be readily compared.",
"This calculation is to alleviate that issue.",
"The calculation is as follows: (Maximum Price−Supplier's Price/Maximum Price)×Supplier's Price×Quantity The process could stop at the point of receiving the sellers quotes, but to add more flexibility to it the buyer is also empowered to negotiate with each proposal as indicated at 625.By negotiating the buyer may persuade a supplier to change certain aspects of the supplier's offering.",
"If the supplier agrees to these changes the buyer has the ability to allow the supplier access to the supplier's proposal and once the change are made the buyer can close the access to the supplier.",
"At this point the real time calculations are refreshed with the changes and the buyer will see at 628 the net effect on the changed proposal.",
"If the buyer needs some leveraging to help in the negotiating process, the buyer can choose an option that allows the supplier to view certain aspects of any other proposals as indicated at 626 and 627.After the supplier reviews this information, then the buyer can again permit access for the supplier to modify his or her offering again.",
"Once the proposals have been negotiated, the buyer can make a final review of the information.",
"After this review the buyer has the ability to award the best offer electronically over the e-mail while also sending out to the other participants a thank you for their participation letter, all at 630.FIG.",
"20 is the administrator, buyer, power buyer's quote view page of a negotiated event affording the ability to award at 810, 811 and 812.Competitive Branded Products Event FIG.",
"21 is a quote view page for comparing quotes on different brands of a product that a buyer seeks to purchase.",
"Shampoo is the product in the example.",
"This page can be used, e.g., to compare promotional programs presented by two, three or more different manufacturers or distributors.",
"The buyer's proposed program is first presented.",
"Retail prices per unit are shown at fields 701.The buyer's gross margins are shown at 702.At 703 the quantity cases of six bottles each is shown for each brand.",
"The proposed price (cost to buyer) that will produce the desired margin appears at 704.Next the seller's or “supplier's” proposals or “quotes” are shown.",
"These include price per case at 706, additional funds at 707, program price or cost to buyer at 708.The resultant gross margin is shown at 710.The buyer's and seller's proposals are then compared.",
"The difference in price appears at 712 and percent difference at 713.As can be seen the buyer's proposed price is less in each instance by the dollar amount shown in parentheses.",
"The best supplier proposal from the buyer's point of view is Brand Z as indicated at 715.At 717 the supplier proposals are compared, using the Brand Z proposal as the basis for a normalized comparison.",
"The difference in percent difference between Brand X and Brand Z is shown at 718.The difference in percent difference between Brand Y and Brand Z is shown at 719.Corresponding dollar differences are at 720 and 721.FIGS.",
"21A and 22B show the quote view page for the competitive branded product event of FIG.",
"21.The administrator, buyer, power buyer's page, FIG.",
"21A, shows all quotes as well as the comparative figures.",
"The seller's page shows just that seller's quotes plus the comparative figures.",
"This then has allowed a comparison between proposals for branded products differing in price, differing in additional funds being offered and in which the buyer has made his or her proposal in one set of units (retail price per unit) and the responding supplier participants have made their proposal in another set of units (price per case).",
"Nevertheless, the differences between sought-after gross margin and gross margin resultant from the suppliers proposals are made apparent.",
"Event Setup Options/Features The following outline sets forth the many various setup options and features that the power buyer or administrator can use in setting up an event.",
"Again, the olive buying event of FIGS.",
"6-9 is the example.",
"Reference is made in FIGS.",
"22 and 23.1.Ownership: a.",
"Event: i.",
"Event Name: The name one allocated for the event on the initial create event page.",
"This field, 501, is automatically populated from what is entered on the initial create event page.",
"However, one may change the event name on the event setup page.",
"ii.",
"Event Type: Indicates, at 502, the type of event and type of purchasing that the host user desires.",
"There are two classifications of events: 1.System, at 503, which can be: a. Live—a binding event with some economic outcome, b. Training—a non binding event with no economic outcome, c. Template—an event that is not actually run but used as the template for setting up similar events.",
"2.Classification, at 504, which can be: a.",
"Contract, b. Replenishment, c. Spot (spot buy) d. Price List, or e. RFP (negotiated event).",
"iii.",
"Parent Event: Identifies at 505, whether or not the event was a copy of another event.",
"If it was a copy, it will list the event name that the current event was created from, i.e.",
"the “parent event.” iv.",
"Host Company Name: Refers, at 506, to the company that is hosting the event.",
"Is automatically populated from what is entered on the create event page.",
"b.",
"Host User: i.",
"Host User Company Name: This field will default to the event host's company name at 507, but can be changed to another company.",
"ii.",
"Host User Name: Provides a drop down list at 508 to select who will be the host user or sponsor of the event.",
"iii.",
"Title: Lists the job title of the host user at 509.Is automatically populated from the user's profile.",
"If incorrect, the user must update their profile to correct it.",
"iv.",
"First Name: Lists the host user's first name.",
"Is automatically populated at 510 from the user's profile.",
"If incorrect, the user must update their profile to correct it.",
"v. Last Name: Lists the host user's last name.",
"Is automatically populated at 511 from the user's profile.",
"If incorrect, the user must update their profile to correct it.",
"vi.",
"Department: Lists the department that the host user works in.",
"Is automatically populated at 512 from the user's profile.",
"If incorrect, an administrator from the company that maintains the website and provides the system (the “Company”) must update the profile to correct it.",
"vii.",
"Phone Number: Lists the host user's phone number at 513.Is automatically populated from the user's profile.",
"If incorrect, the user must update their profile to correct it.",
"viii.",
"Fax Number: Lists the host user's fax number at 514.Is automatically populated from the user's profile.",
"If incorrect, the user must update their profile to correct it.",
"ix.",
"Email: Lists the host user's email address.",
"Is automatically populated from the user's profile.",
"If incorrect, the user must update their profile to correct it.",
"x.",
"Primary Administrating Company: Lists at 515 the host user's primary administrating contact at the Company.",
"Is automatically populated from the user's profile.",
"If a primary Company contact does not appear, one selects one's contact from the drop down list.",
"xi.",
"Secondary Company Contact: Lists at 518 the host user's secondary Company contact.",
"Is automatically populated from the user's profile.",
"If a secondary Company contact does not appear, one may select one's contact from the drop down list.",
"c. Setup: i.",
"Event Setup By: Indicates the user's name that set up the event at 517.This field is automatically populated by the system.",
"ii.",
"Buyer Setup Event: A Yes/No flag that indicates at 518 whether or not a user with the ‘power buyer’ role set up the event.",
"This field is automatically populated by the system.",
"2.Setup: a.",
"Event Start: i.",
"Event Start Date: Indicates at 520 the date that the event will start.",
"Requires entry of a four-digit year, i.e., 2001.ii.",
"Event Start Time: Indicates at 521 the time the event will start.",
"b.",
"Event End: i.",
"Event End Date: Indicates at 522 the date the event will end.",
"Requires entry of a four-digit year, i.e., 2001.ii.",
"Event End Time: Indicates at 523 the time the event will end.",
"This will reflect the actual end time of the event including extensions once the event has completed.",
"c. Contract: i.",
"Contract Start Date: Indicates the date that the contract will begin at 524.Only applies to user event type “Contract”.",
"ii.",
"Contract End Date: Indicates the date that the contract will end at 525.Only applies to user event type “Contract”.",
"iii.",
"Reminder Date: Allows a user to indicate a date at 526 that the system will remind the user who setup the event that the contract is ending and that the event will need to be run again.",
"d. Delivery: i.",
"Delivery Start Date: Indicates at 527 the date that deliveries will begin.",
"ii.",
"Delivery End Date: Indicates at 528 the date that deliveries will end.",
"e. Anticipated Award i.",
"Anticipated Award Date: Indicates at 529 the date that you host user expects to announce the decision on the award of business.",
"f. Event Status: Indicates the status of the event 530.Valid values are: Enabled, Cancelled, Postponed, Awarded, Not Awarded.",
"3.RFP Setup (Referring to FIG.",
"23): a.",
"Contract Period Negotiable: Indicates at 535 whether the buyer is willing to allow the suppliers the ability to input their own proposal period for the contract.",
"b.",
"Total Contract Value Negotiable: Indicates at 536 whether the buyer is willing to allow the suppliers the ability to input their own proposal values for the contract.",
"c. Listed Products are a subset of all items: Indicates at 537 whether the item list for this event will be a subset of all items or a complete list.",
"d. Future Value Discount Rate (%): This figure, at 538, is the percentage rate at which one's company places value on the future rate of money.",
"e. Net Present Value Discount Rate (%): This figure, at 539, is the percentage rate at which one's company places value on the present value rate of money.",
"f. Compounding Period: Indicates, at 540, the compounding period for the discount rates.",
"g. Average Gross Margin (%): This figure, at 541, is the average gross margin percentage for the items or categories which the event is based on.",
"h. Expected Inflation Rate (%): This figure, at 542, represents the inflation rate that is expected to influence this list of items or the category for the event.",
"i.",
"Estimated total Value of Contract ($): This figure, at 543, represents the estimated total of the contract value for the event.",
"This number will give the suppliers an idea of the value for this contract.",
"4.Details: a. Quote/Event Notes: i.",
"Allow Quote\\Event Note: Indicates at 545 whether or not one is willing to allow a supplier to place a single event or item quote notation.",
"ii.",
"View Event Note: Indicates at 546 whether or not one is willing to allow event notes placed by suppliers to be viewed by other suppliers (only applies in a full view event).",
"iii.",
"View Quote Note: Indicates at 547 whether or not one is willing to allow notes placed by suppliers to be viewed by other suppliers (only applies in a full view event).",
"b.",
"Volumes: i.",
"Allow Proxy Volume: Allows at 548 a buyer to increase volume based on targeted price points.",
"ii.",
"Allow Items No Volumes: Allows at 549 a host user to indicate whether or not one will be defining volumes for items in the event.",
"iii.",
"Allow Suppliers Quote Volumes: Allows at 550 a supplier to indicate the volume they have available at the quoted price point.",
"This option cannot be used if the buyer is indicating desired volumes.",
"c. Display: i.",
"Display Reserve Price: Indicates at 551 whether or not a host user wants the reserve price to be displayed on the view page for the suppliers to see.",
"ii.",
"Display Industry Price: Indicates at 552 whether or not the host user wants to display an industry price (such as current USDA or Georgia Dock pricing) to the supplier for an item(s).",
"iii.",
"Display Weighted Quotes: Indicates at 553 who can view weighted supplier quotes.",
"Valid options are Buyer, Supplier or Both.",
"The buyer on the Participant Item page adjusts quotes on a scale from 1 to 100%.",
"iv.",
"Display Supplier Alias: Indicates at 554 whether or not a host user wants to display a supplier alias on the view page.",
"Reduces the amount of characters on the view page in the supplier column header and potentially allows for more supplier data to be viewed on a single page.",
"v. Display Low Net Indicator: Indicates at 555 whether or not a host user wants the lowest total net program value highlighted on the view page.",
"vi.",
"Allow Branded Product Event: Allows at 556 the quote view page to display additional totals that enable a host buyer to make decisions on products that are similar, but different brands (i.e.",
"shampoo) vii.",
"Display Branded Product Details to Suppliers: Displays at 557 to suppliers on the quote view page additional totals that enable a host buyer to make decisions on products that are similar, but different brands (i.e.",
"shampoo) d. Quotes: i. Quote Increment Type: Indicates at 558 what type of quote increment you want the suppliers to place their quotes with.",
"ii.",
"Allow Proxy Quote: Allows at 559 a host user to indicate whether or not a supplier will allow the system place their quotes on their behalf.",
"iii.",
"Allow Matching Quote: Indicates at 560 whether or not a host user will allow a supplier's quote to be matched by another participating supplier (this is for all quotes, not including the low quote).",
"iv.",
"Allow Matching Low Quote: Indicates at 561 whether or not a buyer will allow the low quote for an item to be matched by another participating supplier.",
"v. Max Matching Quotes: Allows at 562 a host user to indicate the maximum number of matching quotes that you will allow (is used in along with the allow matching quote options above).",
"vi.",
"Allow Non-Quote: Indicates at 563 whether or not a host user will allow a supplier to stay in the event without placing any quotes.",
"Prevents a supplier from watching pricing and not participating.",
"vii.",
"Non-Quote Threshold: At 564 works in along with the allow non-quote option and indicates the point in time remaining on the event clock that the system will disable a supplier if they have not placed a quote.",
"For instance, if it is a 15-minute event, and you one has indicated 5 in this field, it means that with 5 minutes remaining on the clock the system will automatically disable any supplier who has not placed at least one quote.",
"The supplier will not be able to access the event again if they are disabled.",
"viii.",
"Allow Quote Increase: Indicates at 565 whether or not a buyer will allow suppliers to increase their current quoted price.",
"ix.",
"Quote Increase Threshold: Indicates at 566 how much time after a supplier places quotes that the buyer will allow a supplier to increase their quoted price.",
"x.",
"Allow Quote Increase Extension: Indicates at 567 whether or not a host user will allow a supplier to increase their pricing during event extensions.",
"If checked, then the Quote Increase Threshold applies.",
"xi.",
"Reset Quotes Prior Start: Allows at 568 the host user to have an event span multiple days and the system will automatically change the date to the following day at midnight each night.",
"In addition, the view page will appear as if it is a new event.",
"xii.",
"Start With Previous Quote: At 569 works in conjunction with “Reset Quotes Prior Start” and allows the suppliers' last quoted prices and notes to be retained on their quote page so that they do not have to re-enter the information the first time.",
"e. Event: i.",
"Event Display Type: Indicates at 570 the view in which the suppliers will be able to watch the event.",
"Valid values are: Full, Low Quote Only, Ranking, Blind, Blind w/Color.",
"ii.",
"Event Review Time: Indicates at 571 the amount of time at the close of the event that a host user will want the suppliers to stay available by phone, in the case of the event needs to be re-opened for some reason.",
"iii.",
"Supplier Award: Indicates at 572 whether one will award the business to a single supplier or multiple suppliers.",
"iv.",
"Items Parent Event: Indicates at 573 whether or not the item “Pick List” will have only those items associated to the parent event.",
"This option does not apply if this event was not a copy of another event.",
"v. Allow Group Buy: Indicates at 574 that the hosting buyer wants to hold an event, which would allow multiple buying organizations to participate in the consolidation of volumes of the selected item(s) and indicate their own volume requirements.",
"vi.",
"Current Price Calc Method: If group buy is checked, indicates at 575 how the current price field should be calculated for each item in the event.",
"Valid values are: Lowest Current Price, Highest Current Price, or an Average of all current prices entered for an item.",
"vii.",
"Allow Extensions: Indicates at 531 whether or not the event will extend if quotes are entered within the specified threshold.",
"viii.",
"Extension Threshold: Indicates at 532 the threshold or at how much time remaining in the event that the event will extend if a significant event happens in the specified threshold.",
"Significant events are: Low quote changes, Any quote going down, An initial quote by a supplier on an item, and any change to terms or funds.",
"For example, if the low quote changes within the last 1-minute remaining on the clock, the event will extend.",
"ix.",
"Extend Time: Indicates at 533 how long the event will extend if a significant event occurs within the specified threshold.",
"This is a repetitive process and will continue until no significant event occurs within the specified threshold.",
"For example, the event will extend 2 minutes if a significant event occurs within the specified threshold.",
"x.",
"Departments In Event: At 579 users in the selected departments are invited to view this event.",
"These departments are based on the departments of the Host Buyer.",
"5.Categories: Indicates at 580 which category to which the products and participants will be sourced from.",
"6.Additional Information: At 581 allows a host user to indicate any additional information that is needed for the supplier to most appropriately prepare for the event.",
"7.Payment Terms: a.",
"Allow Terms: Indicates at 582 whether or not a host user will allow a supplier to quote early payment discount terms (i.e.",
"2% 10, net 30).",
"b.",
"Minimum Terms: Indicates at 583 that the host user is requiring minimum terms and what those required terms are.",
"If this field is populated, the terms will automatically be calculated upon a supplier placing an initial quote.",
"c. Allow Terms Quote: At 584 allows suppliers to quote terms.",
"d. Allow Decrease Terms Quote: At 585 allows suppliers to decrease their term amount.",
"e. Allow Users Add Funds: If present allows a host user to indicate whether or not one is allowing a supplier to quote on funding that one did not specifically request as a part of the program.",
"8.Event Fees: a.",
"Fee Type: Indicates at 587 the type of fee that will be applied to the event.",
"Intesource staff completes this section.",
"9.New Event: a.",
"New Event Name: At 588 allows one to indicate the name one wants the new event to be called when doing a copy.",
"b.",
"New Company Name: At 589 allows a user to change the company name when copying an existing event.",
"c. With Last Quotes: At 590 allows a user to copy an event that has previously been quoted on and not copy the last quote per item to the new event.",
"d. Without Last Quotes: At 591 allows one to copy an event that has previously been quoted without retaining the last quotes per item.",
"From the above outline the breadth of choices given a buyer or power buyer is apparent.",
"Whereas a specific, exemplary embodiment of the invention has been described, it will be readily understood by one skilled in the art that changes may be made without departure from the spirit and scope of the invention as set forth in the claims appended or to be appended hereto."
]
] | Patent_10399196 |
[
[
"Urine test for the diagnosis of prion diseases",
"The present invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject.",
"The method of the invention comprising the steps of: (a) providing a urine sample of said subject; (b) isolating from said sample all proteins, preferably, isolating proteins having a molecular weight higher than about 8 Kda; (c) optionally, and preferably, subjecting the proteins obtained in step (b) to protease digestion, and isolating from the mixture obtained in step (c) any protease resistant proteins; and (d) detecting the presence of PrPSC in the protease resistant fraction obtained in step (c) by a suitable detection technique.",
"Furthermore, the invention further relates to methods for diagnosing a prion disease in a subject and for screening donors of blood samples for the presence of prion diseases.",
"The invention further provides for a diagnostic kit for diagnosing a prion disease in a subject."
],
[
"1-46.",
"(canceled) 47.A method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of: a. providing a urine sample of said subject; b. isolating from said sample proteins; and c. detecting the presence of PrPSC in the protein mixture obtained in step (b) by a suitable detection technique.",
"48.A method according to claim 47 further comprising the step of subjecting the proteins obtained in step (b) to protease digestion.",
"49.A method according to claim 48 for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of: a. providing a urine sample of said subject; b. isolating from said sample all proteins having a molecular weight higher than 8 KDa; c. subjecting the proteins obtained in step (b) to protease digestion; d. isolating from the mixture obtained in step(c) any protease resistant proteins; and e. detecting the presence of PrPSC in the protease resistant fraction obtained in step (d) by a suitable detection technique.",
"50.A method according to claim 49 wherein in step (b) said proteins are isolated by subjecting the urine sample to dialysis and precipitating the proteins from the dialysate.",
"51.A method according to claim 50 wherein step (b) further comprises addition of a carrier to the dialysate, prior to the protein precipitation.",
"52.A method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of: a. providing a urine sample of said subject; b. isolating from said sample all proteins having a molecular weight higher than about 8 KDa by subjecting said sample to dialysis, wherein said dialysis is performed using a membrane having a pore range of from about 6 KDa to 8 KDa; c. precipitating said proteins by ultracentrifuging the dialysate; d. subjecting the proteins obtained in step (b) to protease digestion; e. isolating from the mixture obtained in step (c) any protease resistant proteins; and f. detecting the presence of PrPSC in the protease resistant fraction obtained in step (d) by a suitable detection technique.",
"53.A method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of: a. providing a urine sample of said subject; b. isolating from said sample all proteins having a ;molecular weight higher than about 8 KDa by subjecting said sample to dialysis, wherein said dialysis is performed using a membrane having a pore range of from about 6 KDa to 8 DKa; c. precipitating said proteins by ultracentrifuging the dialysate for 1 hour at 100,000×g at 4° C.; d. subjecting the proteins obtained in step (b) to protease digestion; e. isolating from the mixture obtained in step (c) any protease resistant proteins; and f. detecting the presence of PrPSC in the protease resistant fraction obtained in step (d) by a suitable detection technique.",
"54.A method according to claim 52 wherein the proteins are precipitated by any one of methanol and TCA.",
"55.A method according to claim 54 wherein the proteins are precipitated by methanol.",
"56.A method according to claim 52 wherein said protease is proteinase K. 57.A method according to claim 53 wherein said protease is proteinase K. 58.A method according to claim 56 wherein in step (e) the presence of the PrPSC protease-resistant core in said protease resistant fraction, is detected by immunoassay.",
"59.A method according to claim 58 wherein said immunoassay is by immunoblot SDS PAGE analysis.",
"60.A method according to claim 60 wherein said PrP antibodies are 3F4 or 6H4 monoclonal antibodies.",
"61.A method according to claim 60 wherein said PrP antibodies are 3F4 or 6H4 monoclonal antibodies.",
"62.A method for diagnosing a prion disease in a subject comprising the steps of: a. obtaining a urine sample of said subject; and b. detecting the presence of the abnormal isoform of prion protein (PrPSC) in said urine sample by the method of claim 52; whereby the presence the PrPSC protein in said sample indicates that said subject carries a prion disease.",
"63.A method for diagnosing a prion disease in a subject comprising the steps of: a. obtaining a urine sample of said subject; and b. detecting the presence of the abnormal isoform of prion protein (PrPSC) is said urine sample by the method of claim 53; whereby the presence the PrPSC protein in said sample indicates that said subject carries a prion disease.",
"64.A method of claim 62 wherein said prion disease is a TSE disease.",
"65.A method claim 64 wherein said subject is a human subject.",
"66.A method of claim 64 wherein said subject is a bovine animal.",
"67.A method of claim 66 wherein said prion disease is BSE.",
"68.A method according to claim 62 wherein diagnosing of said prion disease is prior to or after onset of clinical symptoms.",
"69.A method for detecting the presence of metabolites of the abnormal isoform of prion protein (PrPSC) in a urine sample of a human subject, said metabolites being unique for human prion disease carries, said method comprising the steps of: a. providing a urine sample of said subject; b. isolating from said sample all proteins having a molecular weight higher than 8 KDa; and c. detecting the presence of said metabolites of PrPSC in the protein sample obtained in step (b) by an immunoassay comprising the use of 6H4 monoclonal antibodies that specifically bind to the protease-resistant core of PrPSC found in the urine of human prion disease carriers.",
"70.A method according to claim 69 wherein step (b) said proteins are isolated by subjecting the urine sample to dialysis using a membrane having a pore range of from 6 Kda to 8 Kda, and precipitating the proteins from the dialysate by ultracentrifugation.",
"71.A method according to claim 70 wherein the precipitation is performed by ultracentrifuging the dialysate for about 1 hour at 100,000×g at 4° C. 72.A method according to claim 69 wherein the proteins are precipitated by any one of methanol and TCA.",
"73.A method according to claim 72 wherein the proteins are precipitated by methanol.",
"74.A method according to claim 69 wherein said human prion disease is CJD.",
"75.A method for diagnosing a human prion disease in a subject comprising the steps of: a. obtaining a urine sample of said subject; and b. detecting the presence of metabolites of the abnormal isoform of prion protein (PrPSC) that are unique for human prion disease patients in said urine sample by the method of claim 69; whereby the presence of said PrPSC protein metabolites in said sample indicates that said subject carries a human prion disease.",
"76.A method according to claim 75 wherein said human prion disease is CJD.",
"77.A diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said kit comprising: a. means for isolating from said urine sample proteins; b. optionally, suitable carrier for stabilizing the PrPSC in the urine sample.",
"c. a protease for digesting the protein isolate obtained by (a) or (b); d. means for isolating from the digest by (c) any protease resistant proteins; e. means for detecting the presence of PrPSC in the protease resistant fraction obtained by (d) and f. instructions for carrying out the detection of the presence of PrPSc in the urine sample according to the method of claim 52.78.A kit according to claim 77 wherein said means for isolating proteins is for isolating proteins having a molecular weight higher than 8 Kda.",
"79.A kit according to claim 78 wherein said protease is proteinase K. 80.A kit according to claim 77 wherein said means for detecting the presence of PrPSC comprise reagents for detecting PrPSC by immunoassay.",
"81.A kit according to claim 80 wherein said immunoassay reagents comprise antibodies that specifically react with the protease-resistant core of PrPSC.",
"82.A diagnostic kit for detecting the presence of metabolites of the abnormal isoform or prion protein (PrPSC) that are unique for human prion disease carriers in a urine sample of a human subject, said kit comprising: a. means for isolating from said urine sample all proteins having a molecular weight higher than about 8 KDa; b. means for detecting the presence of PrPSC metabolites that are unique for human prion disease carriers in the protein sample obtained by step (a) by immunoassay comprising antibodies that specifically react with the metabolites of PrPSC that are unique for human prion disease carriers; and c. instructions for carrying out the detection of the presence of PrPSC in the urine sample according to the method of claim 69.83.A kit according to claim 82 wherein said human prion disease is CJD."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Prion diseases, also known as TSEs (transmissible spongiform encephalopathies), are a group of fatal neurodegenerative diseases of animals and humans.",
"Among the animal diseases, the most prevalent today is BSE (bovine spongiform encephalopathy) also known as the “Mad Cow Disease”.",
"Although less than 100 patients have been diagnosed to date to be BSE-infected, the number of individuals incubating the disease may be millions.",
"Another animal prior disease is scrapie in sheep, which after transmission to rodents constitutes the main experimental prior animal model.",
"In humans, the most prevalent prion disease is CJD (Creutzfeldt Jakob Discase), which can be manifested either sporadically (about 1 patent per year); genetically (via mutations in the prion protein PrP gene); or in transmissible form, as in the BSE affected cases.",
"It is a well known experimental fact that the incubation of prior diseases in humans and large animals can last for decades.",
"Prion diseases are believed to be caused by the accumulation in the brain of PrP SC , an abnormally folded isoform of PrP C , a GPI anchored protein of unknown function.",
"It has been postulated that prion diseases propagate by the conversion of PrP C molecules into protease-resistant and insoluble PrP SC by an as yet unknown mechanism.",
"The proteinase K (PK) resistant PrP in prion diseases was described by McKinley et al.",
"[Cell 35(1):57-62 (1988)].",
"Immunoblotting of a Proteinase K-digested brain sample infected with a prion disease with an anti-PrP antibody, reveals a characteristic N-terminally truncated PrP protein (the protease resistant core of PrP SC , denominated PrP 27-30), which is not present in controls or in individuals affected with any other neurological disease.",
"To date, the diagnosis of prion diseases was based on the presence of this characteristic protease-resistant PrP in brain biopsies, as well as on clinical criteria.",
"Current methods for the conclusive identification of Prion diseases include mostly a post-mortem analysis of the patient's brain homogenate.",
"Clinical symptoms of the disease can many times be misleading.",
"Evidently, sampling brain tissue from the living patient involves a painful and risky surgical procedure and, moreover, does not give a definite answer since the distribution of PrP SC in the brain is not homogenous.",
"All commercial tests used to date are based on brain presence of protease resistant PrP, for example the Prion-Test of Prionics AG, Switzerland (which company is in charge of Most European active surveillance for BSE cases), which is an immunological test for the detection of prions in brain and spinal cord tissue, and is mainly used for BSE and scrapie diagnostics.",
"Since the incubation period in prion diseases is very long (years), it is possible that there is a large number of asymptomatic human and animal carriers.",
"There exists therefore a need for developing a simple and readily available pre-clinical and clinical diagnostic test for the disease.",
"The need for such an in-vivo test has been reinforced since the reports of the first cases of variant Creutzfeldt Jakob disease (vCJD) in 1996 [Zeidler, M., et al., Lancet 350(9082), 908-10 (1997); Bruce, M. E., et al., Nature 389(6650), 498-501 (1997); Ironside, J. W., et al., Histopathology 37(1), 1-9 (2000)].",
"vCJD is a fatal neurodegenerative disease believed to be caused by the consumption of BSE contaminated meat, and the incubation time between infection to clinical symptoms may be as long as decades [Bruce, M. E., et al., Nature ibid (1997)].",
"As opposed to cattle, the incubating individuals will be present for many years, donating blood and in some cases other organs to the non-affected population.",
"Additionally, such test is important for the food industry, and would enable detecting BSE in bovine animals such as cows and sheep, and to prevent marketing of infected meat and diary products of these animals.",
"Therefore, a major object of the present invention is the development of a reliable, non-invasive method for diagnosing prion diseases which will allow the pre-clinical and clinical diagnosis of the disease in humans and in animals.",
"Since most urine proteins originate from blood, the present inventors speculated that some PrP SC , either from brain or from a peripheral organ, is released during the incubation period into the blood serum in a non-aggregated form, although at low and undetectable concentrations.",
"Due to its protease resistance, PrP SC is not digested by blood proteases.",
"However, since the MW of PrP is below the cutoff size for filtering through kidney cells (about 40 kDA) [Berne, R. M., and Levy, M. N. Physiology, 4th Ed (1998)], PrP may subsequently be secreted into the urine and thereby be concentrated, as other proteins, at about 120 folds of its concentration in blood [Kocisko.",
"D. A., et al., Nature 370(6489), 471-4 (1994)].",
"The concentration by the kidney makes possible to detect PrP SC in urine more easily than in blood.",
"Thus, as will become apparent as the description proceeds, the present inventors have identified a prion specific protease resistant PrP isoform in the urine of prion infected animals and humans (UPrP SC ), which may be used for the in-vivo early diagnosis of ill as well as seemingly healthy but prion infected individuals.",
"Moreover, the present invention shows that this protease resistant isoform UPrP SC , can be detected, following a specific enrichment procedure, in the urine of scrapie-infected hamsters, BSE-infected cattle and humans suffering from CJD.",
"This specific enrichment procedure, according to the present invention may include dialysis of the sample through membrane having a pore range of about 6 KDa to about 8 KDa.",
"The present invention further shows that UPrP SC was also found in urine of hamsters inoculated with prions long before the appearance of clinical signs.",
"These findings strongly indicate the possibility of using the method of the invention also for pre-clinical diagnosis.",
"The theoretical possibility for diagnosis of prion diseases in variety of body fluids, such as urine, has been mentioned in several patent documents.",
"EP 0854364, for example, discloses a diagnostic method for neuro-degenerative disorders such as Alzheimer's disease and prion diseases.",
"This method is based on concentrating a protein associated with the specific neuro-degenerative disease (such as PrP in prion diseases and APP in Alzheimer's disease), in a sample (urine, for example).",
"The concentration is carried out by contacting the sample with a solid, non-buoyant particulate material having free ionic valencies such as calcium phosphate.",
"However, this patent exemplifies the detection of only the Alzheimer's disease associated peptide APP.",
"WO 93/23432 discloses a diagnostic method for prion diseases in different body fluids such as CSF (cerebrospinal fluid) and theoretically, urine.",
"Similarly to EP 0854364, this method is based on concentrating the prion protein by ammonium sulfate precipitation and affinity chromatography.",
"This publication exemplifies CSF as a sample.",
"However, contrary to the prior art methods, the present invention clearly demonstrates the detection of the aberrant protease resistant urine isoform UPrP SC in urine samples of prion infected animals and humans.",
"Furthermore, as shown by the present invention, dialysis of the urine seems to improve the detection procedure.",
"Therefore, the present inventors propose that UPrP SC is present in a semi-denatured form, probably due to the relative high concentrations of urine denaturing agents, and is subsequently re-natured for example by the dialysis step.",
"Thus, the specific enrichment of the urine sample according to the present invention provides a novel and reliable method for the detection of different prion diseases by a non-invasive procedure."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrP SC ) in a urine sample of a subject, said method comprising the steps of: (a) providing a urine sample of said subject; (b) isolating from said sample proteins; and (c) detecting the presence of PrP SC in the protein mixture obtained in step (b) by a suitable detection technique.",
"A preferred embodiment relates to the method of the present invention, further comprising the step of subjecting the proteins obtained in step (b) to protease digestion.",
"In a specifically preferred embodiment, the invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrP SC ) in a urine sample of a subject, said method comprising the steps of (a) providing a urine sample of said subject; (b) isolating from said sample all proteins having a molecular weight higher than about 8 KDa; (c) subjecting the proteins obtained in step (b) to protease digestion; (d) isolating from the mixture obtained in step (c) any protease resistant proteins; and (e) detecting the presence of PrP SC in the protease resistant fraction obtained in step (d) by a suitable detection technique.",
"In step (b) of the method of the invention the proteins are preferably isolated by subjecting the urine sample to dialysis and precipitating the proteins from the dialysate.",
"Optionally, prior to the protein precipitation, a carrier may be added to the dialysate for stabilizing the PrP SC .",
"The dialysis is preferably performed using a membrane having a pore range of from about 6 KDa to about 8 KDa.",
"The proteins may be precipitated from the dialysate by ultracentrifuging the same, for example for about 1 hour at 100,000×g at 4° C. Alternatively the proteins may be precipitated by any suitable protein precipitation technique.",
"As a preferred embodiment proteins according to the invention may be precipitated by any one of methanol, TCA (Trichloracetic acid) or by any other precipitation method.",
"Preferably, proteins may be precipitated by methanol, for example by the addition of methanol and freezing the sample to about −80° C. for about 1 hour, and subsequently centrifuging at 3000×rpm for about 30 minutes.",
"The protein digestion is preferably performed by treating the sample with proteinase K, for example by adding proteinase K in concentration of up to 40 μg/ml and continuing digestion for about 30 min at 37° C. The presence of the PrP SC protease-resistant core in said non-digested fraction is preferably detected by immunoassay, for example by immunoblot SDS PAGE analysis, using monoclonal antibodies that specifically bind to the protease-resistant core of PrP SC , for example 3F4 or GH4 monoclonal antibodies.",
"The invention also relates to a method for diagnosing a prion disease in a subject comprising the steps of (a) obtaining a urine sample of said subject; and (b) detecting the presence of the abnormal isoform of prion protein (PrP SC ) in said urine sample by the method of the invention, whereby the presence the PrP SC protein in said sample indicates that said subject carries a prion disease.",
"In a preferred embodiment, said prion disease may be any TSE disease.",
"The subject may be a human subject, for example a CJD, vCJD, GSS or FFI carrier or an individual infected with BSE.",
"Alternatively the subject may be an animal infected with BSE, scrapie or any other TSE disease.",
"The method of the invention further enables detection of different prion disease prior to or after onset of clinical symptoms.",
"In yet a further embodiment that invention relates to a method for screening donors of blood samples for the presence of a prion disease in said donor comprising the steps of: (a) obtaining a urine sample from said donor; (b) detecting the presence of the abnormal isoform of prion protein (PrP SC ) in said urine sample by the method of the invention; and matching the results of the detection performed in step (b) to said blood sample.",
"Still further, the invention relates to a method for detecting the presence of metabolites of the abnormal isoform which is probably a pathogenic isoform of prion protein (PrP SC ) in a urine sample of a subject, said metabolites being unique for human prion disease carriers.",
"In a preferred embodiment such human prion disease may be CJD or vCJD.",
"This method comprises the steps of: (a) providing a urine sample of said subject; (b) isolating from said sample all proteins having a molecular weight higher than about 8 KDa; and (c) detecting the presence of said metabolites of PrP SC in the protein sample obtained in step (b) by a suitable detection technique.",
"In this embodiment, in step (b) said proteins may be isolated by subjecting the urine sample to dialysis and precipitating the proteins from the dialysate, for example by ultracentrifuging the dialysate, specifically for about 1 hour at 100,000×g at 4° C., or by any other suitable precipitation method.",
"Preferred protein precipitation method may be methods such as methanol or TCA (Trichloracetic acid) precipitation.",
"A specifically preferred technique for precipitation is methanol precipitation, specifically by the addition of methanol to the sample, freezing to about −80° C. for about 1 hour, and subsequently centrifuging at 3000×rpm (rounds per minute) for about 30 minutes.",
"The detection of the presence of the said metabolites of PrP SC protease-resistant core in said protein sample is preferably by immunoassay, particularly SDS PAGE, using monoclonal antibodies that specifically bind to the specific metabolites of PrP SC found in urine of prion disease carriers, for example 6H4 monoclonal antibodies.",
"The invention further relates to a method for diagnosing a prion disease in a subject comprising the steps of: (a) obtaining a urine sample of said subject; and (b) detecting the presence of metabolites of the abnormal isoform of prion protein (PrP SC ) that are unique for piron disease patients in said urine sample by a method of the invention; whereby the presence of said PrP SC protein metabolites in said sample indicates that said subject carries prion disease.",
"According to a preferred embodiment, the method of the invention is intended for detection of the presence of metabolites unique for CJD and vCJD.",
"Still further, the invention relates to a method for screening donors of blood samples for the presence of prion disease in said donor.",
"This method comprises the steps of: (a) obtaining a urine sample from said donor; (b) detecting the presence of metabolites of the abnormal isoform of prion protein (PrP SC ) that are unique for prion disease patients in said urine sample by a method of the invention; and matching the results of the detection performed in step (b) to said blood sample.",
"In another embodiment the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrR SC ) in a urine sample of a subject, said kit comprising means for isolating from said urine sample all proteins; optionally, a carrier for stabilizing the PrP SC ; means for detecting the presence of PrP SC in the non-digested fraction; and instructions for carrying out the detection of the presence of PrP SC in the urine samples.",
"In another preferred embodiment the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrP SC ) in a urine sample of a subject, said kit comprising means for isolating from said urine sample all proteins having a molecular weight higher than about 8 KDa; optionally, a carrier for stabilizing the PrP SC in the dialysate; means for detecting the presence of PrP SC ; and instructions for carrying out the detection of the presence of PrP SC in the urine samples.",
"In yet another specifically preferred embodiment the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrP SC ) in a urine sample of a subject, said kit comprising means for isolating form said urine sample all proteins having a molecular weight higher than about 8 KDa; optionally, a carrier for stabilizing the PrP SC in the dialysate; a protease for digesting the protein isolate; means for isolating from the protein digest any protease digest any protease resistant proteins; means for detecting the presence of PrP SC in the protease resistant fraction; and instructions for carrying out the detection of the presence of PrP SC in the urine samples.",
"In the kit of the invention, protease is preferably proteinase K and said means for detecting the presence of PrP SC comprise reagents for detecting PrP SC by immunoassay, such as antibodies that specifically react with the protease-resistant core of PrP SC .",
"In another embodiment the invention relates to a diagnostic kit for detecting the presence of metabolites of the abnormal isoform of prion protein )PrP SC ) that are unique for human prion disease carriers, in a urine sample of a subject, said kit comprising: means for isolating from said urine sample all proteins having a molecular weight higher than about 8 KDa and means for detecting the presence of PrP SC metabolites that are unique for human prion disease carriers, preferably CJD and vCJD, in the obtained protein sample.",
"The means for detecting the presence of said PrP SC metabolites preferably comprise reagents for detecting said PrP SC metabolites by immunoassay, for example antibodies that specifically react with the metabolites of PrP SC that are unique for human prion disease carriers.",
"According to a preferred embodiment, said human prion disease may be CJD or vCJD."
],
[
"FIELD OF THE INVENTION The present invention is concerned with a method for the diagnosis of prion diseases by detecting the protease-resistant core of PrPSC and/or some metabolites thereof in urine samples.",
"BACKGROUND OF THE INVENTION Prion diseases, also known as TSEs (transmissible spongiform encephalopathies), are a group of fatal neurodegenerative diseases of animals and humans.",
"Among the animal diseases, the most prevalent today is BSE (bovine spongiform encephalopathy) also known as the “Mad Cow Disease”.",
"Although less than 100 patients have been diagnosed to date to be BSE-infected, the number of individuals incubating the disease may be millions.",
"Another animal prior disease is scrapie in sheep, which after transmission to rodents constitutes the main experimental prior animal model.",
"In humans, the most prevalent prion disease is CJD (Creutzfeldt Jakob Discase), which can be manifested either sporadically (about 1 patent per year); genetically (via mutations in the prion protein PrP gene); or in transmissible form, as in the BSE affected cases.",
"It is a well known experimental fact that the incubation of prior diseases in humans and large animals can last for decades.",
"Prion diseases are believed to be caused by the accumulation in the brain of PrPSC, an abnormally folded isoform of PrPC, a GPI anchored protein of unknown function.",
"It has been postulated that prion diseases propagate by the conversion of PrPC molecules into protease-resistant and insoluble PrPSC by an as yet unknown mechanism.",
"The proteinase K (PK) resistant PrP in prion diseases was described by McKinley et al.",
"[Cell 35(1):57-62 (1988)].",
"Immunoblotting of a Proteinase K-digested brain sample infected with a prion disease with an anti-PrP antibody, reveals a characteristic N-terminally truncated PrP protein (the protease resistant core of PrPSC, denominated PrP 27-30), which is not present in controls or in individuals affected with any other neurological disease.",
"To date, the diagnosis of prion diseases was based on the presence of this characteristic protease-resistant PrP in brain biopsies, as well as on clinical criteria.",
"Current methods for the conclusive identification of Prion diseases include mostly a post-mortem analysis of the patient's brain homogenate.",
"Clinical symptoms of the disease can many times be misleading.",
"Evidently, sampling brain tissue from the living patient involves a painful and risky surgical procedure and, moreover, does not give a definite answer since the distribution of PrPSC in the brain is not homogenous.",
"All commercial tests used to date are based on brain presence of protease resistant PrP, for example the Prion-Test of Prionics AG, Switzerland (which company is in charge of Most European active surveillance for BSE cases), which is an immunological test for the detection of prions in brain and spinal cord tissue, and is mainly used for BSE and scrapie diagnostics.",
"Since the incubation period in prion diseases is very long (years), it is possible that there is a large number of asymptomatic human and animal carriers.",
"There exists therefore a need for developing a simple and readily available pre-clinical and clinical diagnostic test for the disease.",
"The need for such an in-vivo test has been reinforced since the reports of the first cases of variant Creutzfeldt Jakob disease (vCJD) in 1996 [Zeidler, M., et al., Lancet 350(9082), 908-10 (1997); Bruce, M. E., et al., Nature 389(6650), 498-501 (1997); Ironside, J. W., et al., Histopathology 37(1), 1-9 (2000)].",
"vCJD is a fatal neurodegenerative disease believed to be caused by the consumption of BSE contaminated meat, and the incubation time between infection to clinical symptoms may be as long as decades [Bruce, M. E., et al., Nature ibid (1997)].",
"As opposed to cattle, the incubating individuals will be present for many years, donating blood and in some cases other organs to the non-affected population.",
"Additionally, such test is important for the food industry, and would enable detecting BSE in bovine animals such as cows and sheep, and to prevent marketing of infected meat and diary products of these animals.",
"Therefore, a major object of the present invention is the development of a reliable, non-invasive method for diagnosing prion diseases which will allow the pre-clinical and clinical diagnosis of the disease in humans and in animals.",
"Since most urine proteins originate from blood, the present inventors speculated that some PrPSC, either from brain or from a peripheral organ, is released during the incubation period into the blood serum in a non-aggregated form, although at low and undetectable concentrations.",
"Due to its protease resistance, PrPSC is not digested by blood proteases.",
"However, since the MW of PrP is below the cutoff size for filtering through kidney cells (about 40 kDA) [Berne, R. M., and Levy, M. N. Physiology, 4th Ed (1998)], PrP may subsequently be secreted into the urine and thereby be concentrated, as other proteins, at about 120 folds of its concentration in blood [Kocisko.",
"D. A., et al., Nature 370(6489), 471-4 (1994)].",
"The concentration by the kidney makes possible to detect PrPSC in urine more easily than in blood.",
"Thus, as will become apparent as the description proceeds, the present inventors have identified a prion specific protease resistant PrP isoform in the urine of prion infected animals and humans (UPrPSC), which may be used for the in-vivo early diagnosis of ill as well as seemingly healthy but prion infected individuals.",
"Moreover, the present invention shows that this protease resistant isoform UPrPSC, can be detected, following a specific enrichment procedure, in the urine of scrapie-infected hamsters, BSE-infected cattle and humans suffering from CJD.",
"This specific enrichment procedure, according to the present invention may include dialysis of the sample through membrane having a pore range of about 6 KDa to about 8 KDa.",
"The present invention further shows that UPrPSC was also found in urine of hamsters inoculated with prions long before the appearance of clinical signs.",
"These findings strongly indicate the possibility of using the method of the invention also for pre-clinical diagnosis.",
"The theoretical possibility for diagnosis of prion diseases in variety of body fluids, such as urine, has been mentioned in several patent documents.",
"EP 0854364, for example, discloses a diagnostic method for neuro-degenerative disorders such as Alzheimer's disease and prion diseases.",
"This method is based on concentrating a protein associated with the specific neuro-degenerative disease (such as PrP in prion diseases and APP in Alzheimer's disease), in a sample (urine, for example).",
"The concentration is carried out by contacting the sample with a solid, non-buoyant particulate material having free ionic valencies such as calcium phosphate.",
"However, this patent exemplifies the detection of only the Alzheimer's disease associated peptide APP.",
"WO 93/23432 discloses a diagnostic method for prion diseases in different body fluids such as CSF (cerebrospinal fluid) and theoretically, urine.",
"Similarly to EP 0854364, this method is based on concentrating the prion protein by ammonium sulfate precipitation and affinity chromatography.",
"This publication exemplifies CSF as a sample.",
"However, contrary to the prior art methods, the present invention clearly demonstrates the detection of the aberrant protease resistant urine isoform UPrPSC in urine samples of prion infected animals and humans.",
"Furthermore, as shown by the present invention, dialysis of the urine seems to improve the detection procedure.",
"Therefore, the present inventors propose that UPrPSC is present in a semi-denatured form, probably due to the relative high concentrations of urine denaturing agents, and is subsequently re-natured for example by the dialysis step.",
"Thus, the specific enrichment of the urine sample according to the present invention provides a novel and reliable method for the detection of different prion diseases by a non-invasive procedure.",
"SUMMARY OF THE INVENTION The invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of: (a) providing a urine sample of said subject; (b) isolating from said sample proteins; and (c) detecting the presence of PrPSC in the protein mixture obtained in step (b) by a suitable detection technique.",
"A preferred embodiment relates to the method of the present invention, further comprising the step of subjecting the proteins obtained in step (b) to protease digestion.",
"In a specifically preferred embodiment, the invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of (a) providing a urine sample of said subject; (b) isolating from said sample all proteins having a molecular weight higher than about 8 KDa; (c) subjecting the proteins obtained in step (b) to protease digestion; (d) isolating from the mixture obtained in step (c) any protease resistant proteins; and (e) detecting the presence of PrPSC in the protease resistant fraction obtained in step (d) by a suitable detection technique.",
"In step (b) of the method of the invention the proteins are preferably isolated by subjecting the urine sample to dialysis and precipitating the proteins from the dialysate.",
"Optionally, prior to the protein precipitation, a carrier may be added to the dialysate for stabilizing the PrPSC.",
"The dialysis is preferably performed using a membrane having a pore range of from about 6 KDa to about 8 KDa.",
"The proteins may be precipitated from the dialysate by ultracentrifuging the same, for example for about 1 hour at 100,000×g at 4° C. Alternatively the proteins may be precipitated by any suitable protein precipitation technique.",
"As a preferred embodiment proteins according to the invention may be precipitated by any one of methanol, TCA (Trichloracetic acid) or by any other precipitation method.",
"Preferably, proteins may be precipitated by methanol, for example by the addition of methanol and freezing the sample to about −80° C. for about 1 hour, and subsequently centrifuging at 3000×rpm for about 30 minutes.",
"The protein digestion is preferably performed by treating the sample with proteinase K, for example by adding proteinase K in concentration of up to 40 μg/ml and continuing digestion for about 30 min at 37° C. The presence of the PrPSC protease-resistant core in said non-digested fraction is preferably detected by immunoassay, for example by immunoblot SDS PAGE analysis, using monoclonal antibodies that specifically bind to the protease-resistant core of PrPSC, for example 3F4 or GH4 monoclonal antibodies.",
"The invention also relates to a method for diagnosing a prion disease in a subject comprising the steps of (a) obtaining a urine sample of said subject; and (b) detecting the presence of the abnormal isoform of prion protein (PrPSC) in said urine sample by the method of the invention, whereby the presence the PrPSC protein in said sample indicates that said subject carries a prion disease.",
"In a preferred embodiment, said prion disease may be any TSE disease.",
"The subject may be a human subject, for example a CJD, vCJD, GSS or FFI carrier or an individual infected with BSE.",
"Alternatively the subject may be an animal infected with BSE, scrapie or any other TSE disease.",
"The method of the invention further enables detection of different prion disease prior to or after onset of clinical symptoms.",
"In yet a further embodiment that invention relates to a method for screening donors of blood samples for the presence of a prion disease in said donor comprising the steps of: (a) obtaining a urine sample from said donor; (b) detecting the presence of the abnormal isoform of prion protein (PrPSC) in said urine sample by the method of the invention; and matching the results of the detection performed in step (b) to said blood sample.",
"Still further, the invention relates to a method for detecting the presence of metabolites of the abnormal isoform which is probably a pathogenic isoform of prion protein (PrPSC) in a urine sample of a subject, said metabolites being unique for human prion disease carriers.",
"In a preferred embodiment such human prion disease may be CJD or vCJD.",
"This method comprises the steps of: (a) providing a urine sample of said subject; (b) isolating from said sample all proteins having a molecular weight higher than about 8 KDa; and (c) detecting the presence of said metabolites of PrPSC in the protein sample obtained in step (b) by a suitable detection technique.",
"In this embodiment, in step (b) said proteins may be isolated by subjecting the urine sample to dialysis and precipitating the proteins from the dialysate, for example by ultracentrifuging the dialysate, specifically for about 1 hour at 100,000×g at 4° C., or by any other suitable precipitation method.",
"Preferred protein precipitation method may be methods such as methanol or TCA (Trichloracetic acid) precipitation.",
"A specifically preferred technique for precipitation is methanol precipitation, specifically by the addition of methanol to the sample, freezing to about −80° C. for about 1 hour, and subsequently centrifuging at 3000×rpm (rounds per minute) for about 30 minutes.",
"The detection of the presence of the said metabolites of PrPSC protease-resistant core in said protein sample is preferably by immunoassay, particularly SDS PAGE, using monoclonal antibodies that specifically bind to the specific metabolites of PrPSC found in urine of prion disease carriers, for example 6H4 monoclonal antibodies.",
"The invention further relates to a method for diagnosing a prion disease in a subject comprising the steps of: (a) obtaining a urine sample of said subject; and (b) detecting the presence of metabolites of the abnormal isoform of prion protein (PrPSC) that are unique for piron disease patients in said urine sample by a method of the invention; whereby the presence of said PrPSC protein metabolites in said sample indicates that said subject carries prion disease.",
"According to a preferred embodiment, the method of the invention is intended for detection of the presence of metabolites unique for CJD and vCJD.",
"Still further, the invention relates to a method for screening donors of blood samples for the presence of prion disease in said donor.",
"This method comprises the steps of: (a) obtaining a urine sample from said donor; (b) detecting the presence of metabolites of the abnormal isoform of prion protein (PrPSC) that are unique for prion disease patients in said urine sample by a method of the invention; and matching the results of the detection performed in step (b) to said blood sample.",
"In another embodiment the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrRSC) in a urine sample of a subject, said kit comprising means for isolating from said urine sample all proteins; optionally, a carrier for stabilizing the PrPSC; means for detecting the presence of PrPSC in the non-digested fraction; and instructions for carrying out the detection of the presence of PrPSC in the urine samples.",
"In another preferred embodiment the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said kit comprising means for isolating from said urine sample all proteins having a molecular weight higher than about 8 KDa; optionally, a carrier for stabilizing the PrPSC in the dialysate; means for detecting the presence of PrPSC; and instructions for carrying out the detection of the presence of PrPSC in the urine samples.",
"In yet another specifically preferred embodiment the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said kit comprising means for isolating form said urine sample all proteins having a molecular weight higher than about 8 KDa; optionally, a carrier for stabilizing the PrPSC in the dialysate; a protease for digesting the protein isolate; means for isolating from the protein digest any protease digest any protease resistant proteins; means for detecting the presence of PrPSC in the protease resistant fraction; and instructions for carrying out the detection of the presence of PrPSC in the urine samples.",
"In the kit of the invention, protease is preferably proteinase K and said means for detecting the presence of PrPSC comprise reagents for detecting PrPSC by immunoassay, such as antibodies that specifically react with the protease-resistant core of PrPSC.",
"In another embodiment the invention relates to a diagnostic kit for detecting the presence of metabolites of the abnormal isoform of prion protein )PrPSC) that are unique for human prion disease carriers, in a urine sample of a subject, said kit comprising: means for isolating from said urine sample all proteins having a molecular weight higher than about 8 KDa and means for detecting the presence of PrPSC metabolites that are unique for human prion disease carriers, preferably CJD and vCJD, in the obtained protein sample.",
"The means for detecting the presence of said PrPSC metabolites preferably comprise reagents for detecting said PrPSC metabolites by immunoassay, for example antibodies that specifically react with the metabolites of PrPSC that are unique for human prion disease carriers.",
"According to a preferred embodiment, said human prion disease may be CJD or vCJD.",
"BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described in more detail on hand of the attached drawings in which: FIG.",
"1 shows an immunoblot analysis of PrPSC in urine samples from Scrapie-infected hamsters (Sc) or from normal controls (N) using 3F4 monoclonal antibodies.",
"Samples were either treated (+) or not treated (−) with proteinase K (PK).",
"FIG.",
"2 shows an immunoblot analysis of PrPSC in urine samples from homozygous (HOZ) or heterozygous (HTZ) human patients suffering from CJD, MS (multiple sclerosis), stroke (str) and healthy individuals (Norm) using 3F4 monoclonal antibodies.",
"Hamster brain extracts (Ham br) was used as positive control.",
"Samples were either treated (+) or not treated (−) with proteinase K (PK).",
"FIG.",
"3 shows an immunoblot analysis of PrPSC in urine samples from homozygous (HOZ) CJD patients and healthy individuals (Norm) using 3F4 monoclonal antibodies.",
"Hamster brain extracts (Ham br) were used as positive control.",
"Samples were either treated (+) or not treated (−) with proteinase K (PK).",
"Blue pre-stained marker (Novex) was used as molecular weight marker (M).",
"FIG.",
"4A-C shows protease resistant PrP in urine or brain samples of TSE affected humans and animals.",
"FIG.",
"4A: shows freshly frozen urine samples from hamsters, humans, and cattle that were enriched for protease resistant PrP as described in the experimental procedures.",
"All samples were digested in the presence or absence of Proteinase K (+ or − PK, respectively), and immunoblotted with either anti-PrP mAb 3F4 (hamster and human samples) or 6H4 (bovine samples).",
"(1) Homozygous E200K CJD patient; (2) Heterozygous E200K CJD patient; (3) Human control; (4) Scrapie sick hamster; (5) Normal hamster; (6) BSE sick cattle; (7) Normal cattle.",
"FIG.",
"4B: shows similar analysis performed using 5 μl of a 10% brain samples (1) Homozygous CJD patient; (2) Heterozygous CJD patient; (3) Human control; (4) Scrapie sick hamster; (5) Normal hamster; (6) Kidney sample from scrapie sick hamster; FIG.",
"4C: shows blocking experiment performed in human brain sample (b) and human urine sample (u).",
"Samples were immunoblotted with mAb 3F4 in the absence (1) or the presence (2) of 10 μg/ml of the peptide comprising the 3F4 epitope.",
"Molecular Weight markers (top to bottom); 36 kDa, 30 kDa.",
"FIG.",
"5 shows scrapie hamster urine samples that were enriched for UPrPSC with and without dialysis step (Dia).",
"Samples were digested in the presence (+) or absence (−) of proteinase K (PK) as described in methods.",
"FIG.",
"6 shows an immunoblot analysis of PrPSC obtained from CJD patients that were either treated or not treated with DMSO for 1 day (+DMSO (1 d)) and healthy individuals (Norm), using 6H4 antibodies as compared to 3F4 antibodies.",
"Hamster brain extracts (Ham br) were used as positive control.",
"Samples were precipitated using methanol and were either treated (+) or not treated (−) with proteinase K (PK).",
"Blue pre-stained marker (Novex) was used as molecular weight marker (M).",
"FIG.",
"7A-B shows that prion specific PrP can be detected during scrapie incubation time (Inc T) in days (d).",
"Urine samples were collected weekly from Syrian hamsters inoculated either i.c.",
"(7A), or i.p.",
"(7B).",
"Samples were immunoblotted with αPrP mAb 3F4.Arrows represent the onset of clinical signs (Clin sig).",
"Molecular weight markers (top to bottom): 36 kDa, 30 kDa.",
"FIG.",
"7A; shows Syrian hamsters inoculated i.c.",
"(intra-cerebally) with hamster 263K prions, and enriched for UPrPSC.",
"FIG.",
"7B: shows Syrian hamsters inoculated i.p.",
"(intraperitoneally) with hamster 263K prions, and enriched for UPrPSC.",
"FIG.",
"8A-B shows i.c.",
"(intra-cerebally) inoculation of Syrian hamsters with UPrPSC.",
"Syrian hamsters were inoculated with equivalent amounts of PK resistant PrP from brain or urine of scrapie infected hamsters.",
"All samples were immunoblotted with 1:5000 mAb 3F4.FIG.",
"8A: shows PK resistant PrPSC equivalents originating from 5μl of 10% hamster brain homogenate (1) as compared to 2 ml scrapie hamster urine (2).",
"FIG.",
"8B: shows brain sample from a scrapie infected hamster (1); urine samples collected (at 60 dpi) from hamsters inoculated with UPrPSc (2); or brain sample of one of the animals inoculated with UPrPSC (3).",
"All samples were digested in the presence (+) or absence (31) of PK.",
"Molecular weight markers (top to bottom); 36 kDa, 30 kDa.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The inventors have now surprisingly found, and this is an object of the invention, that PrPSC, the aberrant isoform and the only known marker for prion diseases, can be identified in the urine of hamsters infected with scrapie, as well as in the urine of humans sick with CJD.",
"In addition, some metabolites of the PrPSC could be detected in the urine of CJD patients, while they were absent from urine of normal individuals.",
"Thus, in a first aspect, the invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of: (a) providing a urine sample of said subject; (b) isolating or concentrating from said sample proteins; and (c) detecting the presence of PrPSC in the protein mixture obtained in step (b) by a suitable detection technique.",
"A preferred embodiment relates to the method of the present invention, further comprising the step of subjecting the proteins obtained in step (b) to protease digestion.",
"In a specifically preferred embodiment, the invention relates to a method for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, said method comprising the steps of (a) providing a urine sample of said subject; (b) isolating or concentrating from said sample all proteins having a molecular weight higher than about 8 KDa; (c) subjecting the proteins obtained in step (b) to protease digestion; (d) isolating from the mixture obtained in step (c) any protease resistant proteins; and (c) detecting the presence of PrPSC in the protease resistant fraction obtained in step (d) by a suitable detection technique.",
"In one preferred embodiment, the proteins may be isolated from the urine sample by subjecting the sample to dialysis and precipitating the proteins from the dialysate.",
"The dialysis may be preferably performed using a membrane having a pore range of from about 6 KDa to about 8 KDa.",
"Following the dialysis, the proteins may be precipitated from the dialysate by ultracentrifuging, for example for about 1 hour at 100,000×g at 4° C., or by any other suitable protein precipitation technique.",
"As a non-limitinig example such protein precipitation techniques may be by any one of methanol or TCA (Trichloracetic acid).",
"Methanol precipitation is preferred for example, by adding methanol and freezing the sample to about −80° C. for about 1 hours, and subsequently centrifuging at 3000×rpm for about 30 minutes.",
"In yet another preferred embodiment proteins are precipitated using TCA.",
"Briefly according to a modified protocol, sample is diluted with 10% TCA, kept for two hours on ice, and subsequently centrifuged at 14000 rpm at 4° C. After discarding the supernatant the pellet is subjected twice to ethanol precipitation [the modified protocol is based upon the TCA precipitation protocol on: Antibodies, a laboratory manual, editors: Ed Harlow, David Lane, Cold spring harbor laboratory (1988)].",
"Optionally, prior to the protein precipitation, the PrPSC may be stabilized by adding a carrier to the dialysate.",
"For example, such carrier may be brain extract of PrP ablated mice.",
"After the said proteins are isolated, they are subjected to digestion by a protease, preferably by proteinase K, for example by adding to the sample proteinse K at a concentration of about 40 μg/ml and continuing digestion for about 30 min at 37° C. Preferably, the presence of the PrPSC protease-resistant core in said non-digested fraction is detected by immunoassay, for example by immunoblot SDS PAGE, employing monoclonal antibodies that specifically bind to the protease-resistant core of PrPSC, preferably the monoclonal antibodies 3F4 or 6H4.The presence of this protein can also be identified by dot blot immunoassays, and by specifically adapted ELISA test.",
"The invention thus provides an efficient, non-invasive method for the diagnosis of prion diseases.",
"It may be appreciated that while the rationale underlying the method of the present invention is yet unclear, it is possible that the PrPSC is secreted from the brain cells during the pre-clinical or clinical stage of the disease, and since this protein is protease-resistant, it is cleared into the urine before it can be digested in the blood.",
"In a further embodiment, the method of detection of the present invention may be used for diagnosing a prion disease in a human or animal subject, by obtaining a urine sample of the subject and detecting the presence of the abnormal isoform of prion protein (PrPSC) in said urine sample by the detection method the invention, the presence of the PrPSC protein in the urine of the subject indicating that said subject carries a prion disease.",
"This abnormal isoform is probably a pathogenic isoform of the prion protein.",
"Thus, the invention provides a method for the detection of different prion diseases before or after onset of clinical symptoms.",
"The prion disease according to a preferred embodiment is a TSE disease.",
"TSE disease may be as a non limiting example any one of CJD, FFI, GSS in human.",
"In a non-human animal TSE disease may be any one of BSE, Scrapie, CWD (Chronic Wasting Disease) of mule, deer and elk and TME (Transmissible Mink Encephalopathy).",
"The diagnostic method of the invention is particularly important for detecting carriers of CJD, for monitoring treatment of CJD patients and for estimating the patients clinical stage as well as the severity of the disease.",
"It is to be noted that when referring to CJD, all other TSE's are also included.",
"Suspected carriers of pathogenic prion mutations are tested by molecular method for the presence of the mutation, which defines their carrier status.",
"However, and since the age of disease onset can be between 35-85 or more, there is no test to establish at early stages whether the disease is manifesting.",
"Such test could be crucial for early or prophylactic treatment.",
"The detection of carriers of the mutation leading to CJD disease may be used, for example, in genetic counseling.",
"The method of the invention for detecting and diagnosing human prion disease carriers, preferably CJD carriers, may be modified to detect the presence in urine of specific metabolites of PrPSC, which have now been identified in the urine of CJD patients.",
"Being specific for the human prion diseases, these metabolites can be identified in the urine of the patients, without first subjecting the protein sample obtained from the urine to protease digestion.",
"This modification can employ antibodies specific for PrPSC, as shown in the Examples, or antibodies that are specific for the metabolites that are unique for human prion disease.",
"For example, 6H4 antibodies can bind to a metabolite of PrP which is not present in normal urine.",
"Therefore, the protease digestion could be omitted, and suitable antibodies, for example, the primary antibody 6H4 used, to bind CJD specific metabolites of PrPSC, found only in sick individuals.",
"Additionally, the diagnostic method of the invention is useful in identifying infection of BSE, particularly in individuals that have been exposed to the disease.",
"Identifying human carriers of BSE has importance, inter alia, in screening blood samples of human donors for the presence of a prion disease in the donors.",
"Screening can be carried out, for example, by obtaining a urine sample from the donor, detecting the presence of the abnormal isoform of prion protein (PrPSC) in the urine sample by the detection method of the invention and ascribing the results of the detection to said blood sample.",
"Such screening would prevent the use of prion-infected blood, thus diminishing risks of blood transfusions.",
"Additionally, the diagnostic method of the invention, when applied to bovine animals, and also to other domestic animals like sheep and goats or any other animal of interest susceptible to BSE or any other prion disease, may assist in screening food products originating from the tested animals, like meat and dairy products, and reduce the risk of infection of human consumers.",
"Some steps of the method of the invention may preferably be adapted when applied to bovine animals.",
"First, larger urine volume (about 20-30 ml) should be tested.",
"After dialysis, it is preferred to stabilize the proteins of the dialysate by adding a carrier.",
"Such a carrier may preferably be brain extracts of PrP ablated mice.",
"The dialysate is next precipitated using ultracentrifuge, or preferably methanol precipitation, as indicated above.",
"Following the PK digestion, samples are separated on SDS PAGE, and the blots are blocked using human serum albumin.",
"It is to be appreciated that any other suitable protein precipitation methods such as TCA (Trichloracetic acid), may be used by the method of the invention.",
"In yet a further embodiment, the invention relates to a diagnostic kit for detecting the presence of the abnormal isoform of prion protein (PrPSC) in a urine sample of a subject, which comprises means for isolating from said urine sample all proteins having a molecular weight higher than about 8 KDa, optionally a carrier for stabilizing the dialysate proteins, a protase for digesting the protein isolate, means for isolating from the digest any non-digested proteins, means for detecting the presence of PrPSC in the non-digested fraction; and instructions for carrying out the detection of the presence of PrPSC in the urine sample.",
"Specifically, the protease comprised in the kit of the invention may be proteinase K, and the means for detecting the presence of PrPSC may comprise reagents for detecting PrPSC by immunoassay, particularly antibodies that specifically react with the protease-resistant core of PrPSC, such as 3F4 and 6H4.In another embodiment, the kit of the invention may be modified to exclude the protease, and include antibodies that specifically bind to metabolites of PrPSC that are uniquely found in urine of human prion disease patients and carriers.",
"Preferably, said human prion disease may be CJD or vCJD.",
"A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art.",
"Such methods include, for example, detection and analysis of naturally occurring, synthetic and recombinant proteins or peptides and the like.",
"Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology, by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al.",
"(eds.)",
"3rd ed.",
"John Wiley & Sons; ISBN: 0471137812, 1995.These publications are incorporated herein in their entirety by reference.",
"Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art.",
"See e.g., Current Protocols in Immunology, Coligan et al.",
"(eds.",
"), John Wiley & Sons, Inc., New York, N.Y. Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat.",
"It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended Claims and equivalents thereof.",
"Throughout this specification and the Claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.",
"It must be noted that, as used in this specification and the appended Claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.",
"The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention.",
"It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the intended scope of the invention.",
"EXAMPLES Reagents Antibodies 3F4—monoclonal anti PrP antibody, detects the 108-111 amino acids residues in the sequence of the PrP protein [Oesch et al., Cell 40(4):735-736 (1985), Kascsak R J Et al., J. Virol.",
"61(12):3688-93 (1987)].",
"6H4—monoclonal anti PrP antibody, which binds to the 144-152 amino acid residues of the PrP protein, purchased from Prionics AG, Switzerland.",
"Buffers Sarkosyl/STE burrer—10 mM Tris HCl pH 7.5, 10 mM NaCl, 1 mM EDTA.",
"Homogenization buffer—10 mM Tris HCl pH 7.5, 300 mM Sucrose.",
"TBST buffer—10 mM Tris HCl Ph 8.0, 150 mM NaCl, 0.05% Tween 20.Experimental Procedures Analysis of Urine Samples Urine samples (2 ml for hamster; 10 ml for human; 15 ml for bovine) were sedimented for 5 min at 3000 rpm to discard occasional cell debris, and then dialyzed over night in a cellulose tubular membrane (pore range 6000-8000 Dalton, FPI; Texas, U.S.A.) against 5 liters of saline at 4° C. (saline was changed twice during dialysis).",
"For experimental purposes, the dialysis step was omitted in some cases.",
"Subsequently, urine samples were centrifuged at high speed (100000 gav *1 hr*4° C.).",
"Pellets were resuspended in 100 μl 2% Sarkosyl/STE buffer.",
"Samples were divided and digested in the presence or absence of proteinase K (PK).",
"Digestion conditions were optimized for each species.",
"For hamster urine: 40 μg/ml PK for 60 min at 37° C.; for human urine: 40 μg/ml PK for 30 min at 37° C.; for bovine urine: 20 μg/ml for 30 min at 37° C. Following protease digestion, urine samples were boiled in SDS sample buffer.",
"Western Blot Analysis Samples boiled in SDS sample buffer were applied to a 12% SDS PAGE and subsequently transferred to a nitrocellulose membrane.",
"Membranes were blocked with 3% fat milk except for the bovine samples which were blocked with 5% HSA (Human Serum Albumin, Sigma).",
"A second blocking step was performed with a mixture of 1:3000 anti mouse IgG and 1:3000 anti-rabbit 1 gG in TBST buffer (for 30 min) to avoid non specific binding of the secondary Ab to IgG light chain present in some urine samples.",
"Membranes were then rinsed in TBST for 15 min and immunoblotted either with α PrP mAb 3F4 or 6H4 (Hamster, Human) at 1:5000 or 6H4 (Bovine) at 1:5000.In Vivo Experiments Syrian hamsters were inoculated with samples containing urine PrP from normal or scrapie sick hamsters.",
"For inoculation, urine samples were prepared as described above (including PK digestion but not SDS boiling) and diluted as required in 1% BSA/PBS.",
"Brain samples from scrapie infected hamsters were diluted to contain comparable concentrations of PrPSC and inoculated to additional groups of hamsters.",
"To achieve similar concentrations of protease resistant PrP of brain and urine inoculi, each animal was inoculated, depending on the appropriate experimental group, with 50 μl sample containing PrP originating from either 0.5 ml urine or from 1.25 μl of 10% brain homogenate of scrapie hamster.",
"Hamster Samples Following inoculation, animals were examined daily for scrapie associated symptoms.",
"For time course experiments, groups of 3 hamsters in an equivalent stage of disease incubation, were housed each week in a metabolic cage for urine collection from 15:00 p.m. to 08:00 a.m. of the next day.",
"Urine was collected in the morning and immediately was frozen at −80° C. Food and water were supplied ad libitum.",
"Similar procedure was applied to scrapie sick hamsters.",
"Human Urine Samples Most of CJD patients tested (6 out of 8) were genetic patients carrying the E200K mutation [Hsiao, K., et al., N Engl J Med 324(16), 1091-7 (1991); Gabizon, R., et al., Nat Med 2(1), 59-64 (1996); Gabizon, R., et al., Am J Hum Genet 53(4), 828-35 (1993) Goldfarb, L. G., et al., Lancet 336(8715), 637-8 (1990)].",
"One of the patients was a 52 year old individual homozygous for this mutation [Simon, E. S., et al., Ann Neurol 47(2), 257-60 (2000)].",
"Among the other genetic patients, 4 were MM at codon 129 and one was MV.",
"The E200K mutation is located at a Methionine 129 allele [Gabizon, R., et al., (1993) ibid].",
"The human controls (n=15), were either healthy individuals (n=7) or patients suffering from diverse neurological disorders, such as Alzheimer's disease (n=3), multiple sclerosis (n=2) and stroke (n=3).",
"Whenever possible, human samples from CJD patients and controls were the first morning urine.",
"Some CJD and post stroke patients were bearing catheters, and in these cases urine was collected for a period of up to 8 h in a urine collecting bag.",
"All samples were frozen until further use.",
"Bovine Urine Samples All BSE and most control bovine urine samples were obtained from the Veterinary Laboratory Agency (VLA) in London.",
"The VLA samples constituted 51 samples of 24 cows, all coded for blind testing.",
"Additional freshly frozen control samples were obtained from the Hebrew University Veterinary School.",
"According to VLA records, most samples were frozen following collection while some were kept chilled.",
"No information was provided regarding time of day for sample collection.",
"Tissue Homogenates Whole brain or kidney samples were homogenized in ten volumes homogenization buffer.",
"Following centrifugation [2000 rpm (rounds per minute), 15 min at 4° C.), the supernatant was frozen (−80° C.).",
"Example 1 Immunoblot Analysis of PrPSC in Urine Samples In order to develop a non-invasive method for diagnosis of different prion diseases, the possibility of detecting the prion protein UPrPSC in urine samples of different mammalian subjects was examined.",
"Urine samples from scrapie infected hamsters, CJD patients, and BSE infected cattle, as well as from their appropriate controls, were processed for enrichment of UPrPSC, and subsequently immunobloted for PrP peptides as described herein above.",
"Human and hamster urine samples were immunoblotted with either mAb 3F4 or 6H4, while bovine samples were blotted only with mAb 6H4.Parallel samples were blotted only with secondary α mouse antisera and showed no interfering signals.",
"As shown in FIGS.",
"1, 2, 3 and 4, a precipitable and protease resistant form of PrP could be detected only in the dialyzed urine of prion disease affected humans and animals.",
"However, in urine samples of the appropriate controls, the resistant form of PrP could not be detected.",
"PrPSC was not found in samples from MS (Multiple Sclerosis) patient, Stroke patients as well as in healthy individuals (FIGS.",
"2 and 3).",
"The differences in band strength of the two patients (FIG.",
"2) is probably the result of the different clinical status of the patients.",
"Similar results were obtained with other CJD homozygous and heterozygous patients (FIGS.",
"3 and 4).",
"In order to verify that the observed signal was specific to PrP, a blocking experiment was next performed.",
"As shown in FIG.",
"4C, PrP signal in urine could be blocked by the 3F4 peptide, providing strong evidence that this signal belongs to a PrP peptide.",
"An essential element in developing the method for detecting UPrPSC in urine was concentrating the samples by dialyzing the urine samples prior to their centrifugation and digestion.",
"FIG.",
"5 demonstrates the importance of dialysis of urine samples from scrapie infected hamsters.",
"The protease resistant UPrPSC could be detected after PK digestion only in dialyzed sample.",
"A surprising result depicted above is that a protease sensitive PrP isoform is present in the precipitable fraction of the normal urine samples, as opposed to what is expected for PrPC.",
"It is to be noted however, that no detergent was added to the urine before ultracentrifugation as performed in membrane extractions that result in a soluble PrPC [Meyer, R. K., et al., Proc Natl Acad Sci U.S.A. 83)8), 2310-4 (1986); Gabizon, R., et al., Proc Natl Acad Sci U.S.A. 84(12), 4017-21 (1987)].",
"It is also possible that all PrP molecules are present in urine in a partially denatured state due to the presence of variety of denaturing agents such as urea.",
"Also dialysis of normal urine may induce the aggregation of the PrPC isoform which, as opposed to UPrPSC, is protease sensitive.",
"Although the exact chemical nature of UPrPSC is yet to be determined, its molecular weight seems to be slightly higher than full length and fully glycosylated PrPC or PrPSC.",
"In addition, the pattern of UPrPSC in the immunoblots suggest it may be composed mostly of the higher molecular band of PrP, and not of the less glycosylated species.",
"This may indicate that partially or non-glycosylated PrP is less resistant to the conditions encountered by PrPSC before it is excreted in urine as UPrPSC.",
"It is conceivable that at least for hamsters, UPrPSC did not originate directly from the kidneys, since no PrPSC could be identified in the kidney tissue of scrapie infected hamsters (FIG.",
"4B, sample 6).",
"This suggests UPrPSC originates from other organs and arrives to the urine from blood.",
"Example 2 Diagnosis of BSE in Urine Samples of Cattle Twenty-four different samples of cattle urine obtained from England were double blind tested for the presence of PrPSC.",
"Briefly, different samples of 20 ml of cattle urine were processed by dialysis against saline, as described above.",
"The dialyzed samples were further stabilized by adding different concentrations 10-5 μl of a 10% homogenate of brain extracts of PrP ablated mice.",
"Addition of the PrP ablated mice extracts as a carrier, improved the ability to obtain a more concentrated protein precipitate due to the presence of molecules in said extract which bind and stabilize the urine PrP.",
"However, it is to be appreciated that addition of the PrP ablated mice extracts as a carrier is not necessary and the test is feasible also without this additional step.",
"The dialyzed samples were then precipitated with methanol (1:4 volume to volume sample/methanol) and subsequently digested in the presence or absence of PK as described above.",
"Digested samples were then subjected to Western blot analysis (12% SDS PAGE), and blots were blocked using 5% Human Serum Albumin (HSA) in TBST buffer prior to addition of the primary antibody.",
"Table 1 presents the obtained results, compared with clinical diagnosis of the same samples by brain histopatholgy.",
"As demonstrated in Table 1, the results obtained by this experiment were highly significant.",
"All the negative samples were properly diagnosed and most of the clinically affected animals were diagnosed as BSE positives (10 out of 12) by the method of the present invention.",
"Only four samples (Nos.",
"4, 9, 14 and 22) were inconclusive, probably due to non-optimized storage and shipment conditions.",
"Further optimization of the sample storage and handling conditions is within the scope of the present invention.",
"TABLE 1 BSE diagnosis of cattle urine samples Urine Test according to Sample # the Invention Brain Histopathology 1 a + + b + 2 a − − b − 3 a − − b − 4 a +/− b +/− − c + 5 a − − b − 6 a − − b − 7 a + + b + 8 a − − b − 9 a +/− b + − c +/− 10 a + + b + 11 a + + b + 12 a − b − − c − 13 a + + b + 14 a +/− + b +/− 15 a − b − − c − 16 a − − b − 17 a + + b + 18 a +/− + b +/− 19 a + + b + 20 a + + b + 21 a + + b + 22 a Ns + b Ns 23 − − 24 a − − b − (+) positive (−) negative (+/−) suspected (low signal) Ns—non specific background Example 3 Comparison Between 3F4 and 6H4 Antibodies in the Analysis of PrPSC in Urine Samples 3F4 and 6H4 monoclonal antibodies were used to detect PrPSC in urine samples of CJD patients.",
"To precipitate the samples, methanol was used instead of ultracentrifugation.",
"As shown in FIG.",
"6, 6H4 antibodies could detect two additional lower bands, probably representing two additional metabolites of PrP that are PK-resistant and are present only in CJD patients.",
"The additional metabolites detected by the 6H4 antibodies were found only in CJD patients, even when the treatment with PK was omitted [FIG.",
"6].",
"A considerable increase in the amount of PrP secreted in the urine was found when dimethylsulfoxide (DMSO) was administrated to CJD patients prior to examination.",
"FIG.",
"6 shows the result of Western blot analysis of CJD patient with and without DMSO administration (5 ml, three times daily) for one day.",
"As depicted in the right panel of FIG.",
"6, DMSO led to the enhancement of PrP secretion to the urine.",
"Example 4 Detection of the Protease Resistant UPrPSC in a Urine Sample Prior to Onset of Clinical Symptoms Detection of PrPSC at the final stages of prion disease may result from some degree of blood brain barrier disruption by brain degeneration [De Armond, S. J., et al., Prog Clin Biol Res 317, 601-18 (1989)].",
"However, the presence of PrPSC in prion infected urine in early stage of the incubation time, suggests a clearance pathway for the aberrant PrP protein either from brain or from peripheral organ, through its excretion into urine.",
"To address this question, Syrian hamsters were inoculated either intra-cerebrally (i.c.)",
"or intraperitoneally (i.p.)",
"with hamster prions.",
"Urine samples were collected every week during the incubation period, as described in experimental procedures and each sample was frozen immediately after collection.",
"At the end of the experiment, similar volumes of these urine samples were thawed, enriched for PK resistant UPrPSC as described above and subsequently immunoblotted with the anti PrP mAb 3F4.As can be seen in FIG.",
"7, a light signal of prion specific protease resistant PrP was detected in urine samples of i.c.",
"inoculated hamsters after only 17 days (FIG.",
"7A), following by the disappearance of the PrP signal until day 35.Subsequently, this signal increased from day 35 until the appearance of clinical signs.",
"Similar results were obtained for i.p.",
"inoculated hamsters (FIG.",
"7B).",
"A PrP signal was detected in the first weeks following inoculation, disappeared at later dates and reappeared at about 60 days.",
"These results may infer that some of the prion inoculum is immediately secreted following inoculation.",
"Thereafter, until the first stages of prion protein accumulation in brain, no PrP signal appeared in urine.",
"It is to be noted that the reported incubation time for i.c.",
"or i.p.",
"scrapie inoculated hamsters with the 263 strain, is about 75 and 120 days respectively, and PrPSc can be identified in enriched brain samples of these hamsters at about 40 (i.c.)",
"or 70 (i.p.)",
"days [Czub, M., Braig, H. R., and Diringer, H. J Gen Virol 69 (Pt 7), 1753-6 (1988); Czub, M., Braig, H. R., and Diringer, H. J Gen Virol 67 (Pt 9), 2005-9 (1986); Taraboulos, A., et al., Proc Natl Acad Sci U.S.A. 89, 7620-7624 (1992)].",
"These results demonstrate that UPrPSC is excreted in urine parallel to its accumulation in brain.",
"These results clearly indicate that urine testing for protease resistant PrP can be used to diagnose prion diseases in animals and humans at terminal stages of the disease and also can be used to diagnose prion diseases in subclinical stages of infection.",
"Detection of the PrP signal at the first weeks post infection is due to clearance of the inoculum and therefore, the PrP urine test may serve as a powerful tool to diagnose a potential new occurrence of infection.",
"This may provide in future, an effective anti-prion therapy for the treatment of individuals at risk of a new prion exposure.",
"Example 5 Can Prion Disease Be Transmitted by UPrPSC Detection of UPrPSC in urine during early stages of incubation time and before the appearance of clinical signs, as was showed herein above, raises the alarming possibility that transmission of prion diseases may occur via urine of either ill animals or of animals during the incubation time of the prion diseases.",
"This prospect is especially disturbing in the case of BSE infected cattle as well as in natural scrapie in sheep, since the mechanism by which these diseases are transmitted among animals within the herd was never elucidated [Chatelain, J., and Dautheville Guibal, C. Eur J Epidemiol 5(1), 113-6 (1989); Berne, R. M., and Levy, M. N. Physiology, 4th Ed.",
"(1998)].",
"Thus, it is conceivable that urine may contaminate the dwelling areas of these animals.",
"To investigate whether urine from TSE infected animals can be infectious, twenty hamsters were inoculated with UPrPSC pooled and enriched from urine of 10 hamsters terminally will with scrapie.",
"Twenty hamsters were inoculated with similarly prepared samples from 10 normal hamsters as negative control.",
"Brain samples from scrapie infected hamsters, diluted to PrPSC concentrations (1.25 μl of 10% homogenate) comparable to those of the enriched UPrPSC (from 0.5 ml urine), were inoculated to additional groups of hamsters (FIG.",
"8A) as a positive control.",
"Hamsters were observed daily for symptoms of scrapie infection and urine was collected periodically from animals inoculated with UPrPSC.",
"Some of the hamsters inoculated with UPrPSC were sacrificed, at different time points during the experiment and tested for the presence of PrPSC in their brains.",
"As expected, animals inoculated with scrapie infected brain samples suffered from fatal disease symptoms at about 80 days post inoculation (dpi).",
"Contrarily, none of the animals inoculated with urine samples (normal or scrapie infected) developed clinical symptoms of prion disease to date (270 dpi).",
"Twelve hamsters (4 groups of 3) were tested for the presence of UPrPSC and all were found positive from about 60days post inoculation (FIG.",
"8B, lane 2).",
"In addition, low concentrations of PrPSC could be identified in brain of one out of three urine infected hamsters that were sacrificed at about 120 days (FIG.",
"8B, lane 3).",
"All other hamsters in this experiment are still under observation to determine whether they will develop a fatal prion disease at a later date.",
"These results suggest that UPrPSC inoculation can result in a subclinical or carrier state prion infection.",
"While specific embodiments of the invention have been described for the purpose of illustration, it will be understood that the invention may be carried out in practice by skilled persons with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the Claims."
]
] | Patent_10399321 |
[
[
"Method of and system for controlling the ratio of a variable lead parameter and an adjustable lag parameter for a lag-lead process",
"In a method of and system for controlling the air/gas ratio in a lag-lead combustion plant the lead and lag parameters are monitored to provide lead and lag signals representative of the values of the parameters.",
"These are compared to provide an error signal representative of the deviation of the ratio of the lead and lag parameters from a preselected ratio.",
"The lag parameter is then adjusted to reduce the deviation in response to the deviation exceeding a preselected deviation."
],
[
"1.A method of controlling the ratio of a variable lead parameter and an adjustable lag parameter for a lag-lead process, the method comprising: monitoring said lead parameter and providing a lead signal representative of the value of said lead parameter; monitoring said lag parameter and providing a lag signal representative of the value of said lag parameter; comparing said lead and lag signals and providing an error signal representative of the deviation of the ratio of said lead and lag parameters from a preselected ratio; and adjusting said lag parameter to reduce said deviation in response to said deviation exceeding a preselected deviation.",
"2.A method as claimed in claim 1 comprising comparing said error signal with a preselected threshold value and adjusting said lag parameter in response to said error signal exceeding said preselected threshold value.",
"3.A method as claimed in claim 1 comprising comparing said error signal with an error range defined by a first, upper preselected threshold value and a second, lower preselected threshold value and adjusting said lag parameter in response to said error signal falling outside said error range.",
"4.A method as claimed in claim 3 comprising: adjusting said lag parameter to reduce said ratio in response to said error signal being above said error range; and adjusting said lag parameter to increase said ratio in response to said error signal being below said error range.",
"5.A method as claimed in claim 4 wherein: said first, upper preselected threshold value is a positive value and said second, lower preselected threshold value is a negative value; a positive error signal indicates an increase in said ratio from said preselected ratio and a negative error signal indicates a decrease in said ratio from said preselected ratio; and the method comprises: adjusting said lag parameter to reduce said ratio in response to said error signal being positive and exceeding said first, upper preselected threshold value; and adjusting said lag parameter to increase said ratio in response to said error signal being negative and exceeding said second, lower preselected threshold value.",
"6.A method as claimed in claim 2 wherein the or each said threshold value is a fixed value.",
"7.A method as claimed in claim 2 wherein the or each said threshold value is a function of the value of said lag parameter.",
"8.A method as claimed in claim 7 wherein the step of comparing said error signal with the or each threshold value comprises adjusting said threshold value in dependence on the value of said lag parameter and comparing said error signal with said adjusted threshold level.",
"9.A method as claimed in claim 8 comprising: providing a look up table for storing a plurality of threshold values; and the step of adjusting said threshold value comprising selecting one of said threshold values in dependence on the value of said lag parameter.",
"10.A method as claimed in claim 7 wherein: the or each threshold value comprises a plurality of threshold levels; and the step of comparing said error signal with the or each threshold value comprises selecting a threshold level for said threshold value in dependence on the value of said lag parameter and comparing said error signal with said selected threshold level.",
"11.A method as claimed in claim 10 comprising: providing a look up table for storing said plurality of threshold levels; and the step of adjusting said threshold value comprising selecting one of said threshold levels in dependence on the value of said lag parameter.",
"12.A method as claimed in claim 1 wherein: said lead and lag parameters are lead and lag fluid flow rates for said industrial process; and said method comprises: providing lag valve means for controlling flow of said lag fluid; monitoring the position of said lag valve means during opening or closing of said valve means; monitoring the flow rate of said lag fluid during opening or closing of said valve means; comparing the change in position of said valve means with the change in said flow rate of said lag fluid; and adjusting said threshold value in response to said comparison indicating a change in the characteristic of said valve means.",
"13.A method as claimed in claim 29 wherein the step of adjusting said threshold value comprises adjusting the threshold values or levels in said look up table.",
"14.A method as claimed in claim 1 wherein: said lead parameter is the flow rate of a combustion gas; said lag parameter is the flow rate of air; and said process is a combustion process.",
"15.A control system for providing lag-lead control of a process having a variable lead parameter and an adjustable lag parameter, the system comprising: lead monitoring means for monitoring said lead parameter and providing a lead signal representative of the value of said lead parameter; lag monitoring means for monitoring said lag parameter and providing a lag signal representative of the value of said lag parameter; comparator means for comparing said lead and lag signals and providing an error signal representative of the deviation of the ratio of said lead and lag parameters from a preselected ratio; and adjusting means for adjusting said lag parameter to reduce said deviation in response to said deviation exceeding a preselected deviation.",
"16.A control system as claimed in claim 15 further comprising: threshold value means for providing a preselectable threshold value; comparator means for comparing said error signal with said preselectable threshold value; and wherein said adjusting means is operable to adjust said lag parameter in response to said error signal exceeding said preselectable threshold value.",
"17.A control system as claimed in claim 16 wherein: said threshold value means comprises a first, upper threshold value means for providing a first, upper preselected threshold value and a second, lower threshold value means for providing a second, lower preselectable threshold value, thereby to define an error range; said comparator means is operable to compare said error signal with said upper and lower preselectable threshold values; and said adjusting means is operable to adjust said lag parameter in response to said error signal falling outside said error range.",
"18.A control system as claimed in claim 17 wherein: said adjusting means is operable to adjust said lag parameter to reduce said ratio in response to said error signal being above said error range and to adjust said lag parameter to increase said ratio in response to said error signal being below said error range.",
"19.A control system as claimed in claim 18 wherein: said first, upper preselected threshold value is a positive value and said second, lower preselected threshold value is a negative value; a positive error signal indicates an increase in said ratio from said preselected ratio and a negative error signal indicates a decrease in said ratio from said preselected ratio; and said adjusting means is operable reduce said ratio in response to said error signal being positive and exceeding said first, upper preselected threshold value and to increase said ratio in response to said error signal being negative and exceeding said second, lower preselected threshold value.",
"20.A control system as claimed in claim 16 wherein the or each said threshold value is a fixed value.",
"21.A control system as claimed in claim 16 wherein the or each said threshold value is a function of the value of said lag parameter.",
"22.A control system as claimed in claim 21 further comprising adjusting means for adjusting said threshold value in dependence on the value of said lag parameter and wherein said comparator means is operable to compare said error signal with said adjusted threshold value.",
"23.A control system as claimed in claim 22 wherein: said threshold value means comprises a look up table for storing a plurality of threshold values; and said threshold value adjusting means is operable to adjust said threshold value by selecting one of said threshold values in dependence on the value of said lag parameter.",
"24.A control system as claimed in claim 21 further comprising: adjusting means for adjusting said threshold value in dependence on the value of said lag parameter; and wherein: the or each threshold value comprises a plurality of threshold levels; said threshold value adjusting means is operable to adjust said threshold value by selecting one of said threshold levels in dependence on the value of said lag parameter; and said comparator means is operable to compare said error signal with said selected threshold level.",
"25.A control system as claimed in claim 24 wherein: said threshold value means comprises a look up table for storing a plurality of threshold values; and said threshold value adjusting means is operable to adjust said threshold value by selecting one of said threshold values in dependence on the value of said lag parameter.",
"26.A control system as claimed in claim 15 wherein: said lead and lag parameters are lead and lag fluid flow rates for said process; and said control system comprises: lag valve means for controlling the flow of said lag fluid; position monitoring means for monitoring the position of said lag valve means during opening or closing of said lag valve means and providing a position signal representative thereof; wherein: said lag monitoring means is operable to monitor the flow rate of said lag fluid during opening or closing of said lag valve means and provide a flow rate signal representative thereof; and said control system further comprises: storage means for storing sampled values of said position and flow rate signals representing a preselected characteristic of said lag valve means; second comparator means for comparing the valve position of said lag valve means and lag fluid flow rate during movement of said lag valve means with the stored values; and second adjusting means for adjusting said threshold value in response to said comparison indicating a change in the characteristic of said lag valve means.",
"27.A control system as claimed in claim 15 wherein: said lead and lag parameters are lead and lag fluid flow rates for said process; and said control system comprises: lag valve means for controlling the flow of said lag fluid; position monitoring means for monitoring the position of said lag valve means during opening or closing of said lag valve means and providing a position signal representative thereof; wherein: said lag monitoring means is operable to monitor the flow rate of said lag fluid during opening or closing of said valve means and provide a flow rate signal representative thereof; and said control system further comprises: storage means for storing sampled values of said position and flow rate signals representing a preselected characteristic of said lag valve means; second comparator means for comparing the lag fluid flow rate during movement of said valve means with stored value corresponding to the monitored position of said lag valve means; and second adjusting means for adjusting said threshold value in response to said comparison indicating a change in the characteristic of said valve means.",
"28.A control system as claimed in claim 15 wherein: said lead parameter is the flow rate of a combustion gas; said lag parameter is the flow rate of air; and said process is a combustion process.",
"29.A method as claimed in claim 8 wherein: said lead and lag parameters are lead and lag fluid flow rates for said industrial process; and said method comprises: providing lag valve means for controlling flow of said lag fluid; monitoring the position of said lag valve means during opening or closing of said valve means; monitoring the flow rate of said lag fluid during opening or closing of said valve means; comparing the change in position of said valve means with the change in said flow rate of said lag fluid; and adjusting said threshold value in response to said comparison indicating a change in the characteristic of said valve means."
],
[
"The present invention relates to a method of and system for controlling the ratio of a variable lead parameter and an adjustable lag parameter for a lag-lead process and particularly, but not exclusively, to apparatus for controlling the air/gas ratio in a-gas combustion plant.",
"It is known that the air/gas ratio (AGR) in a gas combustion plant should be maintained substantially constant to achieve optimum combustion efficiency of the plant.",
"Air/gas ratio controllers are used in the plant to maintain the air/gas ratio when the gas flow rate is increased or decreased.",
"To achieve this, the air/gas ratio controller monitors the gas flow rate and adjusts the air flow rate accordingly, usually by adjusting a valve in an air supply line.",
"A problem with existing air/gas ratio control is the difficulty in adjusting the air flow rate to match accurately the gas flow rate.",
"The present invention aims to provide an improved method and system for air/gas ratio control.",
"Accordingly, the present invention provides a method of controlling the ratio of a variable lead parameter and an adjustable lag parameter for a lag-lead process, the method comprising: monitoring said lead parameter and providing a lead signal representative of the value of said lead parameter; monitoring said lag parameter and providing a lag signal representative of the value of said lag parameter; comparing said lead and lag signals and providing an error signal representative of the deviation of the ratio of said lead and lag parameters from a preselected ratio; and adjusting said lag parameter to reduce said deviation in response to said deviation exceeding a preselected deviation.",
"In a preferred form of the invention said error signal is compared with a preselected threshold value and said lag parameter is adjusted in response to said error signal exceeding said preselected threshold value.",
"Advantageously, the error signal is compared with an error range defined by a first, upper preselected threshold value and a second, lower preselected threshold value and said lag parameter is adjusted in response to said error signal falling outside said error range.",
"The present invention also provides a control system for providing lag-lead control of a process having a variable lead parameter and an adjustable lag parameter, the system comprising: lead monitoring means for monitoring said lead parameter and providing a lead signal representative of the value of said lead parameter, lag monitoring means for monitoring said lag parameter and providing a lag signal representative of the value of said lag parameter; comparator means for comparing said lead and lag signals and providing an error signal representative of the deviation of the ratio of said lead and lag parameters from a preselected ratio; and adjusting means for adjusting said lag parameter to reduce said deviation in response to said deviation exceeding a preselected deviation.",
"Advantageously, the system further comprises threshold value means for providing a preselectable threshold value and comparator means for comparing said error signal with said preselectable threshold value.",
"The adjusting means is operable to adjust said lag parameter in response to said error signal exceeding said preselectable threshold value.",
"Preferably, said threshold value means comprises a first, upper threshold value means for providing a first, upper preselected threshold value and a second, lower threshold value means for providing a second, lower preselectable threshold value, thereby to define an error range; said comparator means is operable to compare said error signal with said upper and lower preselectable threshold values; and said adjusting means is operable to adjust said lag parameter in response to said error signal falling outside said error range.",
"The present invention will now be described, by way of example only, with reference to the accompanying drawings in which: FIG.",
"1 is a schematic block diagram showing a typical gas combustion plant; FIG.",
"2 is a schematic block diagram of an air/gas ratio controller used in the plant of FIG.",
"1; FIG.",
"3 is a schematic block diagram of a control system having a preferred form of air/gas ratio controller according to one aspect of the present invention; FIG.",
"4 is a schematic block diagram of a preferred form of air/gas ratio controller according to another aspect of the present invention; FIG.",
"5 is a schematic block diagram of a modification to the controller of FIG.",
"4; FIG.",
"6 is a graph shoving Me change in valve position with applied control voltage FIG.",
"7 is a graph showing the derivative of the valve characteristic of FIG.",
"6; and FIG.",
"8 is a graph showing the relationship between the valve derivative and total deadband value.",
"A typical gas combustion plant 10 is shown in FIG.",
"1.The plant 10 consists of three main parts, a temperature controller 12, an air/gas ratio control system 20 and a burner 40 within, for example, a kiln or furnace 41.The temperature controller 12 is able to control the temperature of the furnace 41, either by following a predetermined temperature profile or by allowing a user to define the desired temperature profile.",
"To increase the temperature of the furnace, for example, the controller 12 adjusts the valve in the gas supply line to increase the flow rate of the gas supplied to the burner and the air/gas ratio control system 20 adjusts the air flow rate to attempt to maintain the ratio between the flow rates of the air and the gas supplied to the burner substantially constant.",
"A typical configuration for an air/gas ratio control system is shown in FIG.",
"2.The system 20 includes a gas valve 22 connected to the gas supply line 24 for varying the gas flow rate along the line.",
"A gas flow measurement sensor 26 is positioned downstream of the gas valve 22 for monitoring the gas flow rate along the line.",
"Similarly, an air valve 28 is positioned at a point in the air supply line 30 for varying the air flow rate along the line and an ail flow measurement sensor 32 is positioned downstream of the air valve 28 for monitoring the air flow rate along the air line.",
"The gas valve 22 is connected to receive an input signal from the temperature controller 12 to adjust the flow rate of the gas.",
"The air valve 28 is connected to receive an input from an air/gas ratio controller 34 to adjust the flow rate of the air in dependence on the gas flow rate.",
"The air/gas ratio controller 34 receives an input from both of the gas and air measurement sensors 26, 32 and compares the flow rates of the gas and the air and adjusts the air valve to maintain the required air/gas ratio.",
"It can be seen that if the combustion process is to function with the maximum possible efficiency, the air/gas ratio controller 34 must control the air valve to follow changes in the gas valve as closely as possible.",
"Such a system is commonly known as a lag-lead system.",
"In a lag-lead system when a lead parameter (the gas flow rate) varies, a lag parameter (in this case the air flow rate) is adjusted to maintain the ratio of the parameters substantially constant.",
"The flow rate of the air and the gas are monitored by the measurement sensors of the air/gas ratio control system 20.These preferably sample the flow rate at a predetermined sampling rate.",
"The lead parameter (ere the gas flow rate) and the lag parameter (here the air flow rate) are sampled at regular time intervals.",
"The lead parameter is sampled usually at a faster rate than the lag parameter and can be sampled as fast as once every 20 ms.",
"The sample rate of the lag parameter would be adjusted to suit the lead parameter sample rate and in this instance would be typically once every 120 ms. A typical sample range for the lag parameter would be between 100 ms and 500 ms.",
"In a natural gas combustion system the air/gas ratio is typically required to be maintained in the order of 10:1, known as the stoichiometric/gas ratio.",
"Changes in the temperature reference signal result in the gas valve being adjusted by the temperature controller 12.This changes the gas flow rate and thus the air/gas ratio from the desired value.",
"The change in the gas flow rate is monitored by the controller 34 which acts to adjust the air valve 28 to return the air/gas ratio to the desired value.",
"If a change in the air/gas ratio is detected (i.e.",
"the air/gas ratio moves away from the desired value) by the air/gas ratio controller during a particular sampling of the air and gas flow the controller will move the air valve in the required direction (either towards its fully open or fully closed position) until the next sample is taken.",
"However, if the error in the air/gas ratio is smaller than the change in the air/gas ratio effected by the air valve movement over one sample interval (the period between one sample time and the next) the valve will overshoot the desired position and the desired air flow rate will not be achieved.",
"At the next sampling, the controller 34 will detect a reverse error and will move the valve in the opposite direction i.e.",
"it will move the valve towards its closed position if the previous error caused the valve to be moved towards its open position, and vice versa.",
"Again, the valve will be moved too far the reverse direction during the sample interval and will stop at or close to its initial position i.e.",
"the position from which it was first moved in response to the originally monitored error in the air/gas ratio.",
"This opening and closing of the valve, known as hunting, will repeat for as long as the error in the air/gas ratio remains substantially the same as or smaller than the chance effected by movement of the air valve over one sampling interval.",
"The air valve and consequently the air flow rate will thus oscillate about the level required to achieve the desired air/gas ratio.",
"These oscillations are known as limit cycles.",
"It can be seen that if the error in the air/gas ratio exceeds a particular threshold level (being defined by the change in the air/gas ratio effected by the air valve movement over one sample interval) then no limit cycling will occur.",
"However, if the error lies below the threshold level then limit cycling will occur.",
"For valves with linear characteristics i.e.",
"which exhibit a linear response, the threshold level is constant throughout the valve's operating range.",
"However, many electromagnetically operated valves exhibit a non-linear response where the air flow rate through the valve varies non-linearly in relation to the applied control signal.",
"Thus, the change in the air flow through the valve during movement of the valve towards its fully open or fully closed position over a single sample interval will be different depending on the position of the valve within its operating range (FIG.",
"6).",
"Consequently, the threshold level defining the area value below which limit cycling occurs will vary over the operating range of the valve.",
"Since the motor driving the valve acts as an integrator, the change in flow over one sampling interval can be found by differentiating the valve characteristic (FIG.",
"7).",
"The differential curve of the valve characteristic shows how much the valve moves (and thus by how much the air flow rate will alter) during one sample interval, depending on the initial position of the valve in the valve operating range.",
"Since errors in the air/gas ratio can have negative as well as positive values, it is necessary to establish both positive and negative derivative curves centred around a zero value in order to establish the threshold level.",
"As shown in FIG.",
"8, this effectively produces an “error envelope” within which limit cycling occurs (FIG.",
"8).",
"Thus limit cycling will occur where: |ε(IS, u)|<|δ(u)| (1) where: δ(u) is the derivative of the valve characteristic at any given valve position (u) and 2*δ(u) represents the deadband value; Ts is the sample time; and u is the valve position.",
"Conversely, limit cycling will not occur where: |ε(Ts, u)|≧|δ(u)| (2) In order to reduce or substantially eliminate limit cycling in the air/gas ratio controller, it is therefore desirable to ensure that the air valve is not adjusted when the error lies within the error envelope of the valve.",
"In other words when equation 1 applies.",
"In a preferred form of the invention, this is achieved by implementing a so-called “deadband” as described below.",
"FIG.",
"3 is a schematic block diagram of part of a control system 90 having a preferred form of air/gas ratio controller 100.The controller 100 has a first comparator 102 which is connected to receive two input signals, the first from the gas flow sensor 26 being connected to a non-inverting input of the comparator 102 and the second from the air flow sensor 32 connected to an inverting input.",
"An output of the first comparator 102 is connected firstly to a non-inverting input of a second comparator 104 and secondly to an inverting input of a third comparator 106.Positive and negative fixed threshold value circuits 108, 110; the purpose of which is described below, are connected to non-inverting and inverting inputs of the second and third comparators 104, 106 respectively.",
"An output of each of the second and third comparators 104, 106 is connected to a respective operational amplifier 112, 114.An output of each operational amplifier is connected to a respective relay 116, 118 which actuate movement of the air valve 28.During operation of the combustion plant 10, the flow rates of the gas and the air supplied to the burner 40 are measured by the flow sensors 26, 32 each of which generates a signal Sg, Sa corresponding to the respective flow rate and sends the signal to the air/gas ratio controller 100.The gas flow signal Sg and the air flow signal Sa are fed to the first comparator 102, the gas flow signal Sg to the non-inverting input and the air flow signal S2 to the inverting input.",
"The comparator 102 compares the two signals and generates an error signal ε as a function of the comparison.",
"The error signal ε represents the difference between the actual air flow measured by the sensor 32 and the desired air flow to produce a stoichiometric air/gas ratio with the current gas flow rate.",
"Since the sensor 32 would normally produce an air signal Sa which is a magnitude of 10 greater than the gas signal Sg produced by the gas sensor 26 for a stoichiometric ratio (i.e.",
"an airflow rate which is a magnitude of 10 greater than the gas flow rate) the value of the air signal S2 is adjusted to the same level as the gas signal Sg for a stoichiometric ratio.",
"This can be effected by a simple voltage divider in the air flow sensor 32.The error signal ε is fed to the non-inverting input of the second comparator 104 and to the inverting input of the third comparator 106, each of which compares the error signal ε value with fixed positive and negative threshold values generated by the positive and negative threshold value circuits 108, 110 respectively.",
"If the error signal value is greater than or equal to the positive threshold value, then the comparator 104 applies an actuation signal through the first operational amplifier 112 to the first relay 116 which energises the air valve 28 to move in a first direction, towards its fully closed position.",
"Similarly, if the error signal value is less than or equal to the negative threshold value, the comparator 106 applies an actuation signal through the second operational amplifier 114 to the second relay 116 which energises the air valve 28 to move in the opposite direction towards its fully open position.",
"If, however, the error signal value is less than the positive threshold value and greater than the negative threshold value, the second and third comparators are unaffected and the air valve is not adjusted.",
"The threshold value circuits 108, 110 set an error signal range within which the controller 100 takes no corrective action.",
"Thus, if the gas flow rate is changed in order to increase or decrease the temperature of the burner 40 this will result in an air/gas ratio which moves away from the desired value.",
"This will result in an error signal being generated by the comparator 102, the error signal representing the difference between the actual air/gas ratio and the desired air/gas ratio.",
"It will therefore be appreciated that if the change in the air flow rate which is required to return the air/gas ratio to the desired level is less than the change represented by the error signal range set by the threshold value circuits 108, 110 then the error signal ε will fall within this range and the air valve 28 will remain unactuated.",
"The error is in effect deemed to be zero and the air valve is not adjusted.",
"The threshold range set by the threshold circuits 108, 110 is termed a “deadband”.",
"In practice, this reduces the occurrence of limit cycling in the air flow and allows the desired air/gas ratio to be maintained more closely.",
"The value of the deadband affects the performance of the air/gas ratio controller 100 which in turn affects the efficiency of the combustion plant.",
"Selection of the correct value for the deadband is therefore important.",
"By making the deadband value high, limit cycle oscillations are reduced, but the accuracy of control of the air valve to provide the desired air/gas ratio is reduced.",
"Conversely, a low threshold value gives good accuracy but increases the occurrence of limit cycling.",
"It is preferable, therefore to make the deadband as small as possible, without causing limit cycling.",
"It is apparent from the above description that if the deadband value represents a change in air flow rate which is slightly larger than the movement of the air valve (change in air flow rate) in a single sample interval, then adjustment of the valve can be made without limit cycling occurring.",
"A constant deadband value can therefore be used for valves with linear characteristics.",
"However, for non-linear valves having an error envelope such as that shown in FIG.",
"8, the use of a constant deadband value is ineffective since limit cycling may occur in some parts of the operating range of the valve even though a deadband is used.",
"A solution is to vary the value of the deadband according to the valve characteristic over the valve's operating range.",
"It is found that the optimum deadband value for a given valve position is equal to twice the value of the differential of the valve characteristic at that position Since the deadband is centred around a zero value the upper and lower threshold levels of the deadband (set by the positive and negative threshold circuits 108, 110) correspond to the positive and negative derivative curves of the valve.",
"Thus, the deadband is chosen to map exactly the error envelope of the valve.",
"Thus, the controller will adjust the air valve in the instance where: ∈ ( T δ , u ) ≥ D ( u ) 2 where D(u)=δ(u) and represents the deadband value defined by the error envelope at a given valve position (u) which is the region within which limit cycling does not occur even in the absence of a deadband since the value of an error within which region is greater than or equal to the change in flow caused by adjustment of the valve during one sample interval.",
"Conversely, the controller will not adjust the air valve in the instance where: ∈ ( T δ , u ) < D ( u ) 2 In this case, the error lies within the deadband which is the region in which limit cycling would occur if the air valve were adjusted and the deadband were not present A solution is to vary the value of the deadband in dependence on the valve characteristic over the operating range of the valve.",
"FIG.",
"4 shows a second embodiment of air/gas ratio controller 200 as part of a control system 190.In FIG., 3, 4 and 5 like reference numerals indicate like parts.",
"As can be seen, the controller 200 is similar in form to the controller 100 of FIG.",
"3 but with the fixed threshold value circuits replaced by variable threshold value circuits 208, 210 each of which comprises a look-up table.",
"The variable threshold value circuits 208, 210 are connected to receive a signal from an air valve position sensor 222 via an operational amplifier 220.The valve position sensor 222 can be of the form which simply monitors the voltage applied to the valve to drive the valve between its open and closed positions.",
"Before the control system is put into operation the characteristic of the air valve is measured and the differential curve shown in FIG.",
"7 determined for the valve in order to provide the error envelope shown in FIG.",
"8.A number of different threshold values or levels are then taken from the envelope of FIG.",
"8, a positive and a negative value for selected valve positions.",
"The positive values are stored in the look-up table of the threshold value circuit 208 and the negative values are stored in the look-up table of the threshold value circuit 210.During operation, as the valve position changes, the threshold value in the look-up table which is compared with the error signal is selected according to the position signal from the air valve position sensor.",
"The value generated by each variable threshold value circuit 208, 210 is thus a function of the position of the air valve 28 and thus of the air flow rate.",
"As the position of the air valve varies, the change in the air flow rate which occurs during each sample interval also varies.",
"The air valve characteristics are effectively stored in the look-up table in each threshold circuit 208, 210.The look-up table therefore gives the characterstic at a given valve position and thus determines the deadband value for that position.",
"The deadband is thus varied according to the instantaneous position of the air valve 28.As in the previous embodiment, if the error signal E calculated by comparator 202, lies within the range defied by the instantaneous positive and negative threshold values generated by the threshold circuits 208, 210, then the error is deemed to be zero and no corrective action is made to air valve 28.If, however, the error value lies on or outside the error envelope, the air valve 28 is adjusted as described previously.",
"Since the deadband value is always greater than the change in air flow effected by movement of the air valve during one sample interval, the occurrence of limit cycling is used.",
"In addition, the accuracy of the air/gas ratio controller 200 is increased.",
"This results in a significant improvement in combustion efficiency of the gas combustion plant since the air/gas ratio is maintained at an optimum.",
"It will be apparent that various modifications and improvements can be made to the present invention.",
"The present invention may be modified such that the movement of the air valve 28 is continuously monitored to determine whether the characteristics of the valve have changed owing to wear, for example.",
"If the valve characters tics have changed, this information can be fed to the variable threshold value circuits to modify the deadband value for each position of the valve.",
"An example of such a modification to the present invention is shown in FIG.",
"5 in which like reference numerals indicate like parts.",
"In FIG.",
"5 one of the relays, in this case relay 116 which actuates the valve towards its fully closed position, is connected to an input of a multiplexer 300.An output of the air flow sensor 32 and the valve position sensor 222 are also connected to the multiplexer 300.The output from the multiplexer 300 is connected to a parameter estimator 302 whose output in turn is connected to the variable threshold value circuits 208, 210.The parameter estimator 302 may be a microprocessor running, for example, MATLAB.",
"Before the control system is put into operation the characteristic of the air valve is measured and the response curve shown in FIG.",
"6 is stored in a store in the parameter estimator 302.This can be effected by moving the valve from one of a fully open and closed position to the other and monitoring the signals from the valve position sensor 222 and the air flow rate sensor 32 which are then stored in the parameter estimator 302 as continuously variable values or discrete values.",
"When relay 116 is actuated to move the air flow valve 28 towards its fully closed position the parameter estimator 302 is also enabled During closing of the valve 29 the parameter estimator 302 processes the outputs from the air flow rate and position sensors 32, 222 and compares the monitored flow rate with the previously stored flow rate.",
"If there is a deviation between the monitored flow rate with the previously stored flow rate this would suggest, for example, wear in the valve mechanism.",
"The parameter estimator 302 then adjusts the threshold values in the look up tables in the threshold value circuits 208, 210 which relate to the monitored valve position to ale account of changes in the valve characteristics which have occurred.",
"It will be appreciated that equally the movement of the valve towards its fully open position maybe used to update the look up tables to take account of wear, in which case the estimator 302 would be enabled with relay 118.Whilst the above description is made with reference to a lag-lead control system wherein the lead parameter is the gas flow rate and the lag parameter is the air flow rate, it will be appreciated that the invention is equally applicable to systems wherein the lead parameter is the air flow rate and the lag parameter is the gas flow rate, or any other lag-lead system.",
"It Will also be appreciated that,whilst the preferred form of the invention has been described with reference to an air/gas combustion plant or furnace, the invention is equally applicable to lag-lead control systems for controlling the ratio of two fluids where the fluids may be in gas or liquid form."
]
] | Patent_10399374 |
[
[
"Gel-type polymer electrolyte and use thereof",
"A gel-type polymer electrolyte, wherein said polymer comprises (A) an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof and (B) a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof, which are bonded together by an ester bond.",
"The gel-type polymer electrolyte has a high ionic conductivity, and makes it possible to provides a cell which has excellent charge/discharge characteristics at low temperatures as well as at high temperatures."
],
[
"1.A gel-type polymer electrolyte, wherein said polymer comprises (A) an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof and (B) a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof, which are bonded together by an ester bond.",
"2.A gel-type polymer electrolyte according to claim 1, wherein said ester bond is formed by the esterification of a carboxylic acid group of the ethylene-unsaturated carboxylic acid copolymer with the polyalkylene oxide having a hydroxyl group at one terminal thereof or with a derivative thereof.",
"3.A gel-type polymer electrolyte according to claim 1, wherein said ester bond is formed by the ester interchange reaction of an alkyl ester of the ethylene-unsaturated carboxylic acid copolymer or an alkyl ester derivative thereof with the polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof.",
"4.A gel-type polymer electrolyte according to claim 1, wherein said polymer is formed by reacting the ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) with the polyalkylene oxide having a hydroxyl group at one terminal thereof or the derivative thereof (B) at a-molar ratio expressed by the following formula, BHYD/ACAR wherein BHYD is a number of moles of hydroxyl groups of the polyalkylene oxide having a hydroxyl group at one terminal thereof or of the derivative thereof, and ACAR is a number of moles of carboxylic acid groups of the ethylene-unsaturated carboxylic acid copolymer or of the derivative thereof, of from 0.3 to 2.5.5.A gel-type polymer electrolyte according to claim 1, wherein the remaining amount of the unreacted carboxylic acid groups in said polymer is not larger than 30 mol % on the basis of the carboxylic acid group of the ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A).",
"6.A gel-type polymer electrolyte according to claim 2, wherein said ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) has a composition containing ethylene in an amount of from 50 to 98% by weight, an unsaturated carboxylic acid or an anhydride thereof in an amount of from 2 to 50% by weight, and other monomers in an amount of from 0 to 30% by weight.",
"7.A gel-type polymer electrolyte according to claim 3, wherein the alkyl ester of said ethylene-unsaturated carboxylic acid copolymer or the alkyl ester derivative thereof (A) has a composition containing ethylene in an amount of from 50 to 98% by weight, and an alkyl ester of an unsaturated carboxylic acid or an alkyl ester derivative thereof in an amount of from 2 to 50% by weight.",
"8.A gel-type polymer electrolyte according to claim 7, wherein the alkyl ester of said ethylene-unsaturated carboxylic acid copolymer is a methyl ester or an ethyl ester.",
"9.A gel-type polymer electrolyte according to claim 1, wherein said ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) is an ionomer of which the carboxylic acid is partly neutralized with a monovalent metal or a multi-valent metal, the neutralization degree being in a range of from 0.5 to 60 mole %.",
"10.A gel-type polymer electrolyte according to claim 1, wherein said ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) has a melt flow rate of from 0.1 to 500 g/10 min.",
"at 190° C. under a load of 2160 g. 11.A gel-type polymer electrolyte according to claim 1, wherein the polyalkylene oxide having a hydroxyl group at one terminal thereof or the derivative thereof (B) has a number average molecular weight of from 200 to 100,000 and contains the ethylene oxide in an amount of from 30 to 100 mol %.",
"12.A gel-type polymer electrolyte according to claim 1, wherein a hydroxyl group at the other terminal of the polyalkylene oxide or the derivative thereof (B) is blocked by the etherification, esterification or by the reaction with a monoisocyanate.",
"13.A gel-type polymer electrolyte according to claim 2, wherein the esterification of said ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) with the polyalkylene oxide having a hydroxyl group at one terminal thereof or the derivative thereof (B) is conducted in the presence of an acid catalyst.",
"14.A gel-type polymer electrolyte according to claim 3, wherein the formation of the ester bond by the ester interchange reaction is conducted in the presence of an organometal catalyst.",
"15.A gel-type polymer electrolyte according to claim 1, wherein said polymer is partly crosslinked in the presence of at least one kind of crosslinking agent selected from the group consisting of polyhydric alcohol, mono(meth)acrylic acid or an ester thereof, polyethylene glycol di(meth)acrylate, unsaturated higher fatty acid or an ester thereof and polyethylene glycol diglycidyl ether.",
"16.A gel-type polymer electrolyte according to claim 15, wherein said crosslinking agent is present in the reaction system in an amount of from 0.1 to 30% by weight.",
"17.A gel-type polymer electrolyte according to claim 1, wherein said gel-type polymer takes the form of a powder, a film or a sheet.",
"18.A gel-type polymer electrolyte according to claim 1, wherein said gel-type polymer is impregnated with an electrolytic solution comprising an electrolytic salt and a non-aqueous electrolytic solution.",
"19.A gel-type polymer electrolyte according to claim 18, wherein the solvent exists in the electrolytic solution at a ratio of from 30 to 95% by weight on the basis of the sum of said polymer and said electrolytic solution; 20.A gel-type polymer electrolyte according to claim 18, wherein the electrolyte exists in the electrolytic solution at a ratio of from 1 to 30% by weight on the basis of the sum of said polymer and said electrolytic solution; 21.A gel-type polymer electrolyte according to claim 18, wherein the electrolyte in the electrolytic solution is a lithium salt.",
"22.A gel-type polymer electrolyte according to claim 18, wherein the solvent in the electrolytic solution is a nonaqueous electrolytic solvent.",
"23.A polymer for polymer electrolyte comprising (A) an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof and (B) a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof, the two being bonded together through an ester bond.",
"24.A secondary cell equipped with a layer of a gel-type polymer electrolyte of claim 1.25.A secondary cell according to claim 24, wherein said secondary cell is a lithium cell.",
"26.A capacitor equipped with a layer of a gel-type polymer electrolyte of claim 1."
],
[
"<SOH> TECHNICAL FIELD <EOH>The present invention relates to a gel-type polymer electrolyte comprising a non-halogen type polymer, and, particularly, to a polymer electrolyte that can be molded into a self-supported (self-erected) film and can, particularly, be used for polymer lithium cells suppressing the formation of lithium dendrite (tree-like traces) that occurs on the negative electrode surface when being electrically charged.",
"The electrolyte can further be used for the capacitor."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.",
"1 is a perspective view of a test cell for measuring the ionic conductivity and lithium ion transport number of the polymer electrolytes; FIG.",
"2 is a perspective view of a test cell for testing the charge/discharge characteristics; FIG.",
"3 is a graph showing the results of Example 1, i.e., showing changes in the weight of the polymers immersed in the electrolytic solution with the passage of time (Wo is the weight of the polymer only, and W is the weight of the polymer gel at that time); and FIGS.",
"4 a and 4 b are graphs showing the results of Example 23 (solid line) and results of Comparative Example 5 (broken line), wherein FIG.",
"4 ( a ) illustrates charge/discharge cycle characteristics and FIG.",
"4 ( b ) illustrates a charge/discharge curve at the eighth cycle.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to a gel-type polymer electrolyte comprising a non-halogen type polymer, and, particularly, to a polymer electrolyte that can be molded into a self-supported (self-erected) film and can, particularly, be used for polymer lithium cells suppressing the formation of lithium dendrite (tree-like traces) that occurs on the negative electrode surface when being electrically charged.",
"The electrolyte can further be used for the capacitor.",
"PRIOR ART The lithium secondary cells that have now been placed in the market are using, as electrode materials, a carbon-intercurlated material as a negative electrode and lithium cobaltate (LiCoO2) as a positive electrode to reversibly release and receive lithium ions accompanying the electric charge and discharge.",
"Further, the electrolyte is used being dissolved in a lithium salt.",
"What is generally called polymer lithium cell is a so-called polymer electrolyte cell using a polymer electrolyte instead of a liquid electrolyte.",
"The polymer electrolyte cells are all of the solid type and are free from the leakage of liquid, features high degree of safety, excellent workability, making it possible to decrease the thickness of the cell or to laminate the cells.",
"The polymer electrolyte cell must use a polymer material that exhibits an ionic conductivity of the order of as high as 10−3 S/cm.",
"As for polymerizing the electrolyte, development and study have been forwarded concerning chiefly polyethylene oxide (PEO), polyacrylonitrile (PAN), polymethyl methacrylate (PMMA) and polyvinylidene fluoride (PVDF).",
"In particular, there have been developed several polymer gel electrolytes of the type of PAN and PVDF owing to that the polymer that holds a solution containing 40 to 80%, preferably 40 to 70% of a lithium salt exhibits excellent film strength.",
"In Japan, polymer gel electrolyte lithium cells using fluorine-contained polymer gel electrolyte have already been mass-produced since 1999.Owing to their thin thickness and light weight, the lithium polymer secondary cells have been realized satisfying the conditions for use in mobile equipment and, particularly, cellular phones, note PC, PDA and are reliably finding applications.",
"It is no longer a dream that they can be applied to electric vehicles (EVs) in the near future.",
"This is because, with the conventional liquid cells, the energy density and the output density were not compatible with each other.",
"However, it has recently been learned that the lithium polymer secondary cell is an ideal cell offering a high energy density of not smaller than 165 W/kg and a high output density of not smaller than 1200 W/kg.",
"However, the polymer electrolyte used for the lithium polymer secondary cells must satisfy further improved properties such as (1) it does not leak the liquid, (2) it is flame-resistant, (3) it favorably possess high heat conductivity, (4) it favorably possess high ionic conductivity over a wide temperature range, (5) it has a large mechanical strength and (6) it is chemically inert.",
"As for the fluorine-contained polymer materials that have now been widely used, there remains a problem concerning the treatment after the cells are used, since fluorine is a component constituting the polymer matrix.",
"It has therefore been desired to provide a nonhalogen type polymer electrolytic material having electrolytic properties comparable to, or superior to, those of the fluorine-contained polymer gel electrolyte.",
"Poly(acrylnitrile) (PAN) type and poly(methyl methacrylate) (PMMA) type polymer materials are candidates of the nonhalogen type polymer gel electrolyte.",
"However, these self-supported films are not capable of absorbing and holding large amounts of the electrolytic solution unlike the film of polyvinylidene fluoride (PVDF) type, and cannot be used by being simply sandwiched as a self-supported film electrolyte between the positive electrode and the negative electrode at the time of fabricating the cell.",
"The lithium ion cells placed in the market are using a negative electrode material of carbon capable of intercurlating lithium ions.",
"However, lithium metal secondary cells, too, using a single metal of lithium or an alloy of lithium and other metals as the negative electrode, have been expected as secondary cells that provide large energy densities.",
"At present, however, they have not yet been realized as commercial products being hindered by several problems.",
"Among them, what is most desired to be solved is the problem related to the formation and growth of lithium dendrite accompanying the electric charge and discharge.",
"Even the negative electrode of a lithium-inserted carbon material involves a problem that dendrite occurs under a quickly charging condition.",
"If allowed to grow continuously, lithium dendrite multiplies to cause a short-circuit inside the cell.",
"If an internal short-circuit takes place, a heavy current flows instantaneously through the dendrite producing sparks, combustion occurs, high temperature and high pressure are generated, which may result in the occurrence of explosion.",
"Therefore, a variety of methods have been studied to prevent the internal short-circuit.",
"If the internal short-circuit could be prevented, the life of the cell can be extended and can be further increased.",
"Japanese Unexamined Patent Publication (Kokai) No.",
"167280/1985 discloses a rechargeable electrochemical device that suppresses the occurrence of lithium dendrite by using, as the negative electrode, an alloy of lithium and other metals.",
"Methods have further been studied for suppressing the occurrence of lithium dendrite by using an ion-conducting inorganic solid electrolyte, a polymer gel electrolyte or a solid polymer electrolyte.",
"For example, Oyama et al.",
"reports that the polyacrylonitrile (PAN) gel electrolyte (not less than 5% by weight relative to the non-aqueous solvent) suppresses the occurrence of metal lithium dendrite (Report of the Results of Study, New Energy/Industrial Technology Overall Development Organization (NEDO) of the year 1996, reported on March, 1997).",
"Concerning the new lithium cells, further, it has been desired that the cells swiftly operate within limited charging/discharging time accompanying an increase in the energy density of the cells.",
"In particular, it has been desired to provide cells that work to a sufficient degree at low temperatures.",
"The same characteristics are also desired for the capacitors.",
"Due to their principle of operation, in general, the properties of the cells and capacitors are limited by the migrating speed of ions and distance of migration.",
"In the case of the cells, it is impossible to greatly increase the speed of migration of ions in the electrolyte and in the active substance of electrode.",
"To solve the problem, therefore, the distance of migration of ions must be shortened and the cell must be constituted by using a material having wide reaction areas.",
"In the case of the capacitors, too, the charging/discharging time can be conspicuously shortened if carrier ions move fast.",
"In order to enhance the performance, therefore, the distance between the electrodes must be shortened and the reaction area must be widened like the case of constituting the cells.",
"For this purpose, it is necessary to prepare a thin electrolyte film having a very small thickness and a large mechanical strength.",
"Even when the gel-like polymer is used for forming the electrolyte, first, lithium ions are transported through the electrolyte phase in the polymer matrix.",
"Like in the solution electrolyte, therefore, the reaction current concentrates on a portion on the surface of the negative electrode, and lithium locally precipitates inducing the precipitation of lithium like dendrite.",
"Second, the mechanical strength is weaker than that of the solid polymer.",
"In the conventional totally solid and gel-like polymer electrolytes, the ions were not conducted to a sufficient degree.",
"In the case of the gel-like polymers, further, the liquid could not be favorably retained at high temperatures.",
"DISCLOSURE OF THE INVENTION It is a first object of the present invention to provide a novel polymer electrolyte having a high ionic conductivity, which is useful for forming cells that exhibit excellent charging/discharging characteristics at low temperatures as well as at high temperatures.",
"It is a second object of the present invention to provide a polymer electrolyte capable of suppressing the precipitation of lithium in the form of dendrite.",
"According to the present invention, there is provided a gel-type polymer electrolyte, wherein said polymer comprises (A) an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof and (B) a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof, which are bonded together by an ester bond.",
"The ester bond is formed, for example, by the esterification of a carboxylic acid group of the ethylene-unsaturated carboxyl acid copolymer with the polyalkylene oxide having a hydroxyl group at one terminal thereof or with a derivative thereof, or is formed by the transesterification reaction of an alkyl ester of the ethylene-unsaturated carboxylic acid copolymer or an alkyl ester derivative thereof with the polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof.",
"Hereinafter, the invention is described chiefly with reference to the case of introducing the ester bond by the esterification reaction.",
"When the preferred embodiment differs depending upon the esterification reaction and the transesterification reaction, the introduction of the ester bond by the transesterification reaction will be described each time.",
"In the gel-type polymer electrolyte of the present invention, it is desired that: 1.The ester bond is formed by reacting (esterifying) an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof (A) (compound (A)) with a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof (B) (compound (B)), at a molar ratio (BHYD/ACAR) expressed by the following formula, BHYD/ACAR wherein BHYD is a number of moles of hydroxyl groups of the polyalkylene oxide having a hydroxyl group at one terminal thereof or of the derivative thereof, and ACAR is a number of moles of carboxylic acid groups of the ethylene-unsaturated carboxylic acid copolymer or of the derivative thereof, of from 0.3 to 2.5; or in the case of the transesterification reaction, an alkyl ester of the ethylene-unsaturated carboxylic acid copolymer or an alkyl ester derivative thereof (containing neither carboxyl group nor carboxylic anhydride group) is used as the compound (A) and is reacted (transesterification reaction) with the polyalkylene oxide having a hydroxyl group at one terminal thereof or with the derivative thereof (B) so as to satisfy the above molar ratio (BHYD/ACAR); 2.The remaining amount of the unreacted carboxylic acid groups in the polymer is not larger than 30 mol % on the basis of the carboxylic acid of the ethylene-unsaturated carboxylic acid copolymer or of the derivative thereof (A); or in the case of the transesterification reaction, the amount of the residual carboxylic acid group is substantially 0 mole %; 3.In forming the ester bond by the esterification reaction, the compound (A) has a composition containing ethylene in an amount of from 50 to 98% by weight, an unsaturated carboxylic acid or an anhydride thereof in an amount of from 2 to 50% by weight, and other monomers in an amount of from 0 to 30% by weight; or in the case of the transesterification reaction, the compound (A) has a composition containing ethylene in an amount of from 50 to 98% by weight, and an alkyl ester of an unsaturated carboxylic acid or an alkyl ester derivative thereof in an amount of from 2 to 50% by weight; 4.The ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) is an ionomer of which the carboxylic acid is partially neutralized with a monovalent metal or a multi-valent metal, the neutralization degree being from 0.5 to 60 mole %; 5.The ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) has a melt flow rate of from 0.1 to 500 g/10 min.",
"at 190° C. under a load of 2160 g; 6.The polyalkylene oxide having a hydroxyl group at one terminal thereof or the derivative thereof (B) has a number average molecular weight of from 200 to 100,000 and contains the ethylene oxide in an amount of from 30 to 100 mol %; 7.A hydroxyl group at the other terminal of the polyalkylene oxide or of the derivative thereof (B) is blocked by the etherification, esterification or by the reaction with a monoisocyanate; 8.The esterification of the compound (A) with the compound (B) is conducted in the presence of an acid catalyst; or the transesterification of the compound (A) with the compound (B) is conducted in the presence of an organometal catalyst (particularly, a metal alkoxide); 9.The polymer takes the form of a powder, a film or a sheet; and 10.The esterification reaction or the transesterification reaction is conducted in the presence of a crosslinking agent of a small amount (pre-crosslinking), and the polymer has been partially crosslinked.",
"According to the present invention, the gel-type polymer electrolyte is generally used in a form in which the polymer matrix is impregnated with an electrolytic solution.",
"In this case, it is desired that: 1.The solvent exists in the electrolytic solution at a ratio of from 30 to 95% by weight, preferably 30 to 90% by weight, on the basis of the sum of said polymer and said electrolytic solution; 2.The electrolyte exists in the electrolytic solution at a ratio of from 1 to 30% by weight on the basis of the sum of said polymer and said electrolytic solution; 3.The electrolyte species in the electrolytic solution is a lithium salt; and 4.The solvent in the electrolytic solution is a nonaqueous electrolytic solvent.",
"In having the electrolytic solution absorbed, further, the polymer may be blended with a crosslinking agent such as an acrylic derivative to have the electrolytic solution absorbed and to obtain the gel polymer in a form that is crosslinked (after-crosslinking).",
"According to the present invention, further, there is provided a secondary cell and, particularly, a lithium secondary cell equipped with a layer of the gel-type polymer electrolyte.",
"According to the present invention, further, there is provided a capacitor equipped with the layer of the gel-type polymer electrolyte.",
"BRIEF DESCRIPTION OF DRAWINGS FIG.",
"1 is a perspective view of a test cell for measuring the ionic conductivity and lithium ion transport number of the polymer electrolytes; FIG.",
"2 is a perspective view of a test cell for testing the charge/discharge characteristics; FIG.",
"3 is a graph showing the results of Example 1, i.e., showing changes in the weight of the polymers immersed in the electrolytic solution with the passage of time (Wo is the weight of the polymer only, and W is the weight of the polymer gel at that time); and FIGS.",
"4a and 4b are graphs showing the results of Example 23 (solid line) and results of Comparative Example 5 (broken line), wherein FIG.",
"4(a) illustrates charge/discharge cycle characteristics and FIG.",
"4(b) illustrates a charge/discharge curve at the eighth cycle.",
"BEST MODE FOR CARRYING OUT THE INVENTION In order to solve the above-mentioned problems, the present invention pays attention to polyolefin materials which are chief components of the separator material, but which were not so far regarded as the electrolyte.",
"These materials, however, are not capable of absorbing and holding an organic solvent that is used for the lithium secondary cell.",
"The present inventors have previously discovered that a diacrylate compound having oligo(oxyethylene) groups at both terminals thereof and grafted with PMMA, exhibits a property of transporting lithium ions, and that compatibility is markedly enhanced between the polymer chain thereof and the electrolytic solution (patent application therefor is pending, Laid-Open Patent Publication No.",
"189166/2001).",
"The present inventors have further expanded the discovery, paid attention-to a carboxylic acid-containing polyethylene, i.e., to an ethylene-unsaturated carboxylic acid (inclusive of unsaturated acid anhydride thereof) copolymer as a polyolefin, have succeeded in esterifying it with the carboxylic acid (or acid anhydride thereof) by using a polyethylene oxide having hydroxyl group at one terminal thereof or a derivative thereof to introduce the polyethylene oxide like a comb into the polyethylene side chains relying on the ester bond, i.e., have succeeded in synthesizing a polyethylene unsaturated carboxylic acid copolymer grafted with the polyethylene oxide, and have studied electrolytic properties of the polymer.",
"As a result, the inventors have discovered that the polymer can be easily formed into a self-supported film and exhibits properties for absorbing and holding large amounts of the electrolytic solution used in the lithium ion cells.",
"The inventors have further discovered that the polymer having properties same as those described above is obtained even when the ester bond is introduced by the transesterification reaction with the polyethylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof by using an alkyl ester of an ethylene-unsaturated carboxylic acid copolymer or an alkyl ester derivative thereof, instead of relying upon the above esterification reaction.",
"[Polymer Matrix] The polymer material used for the gel-type polymer electrolyte of the present invention comprises, as a basic constitution, an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof (A) and a polyalkylene oxide having a hydroxyl group at one terminal thereof or a derivative thereof (B), which are bonded (grafted) together by an ester bond.",
"That is, the polymer material is the one in which the polyalkylene oxide is introduced like a comb into the ethylene-unsaturated carboxylic acid copolymer by the ester bond.",
"Preferably, the polymer material is obtained by esterifying the ethylene-unsaturated carboxylic acid copolymer with the polyalkylene oxide having a hydroxyl group at one terminal thereof.",
"It is, of course, allowable to obtain the polymer material by introducing the polyalkylene oxide (single-terminated alkoxypolyalkylene oxide) by the transesterification reaction by using a derivative of the ethylene-unsaturated carboxylic acid ester copolymer (alkyl ester or alkyl ester derivative).",
"(1) Ethylene-unsaturated carboxylic acid copolymer or Derivatives Thereof (A) It is desired that the ethylene-unsaturated carboxylic acid copolymer or the derivative thereof (A) (hereinafter often referred to as compound (A)) has a composition containing an ethylene in an amount of from 50 to 98% by weight and, particularly, from 60 to 95% by eight, an unsaturated carboxylic acid in an amount of from 2 to 50% by weight and, particularly, from 5 to 25% by weight, and other monomers in an amount of from 0 to 30% by weight and, particularly, from 0 to 20% by weight.",
"When the ethylene content becomes too small in the copolymer, the resin exhibits an increased melting temperature and a decreased melt fluidity, which is not desirable in the esterification reaction with the polyalkylene oxide from the standpoint of temperature that must be elevated and the stirring efficiency.",
"Examples of the unsaturated carboxylic acid include acrylic acid, methacrylic acid, ethacrylic acid, fumaric acid, maleic acid, itaconic acid, monomethyl maleate, monoethyl maleate, maleic anhydride and itaconic anhydride.",
"Among them, it is most desired to use acrylic acid or methacrylic acid.",
"As other monomers that are arbitrarily added for constituting the ethylene-unsaturated carboxylic acid copolymer, there can be exemplified vinyl esters such as vinyl acetate and vinyl propionate; unsaturated carboxylic acid esters such as methyl acrylate, ethyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, isooctyl acrylate, methyl methacrylate, isobutyl methacrylate, dimethyl maleate and diethyl maleate; as well as carbon monoxide and sulfur dioxide.",
"In the present invention, the compound (A) used for the transesterification method is an alkyl ester of the ethylene-unsaturated carboxylic acid copolymer or an alkyl ester derivative thereof.",
"Further, the unsaturated carboxylic acid ester or a derivative constituting the compound (A) is the above-mentioned unsaturated carboxylic acid ester or a derivative thereof and, particularly, methyl ester or ethyl ester.",
"In the present invention, the ethylene-unsaturated carboxylic acid copolymer itself may be used, but it is still also allowable to use a derivative thereof, such as an ionomer in which some carboxyl groups are replaced by a metal salt.",
"As a metal of a metal salt constituting the ionomer, there can be exemplified a monovalent metal such as lithium, sodium or potassium, or a multi-valent metal such as magnesium, calcium, zinc, copper, cobalt, manganese, lead or iron.",
"Lithium salt is preferably used for the polymer electrolyte material for the polymer lithium cells.",
"When the ionomer has a too large degree of neutralization, it becomes difficult to obtain a homogeneous composition when the polyalkylene oxide is grafted thereon.",
"It is therefore desired that the ionomer has a degree of neutralization of from 0.5 to 60 mol % and, particularly, a degree of neutralization of as low as from 1 to 30 mol %.",
"The ethylene-unsaturated carboxylic acid copolymer, according to the present invention, is obtained by the radical copolymerization of an ethylene with an unsaturated carboxylic acid or with other monomers under the conditions of a high temperature and elevated pressure.",
"The metal ionomer is obtained by neutralizing the above copolymer with a metal salt under the conditions that have been known per se.",
"It is desired that the above ethylene-unsaturated carboxylic acid copolymer or the ionomer thereof has a melt flow rate of from 0.1 to 500 g/10 min.",
"and, particularly, from 0.5 to 300 g/10 min.",
"at 190° C. under a load of 2160 g. It is desired that the weight average molecular weight (Mw) reckoned as polystyrene molecular weight is from 2,000 to 800,000 and, particularly, from 4,000 to 500,000.The above weight average molecular weight (Mw) corresponds to a number average molecular weight (Mn) of from 500 to 150,000 and, preferably, from 1,000 to 100,000.In the present invention, further, the ethylene-unsaturated carboxylic acid copolymers or derivatives thereof of two or more kinds may be used being blended together.",
"(2) Polyalkylene oxide Having a hydroxyl Group at one Terminal Thereof or Derivatives Thereof (B) The polyalkylene oxide having a hydroxyl group at one terminal thereof (often referred to as compound (B)) has the hydroxyl group at the other end thereof that is blocked and, desirably, has a number average molecular weight of from 200 to 100,000 and contains an ethylene oxide in an amount of 30 to 100 mol %.",
"As the comonomer, there is used propylene oxide, n-butylene oxide and/or isobutylene oxide, and there can be used an ethylene oxide and a random copolymer thereof, a grafted copolymer thereof or a block copolymer thereof as the compound (B).",
"It is allowed that the polyalkylene oxide contains the propylene oxide component in an amount of from 0 to 60 mol % and a butylene oxide component in an amount of from 0 to 20 mol %.",
"From the standpoint of structure, it may be a linear homopolymer of which the one terminal is blocked, a copolymer and a branched grafted product based on a polyhydric alcohol (such as glycerin, pentaerythritol).",
"In this case, other hydroxyl groups are stoichiometrically blocked such that each molecule has a hydroxyl group.",
"It is also allowable to use, in combination, two or more kinds of the polyalkylene oxides having a hydroxyl group at one terminal thereof.",
"The hydroxyl group at the other terminal can be blocked by etherification, esterification or by the reaction with a monoisocyanate.",
"In the case of the etherification, there is used, as a substituent, an alkyl group having 1 to 22 carbon atoms (e.g., ethyl group, hexyl group, 2-ethylhexyl group, etc.",
"), phenyl group and benzyl group.",
"In the case of the esterification, there is used a carboxylic acid having 1 to 22 carbon atoms (e.g., acetic acid, maleic acid or terephthalic acid) and a carboxylic anhydride (e.g., maleic anhydride).",
"In blocking the hydroxyl group at one terminal with the monoisocyanate, there is used a methyl isocyanate or a phenyl isocyanate.",
"To obtain the polymer electrolyte comprising a partially crosslinked polymer according to the present invention, a polyhydric alcohol having two or more hydroxyl groups (e.g., glycerin, pentaerythritol, etc.)",
"may be added as a crosslinking agent (pre-crosslinking) prior to effecting the esterification or the etherification.",
"The amount of addition of the crosslinking agent may differ depending upon the molecular weight of the compound (A) or (B) but is, usually, from 0.1 to 30% by weight and, particularly, from 1 to 15% by weight per the sum of the compounds (A) and (B).",
"(3) Grafting and Polymer It is desired that the esterification reaction of the ethylene-unsaturated carboxylic acid copolymer into a carboxylic acid by using the polyalkylene oxide having a hydroxyl group at one terminal thereof (B), is conducted by the bulk reaction without using solvent but using an acid catalyst.",
"Described below next is the basic operation of the esterification reaction.",
"First, an acid catalyst is added to the polyalkylene oxide having a hydroxyl group at one terminal thereof or to the derivative thereof (compound (B)) under the heated and stirring conditions, followed by the addition of an ethylene-unsaturated carboxylic acid copolymer or a derivative thereof (compound (A)) to conduct the reaction for several tens of hours.",
"After the reaction, the reaction product is washed with water and by being immersed in methanol or ethanol to remove unreacted polyalkylene oxide and catalyst, and is dried in vacuum.",
"It is desired that the compounds (A) and (B) are fed at a molar ratio (BHYD/ACAR) expressed by the following formula, BHYD/ACAR wherein BHYD is a number of moles of hydroxyl groups of the polyalkylene oxide of having a hydroxyl group at one terminal thereof or of the derivative thereof, and ACAR is a number of moles of carboxylic acid groups of the ethylene-unsaturated carboxylic acid copolymer or of the derivative thereof (here, however, the carboxylic acid group includes not only a carboxyl group but also a carboxylic ester group and a carboxylic anhydride group, the carboxylic anhydride group being calculated as two carboxyl groups), of from 0.3 to 2.5 and, particularly, from 0.9 to 2.0 (the same also holds even in,the case of the transesterification).",
"It is desired that the reaction temperature is from 80 to 160° C. and, particularly, from 100 to 140° C. Desirably, the reaction time is from 7 to 40 hours and, particularly, from 10 to 30 hours.",
"The acid catalyst for the esterification is desirably sulfuric acid, phosphoric acid, polyphosphoric acid, paratoluenesulfonic acid, benzenesulfonic acid, xylenesulfonic acid or dodecylbenzenesulfonic acid, and the amount of addition is from 0.0001 to 0.1 mole and, particularly, from 0.001 to 0.05 moles per mole of the hydroxyl group.",
"When the transesterification reaction is to be conducted, there can be used, as a catalyst, for example, a metal alkoxide (sodium methoxide, potassium ethoxide, rare earth triisopropoxide, potassium t-butoxide, titanium tetrabutoxide, dibutyltin oxide, etc.",
"), a sodium hydroxide, a potassium hydroxide or a heteropoly-acid (molybdo-phosphoric acid, tungsto-phosphoric acid, etc.).",
"The amount of addition is preferably from 0.0001 to 0.5 mols and, particularly, from 0.001 to 0.1 mol per mole of the hydroxyl groups.",
"The reaction system is maintained under a reduced pressure (e.g., 0.067 MPa=500 mmHg) to efficiently carry out the esterification reaction.",
"In this esterification reaction, it is also allowable to use an organic solvent (e.g., toluene, xylene, ethylene or glycol diethyl ether) and an antioxidizing agent (e.g., hydroquinone, hydroquinone monomethyl ether).",
"The same also holds for the transesterification reaction.",
"It is desired that the amount of addition of the antioxidizing agent is from 100 to 10,000 ppm and, particularly, from 500 to 5,000 ppm with respect to the compound (B).",
"The polymer material used in the present invention is melted alone at a high temperature or is melted at a high temperature in combination with a nonvolatile organic solvent, and is molded into a self-supported film or sheet by means that has been known per se.",
"such as casting method, T-die method or inflation method.",
"[Gel-Type Polymer Electrolyte] The gel-type polymer electrolyte of the present invention comprises a matrix of the above polymer and an electrolytic solution with which the matrix is impregnated.",
"In the gel-type polymer electrolyte of the present invention, it is desired that the solvent exists in the electrolytic solution in an amount of, usually, from 30 to 95% by weight and, particularly, preferably from 30 to 90% by weight, especially preferably from 60 to 90% by weight on the basis of the sum of the polymer and the electrolytic solution.",
"When the amount of the solvent is too small, the ionic conductivity decreases.",
"When the amount of the solvent is too large, the strength of the electrolyte tends to decrease.",
"It is further desired that the electrolyte exists in the electrolytic solution in an amount of, usually, from 1 to 30% by weight and, particularly, from 1 to 20% by weight on the basis of the sum of the polymer and the electrolytic solution.",
"When the amount of the electrolyte is larger or is smaller than the above range, the ionic conductivity tends to decrease.",
"The present invention uses any electrolyte salt that is usually used for the gel-type polymer electrolytes of this kind.",
"When the polymer electrolyte of the invention is used for the lithium polymer secondary cell, there is preferably used a lithium salt such as CF3SO3Li, C4F6SO3Li, (CF3SO2)2NLi, (CF3SO2)3CLi, LiBF4, LiPF6, LiClO4 or LiAsF6 as an electrolytic salt, to which only, however, the invention is in no way limited.",
"As the non-aqueous electrolytic solvent (organic solvent), there can be used any non-aqueous solvent that is usually used for the gel-type polymer electrolyte of this type.",
"For example, there can be used at least any one solvent selected from the group consisting of cyclic carbonates such as ethylene carbonate and propylene carbonate; dipropyl carbonates such as dimethyl carbonate, methyl ethyl carbonate, diethyl carbonate and dipropyl carbonate; cyclic esters such as γ-butylolactone and propiolactone; chain-like ethers such as diethoxyethane and dimethoxyethane; amide compounds such as dimethylacetamide, etc.",
"; nitrile compounds such as acetonitrile, propionitrile, etc.",
"; N-methylpyrrolidione, etc.",
"; and mixture solutions thereof.",
"The invention, however, is in no way limited to these examples only, as a matter of course.",
"In the present invention, the electrolytes and the non-aqueous solvents are in no way limited to the above-mentioned examples only.",
"For example, when the polymer electrolyte of the present invention is used for the capacitor, there is used, as the electrolyte, a salt in which lithium of the above lithium salt is substituted by other alkali metal ion (sodium ion, potassium ion, cesium ion or the like ion), an ammonium salt such as tetraalkylammonium, a tetraalkylfluoroborate ((C2H5)4NBF4, etc), a tetraalkylphosphonium fluoroborate ((C2H5)4PBF4, etc), a tetraalkylphosphonium trifluorosulfonate ((C2H5)4PCF3SO3, etc), an alkylpyridinium salt and an N-alkylimidazole salt.",
"As the non-aqueous solvent in this case, there is preferably used an organic solvent having a donor number of not smaller than 0.1 and a dielectric constant of not smaller than 10.0.Examples of such an organic solvent include acid anhydrides such as acetic anhydride; amide compounds such as dimethylformamide and dimethyl sulfoxide; phosphate compounds such as trimethyl phosphate and tributyl phosphate; and amine compounds such as hydrazine, ethylenediamine, ethylamine and triethylamine, in addition to the above-mentioned solvents for the secondary cells.",
"In the present invention; the polymer is molded into a film or a sheet, and the polymer is impregnated with the electrolytic solution in to any order.",
"For example, the film or the sheet of the polymer prepared through the above-mentioned means is immersed in a solution containing the above-mentioned electrolyte salt to obtain a polymer-electrolyte.",
"At this moment, the temperature of the electrolytic solution is controlled to change the rate of impregnation.",
"It is desired that the polymer electrolyte of the present invention is obtained by impregnating a thin polymer film that has been synthesized in advance with the electrolytic solution.",
"According to another method, the above-mentioned polymer material and the above-mentioned electrolytic solution are mixed together, and are heated up to more than 80° C. to obtain a molten solution thereof, which is then casted into a sheet to use it as the polymer electrolyte.",
"For example, a solution of an N-methylpyrrolidione containing 1 mole/L of a lithium tetrafluoroborate using, as a solvent, a chain-like carbonate such as dimethyl carbonate, and the above-mentioned polymer material are mixed together at a weight ratio of (1.5 to 3.5):1, and are heated up to more than 80° C. to obtain a transparent solution.",
"This solution is casted onto the polyethylene terephthalate film and is pressurized to obtain an electrolyte film having a predetermined thickness.",
"In mixing and heating the above polymer material and the electrolytic solution to prepare a molten solution in the present invention, further, it is further allowable to add a crosslinking agent as is widely done even in the polyethylene oxide polymers to promote gelling and to increase the mechanical strength [Reference literature: Industrial Materials, 47(2), 18 (1999) and Yuasa Jiho No.",
"87, Oct. 4 (1999)].",
"For example, the polymer material of the present invention may be blended with a diacrylate compound having an oligo(oxyethylene) group at both terminals, and is dissolved together with the electrolytic solution to carry out the thermal polymerization (after-crosslinking).",
"Further, the polymer material of the present invention has a polyethylene chain.",
"Therefore, the electron-beam (EB) crosslinking and the UV crosslinking that are usually conducted for crosslinking the gel polymer electrolytes, can also be applied to the polymer electrolyte of the present invention.",
"(Use) The gel-type polymer electrolyte of the present invention is useful as a solid electrolyte layer for the secondary cells such as lithium secondary cells and as a solid electrolyte layer for the capacitors.",
"There is no particular limitation on the structure of these secondary cells or the capacitors if they are provided with a layer of the gel-type polymer electrolyte of the present invention.",
"In the case of the lithium secondary cells, it is desired to interpose a thin layer of the gel-type polymer electrolyte of the present invention between the lithium metal negative electrode and the positive electrode active substance (e.g., LiCoO2).",
"The gel-type polymer electrolyte of the present invention has a thickness of, generally, from 1 μm to 1 mm and, particularly, from 5 μm to 0.3 mm, and can be laminated as a layer in the secondary cells or in the capacitors.",
"The secondary cell of the present invention can be modified in a variety of ways as a matter of course.",
"In the liquid electrolyte-type lithium ion cells, for example, a porous film of polyolefin such as polyethylene is used as a separator and is imparted with a function for cutting off the electric current by utilizing the melting property of the polyethylene when heated.",
"The separator film is prepared by blending the polyethylene with the polypropylene, or is formed of a film obtained by laminating the polyethylene and the polypropylene one upon the other.",
"Therefore, the separator film favorably melts together with the polymer material of the present invention.",
"By adhering the above-mentioned electrolyte film on one surface or on both surfaces of the separator, or by applying a molten solution of the polymer electrolyte onto one surface or both surfaces of the separator to use them as a laminated film, there is obtained a film having a separator function and an electrolytic function.",
"In recent years, further, there has been produced, as a separator, a film of a three-layer structure by arranging a fine porous film of polyethylene between the two fine porous films of polypropylene.",
"By mixing the polymer material of the present invention into the polyethylene layer or by applying the polymer material of the invention onto one surface or two surfaces of the polyethylene layer, there can be obtained a polymer electrolyte film having a separator function and stably holding the electrolytic solution therein.",
"Instead of using the separator, further, the polymer material may be directly applied to one electrode followed by after-crosslinking so as to be sandwiched by the other electrode.",
"Various surface-reforming technologies (chemical modification technologies) can be applied to the surfaces of the polymer material of the present invention to improve the property for retaining electrolytic solution, and to improve adhesiveness to the surface of the negative electrode material, to the surface of the positive electrode material and to the surface of the separator.",
"Besides, a chemical modification technology can be employed for the polymer electrolyte itself.",
"In particular, the surfaces of the film or the polymer electrolyte itself can be reformed by applying a thermally or optically crosslinking agent onto the surface of the film of the invention or by mixing the thermally or optically crosslinking agent at the time of dissolving the electrolyte in connection with the after-crosslinking followed by the irradiation with heat or light.",
"It is further allowable to reform the surfaces by the irradiation with an electron beam (EB).",
"By effecting the pre-crosslinking as described above, further, it is allowed to omit or simplify the step of after-crosslinking.",
"EXAMPLES The invention will be further described by way of working examples which, however, are in no way to limit the present invention.",
"Preparation Example 1 183 Gram of a polyethylene glycol monomethyl ether PEG-MME (number average molecular weight of 550, 0.33 mol of hydroxyl groups) and 1.5 g (0.0079 mol) of a hydrate of paratoluenesulfonic acid (molecular weight of 190) were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and, while blowing nitrogen (0.1 m3/hr) through a capillary tube, 100 g of an ethylene-acrylic acid copolymer (0.28 mol of carboxylic acid group, molar ratio (BHYD/ACAR) of hydroxyl group/carboxylic acid group=1.18) was gradually added while confirming the dissolving state (over about 30 minutes).",
"After the addition has been finished, the mixture was reacted at the same temperature for 24 hours.",
"After the reaction, the reaction product was taken out into another container and, after cooled, was cut into a size of about 1 cm3.500 ml of ethanol was added thereto at room temperature.",
"After immersed for 70 hours, the ethanol solution was removed by decantation.",
"Another 500 ml of ethanol was added thereto, and the same operation was repeated.",
"After washed in ethanol, the esterified product was taken out and was dried in a vacuum drier.",
"Observation of FTIR spectrum of the obtained esterified product indicated almost no absorption peak (νCO=1700 cm−1) due to the carbonyl group of the unreacted free carboxylic acid.",
"The obtained esterified product was regarded to be a polymer electrolyte material A.",
"Composition of the polymer electrolyte material A (% by weight): E/AA/PEG-MME=31.5/0.0/68.5 (E: ethylene AA: acrylic acid) Preparation Example 2 154 Gram of the polyethylene glycol monomethyl ether PEG-MME (number average molecular weight of 550, 0.28 mol of hydroxyl group) and 1.28 g (0.0067 mol) of a hydrate of paratoluenesulfonic acid were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and 100 g of an ethylene-acrylic acid copolymer (0.28 mol of carboxylic acid, BHYD/ACAR=1.00) was gradually added.",
"Then, the operation same as that of preparation Example 1 was repeated but changing the reaction time from 24 hours into 16 hours.",
"Observation of FTIR spectrum of the esterified product indicated, as a shoulder, an absorption peak (νCO=1700 cm−1) due to the carbonyl group of the unreacted free carboxylic acid.",
"Due to the titration with KOH, there remained 5 mol % of carboxylic acid of ethylene-acrylic acid copolymer (0.014 mol in 0.28 mol of carboxylic acid).",
"The titration with KOH was conducted by heating and dissolving 5 g of the sample in 200 g of a solution of toluene/ethanol=¼, and effecting the titration in a 0.1 N KOH aqueous solution by using a phenolpthalein indicator.",
"The obtained product was regarded to be a polymer electrolyte material B.",
"Composition of the polymer electrolyte material B (% by weight): E/AA/PEG-MME=32.5/0.4/67.1 Preparation Example 3 123 Gram of the polyethylene glycol monomethyl ether PEG-MME (number average molecular weight of 550, 0.224 mols of hydroxyl group) and 1.02 g. (0.0054 mol) of a hydrate of paratoluenesulfonic acid were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and 100 g of an ethylene-acrylic acid copolymer (0.28 mols of carboxylic acid, BHYD/ACAR=0.8) was gradually added.",
"The operation same as that of preparation Example 1 was repeated but changing the reaction time from 24 hours into 16 hours.",
"Observation of FTIR spectrum of the esterified product indicated, as a shoulder, an absorption peak (νCO=1700 cm−1) due to the carbonyl group of the unreacted free carboxylic acid.",
"Due to the titration with KOH, there remained 24 mol % of carboxylic acid of ethylene-acrylic acid copolymer (0.067 mols in 0.28 mols of carboxylic acid).",
"The obtained product was regarded to be a polymer electrolyte material C. Composition of the polymer electrolyte material C (% by weight): E/AA/PEG-MME=36.8/2.2/61.0 Preparation Example 4 248 Gram of a polyethylene glycol monomethyl ether PEG-MME (number average molecular weight of 750, 0.33 mol of hydroxyl groups) and 1.5 g (0.0079 mol) of a hydrate of paratoluenesulfonic acid (molecular weight of 190) were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and, while blowing nitrogen (0.1 m3/hr) through a capillary tube, 100 g of an ethylene-acrylic acid copolymer (0.28 mols of carboxylic acid, BHYD/ACAR=1.18) was gradually added while confirming the dissolving state (over about 30 minutes).",
"After the addition has been finished, the mixture was reacted at the same temperature for 24 hours.",
"After the reaction, the reaction product was taken out into another container and, after cooled, was cut into a size of about 1 cm3.500 ml of ethanol was added thereto at room temperature.",
"After immersed for 70 hours, the ethanol solution was removed by decantation.",
"Another 500 ml of ethanol was added thereto, and the same operation was repeated.",
"After immersed in ethanol, the esterified product was taken out and was dried in a vacuum drier.",
"Observation of FTIR spectrum of the obtained product indicated almost no absorption peak (νCO=1700 cm−1) due to the carbonyl group of the unreacted free carboxylic acid.",
"The esterified product was regarded to be a polymer electrolyte material D. Composition of the polymer electrolyte material D (% by weight): E/AA/PEG-MME=32.3/0.0/67.7 Preparation Example 5 277 Gram of a polyethylene glycol-block-polypropylene glycol mono-2-methylhexyl ether (PEG-b-PPG-MEHE)(number average molecular weight of 840, polyethylene glycol of 50% by weight, 0.33 mol of hydroxyl group) and 0.63 g (0.0033 mols) of a hydrate of paratoluenesulfonic acid (molecular weight of 190) were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and, while blowing nitrogen (0.1 m3/hr) through a capillary tube, 100 g of an ethylene-acrylic acid copolymer (0.28 mol of carboxylic acid group, BHYD/ACAR=1.18) was gradually added.",
"After the addition has been finished, the mixture was reacted at the same temperature for 30 hours.",
"After the reaction, the reaction product was taken out into another container and, after cooled, was cut into a size of about 1 cm3.500 ml of ethanol was added thereto at room temperature.",
"After immersed for 48 hours, the ethanol solution was removed by decantation.",
"Another 500 ml of ethanol was added thereto, and the same operation was repeated.",
"After immersed in ethanol, the esterified product was taken out and was dried in a vacuum drier.",
"Observation of FTIR spectrum of the obtained esterified product indicated almost no absorption peak (νCO=1700 cm−1) due to the carbonyl group of the unreacted free carboxylic acid.",
"The obtained esterified product was regarded to be a polymer electrolyte material E. Composition of the polymer electrolyte material E (% by weight): E/AA/PEG-b-PPG-MEHE=23.9/0.0/76.1 Table 1 shows the composition ratios (% by weight) of the polymer electrolyte materials A to E prepared in Preparation Examples 1 to 5.TABLE 1 Acrylic PEG- PEG-b- Polymer Ethylene acid MME PPG-MEHE Prep Ex.",
"1 A 31.5 0.0 68.5 — Prep Ex.",
"2 B 32.5 0.4 67.1 — Prep Ex.",
"3 C 36.8 2.2 61.0 — Prep Ex.",
"4 D 32.3 0.0 67.7 — Prep Ex.",
"5 E 23.9 0.0 — 76.1 Preparation Example 6 Transesterification 154 Gram of a polyethylene glycol monomethyl ether (PEG-MME)(number average molecular weight of 550, 0.28 mol of hydroxyl group), 1.90 g (0.028 mol) of a sodium ethoxide (catalyst) and 50 g of a xylene (solvent) were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and, while blowing nitrogen (0.1 m3/hr) through a capillary tube, 56 g of an ethylene-ethyl acrylate copolymer (ethyl acrylate, 25% by weight; melt index, 250 g/10 min; ethyl acrylate, 0.14 mols; BHYD/ACAR=2.0) was gradually added thereto (over a time of 15 minutes).",
"After the addition has been finished, a pipe for cooling the solvent and for lapping was attached, and the reaction was conducted at the same temperature for 30 hours while blowing nitrogen through a capillary tube.",
"When the temperature has dropped down to not higher than 100° C. after the reaction, the reaction mixture was added dropwise to another container containing 1000 ml of water while stirring the reaction mixture to obtain a slurry.",
"After the dropwise addition has been finished, the slurry was stirred for about one hour and was, then, left to stand still.",
"A granular solid was separated by decantation and was introduced into a container containing 500 ml of methanol, stirred for 2 hours and was, then, left to stand still.",
"The mixture was filtered and washed with methanol again to repeat the same operation.",
"After immersed in methanol, the transesterified product was taken out and was dried in a vacuum drier.",
"From the ratio of FTIR spectral absorbence of the obtained product (grafted PEG-MME carboxylic acid ester, ν=1731 cm−1) and the polyethylene (ν=720 cm−1) of the internal reference, the transesterification ratio was 89%.",
"The esterified product was regarded to be a polymer electrolyte material F. Preparation Example 7 Partially Crosslinked Polymer Material 193 Gram of a polyethylene glycol monomethyl ether (PEG-MME)(number average molecular weight of 550, 0.35 mol of hydroxyl group),3.0 g (0.0058 mol) of a polyethylene glycol diacrylate (ethylene oxide recurring unit n=9, molecular weight of 520) and 1.5 g (0.0079 mole) of a hydrate of paratoluenesulfonic acid (molecular weight of 190) were introduced into a 500-ml four neck flask.",
"The mixture was heated up to 140° C. with stirring, and, while blowing nitrogen (0.1 m3/hr) through a capillary tube, 63 g of an ethylene-acrylic acid copolymer (0.18 mol of carboxylic acid group, BHYDACAR=2.0) was gradually added thereto (over a time of about 20 minutes) while confirming the dissolving state.",
"After the addition has been finished, the mixture was reacted at the same temperature for 24 hours.",
"After the reaction, the reaction mixture was added dropwise to another container containing 1000 ml of water with stirring.",
"After cooled, the precipitate was filtered by using a filtering cloth, and to which 1000 ml of ethanol was added at room temperature, and then the precipitate was immersed therein for 20 hours.",
"The ethanol solution was then removed by decantation.",
"Another 500 ml of ethanol was added thereto, and the same operation was repeated.",
"After immersed and washed in ethanol, the partially crosslinked and esterified product was taken out and was dried in a vacuum drier.",
"The obtained product was regarded to be a polymer electrolyte material G. To 10 g of the polymer electrolyte material was added 90 g of a mixture solution of a propylene carbonate (PC) and an ethylene carbonate (EC); PC:EC=1:1 (weight ratio), containing 1 mol/L of LiPF6, and the mixture was heated at 80° C. for 3 hours so as to be homogeneously dissolved, and was cooled down to room temperature to obtain a gel-like polymer electrolyte containing an electrolyte.",
"FIG.",
"1 illustrates a measuring system used for measuring the ionic conductivity and the lithium ion transport number of the polymer electrolyte in the working examples described below.",
"FIG.",
"1 shows the structure of a measuring cell and a connection between the cell and the measuring equipment.",
"The measuring cell has a constitution in which a predetermined polymer electrolyte is inserted between the two pieces of lithium metal foils having a thickness of 0.2 mm and an electrode area of 2.0×2.0 cm2.Nickel foils are inserted among the glass plate and the lithium metal foils to accomplish an electric connection.",
"The measuring device is a potentio/galvanostat electrolytic device (Model 1287 manufactured by Solatron Co.).",
"The impedance is measured by connecting a frequency response analyzer (Model 1250 manufactured by Solatron Co.) to the electrolytic device.",
"FIG.",
"2 illustrates a measuring system used for measuring the charging/discharging characteristics of a lithium secondary cell using a predetermined polymer electrolyte.",
"FIG.",
"2 shows the structure of a measurement cell and a connection between-the cell and the measurement equipment.",
"The measuring cell has a constitution in which a predetermined polymer gel electrolyte is inserted between the lithium metal negative electrode and the positive electrode formed by applying a lithium cobaltate which is a positive electrode active substance onto both surfaces of an aluminum foil in an amount of 327.0 g/m2, the aluminum foil having a thickness of 20 μm and an electrode area of 2.0×2.0 cm2.Nickel foils are inserted among the glass plate and the electrodes to accomplish an electric connection.",
"The measuring device is BS-2500 manufactured by Keisoku Giken.",
"Examples 1 to 6 <Electrolyte-Absorbing Property of Polymer> The polymer electrolyte material of the present invention obtained in Preparation Example 1 was immersed in a predetermined electrolytic solution at room temperature (25° C.) for two days.",
"The weight of the polymer gel (polymer+electrolytic solution) was measured before and after the immersion to find the absorbed amount of the electrolytic solution.",
"The results were as shown in Table 2.TABLE 2 Polymer electrolyte material A. Polymer: Supporting Electrolyte Electrolyte solution salt (weight ratio) Ex.",
"1 dimethyl carbonate 1M LiBF4 37:63 Ex.",
"2 diethyl carbonate same as above 58:42 Ex.",
"3 dimethoxyethane same as above 24:76 Ex.",
"4 propylene carbonate + ethylene same as above 30:70 carbonate Ex.",
"5 γ-Butylolactone same as above 32:68 Ex.",
"6 N-methylpyrrolidione same as above 46:54 Example 7 <Swelling Property of Polymer Gel> The polymer electrolyte material A of the present invention was immersed in a mixture solution of the propylene carbonate and the ethylene carbonate (PC:EC=1:1, containing 1 mol/L of LiPF6) used in Preparation Example 7, heated at 80° C. for 3 hours, and the temperature was then lowered down to room temperature to measure the mass of the polymer gel with the passage of the time.",
"After immersed for 3 hours, swelling of the polymer gel has terminated.",
"At this moment, the solution was contained in an amount of 83.5% by weight, and there was obtained the polymer electrolyte holding the electrolyte in a sufficiently large amount.",
"In this case, the 100 μm-thick film acquired the thickness of 200 to 250 μm after having been impregnated with the polymer electrolyte, which was an increase of 2 to 2.5 times.",
"Example 8 <Swelling Property of Polymer Gel> The polymer electrolyte material A of the present invention was immersed in a mixture solution (the one used in Example 7 above) of the propylene carbonate and the ethylene carbonate at room temperature, and the mass of the polymer gel was measured with the passage of the time.",
"After immersed for 3 hours, swelling of the polymer gel has terminated.",
"At this moment, the solution was contained in an amount of 61.3% by weight.",
"Example 9 <Swelling Property of Polymer Gel> The polymer electrolyte material A of the present invention was immersed in a mixture solution of an ethylene carbonate (EC) and a dimethyl carbonate (DMC) [EC:DMC=1:2 (weight ratio), containing 1 mol/L of LiPF6], heated at 80° C. for 3 hours, and the temperature was then lowered down to room temperature to measure the mass of the polymer gel with the passage of the time.",
"After immersed for 3 hours, swelling of the polymer gel has terminated.",
"At this moment, the solution was contained in an amount of 88.6% by weight.",
"Thus, there was obtained a polymer electrolyte holding the electrolyte in a sufficiently large amount.",
"Example 10 <Swelling Property of Polymer Gel> The polymer electrolyte material A of the present invention was immersed in a mixture solution of the ethylene carbonate and the dimethyl carbonate used in Example 9 at room temperature, and the mass of the polymer gel was measured with the passage of the time.",
"After immersed for 3 hours, swelling of the polymer gel has terminated.",
"At this moment, the solution was contained in an amount of 72.4% by weight.",
"The results of Examples 7, 8, 9 and 10 were as shown in FIG.",
"3 and in Table 3.TABLE 3 Content of electrolyte after 3 Signs Electrolyte Immersion hours of of Ex.",
"No.",
"Polymer solution temp immersion 7 A 1M LiPF6 PC + EC(1:1) 80° C.(3 hr) 83.5% by wt.",
"▴ 8 A 1M LiPF6 PC + EC(1:1) 25° C. 61.3% by wt.",
"▾ 9 A 1M LiPF6 PC + DMC(1:2) 80° C.(3 hr) 88.6% by wt.",
"▪ 10 A 1M LiPF6 PC + DMC(1:2) 25° C. 72.4% by wt.",
"● Examples 11 to 14 <Ionic Conductivity and Lithium ion Transport Number—Depending Upon the Electrolytes> The polymer electrolyte material A of the present invention and the polymer electrolytes of various electrolytic solutions were measured for their ionic conductivities and lithium ion transport numbers at room temperature (25° C.).",
"These polymer electrolytes exhibited ionic conductivities above 1×10−3 S cm−1 and lithium ion transport numbers of about 0.2, which were favorable properties as electrolytes.",
"The results were as shown in Table 4.The ionic conductivities and the lithium ion transport numbers of the polymer electrolytes were calculated in accordance with the following formulas.",
"Ionic conductivity=bulk resistance/distance between electrodes (thickness of polymer electrolyte) Lithium ion transport number=Is(dV−I0Re0)/I0(dV−IsRes) I0=dV/(Re0+Rb0) I0: current before the constant-voltage electrolysis Re0: interfacial resistance before the constant-voltage electrolysis Rb0: bulk resistance before the constant-voltage electrolysis Is: current after the constant-voltage electrolysis Res: interfacial resistance after the constant-voltage electrolysis dV: voltage applied for electrolysis TABLE 4 Li ion Supporting Ionic transport salt Solvent conductivity number Ex.",
"11 1M LiBF4 PC + EC(1:1) 1.7 × 10−3 S/cm 0.14 Ex.",
"12 1M LiBF4 EC + DMC(1:2) 1.4 × 10−3 S/cm 0.17 Ex.",
"13 1M LiPF6 PC + EC(1:1) 1.5 × 10−3 S/cm 0.12 Ex.",
"14 1M LiPF6 EC + DMC(1:2) 5.2 × 10−3 S/cm 0.09 note: 10−3 means exp.−3.Examples 15 to 20 <Ionic Conductivity and Lithium ion Transport Number—Depending Upon the Kind of Polymers> For polymer electrolytes obtained by impregnating the polymer electrolyte materials B to G obtained in Preparation Examples 2 to 7 with a mixture solution of a propylene carbonate and an ethylene carbonate (PC:EC=1:1, weight ratio, and containing 1 mol/L of LiBF4), their ionic conductivities and lithium ion transport numbers at room temperature were measured.",
"These polymer electrolytes exhibited ionic conductivities above 1×10−3 S cm−1 and lithium ion transport numbers of about 0.2, which were favorable properties as polymer electrolytes.",
"The results were as shown in Table 5.TABLE 5 Li ion Poly- Electrolyte Ionic transport mer solution conductivity number Ex.",
"15 B 1M LiBF4 PC + EC(1:1) 1.5 × 10−3 S/cm 0.20 Ex.",
"16 C 1M LiBF4 PC + EC(1:1) 1.4 × 10−3 S/cm 0.20 Ex.",
"17 D 1M LiBF4 PC + EC(1:1) 1.5 × 10−3 S/cm 0.19 Ex.",
"18 E 1M LiBF4 PC + EC(1:1) 7.8 × 10−3 S/cm 0.08 Ex.",
"19 F 1M LiBF4 PC + EC(1:1) 1.2 × 10−3 S/cm 0.20 EX.",
"20 G 1M LiBF4 PC + EC(1:1) 2.9 × 10−3 S/cm 0.18 Comparative Examples 1 to 4 <Ionic Conductivity and Lithium ion Transport Number—Depending Upon the Kind of Polymers> For the polymer electrolytes obtained by impregnating a polyvinylidene fluoride/hexafluoropropylene copolymer (abbreviated as P(VDF-HFP) and manufactured by Aldrich Co.) with mixture solutions of propylene carbonate and ethylene carbonate used in Examples 15 to 18, for their ionic conductivities and lithium ion transport numbers at room temperature were measured.",
"The results were as shown in Table 6.TABLE 6 Content Comp.",
"of Li ion Ex.",
"electrolyte Ionic transport No.",
"Polymer solution Supporting salt Conductivity number 1 P(VDF-HFP) 70% 1M LiBF4 PC + EC(1:1) 1.7 × 10−3 S/cm 0.20 2 P(VDF-HFP) 65% 1M LiBF4 PC + EC(1:1) 1.5 × 10−3 S/cm 0.20 3 P(VDF-HFP) 60% 1M LiBF4 PC + EC(1:1) 1.3 × 10−3 S/cm 0.21 4 P(VDF-HFP) 50% 1M LiBF4 PC + EC(1:1) 0.7 × 10−3 S/cm 0.22 From Examples 15 to 20 and Comparative Examples 1 to 4, it was demonstrated that the polymer materials of the present invention possessed the properties comparable to those of the PVDF polymer electrolyte.",
"Examples 21 to 23 <Ionic Conductivities—Temperature Dependence> For the polymer electrolytes obtained by impregnating the polymer electrolyte materials B to D of the present invention with a mixture solution of the propylene carbonate and the ethylene carbonate used in Example 15, their ionic conductivities at temperatures of −20, −10, 0, 10, 25, 40, 55, 70 and 85° C. were measured.",
"All of these polymer gel electrolytes exhibited ionic conductivities above 1×10−3 S cm−1 at temperatures above 10° C. It was thus demonstrated that favorable ionic conductivities were obtained even at low temperatures.",
"The results were as shown in Table 7.TABLE 7 Ionic conductivity (mS/cm) Polymer −20° C. −10° C. 0° C. 10° C. 25° C. 40° C. 55° C. 70° C. 85° C. Ex.",
"21 B 0.40 0.49 0.70 1.03 1.53 2.09 2.79 3.36 4.34 Ex.",
"22 C 0.47 0.54 0.82 1.13 1.69 2.26 2.73 3.73 5.17 Ex.",
"23 D 0.36 0.43 0.66 0.93 1.38 1.93 2.69 3.73 4.29 Example 24 <Effect for Suppressing the Formation of Dendrite> The electrolysis was conducted at a constant current of 3 mA/cm2 for one hour and, then, the interface between lithium and the polymer electrolyte was observed by using a CCD camera.",
"The surface of the lithium metal electrode was smooth, and it was learned that the polymer electrolyte of Example 15 suppressed dendrite formation to a conspicuous degree on the interface of the lithium electrode.",
"Example 25 <Charge/Discharge Cycle Characteristics> A test cell was fabricated by using lithium cobaltate applied onto an aluminum collector as a positive electrode, a lithium metal as a negative electrode and the polymer electrolyte material B of Example 15, and was subjected to the charging/discharging testing at 20° C. The cell was electrically charged in a CC mode at a current density of 1.025 mA/4 cm2 while setting the cut-off voltage to be 4.3 V, and was discharged at a current density of 1.025 mA/4 cm2 while setting the cut-off voltage to be 2.5 V. The secondary cell produced an output voltage of as high as 3.8 to 4.0 V and a high coulomb efficiency, from which it was learned that the polymer electrolyte of the present invention can be used as an excellent electrolyte material for the high-performance lithium secondary cells.",
"The results were as shown in FIG.",
"4.This cell maintained not less than 90% of the initial characteristics even after charge/discharge operation was repeated more than 100 times, proving the polymer electrolyte to be highly stable.",
"Example 26 <Charge/Discharge Cycle Characteristics> Experiment same as that of Example 25 was conducted by using the polymer electrolyte material D of Example 17 and nearly the same charge/discharge characteristics as those of Example 25 was obtained.",
"Reference Example <Charge/Discharge Cycle Characteristics> The polymer electrolyte of Example 16 was used for the test cell same as that of Example 25.The charge/discharge characteristics deteriorated with an increase in the number of cycles.",
"This was presumably due to that the electrolyte was affected by the unreacted acrylic acid remaining in the synthesis of the polymer.",
"It is therefore desired to use the polymer material that has been completely esterified.",
"Example 27 <Charge/Discharge Cycle Characteristics> The polymer electrolyte material B prepared in Preparation Example 2 was impregnated with a mixture solution of the propylene carbonate and the ethylene carbonate used in Example 15 (containing 1 mol/L of LiBF4).",
"The film was then heated at 85° C. to prepare a transparent solution.",
"The solution was then applied onto both surfaces of a polyolefin separator (trade name: TN0028 or TN0029) of Asahi Kasei Co, pressurized, and cooled to obtain a polymer electrolyte film having a thickness of about 100 to 150 μm.",
"By using this film as an electrolyte, a test cell same as that of Example 23 was prepared.",
"In this case, too, the charge/discharge characteristics were nearly the same as those of Example 25.Example 28 A polymer electrolyte was prepared by impregnating the polymer material A of Example 2 with a γ-butylolactone containing 1 mol/L of (C2H5)4NCF3SO3.Nickel substrates were used as both electrodes which were then coated with ruthenium oxide and polyaniline film (10 μm thick).",
"A test cell was fabricated by using these electrodes and the above polymer electrolyte to measure the capacitor characteristics.",
"The obtained electric amount was 0.3 F/cm2.To evaluate the properties, the test capacitor was electrically charged by a constant-current method at a 10 C rate.",
"As a result of charge/discharge testing, a discharge capacity of 99% of the charged capacity could be obtained, from which it was learned that the charge and discharge could be accomplished at a high rate.",
"It was learned that excellent capacitor characteristics were exhibited when the polymer electrolyte was prepared by using, in combination, a solvent having a high dielectric constant (ε=39) and a high boiling point (202° C./room temperature) like γ-butylolactone and a salt (C2H5)4NCF3SO3.EFFECT OF THE INVENTION The present invention provides a novel olefinic polymer electrolyte material having a high ionic conductivity (having a lithium ion transport number comparable to that of PVDF), suppressing the precipitation of dendrite-like lithium, offering a favorable electrolyte solution-absorbing quality (retaining property), and making it possible to fabricate a cell that features excellent charge/discharge characteristics at low temperatures as well as at high temperatures."
]
] | Patent_10399377 |
[
[
"Urotensin-II agonists and antagonists",
"The present invention features a novel class of cyclic polypeptides that have U-II antagonist and agonist activity.",
"The invention also features methods for treating physiological or psychological conditions characterized by an excess or under expression of Ur4otensin-II."
],
[
"1.A polypeptide or a variant thereof, said polypeptide having the formula: (R1)a-AA1-cyclo[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 wherein AA1 is the L isomer of an aromatic amino acid; AA2 is the L or D isomer of Cys; AA3 is an L isomer of an aromatic amino acid; AA4 is the L or D isomer of Trp; AA5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R1 is H, lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R2 is OH, OR3, N(R3)2 or NHR3, where R3 is H, a lower alkyl, or arylalkyl; provided said peptide is not Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-NH2; or a pharmaceutically acceptable salt of said polypeptide or variant.",
"2.The polypeptide of claim 1, wherein said aromatic amino acid has the formula: wherein X is H or a bond, and Ar represents a moiety selected from the group consisting of wherein n is 0, 1, 2, or 3 and each substituent Y independently represents NO2, CN, Cl, Br, I, F, Me, COR4, COOR4, or OR4, groups, where R4 is H or C1-C8 alkyl.",
"3.The polypeptide of claim 1, wherein AA3 is selected from the group consisting of Phe, Trp, Pal, His, β-Nal, 3-pyridyl-Ala, 4-pyridyl-Ala, 2,4-dichloro-phe, pentafluoro-Phe, p-Z-Phe, and o-Z-Phe, wherein Z is selected from the group consisting of Me, Cl, Br, F, OH, OMe, and NO2.4.The polypeptide of claim 1, wherein AA4 is L-Trp.",
"5.The polypeptide of claim 1, wherein AA2 is D-Cys.",
"6.The polypeptide of claim 5, wherein AA3 is Phe, AA4 is Trp, AA5 is Lys, AA6 is Thr, AA7 is Val, and AA1 is Cpa.",
"7.The polypeptide of claim 6, wherein said polypeptide has the formula Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]-Val-NH2 (SEQ ID NO: 5).",
"8.A pharmaceutical composition comprising a polypeptide, or a variant thereof, and a pharmaceutically acceptable carrier, said polypeptide having the formula: (R1)a-AA1-cyclo[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 wherein AA1 is the L isomer of an aromatic amino acid; AA2 is the L or D isomer of Cys; AA3 is an L isomer of an aromatic amino acid; AA4 is the L or D isomer of Trp; AA5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R1 is H, lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R2 is OH, OR3, N(R3)2 or NHR3, where R3 is H, a lower alkyl, or arylalkyl; provided said peptide is not Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-NH2; or a pharmaceutically acceptable salt of said polypeptide or variant.",
"9.The pharmaceutical composition of claim 8, wherein said aromatic amino acid has the formula: wherein X is H or a bond, and Ar represents a moiety selected from the group consisting of wherein n is 0, 1, 2, or 3 and each substituent Y independently represents NO2, CN, Cl, Br, I, F, Me, COR4, COOR4, or OR4, groups, where R4 is H or C1-C8 alkyl.",
"10.The pharmaceutical composition of claim 8, wherein AA3 is selected from the group consisting of Phe, Trp, Pal, His, β-Nal, 3-pyridyl-Ala, 4-pyridyl-Ala, 2,4-dichloro-phe, pentafluoro-Phe, p-Z-Phe, and o-Z-Phe, wherein Z is selected from the group consisting of Me, Cl, Br, F, OH, OMe, and NO2.11.The pharmaceutical composition of claim 8, wherein AA4 is L-Trp.",
"12.The pharmaceutical composition of claim 8, wherein AA2 is D-Cys.",
"13.The pharmaceutical composition of claim 12, wherein AA3 is Phe, AA4 is Trp, AA5 is Lys, AA6 is Thr, AA7 is Val, and AA1 is Cpa.",
"14.The pharmaceutical composition of claim 13, wherein said polypeptide has the formula Cpa-c[D-Cys-Phe-Tip-Lys-Thr-Cys]-Val-NH2, (SEQ ID NO: 5).",
"15.The pharmaceutical composition of claim 8, wherein said carrier is selected from the group consisting of saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.",
"16.A method of preventing or treating an abnormal condition characterized by an excess of Urotensin-II activity, said method comprising administering to a subject a therapeutically effective amount of a polypeptide, or variant thereof, said polypeptide having the formula: (R1)a-AA1-cyclo[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 wherein AA1 is the L isomer of an aromatic amino acid; AA2 is the L or D isomer of Cys; AA3 is an L isomer of an aromatic amino acid; AA4 is the L or D isomer of Trp; AA5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R1 is H, lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R2 is OH, OR3, N(R3)2 or NHR3, where R3 is H, a lower alkyl, or arylalkyl; or a pharmaceutically acceptable salt thereof.",
"17.The method of claim 16, wherein said condition is selected from the group consisting of ischaemic heart disease, congestive heart failure, portal hypertension, variceal bleeding, hypotension, angina pectoris, myocardial infarction, ulcers, anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.",
"18.The method of claim 17, wherein said condition is ischaemic heart disease.",
"19.The method of claim 17, wherein said condition is congestive heart failure.",
"20.The method of claim 17, wherein said condition is portal hypertension 21.The method of claim 17, wherein said condition is variceal bleeding.",
"22.A method of modulating the effect of a Urotensin-II (U-II) peptide, said method comprising administering to a subject a polypeptide, or variant thereof, said polypeptide having the formula: (R1)a-AA1-cyclo[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 wherein AA1 is the L isomer of an aromatic amino acid; AA2 is the L or D isomer of Cys; AA3 is an L isomer of an aromatic amino acid; AA4 is the L or D isomer of Trp; AA5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R1 is H, lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R2 is OH, OR3, N(R3)2 or NHR3, where R3 is H, a lower alkyl, or arylalkyl; or a pharmaceutically acceptable salt thereof.",
"23.The method of claim 22, wherein said modulating comprises decreasing the effect of said U-II peptide.",
"24.A urotensin II agonist polypeptide, or variant thereof, said polypeptide having the formula: Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys)-Val-OH (SEQ ID NO: 3).",
"25.A method of modulating the effect of a Urotensin-II (U-II) peptide, said method comprising administering to a subject the polypeptide of claim 24.26.The method of claim 25, wherein said modulating comprises increasing the effect of said U-II peptide.",
"27.A method of preventing or treating an abnormal condition characterized by an under expression of Urotensin-II activity, said method comprising administering to a subject a therapeutically effective amount of the polypeptide of claim 24."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Urotensin-II (U-II) is a cyclic neuropeptide with potent cardiovascular effects.",
"Originally isolated from the caudal neurosecretory system of teleost fish, the primary structure of U-II has been established for several species of vertebrates, including various fish species, frogs, and humans.",
"Sequence analysis of various U-II peptides from different species has revealed that, while the N-terminal region is highly variable, the C-terminal cyclic region of U-II is strongly conserved.",
"Indeed, this cyclic region, which is responsible for the biological activity of U-II, is fully conserved from fish to humans (Coulouran, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA (physiology), 95:15803-15808 (1998)).",
"The fact that evolutionary pressure has acted to fully conserve the biologically active sequence of U-II suggests that this polypeptide plays an important role in human physiology.",
"The cyclic region of U-U includes six amino acid residues (-Cys-Phe-Trp-Lys-Tyr-Cys-(SEQ ID NO: 1)) and is structurally similar to the biologically important central region of somatostatin-14 (-Phe-Trp-Lys-Thr-(SEQ ID NO: 2)).",
"However, molecular cloning and sequence analysis of the carp preprourotensin II gene suggests that U-II and somatostatin are not derived from a common ancestor (Ohsako, S., et al., J.",
"Neurosci., 6:2730-2735 (1986)).",
"In fish, U-II peptides have been shown to exhibit several activities, including general smooth muscle contracting activity, although responses vary between species and vascular beds (Davenport, A., and Maquire, J., Trends in Pharmacological Sciences, 21:80-82 (2000); Bern, H. A., et al., Recent Prog.",
"Horm.",
"Res., 45:533-552 (1995)).",
"Fish U-II has also been shown to possess constrictor activity in mammals, including major arteries in rats, but the receptor(s) mediating these peptide actions are not fully characterized.",
"Recent studies have reported that an orphan human G-protein-coupled receptor, homologous to the rat GPR14 and expressed predominantly in cardiovascular tissue, functions as an U-II receptor (Ames, H., et al., Nature, 401:282-286 (1999)).",
"Fish (goby) and human U-II reportedly bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization.",
"Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates (Ames, H., et al., supra).",
"The potency of vasoconstriction of U-II is substantially greater than that of endothelin-1, making human U-II one of most potent mammalian vasoconstrictors currently known.",
"In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction (Ames, H., et al., supra).",
"Since human U-II-like immunoreactivity is found within cardiac and vascular tissue (including coronary atheroma), U-II is believed to influence cardiovascular homeostasis and pathology (e.g., ischemic heart disease and congestive heart failure).",
"Furthermore, the detection of U-II immunoreactivity within spinal cord and endocrine tissues suggests that U-II may have additional activities, including modulation of central nervous system and endocrine function in humans (Ames, H., et al., supra).",
"Indeed, a number of maladies have been potentially linked to an excess or an under expression of U-II activity, including ischemic heart failure, hypotension, portal hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders.",
"Thus, there is a strong need for the development of potent compounds capable of modulating U-II activity, including U-II inhibitors or antagonists."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention features a novel class of cyclic polypeptides that have U-II antagonist activity.",
"The polypeptides of the invention are octapeptides having the general formula: (R 1 ) a -AA 1 -cyclo[AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -Cys]-AA 7 -R 2 (Formula I), wherein AA 1 is the L isomer of an aromatic amino acid; AA 2 is the L or D isomer of Cys; AA 3 is an L isomer of an aromatic amino acid; AA 4 is the L or D isomer of Trp; AA 5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA 6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA 7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R 1 is H, a lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R 2 is OH, OR 3 , N(R 3 ) 2 , or NHR 3 , where R 3 is H, a lower alkyl, or arylalkyl; provided that the peptide is not Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-NH 2 .",
"In a preferred embodiment, AA 2 and AA 4 are D-Cys and L-Trp, respectively.",
"In another preferred polypeptide, AA 1 is Cpa, AA 2 is D-Cys, AA 3 is Phe, AA 4 is Trp, AA 5 is Lys, AA 6 is Thr, and AA 7 is Val.",
"In a particularly preferred embodiment, the polypeptide is an octapeptide having the formula Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]-Val-NH 2 .",
"The invention also provides a Urotensin-II agonist polypeptide, and variants thereof, having the formula Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH.",
"The polypeptides of the present invention are capable of altering U-II activity and can affect the binding of U-II to a receptor.",
"Thus, these polypeptides may be administered to a subject as a means for preventing or treating medical or psychological conditions characterized by an excess or deficiency or under expression of Urotensin-It activity.",
"Such conditions include, but are not limited to, ischaemic heart disease, congestive heart failure, portal hypertension, variceal bleeding, hypotension, angina pectoris, myocardial infarction, ulcers, anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.",
"The present invention also provides pharmaceutical compositions that include a therapeutically effective amount of a polypeptide of Formula I in combination with a pharmaceutically acceptable carrier.",
"Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.",
"The composition can be adapted for the mode of administration and can be in the form of a pill, tablet, capsule, spray, powder, or liquid.",
"Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims."
],
[
"FIELD OF THE INVENTION The invention relates to urotensin-II polypeptide agonists and antagonists and methods of their use.",
"BACKGROUND OF THE INVENTION Urotensin-II (U-II) is a cyclic neuropeptide with potent cardiovascular effects.",
"Originally isolated from the caudal neurosecretory system of teleost fish, the primary structure of U-II has been established for several species of vertebrates, including various fish species, frogs, and humans.",
"Sequence analysis of various U-II peptides from different species has revealed that, while the N-terminal region is highly variable, the C-terminal cyclic region of U-II is strongly conserved.",
"Indeed, this cyclic region, which is responsible for the biological activity of U-II, is fully conserved from fish to humans (Coulouran, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA (physiology), 95:15803-15808 (1998)).",
"The fact that evolutionary pressure has acted to fully conserve the biologically active sequence of U-II suggests that this polypeptide plays an important role in human physiology.",
"The cyclic region of U-U includes six amino acid residues (-Cys-Phe-Trp-Lys-Tyr-Cys-(SEQ ID NO: 1)) and is structurally similar to the biologically important central region of somatostatin-14 (-Phe-Trp-Lys-Thr-(SEQ ID NO: 2)).",
"However, molecular cloning and sequence analysis of the carp preprourotensin II gene suggests that U-II and somatostatin are not derived from a common ancestor (Ohsako, S., et al., J.",
"Neurosci., 6:2730-2735 (1986)).",
"In fish, U-II peptides have been shown to exhibit several activities, including general smooth muscle contracting activity, although responses vary between species and vascular beds (Davenport, A., and Maquire, J., Trends in Pharmacological Sciences, 21:80-82 (2000); Bern, H. A., et al., Recent Prog.",
"Horm.",
"Res., 45:533-552 (1995)).",
"Fish U-II has also been shown to possess constrictor activity in mammals, including major arteries in rats, but the receptor(s) mediating these peptide actions are not fully characterized.",
"Recent studies have reported that an orphan human G-protein-coupled receptor, homologous to the rat GPR14 and expressed predominantly in cardiovascular tissue, functions as an U-II receptor (Ames, H., et al., Nature, 401:282-286 (1999)).",
"Fish (goby) and human U-II reportedly bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization.",
"Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates (Ames, H., et al., supra).",
"The potency of vasoconstriction of U-II is substantially greater than that of endothelin-1, making human U-II one of most potent mammalian vasoconstrictors currently known.",
"In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction (Ames, H., et al., supra).",
"Since human U-II-like immunoreactivity is found within cardiac and vascular tissue (including coronary atheroma), U-II is believed to influence cardiovascular homeostasis and pathology (e.g., ischemic heart disease and congestive heart failure).",
"Furthermore, the detection of U-II immunoreactivity within spinal cord and endocrine tissues suggests that U-II may have additional activities, including modulation of central nervous system and endocrine function in humans (Ames, H., et al., supra).",
"Indeed, a number of maladies have been potentially linked to an excess or an under expression of U-II activity, including ischemic heart failure, hypotension, portal hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders.",
"Thus, there is a strong need for the development of potent compounds capable of modulating U-II activity, including U-II inhibitors or antagonists.",
"SUMMARY OF THE INVENTION The present invention features a novel class of cyclic polypeptides that have U-II antagonist activity.",
"The polypeptides of the invention are octapeptides having the general formula: (R1)a-AA1-cyclo[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 (Formula I), wherein AA1 is the L isomer of an aromatic amino acid; AA2 is the L or D isomer of Cys; AA3 is an L isomer of an aromatic amino acid; AA4 is the L or D isomer of Trp; AA5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R1 is H, a lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R2 is OH, OR3, N(R3)2, or NHR3, where R3 is H, a lower alkyl, or arylalkyl; provided that the peptide is not Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-NH2.In a preferred embodiment, AA2 and AA4 are D-Cys and L-Trp, respectively.",
"In another preferred polypeptide, AA1 is Cpa, AA2 is D-Cys, AA3 is Phe, AA4 is Trp, AA5 is Lys, AA6 is Thr, and AA7 is Val.",
"In a particularly preferred embodiment, the polypeptide is an octapeptide having the formula Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]-Val-NH2.The invention also provides a Urotensin-II agonist polypeptide, and variants thereof, having the formula Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH.",
"The polypeptides of the present invention are capable of altering U-II activity and can affect the binding of U-II to a receptor.",
"Thus, these polypeptides may be administered to a subject as a means for preventing or treating medical or psychological conditions characterized by an excess or deficiency or under expression of Urotensin-It activity.",
"Such conditions include, but are not limited to, ischaemic heart disease, congestive heart failure, portal hypertension, variceal bleeding, hypotension, angina pectoris, myocardial infarction, ulcers, anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.",
"The present invention also provides pharmaceutical compositions that include a therapeutically effective amount of a polypeptide of Formula I in combination with a pharmaceutically acceptable carrier.",
"Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.",
"The composition can be adapted for the mode of administration and can be in the form of a pill, tablet, capsule, spray, powder, or liquid.",
"Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.",
"Definitions By “polypeptide” is meant any peptide (including cyclic peptides) or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.",
"“Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins.",
"Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.",
"“Polypeptides” include amino acid sequences modified either by natural processes, or by chemical modification techniques which are well known in the art.",
"Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.",
"The notations used herein for the polypeptide amino acid residues are those abbreviations commonly used in the art.",
"The less common abbreviations Abu, Cpa, Nle, Pal, Tle, Dip, 4-Fpa, and Nal stand for 2-amino-butyric acid, p-chloroPhenylalanine, norleucine, 3-pyridyl-2-alanine, tert-leucine, 2,2-diphenylalanine, 4-fluoro-phenylalanine, and 3-(2-naphthyl)-alanine or 3-(1-naphthyl)-alanine, respectively By “alkyl” is meant an aliphatic branched or straight chain hydrocarbon group.",
"An alkyl is optionally substituted with one or more substituents which may be the same or different, and include, but are not limited to, halo, cycloalkyl, hydroxy, alkoxy, amino, carbamoyl, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkyloxycarbonyl, or heteroaralkyloxycarbonyl groups.",
"Representative alkyl groups include, but are not limited to, methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, methoxyethyl, and carboxymethyl.",
"By “lower alkyl” is meant a branched or straight chain alkyl group having less than 11 carbon atoms, preferably a C1-C8 alkyl.",
"By “acyl” is meant a group having the structure wherein R is H or an alkyl group as described herein.",
"By “lower acyl” is meant an acyl group having less than 11 carbon atoms (either branched or straight chain), preferably between 1-8 carbon atoms (i.e., R is H or a lower alkyl).",
"By “lower alkanoyl” is meant an acyl group as described above wherein R is a lower alkyl.",
"By “aryl” is meant a monocyclic or bicyclic aromatic group containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, napthyl or tetrahydronaphthyl.",
"By “arylalkyl” is meant an alkyl group as described herein having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.",
"By “pharmaceutically acceptable salt” is meant non-toxic acid addition salts or metal complexes which are commonly used in the pharmaceutical industry.",
"Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.",
"Metal complexes include zinc, iron, and the like.",
"By “variant” is meant a polypeptide that differs from a reference polypeptide, but retains essential properties.",
"Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.",
"A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions, in any combination.",
"A substituted or inserted amino acid residue may or may not be one encoded by the genetic code.",
"A variant of a polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.",
"Non-naturally occurring variants of polypeptides may be made by mutagenesis techniques or by direct synthesis.",
"Generally, the variant differs from the reference polypeptide by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics (e.g.",
"acidic, basic, aromatic, etc.).",
"Typical substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.",
"By “subject” is meant an animal or human suffering from a U-II-related physiological or psychological condition.",
"The subject may be a mammal, including, but not limited to, humans and non-human mammals such as primates, dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, and the like.",
"By “pharmaceutically acceptable carrier” is meant a carrier that is physiologically acceptable to an administered animal while retaining the therapeutic properties of the compound with which it is administered.",
"One exemplary pharmaceutically acceptable carrier is physiological saline.",
"Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18th edition), ed.",
"A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. By “aromatic amino acid” is meant an amino acid that contains an aromatic group.",
"In preferred embodiments, the aromatic amino acid has the following formula: (Formula II), where X represents a bond or H, and Ar is a moiety containing an optionally substituted aromatic ring.",
"Examples of Ar, include but are not limited to, the following structures wherein Y11 represents n optional substituents and n is 0, 1, 2, or 3: In preferred embodiments, each substituent Y independently represents NO2, CN, Cl, Br, I, F, Me, COR4, COOR4, or OR4, groups, where R4 is H or C1-C8 alkyl.",
"Examples of aromatic amino acids include, but are not limited to, Phe, Cpa, Trp, Pal, His, β-Nal, 3-pyridyl-Ala, 4-pyridyl-Ala, 2,4-dichloro-phe, pentafluoro-Phe, p-Z-Phe, and o-Z-Phe, wherein Z is selected from the group consisting of Me, Cl, Br, F, OH, OMe, and NO1.DETAILED DESCRIPTION We found that the minimum portion of the U-II sequence which retained full biological activity was the octapeptide Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH (SEQ ID NO: 3), which corresponds to hUII(4-7).",
"This octapeptide actually possess greater potency than the full human and fish U-II sequences in inducing rat aorta contraction and in binding to this tissue.",
"Based on this parent sequence, a series of cyclic octapeptides have been synthesized which have U-II antagonist activity.",
"These peptides were discovered to have moderate affinity for U-II receptors and were able to block U-II-induced phasic contracts in circular rat thoracic aorta strips.",
"The polypeptides of the present invention have the general formula: (R1)a-AA1-cyclo[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 (Formula I), wherein AA1 is the L isomer of an aromatic amino acid; AA2 is the L or D isomer of Cys; AA3 is an L isomer of an aromatic amino acid; AA4 is the L or D isomer of Trp; AA5 is the L or D isomer of Lys, N-Me-Lys, or Orn; AA6 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; AA7 is the L or D isomer of Val, Thr, Leu, Ile, tert-Leu, Abu, Nle, or an aromatic amino acid; R1 is H, a lower alkyl, lower alkanoyl, or a lower acyl; a is 1 or 2; and R2 is OH, OR3, N(R3)2, or NHR3, where R3 is H, a lower alkyl, or arylalkyl.",
"One of the most potent U-II inhibitors tested was the SRIF antagonist Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide (SEQ ID NO: 4), which had an IC50 of about 100 nM and Kd of 240.Another potent U-II antagonist was Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]-Val-NH2 (SEQ ID NO: 5) which had an IC50 of about 2 nM.",
"Other SRIF antagonists that were tested are summarized in Example 2 below (see table 1).",
"The polypeptides of the invention are capable of modulating U-II activity and are, therefore, useful for treating physiological and psychological conditions related to either an excess of or an under expression of U-II activity within a subject.",
"Such conditions include, for example, acute heart failure, hypotension, hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation and dyskinesias.",
"If the condition stems from an excess of U-II activity, one approach to treatment is to administer to a subject in need thereof an inhibitor compound (antagonist), optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of U-II.",
"Alternatively, for treating conditions related to under expression of U-II activity, a compound which activates U-II (agonist) is administered.",
"A therapeutically effective amount of a polypeptide of Formula I, or a variant or pharmaceutically acceptable salt-thereof, can be administered orally, parenterally (e.g.",
"intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasally, vaginally, rectally, sublingually or topically, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.",
"Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences (18th edition), ed.",
"A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions.",
"The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation.",
"Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.",
"In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient.",
"These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin.",
"Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used.",
"Tablets and pills can additionally be prepared with enteric coatings.",
"Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules.",
"These forms contain inert diluents commonly used in the art, such as water or an oil medium.",
"Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.",
"Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.",
"Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate.",
"Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents.",
"Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.",
"Other potentially useful parenteral delivery systems for the polypeptides of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.",
"Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions.",
"Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.",
"Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax.",
"Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art.",
"Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.",
"The amount of active ingredient in the compositions of the invention can be varied.",
"One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the polypeptide being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient.",
"In addition, the severity of the U-II-related condition being treated will also have an impact on the dosage level.",
"Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses.",
"Preferably, the general dosage range is between 250 μg/kg to 5.0 mg/kg of body weight per day.",
"Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration.",
"For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection.",
"Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well known in the art.",
"In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above identified factors.",
"The polypeptides of the invention can be administered in a sustained release composition, such as those described in, for example, U.S. Pat.",
"Nos.",
"5,672,659 and 5,595,760.The use of immediate or sustained release compositions depends on the type of condition being treated.",
"If the condition consists of an acute or over-acute disorder, a treatment with an immediate release form will be preferred over a prolonged release composition.",
"Alternatively, for preventative or long-term treatments, a sustained released composition will generally be preferred.",
"Polypeptides of the present invention can be prepared in any suitable manner.",
"The polypeptides may be isolated from naturally occurring sources, recombinantly produced, or produced synthetically, or produced by a combination of these methods.",
"The synthesis of short peptides is well known in the art.",
"See e.g.",
"Stewart et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984).",
"The peptides of the present invention can be synthesized according to standard peptide synthesis methods known in the art and exemplified in Example I below.",
"The present invention is illustrated by the following examples, which are in no way intended to be limiting of the invention.",
"EXAMPLE 1 Preparation of Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide Step 1: Preparation of Boc-4-chlorophenylalanine-S-methylbenzyl-D-cysteine-3-pyridyl-2-alanine-D-tryptophan-Na-benzyloxycarbonyl-lysine-valine-S-methylbenzyl-cysteine-4-chlorophenylalanine-benzhydrylamine resin Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville, Ky.) (1.2 g, 0.5 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced ChemTech peptide synthesizer (Model 200) programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 min.",
"and 25 min.",
"each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f) 10% triethylamine in chloroform.",
"The neutralized resin is stirred with Boc-4-chlorophenylalanine and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hour and the resulting amino acid resin in then cycled through steps (a) through (f) in the above wash program.",
"The following amino acids (1.5 mmole) are then coupled successively by the same procedure: Boc-S-methylbenzyl-Cys, Boc-Val, Boc-Na -benzyloxycarbonyl-lysine, Boc-D-Trp, Boc-Pal, and Boc-S-methylbenzyl-D-Cys and Boc-4-chlorophenylalanine.",
"After washing and drying, the completed resin weighed about 2.0 g. Step 2: Deprotection and Cleavage from Resin The resin described in Step 1 (1.0 g, 0.25 mmole) was mixed with anisole (5 ml), dithiothretol (100 mg), and anhydrous hydrogen fluoride (35 ml) at about 0° C. and stirred for 45 min.",
"Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen, after which free peptide was precipitated and washed with ether.",
"The crude peptide was then dissolved in 500 ml of 90% acetic acid.",
"A concentrated solution of I2/MeOH was then added until a permanent brown color was observed.",
"Excess I2 was removed by the addition of ascorbic acid and the solution evaporated to a small volume which was applied to a column (2.5×90 cm) of VYDAC™ octadecylsilane silica (10-15 μm).",
"This was eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water.",
"Fractions were examined by thin layer chromatography and analytic high performance liquid chromatography and pooled to give maximum purity.",
"Repeated lyophilization of the solution from water gave 125 mg of the desired product as a white, fluffy powder.",
"The product was found to be homogenous by HPLC and TLC.",
"Amino acid analysis of an acid hydrolysate and matrix-assisted laser desorption MS confirmed the composition of the octapeptide.",
"Other peptide of the invention may be made using an analogous procedure with appropriate reactants.",
"EXAMPLE 2 Use of Rat Aorta Circular Strip for Assay U-II Antagonists Male Sprague-Dawley rats (250-350 g), which had been quarantined for 5-7 days prior to the experiments, were sacrificed by decapitation (experiments were approved by the Advisory Committee For Animal Resources, Tulane University School of Medicine).",
"The thoracic aorta was dissected, freed from connective tissue, and cut into rings of about 1.5 mm in width.",
"The rings were suspended in a 15 ml organ bath containing high potassium Kreb's solution (9.15 g/L potassium chloride, 2.1 g/L sodium bicarbonate, 1.0 g/L glucose, 0.16 g/L potassium phosphate monobasic, 0.14 g/L magnesium sulfate (anhydr.",
"), and 0.22 g/L calcium chloride (dihydr.))",
"Optimal tension was applied (0.2 g) to the tissues and the bath medium was maintained at 37° C. and bubbled with a mixture of 95% O2/5% CO2.Prior to mounting in the organ bath, selected preparations were rubbed with a moistened cotton wool swab, in order to remove the endothelial cell layer, and the effect of this procedure was tested using an acetylcholine-relaxation test.",
"(Gibson, A., Br.",
"J. Pharmacol.",
"91:205 (1987)).",
"The aorta rings were allowed to equilibrate for 90 min.",
"at the optimal tensions.",
"During the equilibration period, the bath solution was replaced every 15 min.",
"Contractile responses of aortae rings to various concentrations of peptides were expressed in volts.",
"Changes in arterial smooth muscle tension were recorded isometrically using a force-displacement transducer (Radnoti), and a AcqKnowledge ACK100 Version 3.2 (BIOPAC Systems, Inc., Santa Barbara, Calif.) In siliconized glass tubes, peptides were dissolved in dionized water at a concentration of 1 μg/1 μL (stock solution) and then diluted 1:10 with sterile BSA-saline solution (0.1% BSA, fraction V, Sigma, St. Louis in 0.9% NaCl).",
"All peptide solutions were prepared fresh directly before the experiments.",
"Peptides in the concentration ranges of 10-6 to 10-12 M/L in a final volume of 16-80 μL were direcly introduced into the tested organ bath containing Krebs buffer continuously gassed with 95% O2 and 5% CO1, and the aorta rings at an optimal resting tension (1-0.2 g).",
"Peptide-induced changes in tension of the aorta rings were recorded by force-displacement transducers and processed by the computer system BIOPAC Inc., as described above.",
"Each ring was exposed to one peptide concentration only.",
"Using assay techniques known in the art, we found that the minimally, fully potent sequence of U-II was the octapeptide Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH (SEQ ID NO: 3), which was actually more potent than the full human and fish sequences in inducing rat aorta contracts.",
"Various somatostatin (SRIF) antagonists were discovered to have the ability to block UII-induced phase contractions in the circular rat thoracic aorta strips.",
"One of the most potent inhibitors was the SRIF antagonist Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide (SEQ ID NO: 4), which had an IC50 of about 100 nM and a Kd of 240 nM.",
"The polypeptide Cpa-c[D-Cys-Phe-Trp-Lys-Thr-Cys]-ValNH2 was also a strong U-II antagonist with an IC50 of 2 nM.",
"Other compounds that were tested are summarized in Table I below.",
"TABLE 1 SRIF Antagonist IC50s (nM) against U-II Stimulation of Rat Aorta Phasic Contractions Polypeptide IC50 Nal-D-Cys-His-D-Trp-Lys-Val-Cys-D-Dip-NH2 (SEQ ID NO: 6) 1800 4Fpa-D-Cys-Pal-D-Trp-Lys-Val-Cys-Nal-NH2 (SEQ ID NO: 7) 1090 4Fpa-D-Cys-Pal-D-Trp-Lys-Tle-Cys-Nal-NH2 (SEQ ID NO: 8) 100 Cpa-D-Cys-Tyr-D-Trp-Lys-Thr-Cys-Nal-NH2 (SEQ ID NO: 9) 12 Cpa-D-Cys-Pal-D-Trp-Lys-Tle-Cys-Nal-NH2 (SEQ ID NO: 10) 10 Cpa-D-Cys-Pal-Trp-Lys-Thr-Cys-Cpa-NH2 (SEQ ID NO: 11) 2 Equivalents Although the present invention has been described with reference to preferred embodiments, one skilled in the art can easily ascertain its essential characteristics and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.",
"Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.",
"Such equivalents are intended to be encompassed in the scope of the present invention.",
"All publications and patents mentioned in this specification are herein incorporated by reference."
]
] | Patent_10399542 |
[
[
"Method for verifying a perforation pattern serving as a security characteristic",
"A method of checking the authenticity of a perforation pattern which serves as a security feature and which when viewed in daylight back-lighting shows image information in the substrate of a document in particular in card form, wherein both the front side and also the rear side of the substrate is viewed in the region of the perforation pattern under incident light with light of a defined wavelength and in that case the image which is inherent to the perforation pattern shows on the front side while on the rear side thereof an area corresponding to the region of the perforation pattern shows up only in unspecified fashion."
],
[
"1.A method of checking a perforation pattern which serves as a security feature and which when viewed in daylight back-lighting shows image information in the substrate of a document in particular in card form, characterized in that both the front side and also the rear side of the substrate is viewed in the region of the perforation pattern under incident light with light of a defined wavelength and in that case the image which is inherent to the perforation pattern shows on the front side while on the rear side thereof an area corresponding to the region of the perforation pattern shows up only in unspecified fashion.",
"2.A method as set forth in one of the preceding claims characterized in that viewing in daylight back-lighting is effected both from the front side and also from the rear side of the substrate and the image information must be visible in both cases.",
"3.A method as set forth in one of the preceding claims characterized in that viewing is effected both in daylight back-lighting and/or under incident light with light of a defined wavelength with the naked eye.",
"4.A method as set forth in one of the preceding claims characterized in that the wavelength of the incident light is between 10 nm and 500 nm, in particular between 100 nm and 400 nm and is in particular wide-band UV-light.",
"5.A method as set forth in one of the preceding claims characterized in that on the rear side the mouth openings of the perforation pattern are all of equal size and in particular are arranged in regular relationship with each other, but on the front side the mouth openings are of different sizes.",
"6.A method as set forth in one of the preceding claims characterized in that the diameters of the mouth openings on the front side are between 20 μm and 140 μm.",
"7.A method as set forth in one of the preceding claims characterized in that the perforations were burnt in by means of laser light.",
"8.A method as set forth in one of the preceding claims characterized in that the substrate is a plastic material, in particular a thermosetting plastic material, in particular polycarbonate.",
"9.A method as set forth in one of the preceding claims characterized in that the thickness of the substrate is between 0.1 and 1.0 millimeter.",
"10.A method as set forth in one of the preceding claims characterized in that the wavelength of the laser light used for burning in the perforations is between 6 μm and 12 μm.",
"11.A method as set forth in one of the preceding claims characterized in that the diameters of the mouth openings on the front side are between 100 times and 500 times the wavelength of the incident light used.",
"12.A method as set forth in one of the preceding claims characterized in that the thickness of the substrate is at least 300 thousand times the wavelength of the incident light used.",
"13.A method as set forth in one of the preceding claims characterized in that the wavelength of the laser light used for burning in the perforations is between 10 times and 70 times, in particular between 20 times and 40 times, the wavelength of the incident light used."
],
[
"<SOH> II.",
"TECHNICAL BACKGROUND <EOH>It is known from WO 98/19869 to incorporate a security feature in the form of a perforation pattern into documents such as banknotes, passes, or plastic cards, for example payment cards.",
"In that case a plurality of perforations are applied extensively in side-by-side relationship.",
"In that respect the attempt is made to produce an extensive two-dimensional image by control of the diameters of the perforations, that is to say the mouth openings of the perforations on one of the outward sides, or by way of the spacings between the mouth openings, that is to say the distribution of the mouth openings on the surface.",
"In accordance with WO 00/43216 that is possible even when the arrangement involves blind holes which do not at all reach the viewing side.",
"In practice that method is implemented by a procedure whereby perforations are burnt in by means of laser light in mutually juxtaposed relationship from the rear side of the document, that is to say the substrate, in regular or irregular raster configurations.",
"Accordingly the mouth openings of the perforations on that rear side are all of the same size, but not the mouth openings of the corresponding perforations on the front side.",
"Accordingly, by virtue of the variation in the size of the mouth openings on the front side, when viewing the front side in back-lighting, for example in daylight, it is possible to see an image with gray scales.",
"This image may involve digits, letters, symbols or representations for example of a face.",
"The differing diameters of the mouth openings on the front side are implemented by the duration for which the substrate is subjected to the action of laser light being varied in dependence on the desired diameter of the mouth opening.",
"Although a laser beam is very well focused, ultimately, as it increasingly burns into a substrate from the rear side, it produces a hole which is conical at least in the deepest region, due to the Gaussian distribution of the energy within the cross-section of the laser beam and at least as long as the duration of action of the laser beam is not still substantially prolonged after piercing the substrate.",
"That conicity can be increased or attenuated by suitable optical systems arranged upstream of the point of impingement, for shaping the laser beam.",
"For, after piercing the substrate, the burnt passage is expanded to the full thickness of the laser beam and it is only after this has been achieved that the mouth opening on the exit side is approximately of the same size as the mouth opening on the entry side.",
"As this is precisely not wanted for influencing the size of the mouth openings on the exit side, that is to say the front side of the document, the through openings through the substrate are slightly conical when considered in longitudinal section.",
"In addition, due to the generation of heat by the laser light, the edges of the mouth openings, in particular on the exit side of the laser light, are rounded, at least insofar as the materials involved are relatively soft such as plastic materials.",
"By virtue of the conical shape of the holes which constitute the perforation pattern, the intended image, in contrast to the assertion in the above-mentioned patent application, can be recognized when considered in the back-lighting mode, irrespective of the side from which the substrate is viewed.",
"This is due to the fact that, even when viewing from the rear side, at which in fact the mouth openings are arranged regularly or irregularly in mutually juxtaposed relationship and are of the same diameter, the amount of the light passing thereto is nonetheless determined by the size of the mouth openings on the opposite side, the front side.",
"Accordingly therefore the intended image effect can be recognized when viewing from both sides.",
"Such a security feature however is relatively easy to circumvent: thus for example a corresponding perforation pattern could be produced by means of a kind of sewing machine, for example by using two needles of differing thicknesses, or also by means of one and the same needle which is of a conical configuration at least in the front region, and simultaneous control of the depth of engagement of the needle."
],
[
"I.",
"FIELD OF USE The invention concerns a method of checking a security feature.",
"II.",
"TECHNICAL BACKGROUND It is known from WO 98/19869 to incorporate a security feature in the form of a perforation pattern into documents such as banknotes, passes, or plastic cards, for example payment cards.",
"In that case a plurality of perforations are applied extensively in side-by-side relationship.",
"In that respect the attempt is made to produce an extensive two-dimensional image by control of the diameters of the perforations, that is to say the mouth openings of the perforations on one of the outward sides, or by way of the spacings between the mouth openings, that is to say the distribution of the mouth openings on the surface.",
"In accordance with WO 00/43216 that is possible even when the arrangement involves blind holes which do not at all reach the viewing side.",
"In practice that method is implemented by a procedure whereby perforations are burnt in by means of laser light in mutually juxtaposed relationship from the rear side of the document, that is to say the substrate, in regular or irregular raster configurations.",
"Accordingly the mouth openings of the perforations on that rear side are all of the same size, but not the mouth openings of the corresponding perforations on the front side.",
"Accordingly, by virtue of the variation in the size of the mouth openings on the front side, when viewing the front side in back-lighting, for example in daylight, it is possible to see an image with gray scales.",
"This image may involve digits, letters, symbols or representations for example of a face.",
"The differing diameters of the mouth openings on the front side are implemented by the duration for which the substrate is subjected to the action of laser light being varied in dependence on the desired diameter of the mouth opening.",
"Although a laser beam is very well focused, ultimately, as it increasingly burns into a substrate from the rear side, it produces a hole which is conical at least in the deepest region, due to the Gaussian distribution of the energy within the cross-section of the laser beam and at least as long as the duration of action of the laser beam is not still substantially prolonged after piercing the substrate.",
"That conicity can be increased or attenuated by suitable optical systems arranged upstream of the point of impingement, for shaping the laser beam.",
"For, after piercing the substrate, the burnt passage is expanded to the full thickness of the laser beam and it is only after this has been achieved that the mouth opening on the exit side is approximately of the same size as the mouth opening on the entry side.",
"As this is precisely not wanted for influencing the size of the mouth openings on the exit side, that is to say the front side of the document, the through openings through the substrate are slightly conical when considered in longitudinal section.",
"In addition, due to the generation of heat by the laser light, the edges of the mouth openings, in particular on the exit side of the laser light, are rounded, at least insofar as the materials involved are relatively soft such as plastic materials.",
"By virtue of the conical shape of the holes which constitute the perforation pattern, the intended image, in contrast to the assertion in the above-mentioned patent application, can be recognized when considered in the back-lighting mode, irrespective of the side from which the substrate is viewed.",
"This is due to the fact that, even when viewing from the rear side, at which in fact the mouth openings are arranged regularly or irregularly in mutually juxtaposed relationship and are of the same diameter, the amount of the light passing thereto is nonetheless determined by the size of the mouth openings on the opposite side, the front side.",
"Accordingly therefore the intended image effect can be recognized when viewing from both sides.",
"Such a security feature however is relatively easy to circumvent: thus for example a corresponding perforation pattern could be produced by means of a kind of sewing machine, for example by using two needles of differing thicknesses, or also by means of one and the same needle which is of a conical configuration at least in the front region, and simultaneous control of the depth of engagement of the needle.",
"III.",
"STATEMENT OF THE INVENTION a) TECHNICAL SUPPORT Therefore the object of the present invention is to provide a method of checking the authenticity of such a security feature.",
"b) ATTAINMENT OF THE OBJECT That object is attained by the features of claim 1.Advantageous embodiments are set forth in the appendant claims.",
"The—optionally additional—checking of the perforation pattern in an incident light mode with light of a defined wavelength, for example UV-light, means that a completely different image effect can be detected on the front side and the rear side, with only a very simple aid, for example a UV-lamp: While the desired image effect can be clearly recognized on the front side, insofar as the overall reflecting surface of the perforation pattern or blind hole pattern reflects to a differing degree, according to the gray scales of the image, from the rear side only a surface which reflects to a uniform degree is to be recognized, over the region of the perforation pattern.",
"That effect cannot be achieved with bored perforations.",
"This differing degree of reflection is due to interference of the UV-light used, caused by reflection phenomena in particular in the edge region of the mouth openings and in the peripheral region at the beginning of the through openings of the perforation pattern.",
"For checking the security feature in general the first security step involves light checking by means of daylight.",
"If however incident light viewing is to be implemented directly by means of a light of a defined wavelength as the first checking operation, it is recommended that transillumination by means of daylight be additionally implemented from both sides of the document as an enhanced level of security can be achieved by the combination of the two checking procedures.",
"In both cases viewing was effected with the naked eye so that no aids are required for that purpose, besides shining on the light of a defined wavelength, that is to say for example UV-light.",
"c) EMBODIMENTS An embodiment according to the invention is described in greater detail hereinafter by way of example with reference to the Figures in which: FIG.",
"1 is a cross-sectional view through the substrate, and FIG.",
"2 is a plan view onto the substrate illuminated with UV-light.",
"FIG.",
"1 shows a cross-section through the substrate 1, more specifically along the longitudinal axes of the holes 4a, b, c .",
".",
".",
", thereof, which form the perforation 4.The holes 4 are all introduced into the substrate 1 from the upwardly disposed rear side 3.In that respect the hole 4a shows how, with a progressive time of exposure by virtue of a laser beam, the result produced is a blind hole which is of a conical configuration at least in its respective deepest region, by virtue of distribution of the energy within the laser beam.",
"In a corresponding manner, when piercing a front side 2 which is in opposite relationship to the rear side 3, the mouth opening on that exit side is substantially smaller in diameter than on the entry side.",
"As the hole 4b which passes completely through the substrate 1 shows it is possible in that way to produce through holes whose mouth opening, for example 5b, on the exit side thereof, is definedly smaller than on the entry side of the hole.",
"If, after the exit side, that is to say the front side 2, has been pierced in that way, as has just been effected, the action of the laser light is continued for a longer period of time, in that way the mouth opening on the exit side is enlarged more and more until—as shown in the case of the hole 4c—on the exit side it is of a diameter which is practically identical to the diameter of the mouth opening on the entry side.",
"The Figure shows, by reference to the hole 4d, that in the same way, but only with a reduced period of action of the laser light, in comparison with the structure involved with the hole for example 4b, that is to say until piercing of the oppositely disposed exit side thereof, it is also possible to produce blind holes.",
"In this case the bottom of that blind hole 4d is of an again markedly smaller cross-section than the mouth opening at the entry side of the hole 4d.",
"FIG.",
"1 further shows how, when that front side 2 is illuminated with UV-light 9, the angle of impingement of which can be substantially freely selected, but which preferably should be at an angle relative to the front side 2 of more than 10°, preferably more than 40°, the light is reflected and also diffusely scattered, in particular at the edges 6 of the mouth openings 5 but also at the inside peripheral walls of the holes 4, in the region near the mouth openings.",
"Accordingly, when viewing that front side 2 during illumination with UV-light 9, a viewer perceives each mouth opening 5b, c as a light spot 7a, b whose diameter corresponds to the diameter of the respective mouth opening.",
"In that situation the light spot 7 appears as shining over its entire area although the mouth opening 5 represents a ring.",
"The diameter of the light spot is in particular slightly larger than the diameter of the respective mouth opening, which is to be attributed to diffuse radiation of the UV-light in particular in the regions of the edge 6.As FIG.",
"2 shows, surface-like light spots which are arranged generally regularly along a raster grid 8 but which are also irregularly distributed can produce surface effects insofar as such surfaces which have a higher proportion of shining surfaces than other surfaces are in contrast to those other surfaces.",
"In the case of light spots arranged along a regular raster grid 8 for example that effect is produced by brightly shining surfaces being produced by virtue of the arrangement of large light spots on the intersections of the raster grid 8.When smaller light spots 7b are arranged on the intersection points of the raster grid 8 the surface covered in that way shines less brightly.",
"In that way just two mouth openings 5b, 5c of differing sizes and thus light spots 7a, b of differing sizes already make it possible to produce images whose contrast corresponds to that of a black-and-white photograph.",
"As the sizes of the mouth openings can be varied in more than two stages, it is also possible to provide gray scale representations.",
"In addition it is also possible for blind holes like the hole 4b which do not penetrate the substrate as far as the front side 2b which is being viewed nonetheless to be made visible by means of UV-light insofar as the remaining residual thickness 11 is sufficiently thin to be penetrated by the UV-light.",
"List of References 1 substrate 2 front side 3 rear side 4 perforation 5 mouth opening 6 edge 7 light spot 8 raster grid 9 UV-light 11 residual thickness"
]
] | Patent_10399741 |
[
[
"Lightweight aggregate binder formulation",
"The invention relates to a thermally stable composition, and methods for the preparation thereof, for use in the manufacture of a lightweight aggregate including a continuous bituminous phase, a discontinuous aqueous phase, an anionic oxide suspended in the discontinuous aqueous phase and at least one emulsifying agent.",
"The anionic oxide may be a naturally occurring metal oxide having a needle-like crystal structure such as red oxide (CuO) or yellow oxide.",
"The continuous bituminous phase may be for example grade 170 bitumen or crude oil, hydrocarbons or mixtures thereof.",
"The emulsifying agent may be a clay emulsifier such as sodium bentonite, or a hydrous aluminium silicose or montmorillonite clay.",
"The invention also relates to lightweight aggregate particles coated with such compositions and building materials formed from the lightweight aggregate in combination with a cementitious material."
],
[
"1: A thermally stable composition for use in the manufacture of a lightweight aggregate including: a continuous bituminous phase; a discontinuous aqueous phase; an anionic oxide suspended in the discontinuous aqueous phase; and at least one emulsifying agent.",
"2: A thermally stable composition according to claim 1 wherein the anionic oxide is a naturally occurring metal oxide.",
"3: A thermally stable composition according to claim 1 wherein the anionic oxide is a metal oxide having a needle-like crystal structure.",
"4: A thermally stable composition according to claim 1 wherein the anionic oxide is red oxide (CuO) or yellow oxide.",
"5: A thermally stable composition according to claim 1 wherein the anionic oxide is present in an amount of 20 wt % of the composition.",
"6: A thermally stable composition according to claim 1 wherein the discontinuous aqueous phase is distilled and/or de-ionised water.",
"7: A thermally stable composition according to claim 1 wherein the discontinuous aqueous phase is water present in an amount of 40 wt % of the composition.",
"8: A thermally stable composition according to claim 1 wherein the continuous bituminous phase is grade 170 bitumen.",
"9: A thermally stable composition according to claim 1 wherein the continuous bituminous phase is selected from crude oil, hydrocarbons or mixtures thereof.",
"10: A thermally stable composition according to claim 1 wherein the continuous bituminous phase is present in an amount of 35 wt % of the composition.",
"11: A thermally stable composition according to claim 1 wherein the emulsifying agent is a clay emulsifier.",
"12: A thermally stable composition according to claim 8 wherein the clay emulsifier is selected from sodium bentonites, hydrous aluminum silicose clays, montmorillonite clays, and mixtures thereon.",
"13: A thermally stable composition according to claim 11 wherein the clay emulsifier is a 200 mesh bentonite.",
"14: A thermally stable composition according to claim 1 wherein the emulsifying agent is present in an amount of 5 wt % of the composition.",
"15: A thermally stable composition according to claim 1 wherein the emulsifying agent is a gel having an oleophilic tale and an oleophobic head.",
"16: A thermally stable composition according to claim 1 further including one or more of a thickener, a biocidal inhibitor, and a perfume.",
"17: A thermally stable composition according to claim 16 wherein the biocidal inhibitor is sodium ortho-phenylphenol.",
"18: Lightweight aggregate particles coated with a composition according to claim 1.19: Lightweight aggregate particles according to claim 18 formed from polymer foam.",
"20: Lightweight aggregate particles according to claim 18 formed from polystyrene foam particles.",
"21: A building material formed from a lightweight aggregate according to claim 18 in combination with a cementitious material.",
"22: A method of forming a composition according to claim 1 including the steps of optionally bending one or more of an air entrainment agent, a fragrance and a biocide with water and shearing to form a soap; adding an emulsifier; dispersing naturally occurring anionic oxide particles in the aqueous phase; and dispersing the aqueous phase in a bituminous phase."
],
[
"<SOH> BACKGROUND ART <EOH>The preparation of low density concrete by the incorporation of lightweight aggregates such as vermiculite, cork, slag, asbestos, bagasses and the like in a hydraulic binder such as a cement/sand/water mixture is well known.",
"Low density concrete having much improved properties may be prepared by the incorporation of lightweight aggregate, in particular, foam particles such as polystyrene, into the binder.",
"However, cementitious materials do not readily bond with these lightweight aggregates, which are generally highly hydrophobic.",
"The hydrophobic character and low density of polystyrene beads produces a tendency to float to the surface of the concrete as it sets.",
"To overcome this problem, various bonding agents have been used to facilitate the incorporation of polystyrene foam particles into lightweight aggregates.",
"These agents include bituminous products, coal tars and mixtures of pitch with epoxy resins or phenolic resins.",
"The use of such bonding agents has proved problematic because coating of the polystyrene particles results in a tacky surface causing the particles to coalesce into a mass which is difficult to disperse in the cementitious matrix.",
"Further, the coatings generally retain a strong bituminous odour which remains unacceptably detectable in the end product.",
"More importantly, the bonding strength between the foam particles and the other components in the cured cement has not been ideal, resulting in an inferior product.",
"Applicant's earlier patent AU670754 (see also corresponding U.S. Pat.",
"No.",
"5,472,498) the contents of which are incorporated herein by reference) provided a major advance in the art.",
"By treating polystyrene foam particles with a bonding agent consisting of an emulsion comprising a discontinuous bituminous phase, and a continuous aqueous phase and having ferric oxide suspended in the aqueous phase.",
"The use of this emulsion avoided the problems of surface tackiness and aggregation, provided a lightweight aggregate which was uniformly dispersible in concrete, and strongly bonded therewith providing a lightweight concrete of substantially improved strength.",
"The ferric metal ions kept the lightweight aggregate in suspension in the wet concrete, preventing the aggregate particles from floating to the top, and also led to a strong bond between the aggregate and the concrete in the cured mixture providing a lightweight concrete of improved strength.",
"Prior to application to the lightweight particles, it was usual for the emulsion described in AU670754 to be diluted with water in compliance with specified concentrations.",
"Failure to adhere to the specified concentration range would lead to a deterioration in the performance of the final product.",
"The previous coating agent typically required addition of around 1 part of the emulsion to 2 parts water prior to application.",
"Attempts by users to extend the emulsion by adding more water, contrary to product specifications, could result in an inferior concrete product.",
"Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.",
"Further, the previous bonding agent did not always store well for extended periods of time in conditions of extreme temperature, exhibiting a tendency to settle.",
"Extreme temperature fluctuations can cause stratification of the mixture.",
"Previous mixtures have also sometimes required the addition of a thickening agent to increase viscosity.",
"These thickening agents are typically cellulose based and are thus vulnerable to bacterial attack.",
"It is an object of the present invention to overcome or ameliorate one or more of the disadvantages of the prior art, or to provide a commercial alternative."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 shows a flow chart of a preferred method of forming the coating composition of the present invention detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to aggregate suitable for use in lightweight concrete, and to lightweight concrete including the aggregate.",
"BACKGROUND ART The preparation of low density concrete by the incorporation of lightweight aggregates such as vermiculite, cork, slag, asbestos, bagasses and the like in a hydraulic binder such as a cement/sand/water mixture is well known.",
"Low density concrete having much improved properties may be prepared by the incorporation of lightweight aggregate, in particular, foam particles such as polystyrene, into the binder.",
"However, cementitious materials do not readily bond with these lightweight aggregates, which are generally highly hydrophobic.",
"The hydrophobic character and low density of polystyrene beads produces a tendency to float to the surface of the concrete as it sets.",
"To overcome this problem, various bonding agents have been used to facilitate the incorporation of polystyrene foam particles into lightweight aggregates.",
"These agents include bituminous products, coal tars and mixtures of pitch with epoxy resins or phenolic resins.",
"The use of such bonding agents has proved problematic because coating of the polystyrene particles results in a tacky surface causing the particles to coalesce into a mass which is difficult to disperse in the cementitious matrix.",
"Further, the coatings generally retain a strong bituminous odour which remains unacceptably detectable in the end product.",
"More importantly, the bonding strength between the foam particles and the other components in the cured cement has not been ideal, resulting in an inferior product.",
"Applicant's earlier patent AU670754 (see also corresponding U.S. Pat.",
"No.",
"5,472,498) the contents of which are incorporated herein by reference) provided a major advance in the art.",
"By treating polystyrene foam particles with a bonding agent consisting of an emulsion comprising a discontinuous bituminous phase, and a continuous aqueous phase and having ferric oxide suspended in the aqueous phase.",
"The use of this emulsion avoided the problems of surface tackiness and aggregation, provided a lightweight aggregate which was uniformly dispersible in concrete, and strongly bonded therewith providing a lightweight concrete of substantially improved strength.",
"The ferric metal ions kept the lightweight aggregate in suspension in the wet concrete, preventing the aggregate particles from floating to the top, and also led to a strong bond between the aggregate and the concrete in the cured mixture providing a lightweight concrete of improved strength.",
"Prior to application to the lightweight particles, it was usual for the emulsion described in AU670754 to be diluted with water in compliance with specified concentrations.",
"Failure to adhere to the specified concentration range would lead to a deterioration in the performance of the final product.",
"The previous coating agent typically required addition of around 1 part of the emulsion to 2 parts water prior to application.",
"Attempts by users to extend the emulsion by adding more water, contrary to product specifications, could result in an inferior concrete product.",
"Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.",
"Further, the previous bonding agent did not always store well for extended periods of time in conditions of extreme temperature, exhibiting a tendency to settle.",
"Extreme temperature fluctuations can cause stratification of the mixture.",
"Previous mixtures have also sometimes required the addition of a thickening agent to increase viscosity.",
"These thickening agents are typically cellulose based and are thus vulnerable to bacterial attack.",
"It is an object of the present invention to overcome or ameliorate one or more of the disadvantages of the prior art, or to provide a commercial alternative.",
"DESCRIPTION OF THE INVENTION According to a first aspect, the invention provides a thermally stable composition for use in the manufacture of a lightweight aggregate including: a continuous bituminous phase; a discontinuous aqueous phase an anionic oxide suspended in the discontinuous aqueous phase, and at least one emulsifying agent.",
"Preferably, the anionic oxide is a naturally occurring metal oxide, for example red oxide, such as Cupric Oxide (CuO).",
"It is highly preferred to use metal oxides having a needle like crystal structure.",
"However, other naturally occurring oxides, for instance yellow oxide, may be used.",
"The discontinuous aqueous phase is preferably distilled, or deionized, water.",
"Grade 170 Bitumeni is preferred as the continuous bituminous phase, although other crude oil or hydrocarbons or mixtures thereof may be used.",
"The emulsifying agent is preferably a clay emulsifier, such as the sodium bentonites, hydrous aluminium silicose clays and montmorillonite families.",
"A highly preferred emulsifier is Volclay® premium gel, which is a 200 mesh bentonite.",
"Other gels having a similar structure, i.e.",
"with an oleophilic tail and an oleophobic head have also been found suitable for use in the present invention.",
"Preferably, the composition further includes one or more of a thickener, an inhibitor, and a perfume.",
"The selection of concentrated fragrance used does not appear to be critical.",
"In the present invention, the preferred fragrance is “Eternal”.",
"Similarly, the biocidal concentrate used does not appear to be critical, but sodium ortho-phenylphenol (“opp”) is preferred.",
"According to a second aspect, the invention provides lightweight aggregate particles coated with a composition according to the first aspect.",
"Although the coating composition can be applied to a wide range of light weight aggregate materials, it is preferred to apply the composition to polymer foam (including recycled polymer foam) particles, and it is especially desirable to select polystyrene foam particles as the lightweight aggregate to be coated.",
"According to a third aspect, the invention provides a building material formed from a lightweight aggregate according to the second aspect in combination with a cementitious material.",
"According to a fourth aspect, the present invention provides a method of forming a composition according to the first aspect, including the steps of: optionally blending one or more of an air entrainment agent, a fragrance and a biocide with water and shearing to form a soap; adding an emulsifier; dispersing naturally occurring anionic oxide particles in the aqueous phase; and dispersing the aqueous phase in a bituminous phase.",
"The present invention will now be described by way of example only with reference to the embodiments shown in the accompanying drawings and/or examples.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a flow chart of a preferred method of forming the coating composition of the present invention BEST MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1 Bonding Agent The preferred bonding agent of the present invention has the following composition (all values are wt %): Distilled water 40% (wt %) Bentonite (Volclay ® premium gel) 5% Red Oxide 20% Grade 170 Bitumen 35% sodium ortho-phenylphenol .13% Fragrance .14% Surfactant .25% Compatible chemicals performing a similar function may be substituted for any of those exemplified in the above formulation, and the relative quantities adjusted accordingly in a manner which will be apparent to a skilled formulator based on the teachings hereof.",
"EXAMPLE 2 Preparation of Bonding Agent The binder formulations of the present invention are best formed by an “in line” emulsification procedure according to the following steps: 1.Micro-aire 940, an air entrainment agent; “Eternal”, the desired fragrance; and sodium ortho-phenylphenol, an antibacterial agent (“opp”) are added to distilled water to form a soap.",
"This soap is circulated for 25 minutes while the temperature is raised to 35° C. 2.Volclay® premium gel is then added to the soap at a slow rate, approximately 10 kg per minute.",
"3.Red oxide is then dispersed in the mixture at a rate of around 10 kg per minute 4.The mixture is sheared in a high shear mill while Grade170 Bitumen at 195° C. is added at around 1500 kg per hour.",
"In the above method, the exact sequence of addition in the manufacture of the composition is critical for obtaining the water-in-oil emulsion structure and for providing the coating composition with its useful properties.",
"However, those skilled in the art of emulsification will appreciate that the conditions will vary with equipment employed and formulation used and that other methods for producing the water-in-oil emulsion with a dispersed aqueous phase containing the oxide in suspension may be suitable.",
"EXAMPLE 3 Physical Structure of Emulsion The preferred physical structure of the natural anionic oxides preferred in the present invention may be classified as either “needle-like” or “spherical”.",
"“Needle-like” oxides hold more water, so when they are formed into a paste, this tends to be drier, and thus more viscous than that formed from the corresponding “spherical oxide”.",
"Needle-like oxides are highly preferred.",
"However, the oxides may be either “needle-like” or “spherical”.",
"The droplets in the emulsion of the present invention have been found to be around 25 microns in diameter, which is considerably larger than the 5 micron diameter droplets found in typical emulsions, and contain the oxide as a suspension within the droplets.",
"EXAMPLE 4 Stability of Composition The emulsion has been tested for prolonged exposure to freezing.",
"No noticeable deterioration of the structure or qualities of the composition was observed.",
"EXAMPLE 5 Preparing Coated Aggregate The coating composition (1 part by weight) is mixed with water (8 parts by weight).",
"The formulation can then be used to coat polystyrene balls in a manner similar to that described in AU670754.The coated balls are free flowing when dry and have a pink appearance.",
"EXAMPLE 6 Concrete Preparation The coated polystyrene balls form an aggregate which is mixed with cement, sand and water.",
"Other types of conventional aggregate can be added if required.",
"The concrete produced using the present invention can be worked in the same way as normal concrete.",
"The exact quantities used will depend upon the required properties of the concrete and the nature of the other materials used.",
"EXAMPLE 7 Concrete Produced The concrete produced is compatible with the lightweight concrete described in AU670754 in terms of physical performance.",
"The weight and other physical properties of the concrete produced in accordance with the present invention is similar to that described in AU670754.However, surprisingly, a compressive strength of around 30 MPa may be obtained using aggregate coated with bonding formulations of the present invention, which is unexpectedly 20% higher than that obtainable from the concrete of AU670754.Those skilled in the art will appreciate that lightweight concrete is designed for low load bearing applications.",
"Concrete weights as low as 250 kg/m3 can be obtained, but to achieve the maximum strength, 1200-1800 kg/m3 are used, which is about 50-75% the weight of conventional concrete.",
"Concrete density may be controlled by varying the amount of polystyrene in the mixture.",
"The concrete also has the desirable insulation properties of the lightweight concrete in AU670754, namely a thermal insulation coefficient of 0.065 w/m degree C., and R up to 1.9.The sound insulation properties are also as for the concrete in AU670754, with an STC rating of 40-55.Although the invention has been described with reference to a specific example, it will be appreciated by those skilled in the art that the compositions and methods of manufacture may be varied without departing from the inventive concept herein disclosed and that the invention may be embodied in many other forms."
]
] | Patent_10399873 |
[
[
"Reengineering event-driven field processes with a self-managed team approach",
"The invention provides a globally scalable, business-to-business (B2B) Mobile Process Service (MPS) that creates value for users and furthermore, through a franchise arrangement, allows value to be captured especially by telecom service providers.",
"The MPS is characterized by conducting mass-customized, event-driven processes with teams dynamically maintained by requisition of these resources from virtual competence centers using an information system that notifies team members of changes of state that require activity.",
"The information required, derived from both the field and from back-office, to execute that activity is made available at local, single-user server, distributed-databases maintained by replication over packet-switched radio.",
"This required information is identified from business process object collaboration models in which the parameters (rows/columns) are specified in the modelled messages between the business objects.",
"Main and supporting process frameworks are specified."
],
[
"1.Resolution of the Productivity Paradox of Information Technology by a mobile process service (abbreviated to MPS) that increases the effectiveness and efficiency of any (main or supporting) event-driven, business-to-business (abbreviated to B2B) mobile, processes and is realized by combining three components: object-engineered processes (abbreviated to P), competence-centric organizations (abbreviated to O), and an information handling software that includes distributed relational databases maintained over radio (abbreviated to S); furthermore this claim is made only when all three components (P, O and S) are invoked and offered as a MPS, rather than as individual components, since it is the synergy between multi-disciplines (P, O and S) that is claimed as innovative and not obvious.",
"2.Mass customization of the mobile process service of claim-1 by rapid application development (RAD) wherein the object-oriented model of the intended business process and organization shall semi-automatically create system models (including data models) and these system models shall be one input for semi-automatic creation of the system executable programs (optionally including replication rules and triggers) to be employed as a component of the ultimate mobile process service (MPS) the intention being that such automation makes mass customization (for each individual end-user company) of processes economically feasible.",
"3.An ‘ABC’ refinement of the mobile process service of any preceding claim in which the main processes are enhanced by invoking three engineered supporting processes: Assessing (abbreviated to A), Building team (abbreviated to B), under the conditions that a) participant competence centers are within a single organization, or if b) several companies cooperate by allowing one or more companies to requisition resources from another's virtual, or more formal, competence centers (for example when a company requisitions resources from an up- or down-stream channel member), or if c) partner companies agree that resources in one company will conduct processes traditionally performed by the other (for example, if a service organization spots sales leads for the selling company), or if d) partner companies (for example one being a vendor of capital equipment and the other a vendor of related supplies) share the activities and information related to the customer, or if e) two or more vendor companies co-fund and collaboratively conduct the marketing/sales of a solution that utilizes the products/services from such participating vendors.",
"Coordinating contact occasions (abbreviated to C).",
"furthermore this claim is made only when all three components (A, B and C) are invoked since it is the synergy between these three processes that is claimed as innovative and not obvious.",
"4.An interface database that enables a refinement of the mobile process service (MPS) of any preceding claim, inserted as an information link between the resource team-members as engaging in preceding claims, and the so-called ‘back-office’ (or other) information storage sites the interface database being a replicated subset of the primary database, the latter maintained at the data center, wherein the interface database contains only those data elements flowing between the teams in the field and the back-office (or other) information storage sites, there being an option for the interface database to reside either at the data center of the mobile process service provider or at the premises of the mobile process user (MPU).",
"5.Worldwide deployment of the mobile process service of preceding claims, economically scalable, and achieved by information communication technology (ICT) wherein the data flow is orchestrated by data service (DS) from a single data center, and delivery executed by network service (NS) through, usually national, carriers to the teams in the local, usually national, field sites.",
"6.A selling process that is dependent on any preceding claim and has the following sequence of sub-processes: understanding customer fulfillment (abbreviated to UCF), in which Need and Solution are explicitly represented as business objects and Fulfillment (satisfaction) represents the relationship, with associated ‘states’ between Need and Solution reflecting the customer satisfaction level, inspiring customer (abbreviated to IC), and pursuing prospect (abbreviated to PP) 7.A mobile process service that globally deploys the SELLING process (which is a main, event-driven, B2B process) and is dependent on any preceding claim; the claimed delivering process service supports sub-processes that include, but are not limited to: understanding customer fulfillment (abbreviated to UCF), inspiring customer (abbreviated to IC), and pursuing prospect (abbreviated to PP) 8.A mobile process service that globally deploys the DELIVERING process (which is a main, event-driven, B2B process) and is dependent on any preceding claim; the claimed delivering process service supports sub-processes that include, but are not limited to: recognising (abbreviated to RE), planning (abbreviated to PL), committing and dispatching (abbreviated to CD), delivering (abbreviated to DE), documenting (abbreviated to DO), financial transactions (FT).",
"9.The organization and mechanisms to grow a market for mobile process service as articulated in the preceding claims, then explicit capture of a fair share of the value created during execution of any of the preceding claims within the telecom industry, by establishing telecom infrastructure providers as Franchisers that will: appoint expert resources by certification to design, construct and deploy the mobile process service for the business-to-business (B2B) mobile process users (MPUs); appoint ‘Inspiration Franchisees’ that will build the market for these mobile process services; appoint ‘System Service’ franchisees that will be responsible for constructing and continuously updating the system consisting of processes, organization and software; appoint ‘ICT Service’ franchisees that will be responsible for operating the system that distributes the data by radio and/or wire-line communication as a mobile process service (MPS), all this being explicitly such that the Franchiser may, at its discretion, appoint companies (such as network operators) as ICT Service franchisees that are customers, or potential customers, for products and services (such as base-stations) offered by the Franchiser and thus capture value indirectly.",
"10.A service specific basis for billing the user for mobile process service support to each instance of the process articulated in any of the preceding claims which may include, but are not limited to, a specific charge for supporting each selling instance, or each delivering instance, or each assessing instance, or each building team instance, or each coordinating contact occasion instance.",
"This is in contrast to billing for products (like computer hardware), consulting services (utilized to design the system), horizontal applications (such as E-mail) and low-level service (such as time on the network or packets of data transported).",
"11.An inter-franchisee pricing mechanism, dependent on any of the preceding claims, mandated by the Franchiser such that all members of the channel, including certified expert resources, Inspiration Franchisees, System Service Franchisees and ICT Franchisees will be remunerated partly at the time when services are delivered to the next downstream level in the channel (for example when the system simulator is delivered to ICT provider) and partly at the time when the mobile process service is delivered to the mobile process user."
],
[
"<SOH> E. BACKGROUND OF THE INVENTION <EOH>E.1 Field of Invention The invention concerns the reengineering of event-driven, field business processes (for example sales, or delivery of articles and/or services at customer sites), enabled by teams assembled within a competence-centric organization that is, in turn, enabled by an information system exploiting data distribution over radio.",
"Of particular interest is the deployment as a Mobile Process Service—with emphasis on ‘process’ rather than ‘product’ or ‘application’.",
"Deployment can be worldwide due to advances in ICT (information communication technology).",
"E.2 Background Art and Assessment Thereof E.2.1 Theoretical Background E.2.1.1 Relationship Between Effort and Result The generic S-curve nature of the relationship between effort and result is well established.",
"In the ‘selling process’ case, for example, the aspiration is to optimize the combination of sales revenue and M&S (marketing and selling) costs so as to maximize profit.",
"It's easy to show arithmetically how modest increases in sales revenues and/or slight reductions in M&S costs have high impact on net profit.",
"See Drawing 38 : Creating Value for End-Users Choosing the best point (TODAY) on the current (red) curve is done somewhat intuitively today.",
"It's natural to increase resources (like adding another salesman) if expected additional gross profit generated exceeds the extra selling cost.",
"Increasing M&S costs has little or no effect on Sales revenue once on the plateau.",
"See Drawing 1 : Current Selling Process In the ‘service process’ case companies aspire to providing the highest affordable level of service and try to improve service level both in response time and in delivered quality.",
"Any attempt to increase TODAY's level of service increases cost exponentially unless the service process is re-engineered.",
"See Drawing 2 : Current Service Process Due to economies of scale there is a range in which sales-revenues are highly responsive to applied M&S resources (costs) and in which levels of service can be economically improved (red curve).",
"However, in both the selling and service cases both curves hit an asymptote—sales revenue does not respond to further M&S costs and attempts to deliver higher service causes exponential increases in cost of providing that service.",
"These ‘plateaus’ are due to diminishing returns.",
"Most companies are at a point nearing the plateau—otherwise they would simply move along the curve until they do reach the plateau (red star on diagrams)—in other words they would just do ‘more of the same’ as long as return is greater than cost.",
"See Drawing 3 : Selling—Shift to a Reengineered Process See Drawing 4 : Service—Shift to a Reengineered Process Modest gains might be made by automation of the existing processes.",
"But the ‘automated process’ (orange) curve is identical in shape to today's more manual process—just displaced.",
"Savings from pure automation are often not worth the cost of automating.",
"Significant sales-revenue increases and level-of-service improvements, respectively, require reengineering the processes.",
"Then new curves of a different shape emerge (green) in which increasing sales-revenue and level-of-service occur in an economical manner.",
"E.2.1.2 Processes Conducted by Teams Sales and service processes are event-driven because of the unpredictable behavior of buying organizations and competitors (for sales) and machines (for delivery of service).",
"Therefore it seems reasonable to consider possible ways of responding quickly to these events with appropriate competence.",
"Thus, it is axiomatic (self-evident) that: IF event-driven processes, such as selling and service, are conducted by dynamically formed and informed, self-managed, customer focused teams, consisting of expert team members as appropriate to the progression of the process THEN sales revenues achieved (at a particular M&S cost level) and the cost-of-providing-service (at a particular service-level) can be dramatically improved.",
"For event-driven field processes particularly, there is still a need to find more effective and efficient ways to achieve 1:1 (one person to one person), 1:N (one person to a team), N: 1 (a team to one person) and N:N (team to a team) interactions.",
"Such team: team interactions are desirable within and between the inbound (such as buyer) and outbound (such as seller) process participants.",
"Analogous to a football game, both teams must keep their eye-on-the-ball, being prepared at all times that the ball be passed to them to apply their particular skill or to respond to action by the other team.",
"E.2.2 The State-of-the-Art E.2.2.1 Background Art—the Productivity Paradox of Information Technology.",
"Modern communication technology has resulted in exponential growth of information distribution from the ‘hubs’ of centralized organizations but, on average, the information distributed has low added value beyond quick dispatching.",
"Centralization is also contrary to the way people like to work.",
"The high cost, low speed and the incomplete radio network coverage (except in the cities) are facts that will not go away soon.",
"Productivity corresponding to exponentially increasing information communication traffic is thus not being realized.",
"This is part of the long-standing ‘Productivity Paradox of Information Technology’.",
"E. 2.2.2 Background Art—, Object-Oriented Business Process Engineering Background art includes the availability (for at least five years) of tools to model business processes in an object-oriented manner (such as Rational Rose from the Rational Software company).",
"Business objects, including objects that represent worker role in processes, are identified by a so-called ‘Use-case’ approach.",
"These objects are later mirrored electronically in a supporting information system.",
"A software tool capable of converting an object-oriented business-engineering model to a system model was released to the market only in 1998.Therefore we believe that there are few published cases in which systems have been developed as a waterfall from business model to system model to working system and there are certainly no cases of systems created in such a manner that are an integral part of offerings of entire mobile process service (MPS) delivered over ICT.",
"E. 2.2.3 Background Art—Competence-Centric Organization The literature includes rather vague references to Competence Centers (Beyond Reengineering—Champy).",
"However, there are very few concrete adoptions—for reasons that include the unwillingness to give up ‘turf’ (hierarchical organizations) and the many other human factors such as recruitment, education, motivation and compensation of teams.",
"See Drawing 5 : the Process-Managed Company The need for such organizations applies to, for example, consultative selling processes where there is discussion of need/solution between seller/buyer.",
"In the delivery processes, especially delivery of engineering service that achieve the repair/maintenance of increasingly sophisticated equipment, such organizations are also needed.",
"Hierarchical, departmental organization is still the norm in most companies.",
"This results in allocation of tasks to resources from smaller, and more generally (rather than specially) qualified units.",
"Furthermore, a person's competence (education/training/experience) is still applied repetitively to few (usually one) processes (such as selling) rather than being spread over several processes.",
"In contrast, a competence-centric organization has qualified (educated, trained and experienced) personnel in (virtual) competence centers.",
"An engineer (for example) may be assigned to activities within development, production or marketing/sales processes when/where required as the process progresses.",
"Today such organizations rarely exist because the department (marketing, sales, development, production) ‘owns’ the resource.",
"See Drawing 36 : Matching Competence and Task E.2.2.4 Background Art—Access to Data Stored at a Central Site Wire-line can collect and deliver data as far as the radio base-station.",
"From there it must travel by radio to mobile people.",
"Today communication by radio is still slow, expensive (typically up to 25 cents/minute during business hours).",
"Good radio coverage is limited to cities—and there circuits are becoming overloaded.",
"Wide coverage (especially internationally), convenient, fast, low cost direct radio access to data stored at a central site is still several years away.",
"The development of good coverage has been handicapped by the recent auction of operating licenses by governments at high prices making subsequent investment in more base-stations (needed to give better coverage) a severe financial strain for operators.",
"E. 2.2.5 Background Art—Data Replication Data can be replicated from Data Base Management Systems (DBMS) based on rules that identify which rows/columns of data tables shall be exchanged between stations (for example using Adaptive Server DBMS from Sybase).",
"However, the rules for replication are still primitive—based on intuitive understanding of data requirement.",
"This means that data delivered over radio is far from optimized and has low average value-added.",
"See Drawing 6 : Replication—Publish and Subscribe E.2.2.6 Background Art—Ultra-Lite DBMS Recently DBMSs with minute ‘finger-print’ have appeared (such as from Sybase).",
"These can carry a database on palmtop (Psion) and mobile phone.",
"New operating systems (Windows CE from Microsoft and EPOC from Symbian) capable of working with these DBMSs are available.",
"Present applications are limited to storing small personal databases and two-way paging and certainly do not include the orchestration of self-managed teams.",
"E.2.2.7 Background Art—DBMS Triggers Some DBMSs (data base management systems) have the capability of creating notification messages when the ‘state’ of an object changes.",
"This is achieved by using ‘triggers’.",
"Such capability is hardly exploited today because the critical state-transitions have not been defined in the processes therefore what is notified to whom is ad hoc.",
"E.2.2.8 Background Art—Simple Notification The most advanced combination of appropriate notification to the appropriate person in effect today is a short message sent to a mobile telephone to advise the recipient that there is a waiting E-Mail—the title of the E-Mail may also be displayed.",
"But the E-Mail content itself is generally very unstructured, it is sent manually on a 1:1 or 1:N basis, is expensive to retrieve over radio and the E-Mail becomes yet another fragment in the unorganized in-box of the recipient."
],
[
"<SOH> F. BRIEF SUMMARY OF THE INVENTION <EOH>"
],
[
"E. BACKGROUND OF THE INVENTION E.1 Field of Invention The invention concerns the reengineering of event-driven, field business processes (for example sales, or delivery of articles and/or services at customer sites), enabled by teams assembled within a competence-centric organization that is, in turn, enabled by an information system exploiting data distribution over radio.",
"Of particular interest is the deployment as a Mobile Process Service—with emphasis on ‘process’ rather than ‘product’ or ‘application’.",
"Deployment can be worldwide due to advances in ICT (information communication technology).",
"E.2 Background Art and Assessment Thereof E.2.1 Theoretical Background E.2.1.1 Relationship Between Effort and Result The generic S-curve nature of the relationship between effort and result is well established.",
"In the ‘selling process’ case, for example, the aspiration is to optimize the combination of sales revenue and M&S (marketing and selling) costs so as to maximize profit.",
"It's easy to show arithmetically how modest increases in sales revenues and/or slight reductions in M&S costs have high impact on net profit.",
"See Drawing 38: Creating Value for End-Users Choosing the best point (TODAY) on the current (red) curve is done somewhat intuitively today.",
"It's natural to increase resources (like adding another salesman) if expected additional gross profit generated exceeds the extra selling cost.",
"Increasing M&S costs has little or no effect on Sales revenue once on the plateau.",
"See Drawing 1: Current Selling Process In the ‘service process’ case companies aspire to providing the highest affordable level of service and try to improve service level both in response time and in delivered quality.",
"Any attempt to increase TODAY's level of service increases cost exponentially unless the service process is re-engineered.",
"See Drawing 2: Current Service Process Due to economies of scale there is a range in which sales-revenues are highly responsive to applied M&S resources (costs) and in which levels of service can be economically improved (red curve).",
"However, in both the selling and service cases both curves hit an asymptote—sales revenue does not respond to further M&S costs and attempts to deliver higher service causes exponential increases in cost of providing that service.",
"These ‘plateaus’ are due to diminishing returns.",
"Most companies are at a point nearing the plateau—otherwise they would simply move along the curve until they do reach the plateau (red star on diagrams)—in other words they would just do ‘more of the same’ as long as return is greater than cost.",
"See Drawing 3: Selling—Shift to a Reengineered Process See Drawing 4: Service—Shift to a Reengineered Process Modest gains might be made by automation of the existing processes.",
"But the ‘automated process’ (orange) curve is identical in shape to today's more manual process—just displaced.",
"Savings from pure automation are often not worth the cost of automating.",
"Significant sales-revenue increases and level-of-service improvements, respectively, require reengineering the processes.",
"Then new curves of a different shape emerge (green) in which increasing sales-revenue and level-of-service occur in an economical manner.",
"E.2.1.2 Processes Conducted by Teams Sales and service processes are event-driven because of the unpredictable behavior of buying organizations and competitors (for sales) and machines (for delivery of service).",
"Therefore it seems reasonable to consider possible ways of responding quickly to these events with appropriate competence.",
"Thus, it is axiomatic (self-evident) that: IF event-driven processes, such as selling and service, are conducted by dynamically formed and informed, self-managed, customer focused teams, consisting of expert team members as appropriate to the progression of the process THEN sales revenues achieved (at a particular M&S cost level) and the cost-of-providing-service (at a particular service-level) can be dramatically improved.",
"For event-driven field processes particularly, there is still a need to find more effective and efficient ways to achieve 1:1 (one person to one person), 1:N (one person to a team), N: 1 (a team to one person) and N:N (team to a team) interactions.",
"Such team: team interactions are desirable within and between the inbound (such as buyer) and outbound (such as seller) process participants.",
"Analogous to a football game, both teams must keep their eye-on-the-ball, being prepared at all times that the ball be passed to them to apply their particular skill or to respond to action by the other team.",
"E.2.2 The State-of-the-Art E.2.2.1 Background Art—the Productivity Paradox of Information Technology.",
"Modern communication technology has resulted in exponential growth of information distribution from the ‘hubs’ of centralized organizations but, on average, the information distributed has low added value beyond quick dispatching.",
"Centralization is also contrary to the way people like to work.",
"The high cost, low speed and the incomplete radio network coverage (except in the cities) are facts that will not go away soon.",
"Productivity corresponding to exponentially increasing information communication traffic is thus not being realized.",
"This is part of the long-standing ‘Productivity Paradox of Information Technology’.",
"E. 2.2.2 Background Art—, Object-Oriented Business Process Engineering Background art includes the availability (for at least five years) of tools to model business processes in an object-oriented manner (such as Rational Rose from the Rational Software company).",
"Business objects, including objects that represent worker role in processes, are identified by a so-called ‘Use-case’ approach.",
"These objects are later mirrored electronically in a supporting information system.",
"A software tool capable of converting an object-oriented business-engineering model to a system model was released to the market only in 1998.Therefore we believe that there are few published cases in which systems have been developed as a waterfall from business model to system model to working system and there are certainly no cases of systems created in such a manner that are an integral part of offerings of entire mobile process service (MPS) delivered over ICT.",
"E. 2.2.3 Background Art—Competence-Centric Organization The literature includes rather vague references to Competence Centers (Beyond Reengineering—Champy).",
"However, there are very few concrete adoptions—for reasons that include the unwillingness to give up ‘turf’ (hierarchical organizations) and the many other human factors such as recruitment, education, motivation and compensation of teams.",
"See Drawing 5: the Process-Managed Company The need for such organizations applies to, for example, consultative selling processes where there is discussion of need/solution between seller/buyer.",
"In the delivery processes, especially delivery of engineering service that achieve the repair/maintenance of increasingly sophisticated equipment, such organizations are also needed.",
"Hierarchical, departmental organization is still the norm in most companies.",
"This results in allocation of tasks to resources from smaller, and more generally (rather than specially) qualified units.",
"Furthermore, a person's competence (education/training/experience) is still applied repetitively to few (usually one) processes (such as selling) rather than being spread over several processes.",
"In contrast, a competence-centric organization has qualified (educated, trained and experienced) personnel in (virtual) competence centers.",
"An engineer (for example) may be assigned to activities within development, production or marketing/sales processes when/where required as the process progresses.",
"Today such organizations rarely exist because the department (marketing, sales, development, production) ‘owns’ the resource.",
"See Drawing 36: Matching Competence and Task E.2.2.4 Background Art—Access to Data Stored at a Central Site Wire-line can collect and deliver data as far as the radio base-station.",
"From there it must travel by radio to mobile people.",
"Today communication by radio is still slow, expensive (typically up to 25 cents/minute during business hours).",
"Good radio coverage is limited to cities—and there circuits are becoming overloaded.",
"Wide coverage (especially internationally), convenient, fast, low cost direct radio access to data stored at a central site is still several years away.",
"The development of good coverage has been handicapped by the recent auction of operating licenses by governments at high prices making subsequent investment in more base-stations (needed to give better coverage) a severe financial strain for operators.",
"E. 2.2.5 Background Art—Data Replication Data can be replicated from Data Base Management Systems (DBMS) based on rules that identify which rows/columns of data tables shall be exchanged between stations (for example using Adaptive Server DBMS from Sybase).",
"However, the rules for replication are still primitive—based on intuitive understanding of data requirement.",
"This means that data delivered over radio is far from optimized and has low average value-added.",
"See Drawing 6: Replication—Publish and Subscribe E.2.2.6 Background Art—Ultra-Lite DBMS Recently DBMSs with minute ‘finger-print’ have appeared (such as from Sybase).",
"These can carry a database on palmtop (Psion) and mobile phone.",
"New operating systems (Windows CE from Microsoft and EPOC from Symbian) capable of working with these DBMSs are available.",
"Present applications are limited to storing small personal databases and two-way paging and certainly do not include the orchestration of self-managed teams.",
"E.2.2.7 Background Art—DBMS Triggers Some DBMSs (data base management systems) have the capability of creating notification messages when the ‘state’ of an object changes.",
"This is achieved by using ‘triggers’.",
"Such capability is hardly exploited today because the critical state-transitions have not been defined in the processes therefore what is notified to whom is ad hoc.",
"E.2.2.8 Background Art—Simple Notification The most advanced combination of appropriate notification to the appropriate person in effect today is a short message sent to a mobile telephone to advise the recipient that there is a waiting E-Mail—the title of the E-Mail may also be displayed.",
"But the E-Mail content itself is generally very unstructured, it is sent manually on a 1:1 or 1:N basis, is expensive to retrieve over radio and the E-Mail becomes yet another fragment in the unorganized in-box of the recipient.",
"F. BRIEF SUMMARY OF THE INVENTION F.1 PFCN—Profitable Fulfillment of Customer Needs If customer needs are not fulfilled (satisfied) then there is no way in which vendors can be profitable.",
"However, it can happen that customer needs are fulfilled but, nevertheless, the vendors fail to capture enough of created value for they themselves to be profitable.",
"The acronym PFCN, Profitable Fulfilment of Customer Needs, the philosophy underlying the invention, reflects the intention for win-win.",
"This requires that the vendor: a) creates value for the customer (a Value Chain issue), AND b) captures a fair share of that value (an Industry Structure issue).",
"Thus, projects for customer clients must strike a balance between creation and capture of value.",
"The invention does exactly that—it creates value in a customer team environment, but also assures that the value within the vendor team will be captured in an equitable way for, and between, the vendors (that also work as a team!).",
"See Drawing 44: Balancing Value Creation & Value Capture F.2 The Invention is Recognition, Followed by Realization, of the PFCN Axiom!",
"F.2.1 Recognition of the PFCN Axiom by Inductive Thinking Traditional business education is based on deductive thinking.",
"Deductive thinking starts with a problem and reasons about different ways to solve the problem (the pay-off matrix).",
"Such an approach is analogous to goal achievement (solving the problem) by back-chaining logic as applied in some expert systems: The problem in our case is to make event-driven B2B mobile processes more effective and efficient Such effectiveness and efficiency could be achieved if business process could, somehow, be readily reengineered.",
"Such process reengineering could be achieved if appropriate organization changes could, somehow, be implemented.",
"Such organization changes could be implemented if appropriate systems to support those changes could, somehow, be developed.",
"The invention breakthrough was the sudden recognition of the organizational and process effects that occurred when isolated new technology implementations, originally intended as automations of traditional processes, were made.",
"For example, automation of complex pricing calculations done on notebook PCs (a technology implementation) meant that the billing process could now be done at the customer site (a process change); the central billing department thus disappeared (an organization change).",
"This led to the formulation of the PFCN hypothesis in which synergies were suspected—but not yet rigorously proven.",
"This led to pressure to test the hypothesis, but because of the recent dot-com bubble experience there was extreme resistance to moving ahead as if the hypothesis was true.",
"Even scientific explanation of the risks of missing the next big idea because of inaction failed to provoke modest testing of the hypothesis.",
"See Drawing 45: Errors in Hypothesis Testing However, it soon became self-evident to experienced B2B process observers that the organizational and process effects could be harnessed to yield dramatic changes in effectiveness and efficiency but that this must be done in a structured way—by business process engineering.",
"The PFCN hypothesis was therefore upgraded—leapfrogging the traditional escalation from hypothesis to theory, and hence to law, and jumping directly to axiom status.",
"If the positive effect of actions is obvious it should be done!",
"This is exactly what Lucy Kellaway recommended in her recent article in Financial Times.",
"“I'd like to offer academics considering further studies a tip.",
"If they already know the answer to a question, proving it statistically is not time well spent.",
"Which leaves one wondering how it is possible to be so very clever and so fantastically stupid at the same time.” See Drawing 34: the PFCN Axiom The PFCN axiom was thus recognized by inductive thinking—understanding that new technical capabilities can solve persisting, and perhaps accepted, problems: New technology, such as the instantaneous creation of distributed databases invoking data over radio, means that new systems to be built.",
"Such systems enable self-managed team organization to be sustained in which teams are dynamically formed and informed.",
"Such team-oriented organization enables processes to be reengineered in a manner that creates more value through greater effectiveness and efficiency.",
"The recognition of the axiomatic nature of the PFCN approach is, in fact, due to the well-established fact that ‘need is based on an understanding of what is possible’.",
"Since it is now technically possible to satisfy all the prerequisites of the PFCN axiom through the synergistic combination of systems, organization and processes why not do it?",
"See Drawing 35: Engineered SOP Synergy Thus the invention has been conceived by inductive thinking but, once understood, has been implemented by traditional deductive thinking—as we will now describe .",
".",
".",
".",
"F.2.2 Realization of the PFCN axiom by deductive thinking The invention exploits the understanding that an entire mobile process service (MPS) can be realized by the deductive combination of object-oriented business process reengineering, competence-centric organization and object-oriented system development with final software generation by RAD (rapid application development).",
"Global deployment of reengineered processes with ICT is the icing on the cake.",
"The invention is characterized by conducting event-driven processes with teams dynamically formed and dissolved by the requisition of members from virtual competence centers using an information system whereby each team member is notified of a change of state that requires activity and that exactly the right information required to execute that activity—as indicated by a business object collaboration model (another new technology)—is physically communicated to local, distributed databases by replication over radio.",
"The synergistic combination of isolated new Information system, isolated new Organization ideas and isolated new Business Processes modeling capability is achieved in the following way: F.2.2.1 Information System High integrity distributed data bases (on portable devices called-B) are maintained by replication and asynchronous (message) communication to a remote, primary, database (called-A), including one or more stations operating over radio, often using middle-ware that enables transparent multi-network (e.g.",
"circuit/packet switched) data transport.",
"Automated message notifications of critical changes of state are made over pocket (or smaller) communication devices (called-C) such as mobile phones and/or pagers and/or communicators.",
"When a user has both a distributed database (on device called-B) and a pocket device (called-C) then there may be one- or two-way wire or wireless interaction between A and C and/or between B and C by asynchronous communication.",
"An information system of this nature synergistically facilitates the following organization: F.2.2.2 Organization Virtual, dynamically formed and dissolved, empowered customer-focused teams that conduct marketing or sales or service processes.",
"Teams are formed by requisition of resources from virtual competence centers to manage the state-transitions of key objects.",
"An organization of this nature synergistically facilitates the following business processes: F. 2.2.3 Business Processes Event-driven processes, wherein activities are conducted at remote sites (such as at customer premises), often engineered by object-oriented modeling then conveyed to organization architects and information system builders as descriptions of processes in particular: 1.assessment of the continuously changing (event-driven) situation to determine the value of the resource that should justifiably be applied to the process, 2.formation of the team (by requisitioning resources from competence centers), 3.coordination of contact occasions (in which people and/or equipment, and/or sites such as meeting rooms participate).",
"F.3 ADVANTAGEOUS EFFECTS OF THE INVENTION F.3.1 WHY Does the Invention Give a ‘Result of Value’?",
"The reason for results of value can be traced to value chain effects—in particular: HR Management in the ‘support activities’ area, outbound logistics for vendors and the mirror-image inbound activities for customers, and marketing/selling.",
"See Drawing 41: Value Chain F.3.2 FOR WHOM Does the Invention Give a ‘Result of Value?",
"F.3.2.1 Outbound Process Beneficiary The organization conducting the outbound process—such as selling or performing the service (Dr. Michael Porter terminology) benefits from the following added values: If an organization conducts the marketing/sales or service process at lower cost (efficiency) this leads to higher profit to that organization.",
"Alternatively the lower cost may enable a lower price to be offered resulting in being more competitive (effectiveness).",
"And/or the result may be better (effectiveness) yielding more sales or, in the case of service, a higher quality repair/maintenance job thus improving the reputation of the party conducting the outbound process—and enable charging a higher price.",
"F.3.2.2 Inbound Process Beneficiary The organization conducting the inbound process, such as buying, or being the owner/user of the equipment being serviced, also gain value in the form of: reducing time/cost incurred in reaching the buying decision (efficiency), and/or making a better choice regarding what to buy (effectiveness), and/or getting a prompt (efficiency) higher quality (effectiveness) service repair/maintenance job done.",
"F. 3.2.3 Personal Satisfaction Beneficiary All individuals involved in the superior processes gain value.",
"One source of such value is when the employee work satisfaction is increased.",
"For example—engagement in tasks that are self-planned and at/near the upper limit of competence leads to job satisfaction (value to employee) and consequently lower employee turnover (value to employer).",
"F.3.3 What is The Magnitude of The Benefits of The Invention?",
"Capture of created value (due to SOP synergy) depends on industry structure.",
"See Drawing 37: Five Competitive Forces One option for industry structure is a franchise arrangement in which a FranchisER appoints three types of FranchisEE.",
"This particular franchise structure will be one of the invention claims (see later) because it is the means of: conducting both marketing/selling and delivery processes of the proposed mobile process service (MPS) to create value, and protecting the capture of a fair share of the value created for the ICT franchisee—without such protection it is likely that the ICT service would be relegated to commodity status (this is a dilemma for today's telecom industry).",
"See Drawing 9: Mobile Process Service Industry F. 3.3.1 Benefit Magnitude for Mobile Process Service End-Users (MPUs) See Drawing 38: Creating Value for End Users In this example any company that conducts mobile processes will benefit by using the invented mobile process service because of increased effectiveness of both their marketing/selling and delivery processes.",
"Their customers will be more satisfied with the mobile process user's new processes and thus will buy more from such vendors (or buy the same volume at a higher price).",
"A modest 5% increase in sales results in a 50% increase in profit for the mobile process user.",
"F.3.3.2 Benefit Magnitude for Vendors of the Mobile Process Service The current estimate for the mobile process service market in the NORBA countries (consisting of Denmark, Finland, Iceland, Norway, Sweden and the so-called ‘Baltic States’ of the previous USSR) is over two billion Swedish Kronor (about three hundred million US$) in the fifth year.",
"This could be extrapolated to six billion US$ worldwide.",
"See Drawing 39: Norba Bundled ($1=7Swedish Kronor) Exactly how the revenue might be divided between the vendors also depends on industry structure.",
"The example chosen for illustration shows the split between the proposed SYSTEM resource team-members (Prestudy and Development) and the ICT team-members (Operation).",
"It might be that there will be sufficient revenue generated by what is called ‘Differentiation’ (due mainly to synergy between team-resource expertises) that the Franchiser and Franchisees can live on this.",
"The resource team-members can reap unusually high margins as resource team-members and will not be tempted to compete as ‘new entrants’ to the franchisee's position in the channel (an industry structure insight).",
"See Drawing 40: Norba Unbundled ($1=7 Swedish Kronor) G. BRIEF DESCRIPTION OF THE VIEWS OF THE DRAWING DRAWING 1: CURRENT SELLING PROCESS 40 DRAWING 2: CURRENT SERVICE PROCESS 40 DRAWING 3: SELLING—SHIFT TO A REENGINEERED PROCESS 41 DRAWING 4: SERVICE—SHIFT TO A REENGINEERED PROCESS 41 DRAWING 5: THE PROCESS-MANAGED COMPANY 42 DRAWING 6: REPLICATION—PUBLISH AND SUBSCRIBE 42 DRAWING 7: REENGINEERING v. IMPROVEMENT 43 DRAWING 8: EVENT-DRIVEN PROCESS FULFILLMENT 43 DRAWING 9: MOBILE PROCESS SERVICE INDUSTRY 44 DRAWING 10: DELIVERY—TEAM ROLES.",
"This picture shows the delivery role taken by hypothetical team members.",
"Company logos should be interpreted as “such as”.",
"44 DRAWING 11: MAIN PROCESSES 45 DRAWING 12: USE CASE MODEL 45 DRAWING 13: ‘ASSESSING USE CASE OBJECT COLLABORATION 46 DRAWING 14: NOTIFICATION 46 DRAWING 15: OBJECT COLLABORATION DIAGRAM—ALLOCATING 47 DRAWING 16: ‘BUILDING TEAM’ SUPPORTING USE CASE 47 DRAWING 17: OBJECT COLLABORATION DIAGRAM—COORDINATING 48 DRAWING 18: ‘COORDINATING CONTACT OCCASIONS SUPPORTING USE CASE 48 DRAWING 19: PARTICIPATION OBJECT STATE TRANSITIONS 49 DRAWING 20: SPECIFICATION OF THE PROSPECT MANAGER OBJECT 49 DRAWING 21: RESOURCE BUDGET 50 DRAWING 22: INFORMATION SYSTEM—PHYSICAL DESCRIPTION 50 DRAWING 23: SYSTEM NETWORK AND DEVICES 51 DRAWING 24: MPS PROVIDER AND MPU DOMAINS 51 DRAWING 25: INFORMATION SYSTEM—USER INTERFACE MAIN SCREEN 52 DRAWING 26: DATA MODEL 52 DRAWING 27: INFLUENCE TABLE 53 DRAWING 28: REPLICATION RULE FORMULATION 53 DRAWING 29: COMMUNICATION MIDDLEWARE—PHYSICAL 54 DRAWING 30: COMMUNICATION MIDDLEWARE—LOGICAL 54 DRAWING 31: STATE TRANSITION NOTIFICATION MESSAGE REQUEST 55 DRAWING 32: OBJECT COLLABORATION—EXPEDITE REQUEST 55 DRAWING 33: CUSTOMER MAY ACCESS OVER WEB SERVER 56 DRAWING 34: THE PFCN AXIOM 56 DRAWING 35: ENGINEERED SOP SYNERGY 57 DRAWING 36: MATCHING COMPETENCE AND TASK 57 DRAWING 37: FIVE COMPETITIVE FORCES 58 DRAWING 38: CREATING VALUE FOR END USERS 58 DRAWING 39: NORBA MARKET BUNDLED ($1=7 Swedish Kronor) 59 DRAWING 40: NORBA UNBUNDLED ($1=7 Swedish Kronor) 59 DRAWING 41: VALUE CHAIN 60 DRAWING 42: PFCN COMPETENCE CENTERS 60 DRAWING 43: SE COMPETENCE CENTER PROFILE 61 DRAWING 44: BALANCING VALUE CREATION & VALUE CAPTURE 61 DRAWING 45: ERRORS IN HYPOTHESIS TESTING 62 DRAWING 46: EXPERT SYSTEM LANGUAGE TRANSLATION 62 H. DETAILED DESCRIPTION OF THE INVENTION The invention describes the implementation of a combination of object-engineered process, competence-centric organization and a radio-enabled information system.",
"That combination is then applied through a mobile process service (MPS) to enable field-based teams.",
"This will have a major effect on productivity of the event-driven field processes.",
"The combination is very multi-discipline and unlikely to occur to a person skilled in just one, or even two, of the necessary three disciplines (process, organization and systems).",
"A business process is defined by Ivar Jacobssen as ‘a series of activities that delivers a result of value’.",
"Whether a business process shall be truly reengineered or just ‘improved’ depends on willingness to make fundamental changes in all of: PROCESS, ORGANIZATION, and INFORMATION SYSTEMS.",
"See Drawing 7: Reengineering V. Improvement The invention is the result of combining a sub-set from each of these three mutually dependent and synergistic components namely: Process: object-engineered Organization: competence-centric, and Information system: radio-enabled This combination is then applied not only to main processes but also to the essential supporting process components of event-driven field processes—namely: assessing (the continuously changing situation), building team, and coordinating contact occasions See Drawing 8: Event-Driven Process Fulfillment Performance of processes by a hierarchical (dispatch oriented) organization is dispensed with and the object-engineered process, treated as self-managed collaboration (by the team) between business objects, is conducted by co-operation within and between dynamically formed/dissolved teams composed of empowered members drawn from (virtual) competence centers.",
"Both the organization and process are supported by a radio-enabled information system that provides on-site electronic representation of the progress of the process by mirroring the collaboration between the real objects.",
"The invention virtually eliminates cost, speed and convenience as hindrances because information is communicated by radio to and from locally stored high integrity databases.",
"This solution is: low cost for two reasons: a) efficient relational representation of information, and b) field-by-field update by relatively short data packages over radio, furthermore speed is unimportant because the database is maintained in the background, and access is convenient because information is always available at the finger-tips (must not be retrieved from a remote central site) and arrives at the user before it will be needed (this is achieved by modeling the required information then rifle-shooting it over radio).",
"H.1 Object-Engineered Process With the exception of one claimed proprietary selling process (in which Understanding Customer Fulfillment will be articulated), the invention applies explicitly to three so-called ‘supporting’ sub-processes that are relevant only when supporting event-driven field processes (such as sales and service).",
"Again, the three supporting sub-processes are: Assessing (the situation) Building the team, and Coordinating contact occasions H.2 Competence-Centric Organization The organization has appropriate Competence Centers to which personnel are recruited and assume a Resource role whenever requisitioned by the Process Leader role and assigned by the Allocator role.",
"Such ‘Competence Centers’ are virtual—not physical.",
"H.3 Radio-Enabled Information System The radio-enabled information system has three essential features: distributed databases, maintained by replication asynchronous communication over packet-switched radio, and notification messages.",
"These foregoing three features are especially powerful when combined.",
"For example, in the following ‘one way of implementing the invention’ section there may be: cascading replication (for example from a central database, to a field notebook station to a hand-held device carrying the Ultralite DBMS from Sybase) combined with the emerging Blue-Tooth technology (for communication within short range between a mobile phone and/or palmtop and/or notebook), combined with SMS messages for notification.",
"H.4 One Way to Carry Out the Invention NOTE: Some examples are intentionally repetitively described—perhaps once functionally and later in the text technically.",
"The interaction between System, Organization and Process is fundamental and therefore emphasised.",
"H.4.1 Selection of Example at the Vendor Level The way in which a mobile resource (working in the field) operates is virtually independent of how the MPS (mobile process system) has been: sold to the user company, developed as a system (process, organization and software), deployed invoking ICT as a combination of data service (DS) and network service (NS).",
"Nevertheless, that background is shown below because there are claims associated with the manner in which the technical and process thinking permits innovative industry structure and pricing—and vice versa.",
"H.4.1.1 Mobile Process Service Industry Level The overall industry structure that will be claimed (see later) as innovative and not obvious has a FRANCHISER and three types of FRANCHISEE (Innovation Service, System Service and ICT Service).",
"See Drawing 9: Mobile Process Service Industry.",
"H.4.1.2 Mobility Franchiser (Certification) Level The MOBILITY FRANCHISER has two operation roles: Appoint Inspiration-, System- and ICT-Service franchisees Certify resource Team members Franchisees may sub-contract only certified resources.",
"This guarantees service quality.",
"See Drawing 42: PFCN Competence Centers During certification, the franchiser will assure that Team member resources have the necessary competence profile—at the Basic, Professional or Expert level.",
"For example, the SE resource must be expert in SE, Professional in SIF and Basic in all other areas.",
"See Drawing 43: SE Competence Center Profile H.4.1.3 Inspiration Franchise (Customer ‘Inspiring’) Level The franchiser subcontracts the INSPIRATION FRANCHISEE to conduct the IC (Inspiring Customer) process.",
"H.4.1.4 System Service Franchisee (Development) Level The SYSTEM SERVICE FRANCHISEE will sub-contract resource Team members to construct a customized, production quality, PFCN Process Simulator for the mobile process user.",
"This will be achieved by Business Process Engineering, Organization formulation, Software creation as well as system integration in the field devices and to the back-office architecture of the client MPU.",
"The PFCN Process Simulator is the deliverable.",
"See Drawing 10: Delivery—Team Roles H.4.1.5 ICT Franchisee (Deployment) Level The MPU (mobile process user) company has several options: operate the data service (DS) itself, and/or ask the ICT franchisee to appoint a DS resource team member, and/or ask the ICT franchisee to arrange for network services in various countries.",
"The PFCN architecture is such that the MPU has many options for physical location of the various servers and ‘interface’ databases (the ‘sliding window’ analogy in the drawing).",
"Most important, the ICT service can be arranged so that the ‘System’ designed by the System Franchisee can be deployed worldwide.",
"See Drawing 24: MPS Provider and MPU Domains H.4.2 Selection of Example at the Mobile Process User (MPU) Level The invention is applicable to many event-driven field processes—such as sales, customer service, public emergency services.",
"The PFCN framework has four ‘sub’ main processes (UCF, IC, PP and DAS) from the domain of industrial marketing in which the invention may be carried out.",
"These four sub-processes apply to companies selling capital equipment, consumables or various forms of service ranging from preventive or emergency equipment maintenance to security.",
"We shall show in detail how the invention could be carried out in one of these—the ‘Pursuing Prospect’ (PP) process that pursues an already identified acute, significant, sales opportunity.",
"See Drawing 11: Main Processes The foregoing four sub-processes are event-driven to various extents depending on such things as products/services offered, industry and competitive environment.",
"The modern approach is to customize each of these four processes to individual customers.",
"H.4.3 Terminology Throughout the following scenario all business object names will be capitalized and underlined the first time they are introduced—thus: PROSPECT.",
"Thereafter just the first letter will be capitalized and the object name underlined as a constant reminder that this is a business object.",
"For simplicity in this example we minimize the number of Roles, referring only to PROSPECT MANAGER, ALLOCATOR and RESOURCE.",
"In this scenario we assume that the business process owner has decided that the role ‘Prospect Manager’ shall handle what could have been alternatively named as three separate three sub-roles—namely Assessor, Team-builder and Contact-Coordinator.",
"This is a nice example of retention of a little ‘centralized’ thinking—to some extent the three roles are at the Prospect manager role ‘responsibility hub’—but who shall assume that role can be changed at any time as the process progresses.",
"Retention of some hierarchical and centralized thinking is usual during the transition to the new competence-centric form of organization.",
"H.4.4 Multiple Roles The several roles, for a given Prospect, are often played by one person.",
"Thus, for a given Prospect instance, Annika might take both the Prospect Manager role and a Resource role—that decision will be made by the Allocator role when Annika (in her role of Prospect manager) specifies the required COMPETENCE.",
"H.4.5 How the Example Prospect Pursuit Process Begins The assignment of the Prospect in question to Annika in the first place was the last step of the previous process called ‘Inspiring Customer’.",
"The Business Process Owner (responsible for the specification of the process) has decided that the ‘Pursuing Prospect’ process formally begins when the Prospect Manager role (in this case assumed by Annika) begins the first Assessment task (for the specific Prospect instance).",
"The purpose of the Assessment process is to control release of Funds to pay (by internal accounting) for Resources that will pursue each specific Prospect.",
"H.4.6 Development of the Three Object-Engineered Supporting Processes Recall that the invention is the competence-centric organization and radio-enabled information system required for execution of the Assessing, Building team and Coordinating Contact Occasion supporting processes.",
"H.4.6.1 Use Case Model As its name suggests a ‘Use case’ is used.",
"A Use case is always used by an Actor.",
"See Drawing 12: Use Case Model By definition an Actor must gain a result of value by using the Use case.",
"Delivery of this result is achieved by ‘realization’ of the Use case.",
"The decision Influencer (usually employed within the CUSTOMER team) must find this process efficient and effective in his/her role as an Influencer.",
"Once the Actor-Use case relationships are mapped, the Use case must be ‘realized’ by collaboration between business objects.",
"Objects collaborate by sending messages and receiving messages as the following example shows.",
"H.4.6.2 ‘Assessing’ Supporting Use case Just as diagnosis and treatment often require different competencies so it is with assessment of the Prospect (by the Prospect Manager) and the execution of pursuit action (by the Resource).",
"Assessing can be represented by the collaboration between the Prospect Manager, PROSPECT BANK, Prospect, BUSINESS PARTY, INFLUENCE, PERSON and PROSPECT PURSUIT BUDGET objects in the sequence shown on the connecting arrows.",
"See Drawing 13: ‘Assessing’ Supporting Use Case This process example requires certain capabilities of executing activities.",
"For example Step #6 (Select prospect) requires that the Prospect manager decide which Prospect to prioritize.",
"This is discussed further in section 1.5.6.2.Not surprisingly the ‘Assessing’ process formally begins at ‘Start Assessment’ (see ‘START’ above).",
"One might wonder what triggers Annika to ‘start’?",
"She may get a phone call from a team colleague to inform her that an event has occurred that requires her to ‘Assess’ the Prospect again or, in a highly automated ‘Assessing’ process, she may receive a SMS message on her mobile telephone (how this is achieved technically comes later .",
".",
".",
").",
"See Drawing 14: Notification This was an introduction to object collaboration using the Assessing process as an example.",
"There are corresponding sequences for the other two sub-processes (Building team and Coordinating contact occasion).",
"H.4.6.3 ‘Building Team’ Supporting Use Case In Building team, the Prospect manager role specifies the required Competence of the Resource being requisitioned to the team.",
"But it is the ALLOCATOR role that decides which PERSON shall fill that Resource role by checking the AVAILABILITY (another new object) of the alternative Resources then proceeding to adding a team member.",
"A typical Building team object collaboration diagram is provided in the appendices—this model is not intended to be studied in detail but just to indicate the appearance of several more objects.",
"See Drawing 15: Object Collaboration Diagram—Allocating Building team is a reiterative process.",
"In the example below there is a team to pursue a milking system Prospect at the Dancing Bull Ranch.",
"At any point in time there may be a team (Kristina Lund and Hans Welander) and outstanding requests for further Competence (Product/Service Engineer still to be filled).",
"See Drawing 16: ‘Building Team’ Supporting Use Case H.4.6.4 ‘Coordinating Contact Occasion’ Supporting Use Case A typical Coordinating contact occasion collaboration diagram is provided in the appendices.",
"See Drawing 17: Object Collaboration Diagram—Coordinating Contact Occasion A CONTACT OCCASION may be represented in the following way: See Drawing 18:‘Coordinating Contact Occasions’Supporting Use Case In the Coordinating contact occasion sub-process it has been decided (by the ‘Business Process Owner’) that the Prospect manager role shall take the responsibility for coordinating Contact occasions.",
"This includes deciding who shall be invited and who are the essential participants that must accept the invitation before the meeting is confirmed.",
"Note that two of the meeting participants are from the Customer organization (Dancing Bull Ranch) and one from the Vendor company (Beta Laval).",
"The Customer has a team too!",
"One reason for the anticipated effectiveness and efficiency shifts by the proposed process reengineering is that the Customer will take a much more active role in the overall selling process—for mutual benefit.",
"The information system (see later) must therefore enable dialogue with the Customer—not only in the Pursuing Prospect process but also in other core marketing/sales processes (not elaborated here).",
"See Drawing 19: Participation Object Especially important is that objects can be considered to have states, and that progress in processes may be tracked/measured according to state-transitions.",
"For example, the allowable ‘states’ of a Contact occasion object include ‘intended’, ‘proposed’ and ‘committed’.",
"Allowable ‘states’ of the PARTICIPATION object (shown above) are ‘desired’, ‘invited’, ‘reserved’, ‘confirmed’ and ‘withdrawn’.",
"In order to keep a record of the meeting, ‘attended’ would be another required state.",
"This Participation object thus has the potential to record the time that the Resource was occupied—either for billing the Customer (if the Contact Occasion is a service call for example) and/or for internal cost tracking necessary to determine profitability of each Customer.",
"The factor most responsible for tedious co-ordination of meetings (or scheduling service visits) is delays in responding to the invitation.",
"Therefore when the Participation state is changed from ‘desired’ to ‘invited’ it's important that the invitee is made aware of the invitation and responds quickly.",
"This explains the notation ‘Notification required’ in the diagram.",
"See Drawing 31: State Transition Notification Message Request The system engineer is requested to find a way to alert the invitee and facilitate an accept/decline response that can be incorporated into the database en route to sharing with all team members.",
"Thus, ten minutes after an invitation message is sent the system can check whether a reply has been received.",
"If not further follow-up action can be initiated.",
"See Drawing 32: Object Collaboration_Expedite Request H.4.7 Competence-Centric Organization H.4.7.1 Process-Management Orientation The following diagram illustrates a single instance of a Prospect pursuit in which Resources from two (Product and Sales) of the three competence centers (Application, Product and Sales) participate.",
"See Drawing 5: Process Management Orientation Already the involvement of several ‘roles’ in the Pursuing Prospect process have been mentioned—Prospect manager, Allocator and Resource.",
"In a pure competence-centric organization all of these roles would be filled by requisition from a competence center.",
"A competence center is not a physical site but a virtual grouping of employees based on primary competence into which personnel are recruited, trained, motivated and rewarded based on the performance of the teams of which they become members.",
"The Allocator role would be especially aware of the skills available in the various competence centers.",
"Selection of the appropriate Person as a Resource in any instance would be governed by Competence to perform the ‘activities’ defined by the Business Process Owner.",
"H.4.7.2 Specification of the Prospect Manager Object (For Example) The person who manages the competence center is made aware, in a highly structured way, of which activities are to be performed by each role.",
"This is part of the formal specification of the role (such as Prospect manager) object.",
"A role object must have the Competence to conduct ‘activities’ assigned to that role by the Business Process Owner.",
"Recalling that (in this example) the Prospect manager role covers Assessor, Team Builder and Contact occasion coordinator roles it is not surprising that the Prospect manager specification includes a wide range of activities derived from collaboration in several processes.",
"Notice that all the various activities, of which the Prospect manager role must be capable, occur in one or more of the object collaboration diagrams for the use cases in which the Prospect manager participates.",
"This object-oriented approach clearly gives a ‘language’ (for example UML) in which the business process engineer and the organization consultant may communicate.",
"See Drawing 20: Specification of the Prospect Manager Object The overall Pursuing process ends when the Customer has made a decision.",
"Each sub-process also has an end, as shown in the following Assessing process example that ends when the Pursuit budget has been updated.",
"One can see instantly how much the Account manager (role object) is prepared to invest to secure this business (Ceiling 10 000), how much the Prospect manager has released to the Allocator (Funded 10 000) and what Resources have been ‘allocated’ on a contingent basis (2700).",
"See Drawing 21: Resource Budget H.4.8 The Information System H.4.8.1 Physical Description See Drawing 22: Information System—Physical Description At a high level the information system enables communication in five ways: Mobile distributed databases maintained by replication over radio—here is the primary access by vendor team-members having allocated roles.",
"Internet access to a web-server on which the database is also maintained by replication—this would probably be the main route for Customers.",
"Local LAN for office-resident participants.",
"Dial-up access to the LAN in two situations—firstly, by field resources wishing to access information not replicated to their stations and, secondly, by occasionally participating resources for which it is not considered worthwhile to set up a distributed database.",
"Notification that may be by SMS to mobile phone/communicator, or by the more traditional E-Mail and/or Fax.",
"See Drawing 23: System Network and Devices There is great flexibility in deployment of servers and devices.",
"This is represented in the example by considering the MPS provider and MPU domains as ‘sliding windows’.",
"See Drawing 24: MPS Provider and MPU Domains H.4.8.2 User Interface Since almost all user access to data is local (reading the distributed database) the screen response is instantaneous.",
"The user interface, created automatically from the system object-oriented model is process oriented—so if the user wishes to conduct the Pursuing Prospect process the way in is by clicking on the Pursuing Prospect icon.",
"See Drawing 25: Information System—User Interface Expert system language translation technology can have a dramatic effect on global scalability.",
"Resources working in multi-national account teams can work in their own language and have dialogue with colleagues in other countries automatically translated.",
"See Drawing 46: Expert System Language Translation H.4.8.3 Databases See Drawing 26: Data Model The database within an information system can be considered as a collection of tables.",
"The table T-Person is simply a list of people.",
"T-Prospect is a list of prospects.",
"In addition to ‘self information’ such tables also carry cross-references to related tables, that's why it's called a ‘relational’ database.",
"Some tables are more abstract in nature—for example T-Influence that is simply a list of pairs—a Prospect and a Person: See Drawing 27: Influence Table The T-Participation table is very similar—it shows which people ‘participate’ in meetings.",
"A data table is extremely efficient for representing information.",
"In the context of this invention such relational databases are an important background technicality because the sending of a few characters over radio to update a remote database can convey dramatic changes in the information.",
"Such highly efficient communication of information, especially when necessary over radio, is in contrast to the redundancy of communicating in text form over E-Mail.",
"H.4.8.4 Replication Speed of data transmission is irrelevant for distributed databases—whether it takes ten or twenty seconds to background-update a database has zero negative effect on functionality.",
"If a distributed database approach is taken, ‘fair’ (say 80%) coverage will be adequate in most cases because databases will be synchronized and notification messages will be received as the worker travels into and out of coverage.",
"In the worst case synchronization can occasionally be made over wire-line if out of coverage for long periods of time.",
"The elaboration of which objects participate in which process activities conducted by which roles is also important to ensuring that the right system objects are physically available to the resource that must capture and/or read information.",
"Because the data will be replicated and communicated by radio (which is expensive and slow), distribution rules must be established to ensure that the essential system objects are physically distributed to the appropriate stations.",
"For example, the rules for replication to a resource having role of Allocator may be as follows: In this example neither Contactoccasion, nor Influence, nor Participation data is distributed to the Allocator because it is considered unnecessary for the Allocator to have information about Contactoccasions (meetings with the Customer) or Influence (the reference to which people influence the purchase decision in active Prospects).",
"Some data is conditionally replicated—for example Person is limited to the employees of the Vendor company (“our company”).",
"Other data is entirely replicated—for example the identification of all Prospect managers and all Availability of all Resources.",
"See Drawing 28: Replication Rule Formulation H.4.8.5 Communication Middleware Modern middle-wares, such as EVO from Ericsson, provide transparent inbound/outbound access to a wide variety of radio networks (and wire-line).",
"See Drawing 29: Communication Middle Ware—Physical Today such technology is applied almost exclusively to E-Mail because of its encryption, compression and other efficiency attributes.",
"But the ability to dynamically swap between transport providers, for example between packet- and circuit-switched, is not exploited.",
"See Drawing 30: Communication Middleware—Logical In this implementation example, let's say it's in Sweden, GSM coverage in the cities is highly developed and until recently was the only way to send data over radio from portable devices.",
"GSM is, and will continue to be, the network of choice when sending more than 3K.",
"Relatively slow Mobitex (1.2K) on the other hand has total national coverage but requires heavy equipment and a long antenna—but it's suitable for vehicle-mounted operation.",
"The recent introduction of faster (8K) and more portable (PCMCIA modems in PCs) used with Mobitex 8K in Stockholm and Gothenburg supersedes 1.2K Mobitex—having the further advantage of giving indoor coverage.",
"Middle-ware as described here allows all of these transports, including the soon-to-be-released GPRS, to be invoked—the application is totally shielded from the transport provider.",
"H.4.8.6 Notification Messages In event-driven processes (rather than in reliably/predictably planned processes) changes of state are often beyond the control of the resource conducting the process—then states tend to be ‘observed’ (and responded to) rather than ‘caused’ by the process worker.",
"It is the process by which observations of these states, and communication of such observations within and between process teams, that enables process effectiveness and efficiency gains to be made.",
"In this example the business process engineer asks the system engineer to program a triggered automatic notification (by a mobile phone SMS message) to a resource invited to a meeting—this is because an immediate response of acceptance or decline is needed to speed up the ‘Coordinating contact occasion’ process.",
"See Drawing 31: State Transition Notification Message Request Here the business process engineer emphasizes that both invitation and acceptance should be rapid.",
"See Drawing 32: Object Collaboration—Expedite Request One way of achieving this technically is by DBMS triggers.",
"The application for patents is limited to event-driven processes (such as marketing, sales and service).",
"H.4.8.7 Customer Web Server See Drawing 33: Customer May Access Over Webserver One of the most important aspects of the invention is that the so-called ‘Team approach’ is customer-centric.",
"The creation of customer-intimacy in which the customer is considered to be a team-member is critical.",
"Sales and service personnel working in the field have their own devices on which distributed databases are physically located and maintained.",
"Theoretically every customer, and indeed every employee of every customer, could have her/his own distributed database—but that is not practical.",
"More usual is for customers to read and write information to/from a Customer Web Server.",
"In this manner the customer is empowered (by password and appropriate rules) to read and write to the Customer Web Server's database that resides at the vendor companies' central location.",
"In the example shown here the customer can take the responsibility for maintaining certain data-like telephone number.",
"The customer can now participate directly in processes—for example the ‘Coordinating contact occasion’ process.",
"Either the customer or the salesman can initiate a meeting and proposed participants can dialogue to set mutually appropriate date and time."
]
] | Patent_10405937 |
[
[
"Security feature",
"The invention concerns a process for producing a security feature and a print medium which is equipped in that respect, which affords the possibility of not having to implement any change and in particular mechanical change in the surface of the card body.",
"In that respect the substrate includes at least one change-over substance which by irradiation with light of a given wavelength experiences an irreversible change in colour from a starting colour to a final colour, wherein the substrate in the initial condition is so irradiated with a controlled light beam of that wavelength, in particular a laser light beam that, due to the change in colour caused thereby in the change-over substance, an image which can be recognised with the naked eye is produced within the volume of the substrate."
],
[
"1.A process for producing a security feature, in particular on print media, in particular passes and identity cards, plastic payment cards, credit cards, memory cards etc, wherein the substrate (1, 1a, 1b) includes at least one change-over substance which by virtue of irradiation with light of a given wavelength (λ, λ1, λ2) experiences an irreversible change in color from a starting color to a final color, characterized in that the substrate when in the initial condition is so irradiated by a controlled light beam of that wavelength (λ, λ1, λ2), in particular a laser beam, that due to the change in color caused thereby in the change-over substance an image which can be recognized especially with the naked eye is produced on the substrate (1).",
"2.A process according to claim 1 characterized in that an image is produced within the volume of the substrate (1).",
"3.A process according to claim 1 characterized in that the change in color is a change from opaque to transparent.",
"4.A process according to one of the preceding claims characterized in that the change-over substance is present in the substrate in such a level of concentration that the change in color of the change-over substance appears to the viewer as a corresponding change in color of the substrate, in particular the substrate in the initial condition becomes thereby opaque and in the final condition becomes thereby transparent in the irradiated region.",
"5.A process according to claim 1 characterized in that the substrate is a plastic material, in particular ABS, PVC, PTE.",
"6.A process according to claim 1 characterized in that the change-over substance is distributed, in particular uniformly distributed, in the form of microcapsules or granules (2) or powder in the substrate.",
"7.A process according to claim 1 characterized in that the change-over substance is arranged only in a layer of the substrate, as viewed in cross-section through the substrate, in the interior of the substrate.",
"8.A process according to claim 1 characterized in that the print medium comprises a single substrate layer (1).",
"9.A process according to claim 1 characterized in that the print medium has a single substrate layer (1) having the change-over substance and is covered on at least one side by a cover layer (1′) of transparent material which does not react to irradiation with light of the wavelength (λ, λ1, λ2), and in particular the change-over substance is arranged on one of the outward sides of the substrate (1).",
"10.A process according to claim 1 characterized in that the print medium has a single substrate layer (1) having the change-over substance and said substrate (1) is coated on the rear side remote from the irradiation side with an opaque cover layer (1″).",
"11.A process according to claim 1 characterized in that the print medium comprises at least two interconnected substrate layers (1a, 1b) having different change-over substances which react to different wavelengths (λ, λ1, λ2) and which are so irradiated simultaneously or successively with light beams of different wavelengths (λ, λ1, λ2) that considered in plan view in terms of surface at most partially overlapping images are produced by the color changes in the two substrate layers (1a, 1b).",
"12.A process according to claim 11 characterized in that the starting colors of the substrate layers (1a, 1b) are different and the final colours colors are in particular the same and in particular are transparent."
],
[
"<SOH> II.",
"BACKGROUND OF THE INVENTION <EOH>In order to improve the anti-forgery security features of passes, identity cards, plastic payment cards or credit cards used for payments, various security features will be formed on appropriate print mediums.",
"For example, one possibility consists in having the plastic payment card made with a multilayer part whose medium layer is opaque, and is used for printing the specific data, whereupon at least the front print side, mostly the rear side too, is coated in a transparent cover layer.",
"On the upper side of the opaque medium layer there is printed an image, a symbol or something alike, and in the area of the above arranged transparent cover layer there is printed a surface three-dimensional structure having such a configuration that—by deflection and the lens effect of the elements that structure is consisting of—on viewing it from two different direction, usually forming a right angle between them, the different images and symbols respectively of that colours configuration become visible.",
"Since on the one hand, from a technical point of view, such security features may be forged only by using an expensive equipment, and on the other hand as far as the type of the created image and the symbol respectively are concerned, and in regard of the serial number, the date of fabrication, a.s.o these may be modified, that provides first of all a relatively enhanced protection against short forgeries.",
"However, the drawback of this security feature consists in having at least some parts of the arrangement on the card body surface which may be smeared, deteriorated or otherwise brought into a non-operating situation."
],
[
"<SOH> III.",
"SUMMARY OF THE INVENTION <EOH>a) Technical Object Therefore, the object of this invention is to provide another security feature and a process for producing it, and in particular a security feature which is not implying the necessity of operating modification, in particular mechanical ones, on the surface of the card's body.",
"b) Attainment of the Object This object is attained by features of claim 1 .",
"Advantageous embodiments are set forth in the append ant claims.",
"In this case the basic idea consists in arranging at least a substance in a substrate of the plastic card body or in a layer of that card body which may be made of plastic, paper or a composite material, substance which experiences an irreversible colour change upon being irradiated with a light having a given wavelength, for example a laser beam.",
"Such a changeover substance may be arranged in a substrate layer uniformly distributed in the form of microcapsules or granules or powder.",
"Thereby there is possible to produce in the card a visible colour change for the viewer by irradiating it with a laser beam of a given wavelength the changeover substance reacts to.",
"And as long as this substance is directly arranged in the substrate beneath the visible surface for the viewer, and there is a sufficient amount of it, for the viewer there is not visible the changing of the component colour particles but the colour effect of the substrate itself wherein the area irradiated with light of a given wavelength experiences a changing for the viewer.",
"Thereby there is possible to produce images of a different colour inside a previously monochromatic substrate by irradiating it with light, in particular by means of a focused beam or by preventing it from being irradiated by means of an appropriately shaped shutter.",
"Such an optical effect on a print medium may be indeed theoretically provided by printing on the medium surface too, but however there is another optical effect since on a closer examination there may be noticed that in case of employing a process according to the present invention the optical effect emerges inside the substrate and not on its surface.",
"Even this optical effect may disappear if the change in colour experienced by the changeover substance which will thereby be produced in the entire substrate, is a change in colour from opaque to transparent.",
"Thereby a previously entirely coloured card body may become completely transparent upon being irradiated with a given wavelength if the distribution and the density of the changeover substance are sufficiently high and homogeneous.",
"Thereby the viewer may undertake the examinations relying on the fact that inside the card body there should be a pre-arranged image or a visible symbol which may be best noticed if is viewed in front of a source of light.",
"In spite of this effect, the rear side of this substrate as seen in regard of the viewer may be coated in an opaque layer, preferably having another colour than the substrate in its previous state, thereby providing a different colouring in the irradiated area, which however becomes visible only in the rear side of the card body, therefore inside it.",
"Likewise, such a substrate may be coated on the front and/or the rear side with a transparent, durable cover layer.",
"The advantage of all these features consists in that, on the one hand the surface is always smooth and therefore it should not be mechanically printed or otherwise mechanically influenced, making thus the smearing almost impossible, and on the other hand the employing of this process allows the making of the transparent or otherwise coloured areas, therefore images and symbols, both in different shapes and locations on the initial card.",
"Further on, the image effects thus created, in particular in the shape of transparent areas, may be anytime later improved too by irradiating them with a light of a given wavelength.",
"Thereby the passports may be properly endowed with supplementary security features or the existing security feature may be improved by means of adding supplementary images a.s.o.",
"Another advantage consists in that the print medium has several overlapping substrate layers, whereupon there is in every substrate layer another changeover substance which therefore reacts to another wavelength.",
"Thereby, upon being simultaneously irradiated with different wavelength or with time delayed lights having both the given wavelengths, there may be created separate images in each of the overlapping layers, in particular transparent areas.",
"When there are created partially overlapping transparent areas and the initial colour of those two substrate layers is different, there appears a special image effect because, the card body is transparent in the overlapping area of those two substrate layers, which do not overlap as seen from the front and rear side and provide thus another colour in every case and—according to the volume of the irradiated area—another image contour too.",
"Likewise, there is possible to arrange more different changeover substances which react to different wavelengths in one and the same substrate layer."
],
[
"I.",
"FIELD OF THE INVENTION The invention concerns a process for producing a security feature and a print medium which is equipped in this respect.",
"II.",
"BACKGROUND OF THE INVENTION In order to improve the anti-forgery security features of passes, identity cards, plastic payment cards or credit cards used for payments, various security features will be formed on appropriate print mediums.",
"For example, one possibility consists in having the plastic payment card made with a multilayer part whose medium layer is opaque, and is used for printing the specific data, whereupon at least the front print side, mostly the rear side too, is coated in a transparent cover layer.",
"On the upper side of the opaque medium layer there is printed an image, a symbol or something alike, and in the area of the above arranged transparent cover layer there is printed a surface three-dimensional structure having such a configuration that—by deflection and the lens effect of the elements that structure is consisting of—on viewing it from two different direction, usually forming a right angle between them, the different images and symbols respectively of that colours configuration become visible.",
"Since on the one hand, from a technical point of view, such security features may be forged only by using an expensive equipment, and on the other hand as far as the type of the created image and the symbol respectively are concerned, and in regard of the serial number, the date of fabrication, a.s.o these may be modified, that provides first of all a relatively enhanced protection against short forgeries.",
"However, the drawback of this security feature consists in having at least some parts of the arrangement on the card body surface which may be smeared, deteriorated or otherwise brought into a non-operating situation.",
"III.",
"SUMMARY OF THE INVENTION a) Technical Object Therefore, the object of this invention is to provide another security feature and a process for producing it, and in particular a security feature which is not implying the necessity of operating modification, in particular mechanical ones, on the surface of the card's body.",
"b) Attainment of the Object This object is attained by features of claim 1.Advantageous embodiments are set forth in the append ant claims.",
"In this case the basic idea consists in arranging at least a substance in a substrate of the plastic card body or in a layer of that card body which may be made of plastic, paper or a composite material, substance which experiences an irreversible colour change upon being irradiated with a light having a given wavelength, for example a laser beam.",
"Such a changeover substance may be arranged in a substrate layer uniformly distributed in the form of microcapsules or granules or powder.",
"Thereby there is possible to produce in the card a visible colour change for the viewer by irradiating it with a laser beam of a given wavelength the changeover substance reacts to.",
"And as long as this substance is directly arranged in the substrate beneath the visible surface for the viewer, and there is a sufficient amount of it, for the viewer there is not visible the changing of the component colour particles but the colour effect of the substrate itself wherein the area irradiated with light of a given wavelength experiences a changing for the viewer.",
"Thereby there is possible to produce images of a different colour inside a previously monochromatic substrate by irradiating it with light, in particular by means of a focused beam or by preventing it from being irradiated by means of an appropriately shaped shutter.",
"Such an optical effect on a print medium may be indeed theoretically provided by printing on the medium surface too, but however there is another optical effect since on a closer examination there may be noticed that in case of employing a process according to the present invention the optical effect emerges inside the substrate and not on its surface.",
"Even this optical effect may disappear if the change in colour experienced by the changeover substance which will thereby be produced in the entire substrate, is a change in colour from opaque to transparent.",
"Thereby a previously entirely coloured card body may become completely transparent upon being irradiated with a given wavelength if the distribution and the density of the changeover substance are sufficiently high and homogeneous.",
"Thereby the viewer may undertake the examinations relying on the fact that inside the card body there should be a pre-arranged image or a visible symbol which may be best noticed if is viewed in front of a source of light.",
"In spite of this effect, the rear side of this substrate as seen in regard of the viewer may be coated in an opaque layer, preferably having another colour than the substrate in its previous state, thereby providing a different colouring in the irradiated area, which however becomes visible only in the rear side of the card body, therefore inside it.",
"Likewise, such a substrate may be coated on the front and/or the rear side with a transparent, durable cover layer.",
"The advantage of all these features consists in that, on the one hand the surface is always smooth and therefore it should not be mechanically printed or otherwise mechanically influenced, making thus the smearing almost impossible, and on the other hand the employing of this process allows the making of the transparent or otherwise coloured areas, therefore images and symbols, both in different shapes and locations on the initial card.",
"Further on, the image effects thus created, in particular in the shape of transparent areas, may be anytime later improved too by irradiating them with a light of a given wavelength.",
"Thereby the passports may be properly endowed with supplementary security features or the existing security feature may be improved by means of adding supplementary images a.s.o.",
"Another advantage consists in that the print medium has several overlapping substrate layers, whereupon there is in every substrate layer another changeover substance which therefore reacts to another wavelength.",
"Thereby, upon being simultaneously irradiated with different wavelength or with time delayed lights having both the given wavelengths, there may be created separate images in each of the overlapping layers, in particular transparent areas.",
"When there are created partially overlapping transparent areas and the initial colour of those two substrate layers is different, there appears a special image effect because, the card body is transparent in the overlapping area of those two substrate layers, which do not overlap as seen from the front and rear side and provide thus another colour in every case and—according to the volume of the irradiated area—another image contour too.",
"Likewise, there is possible to arrange more different changeover substances which react to different wavelengths in one and the same substrate layer.",
"C) EMBODIMENTS Embodiment in accordance with the invention are described in greater detail by way of example hereinafter with reference to the figures in which FIG.",
"1 is a partially cross-section representation of a card body, FIG.",
"2 is a cross-section representation of another card body, and FIG.",
"3 is a cross-section representation of a card body having more active layers.",
"On the right side of the image there is represented in FIG.",
"1 a card body having a single layer as it is seen in perspective and in a cross-section through the changeover area 5 in regard of the plane 10 of the card.",
"The card body consists of one piece of a substrate layer 1, as it may for example be produced by means of a continuous casting or a mould casting, in that substrate layer 1 being uniformly distributed microcapsules or granules 2 comprising a change-over substance which produces an irreversible change of colour when irradiated with a light of a given wavelength λ.",
"The granules 2 are arranged here in such a dense distribution that the change of colour from the change-over area 5, which is irradiated with that light of a given wavelength λ. will be seen by the viewer as a change of colour of the whole substrate layer 1, namely not only at the surface of this substrate layer 1 but deep inside it.",
"This means in particular that—as it is presented in FIG.",
"1—as long as the change of colour takes place from opaque to transparent the change-over area 5 becomes totally transparent for the viewer when irradiated with that light of a given wavelength λ. while the areas of substrate layer 1 outside the change-over area remain in a colour corresponding to the initial colour of the granules 2.The changeover area may comprise an image, a symbol representation or a letter/numerical code.",
"As it is represented in the half left side of FIG.",
"1, the front side 3 and/or the rear side 4 of this substrate layer 1 may be covered in a transparent cover layer 1′ which does not comprise any substance to react to that light of a given wavelength λ. and which may be entirely penetrated by it, thus that these cover layers may be applied before irradiation too.",
"Thereby the already made card may be subjected to a later manipulation by means of a light irradiation, in particular a laser beam having a given wavelength λ. which produces an alteration in the change-over substance.",
"In FIG.",
"2 there is represented in its turn another structure of the card body, since on the rear side 4 of the substrate layer 1 comprising granules 2 made of a change-over substance is arranged an opaque cover layer 1″.",
"This cover layer 1″ preferably has another colour than the initial colour of the substrate layer 1.Thereby after the irradiation with that light of a given wavelength λ. there appears in the change-over area 5 another colour in regard of the remaining area, and consequently for the viewer there appears a bicoloured image whereupon is obviously that the colour effect in the change-over area 5 appears inside the card body, more precisely on the contact surface between the substrate layer 1 and the cover layer 1″.",
"In FIG.",
"3 there is represented a card body having two substrate layers 1a, 1b, both of them comprising the granules 2a, 2b which consist of different change-over substances, thereby reacting to different wavelengths λ1 and λ2 respectively by irreversibly changing their colour, in particular from opaque to transparent.",
"These two layers made of card body may—as it is represented in the right side of the image—not be endowed with cover layers.",
"On irradiating or respectively imprinting with a laser beam having different wavelengths λ1 and λ2 respectively, there may be produced—simultaneously and/or consecutively—change-over areas, therefore differently shaped images.",
"First of all, if these change-over areas from the layers are partially overlapping—as it may be seen in FIG.",
"3—this has a gaudy effect for the viewer if the change of colour from these areas always takes place from the initial colour to transparency since there may be altogether created three images by means of these two different irradiated areas.",
"In the overlapping area of those two changeover areas the card body is completely transparent, therefore representing a first transparent image.",
"In the area in which the substrate layer 1b having the initial colour is arranged beneath the transparent change-over area of the substrate layer 1a (on the left overlapping area in FIG.",
"3), the viewer sees an image having the initial colour of the substrate layer 1b if this is looking at the card from the front side 3a of the upper substrate layer 1a.",
"If the user is inverting the card from the rear side 4a of the other substrate layer 1b he may see a third image which—as it may be seen on the left of the overlapping area in FIG.",
"3—is having the initial colour of the substrate layer 1a while the adjoining area has the initial colour of the substrate layer 1b.",
"Of course, this objective may be attained with more than two changeover substance comprising substrate layers 1a, 1b.",
"On the left half side of the image there is further on represented the possibility of supplementary endowing the rear side 4b with an opaque cover layer 1″.",
"However, the above presented tricoloured effect is thus reduced to the bicoloured effect which may be seen from the front side 3a, the overlapping area being thereby opaque as the cover layer 1″.",
"List of References 1.—substrate layer 1′—opaque cover layer 1″—transparent cover layer 2.—granules 3.—front side 4.—rear side 5.—change-over area λ.—wavelength"
]
] | Patent_10415199 |
[
[
"System and method for estimating cash flow at risk for a non-financial institution",
"A method for estimating cash flow at risk for a non-financial entity over a particular future time period, including receiving quarterly data associated with at least two of a plurality of non-financial entities, generating a plurality of data elements, each of the plurality of data elements representing a portion of the quarterly data of an associated one of the at least two of the plurality of non-financial entities, selecting one of the at least two of the plurality of non-financial entities, and estimating the cash flow at risk for the selected one of the at least two of the plurality of non-financial entities based on at least two of the plurality of data elements."
],
[
"1.A computer-implemented method for estimating cash flow at risk for a non-financial entity over a particular future time period, comprising: receiving quarterly data associated with at least two of a plurality of non-financial entities; generating a plurality of data elements, each of said plurality of data elements representing a portion of said quarterly data of said at least two of said plurality of non-financial entities; selecting one of said at least two of said plurality of non-financial entities; estimating by computer analysis said cash flow at risk for said selected one of said at least two of said plurality of non-financial entities based on at least two of said plurality of data elements, wherein one of the at least two of said plurality of data elements represents at least a portion of said quarterly data of said selected one of said at least two of said plurality of non-financial entities, and wherein another of the at least two of said plurality of data elements represents at least a portion of said quarterly data of another of said at least two of said plurality of non-financial entities; and providing an output of said estimate whereby an end user or additional computer processing may make use of the output.",
"2.The method of claim 1, wherein each of said plurality of data elements includes: an indication of a calendar quarter to which the data element pertains; and an indication of a year to which the data element pertains.",
"3.The method of claim 1, wherein each of said plurality of data elements includes: an indication of earnings before interest, taxes, depreciation and amortization for said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; an indication of assets for said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; and an indication of actual earnings before interest, taxes, depreciation and amortization per assets, said indication of actual earnings before interest, taxes, depreciation and amortization per assets calculated by dividing said indication of earnings before interest, taxes, depreciation and amortization for said indicated calendar quarter of said indicated year by said indication of assets for said indicated calendar quarter of said indicated year.",
"4.The method of claim 3, wherein each of the plurality of data elements further comprises: an indication of market capitalization for said indicated calendar quarter of said indicated year of said associated one of said at least two'of said plurality of non-financial entities; an indication of average income to assets for said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; an indication of annualized stock price volatility during said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; an indication of an industry to which said associated one of said at least two of said plurality of non-financial entities belongs; and an indication of cashflow volatility of said industry to which said associated one of said at least two of said plurality of non-financial entities belongs.",
"5.The method of claim 4, wherein each of the plurality of data elements includes an indication of an earnings before interest, taxes, depreciation and amortization per asset forecast error for one quarter in the future for said associated one of said at least two-of said plurality of non-financial entities.",
"6.The method of claim 5, wherein said indication of earnings before interest, taxes, depreciation and amortization for per asset forecast error for one quarter in the future is calculated by generating an earnings before interest, taxes, depreciation and amortization per asset forecast for one quarter in the future for said associated one of said at least two of said plurality of non-financial entities using linear regression, and subtracting said indication of earnings before interest, taxes, depreciation and amortization per asset forecast for one quarter in the future from said indication of actual earnings before interest, taxes, depreciation and amortization per asset.",
"7.The method of claim 4, wherein each of the plurality of data elements includes an indication of an earnings before interest, taxes, depreciation and amortization per asset forecast error for one year in the future for said associated one of said, at least two of said plurality of non-financial entities.",
"8.The method of claim 7, wherein said indication of earnings before interest, taxes, depreciation and amortization for per asset forecast error for one year in the future is calculated by generating an earnings before interest, taxes, depreciation and amortization per asset forecast for one year in the future for said associated one of said at least two of said plurality of non-financial entities using linear regression, and subtracting said indication of earnings before interest, taxes, depreciation and amortization per asset forecast for one year in the future from said indication of actual earnings before interest, taxes, depreciation and amortization per asset.",
"9.The method of claim 8, wherein said plurality of data elements includes one thin of said plurality of data elements, said one third of said plurality of data elements having said indication of market capitalization centered around said indication of market capitalization of one of said plurality of data elements associated with said selected one of the plurality of non-financial entities.",
"10.The method of claim 9, wherein said estimating step further comprises determining a five percent tail event for a forecast error by selecting said quarter ahead forecast error earnings before interest, taxes, depreciation and amortization per assets fox one quarter in the future of one of said one third of said plurality of data elements with the highest forecast error earnings before interest, taxes, depreciation and amortization per assets for one quarter in the future of said one third of said at least two of the; plurality of data elements that is within the lowest five percent of the data elements contained within said one third of said plurality of data elements.",
"11.The method of claim 9, wherein said estimating step further comprises determining a one percent tail event for a forecast error by selecting said quarter ahead forecast error earnings before interest, taxes, depreciation and amortization per assets for one quarter in the future of one of said one third of said plurality of data elements with the highest forecast error earnings before interest, taxes, depreciation and amortization per assets for one quarter in the future of said one third of said at least two of the plurality of data elements that is within the lowest one percent of the data elements contained within said one third of said plurality of data elements.",
"12.The method of claim 9, wherein said estimating step further comprises determining a five percent tail event for a forecast error by selecting said year ahead forecast error earnings before interest, taxes, depreciation and amortization per assets for one year in the future of one of said one third of said plurality of data elements with the highest forecast error earnings before interest, taxes, depreciation and amortization per assets for one year in the future of said one third of said at least two of the plurality of data elements that is within the lowest five percent of the data elements contained within said one third of said plurality of data elements.",
"13.The method of claim 9, wherein said estimating step further comprises determining a one percent tail event for a forecast error by selecting said year ahead forecast error earnings before interest, taxes, depreciation and amortization per assets for one year in the future of one of said one third of said plurality of data elements with the highest forecast error earnings before interest, taxes, depreciation and amortization per assets for one year in the future of said one third of said at least two of the plurality of data elements that is within the lowest one percent of the data elements contained within said one third of said plurality of data elements.",
"14.The method of claim 8, wherein said plurality of data elements includes one third of said plurality of data elements, said one third of said plurality of data elements having said indication of average income to assets centered around said indication of average income to assets of one of said plurality of data elements associated with said selected one of the plurality of non-financial entities.",
"15.The method of claim 8, wherein said plurality of data elements includes one third of said plurality of data elements, said one third of said plurality of data elements having said indication of annualized stock price volatility centered around said indication of annualized stock price volatility of one of said plurality of data elements associated with said selected one of the plurality of non-financial entities.",
"16.The method of claim 8, wherein said plurality of data elements includes one third of said plurality of data elements, said one third of said plurality of data elements having said indication of cashflow volatility of the industry centered around said indication of cashflow volatility of the industry of one of said plurality of data elements associated with said selected one of the plurality of non-financial entities.",
"17.The method of claim 8, wherein said plurality of data elements include each of the plurality of data elements having said indication of said industry which is the same as said indication of said industry of one of said plurality of data elements associated with said selected one of said at least two of said plurality of non-financial firms.",
"18.A computer system comprising: means for receiving quarterly data associated with at least two of a plurality of non-financial entities; means for generating a plurality of data elements, each of said plurality of data elements representing a portion of said quarterly data of an associated one of said at least two of said plurality of non-financial entities; means for selecting one of said at least two of said plurality of non-financial entities; and means for estimating said cash flow at risk for said selected one of said at least two of said plurality of non-financial entities based on at least two of said plurality of data elements, wherein one of the at least two of said plurality of data elements represents at least a portion of said quarterly data of said selected one of said at least two of said plurality of non-financial entities, and wherein another of the at least two of said plurality of data elements represents at least a portion of said quarterly data of another of said at least two of said plurality of non-financial entities.",
"19.The system of claim 18, wherein each of the plurality of data elements includes: an indication of earnings before interest, taxes, depreciation and amortization for said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; an indication of assets for said indicated calendar quarter of said indicated year said associated one of said at least two of said plurality of non-financial entities; and and indication of actual earnings before interest, taxes, depreciation and amortization per assets, said indication of actual earnings before interest, taxes, depreciation and amortization per assets calculated by dividing said indication of earnings before interest, taxes, depreciation and amortization for said indicated calendar quarter of said indicated year by said indication of assets for said indicated calendar quarter of said indicated year.",
"20.The system of claim 19, wherein each of the plurality of data elements further includes: an indication of market capitalization for said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; an indication of average income to assets for said indicated calendar quarter of said indicated year of said associated one of said at least two of said plurality of non-financial entities; 21.The system of claim 20, wherein each of the plurality of data elements includes an indication of an earnings before interest, taxes, depreciation and amortization per asset forecast error for one quarter in the future for said associated one of said at least two of said plurality of non-financial entities.",
"22.The system of claim 21, wherein said plurality of data elements includes one third of said plurality of data elements, said one third of said plurality of data elements having said indication of market capitalization centered around said indication of market capitalization of one of said plurality of data elements associated with said selected one of the plurality of non financial entities.",
"23.The system of claim 22, wherein said estimating step further comprises determining a five percent tail event for a forecast error by selecting said quarter.",
"ahead forecast error earnings before interest, taxes, depreciation and amortization per assets for one quarter in the future of one of said one third of said plurality of data elements with the highest forecast error earnings before interest, taxes, depreciation and amortization per assets for one quarter in the future of said one third of said at least two of the plurality of data elements that is within the lowest five percent of the data elements contained within said one third of said plurality of data elements."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Financial institutions use value-at-risk (VaR) measures to project asset values in the near future, for example by days or weeks, so that the financial institutions can balance their respective debt-equity ratio, or utilize derivatives to hedge commodity-price exposures to manage risk over the short term.",
"Typically VaR is estimated using a “bottom up” method.",
"The bank begins by enumerating each of the bank's assets, for example, each loan, trading position, etc.",
"The risk exposures for each of the bank's assets, i.e.",
"to interest rate shocks, credit risk, foreign exchange movements, etc., are then quantified.",
"The risks are aggregated across the bank's entire portfolio of assets and VaR is calculated.",
"VaR works well to the extent that a bank can identify each of its main sources of risk, and these sources of risk correspond, either directly or indirectly, to traded assets, for which there is good historical data on price movements.",
"In particular, this method is very well suited to evaluating the risks of a trading desk that deals in relatively liquid instruments.",
"A “bottom-up” VaR type analysis of the cash flows at risk for non-financial institutions has also been used so that the non-financial institution can balance its debt-equity ratio, or utilize derivatives to hedge commodity-price exposures to manage risk over the short term.",
"However, conducting a “bottom-up” VaR type analysis of non-financial institutions is complicated by the fact that each risk faced by a non-financial institution may not be related to a clearly identifiable asset.",
"Additionally, the asset held by the non-financial institution is not likely to be traded frequently, nor is it likely to have good historical data on price movements.",
"For example, one of Company X's assets may be a marketing campaign, and one of the risks associated with the marketing campaign may be public approval of Company X's marketing campaign.",
"Company X's marketing campaigns are not traded and good historical data on Company X's marketing campaigns may not be readily available, thus making it all but impossible to calculate an accurate VaR which reflects the risk to Company X's marketing effort.",
"A “top down” analysis of the cash flows at risk for a non-financial institution has also been used so that the non-financial institution can balance its debt-equity ratio, or utilize derivatives to hedge commodity-price exposures to manage risk over the mid-term.",
"The advantage of conducting a “top down” analysis for non-financial institutions is that a “top down” analysis should summarize the combined effect of all relevant risks facing a particular non-financial institution.",
"Unfortunately, data availability presents a problem.",
"Generally, only quarterly data is available for a non-financial institution, such that to obtain a statistically valid number of samples of cash flow at risk for a particular non-financial institution more than two decades of data will have to be used.",
"This presents a problem because many non-financial institutions did not exist twenty years ago, and even for the non-financial institutions that did exist twenty years ago, the cash flows at risk twenty years ago are not relevant to the cash flows at risk currently."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention in one aspect is a method for estimating cash flow at risk for a non-financial entity over a particular future time period.",
"In accordance with a first exemplary embodiment of the method of the present invention, there is provided a method for estimating cash flow at risk for a non-financial entity over a particular future time period, including receiving quarterly data associated with at least two of a plurality of non-financial entities, generating a plurality of data elements, each of the plurality of data elements representing a portion of the quarterly data of an associated one of the at least two of the plurality of non-financial entities, selecting one of the at least two of the plurality of non-financial entities, and estimating the cash flow at risk for the selected one of the at least two of the plurality of non-financial entities based on at least two of the plurality of data elements.",
"In accordance with a first exemplary embodiment of the system of the present invention, there is provided a computer system including means for receiving quarterly data associated with at least two of a plurality of non-financial entities, means for generating a plurality of data elements, each of said plurality of data elements representing a portion of said quarterly data of an associated one of said at least two of said plurality of non-financial entities, means for selecting one of said at least two of said plurality of non-financial entities, and means for estimating said cash flow at risk for said selected one of said at least two of said plurality of non-financial entities based on at least two of said plurality of data elements."
],
[
"RELATED APPLICATIONS This invention claims priority from (and incorporates in whole by reference Provisional Application No.",
"60/243,460, filed Oct. 26, 2000.FIELD OF THE INVENTION The present invention relates to corporate finance.",
"In particular, the present invention relates to the estimation of cash flow at risk for a non-financial institution.",
"BACKGROUND OF THE INVENTION Financial institutions use value-at-risk (VaR) measures to project asset values in the near future, for example by days or weeks, so that the financial institutions can balance their respective debt-equity ratio, or utilize derivatives to hedge commodity-price exposures to manage risk over the short term.",
"Typically VaR is estimated using a “bottom up” method.",
"The bank begins by enumerating each of the bank's assets, for example, each loan, trading position, etc.",
"The risk exposures for each of the bank's assets, i.e.",
"to interest rate shocks, credit risk, foreign exchange movements, etc., are then quantified.",
"The risks are aggregated across the bank's entire portfolio of assets and VaR is calculated.",
"VaR works well to the extent that a bank can identify each of its main sources of risk, and these sources of risk correspond, either directly or indirectly, to traded assets, for which there is good historical data on price movements.",
"In particular, this method is very well suited to evaluating the risks of a trading desk that deals in relatively liquid instruments.",
"A “bottom-up” VaR type analysis of the cash flows at risk for non-financial institutions has also been used so that the non-financial institution can balance its debt-equity ratio, or utilize derivatives to hedge commodity-price exposures to manage risk over the short term.",
"However, conducting a “bottom-up” VaR type analysis of non-financial institutions is complicated by the fact that each risk faced by a non-financial institution may not be related to a clearly identifiable asset.",
"Additionally, the asset held by the non-financial institution is not likely to be traded frequently, nor is it likely to have good historical data on price movements.",
"For example, one of Company X's assets may be a marketing campaign, and one of the risks associated with the marketing campaign may be public approval of Company X's marketing campaign.",
"Company X's marketing campaigns are not traded and good historical data on Company X's marketing campaigns may not be readily available, thus making it all but impossible to calculate an accurate VaR which reflects the risk to Company X's marketing effort.",
"A “top down” analysis of the cash flows at risk for a non-financial institution has also been used so that the non-financial institution can balance its debt-equity ratio, or utilize derivatives to hedge commodity-price exposures to manage risk over the mid-term.",
"The advantage of conducting a “top down” analysis for non-financial institutions is that a “top down” analysis should summarize the combined effect of all relevant risks facing a particular non-financial institution.",
"Unfortunately, data availability presents a problem.",
"Generally, only quarterly data is available for a non-financial institution, such that to obtain a statistically valid number of samples of cash flow at risk for a particular non-financial institution more than two decades of data will have to be used.",
"This presents a problem because many non-financial institutions did not exist twenty years ago, and even for the non-financial institutions that did exist twenty years ago, the cash flows at risk twenty years ago are not relevant to the cash flows at risk currently.",
"SUMMARY OF THE INVENTION The present invention in one aspect is a method for estimating cash flow at risk for a non-financial entity over a particular future time period.",
"In accordance with a first exemplary embodiment of the method of the present invention, there is provided a method for estimating cash flow at risk for a non-financial entity over a particular future time period, including receiving quarterly data associated with at least two of a plurality of non-financial entities, generating a plurality of data elements, each of the plurality of data elements representing a portion of the quarterly data of an associated one of the at least two of the plurality of non-financial entities, selecting one of the at least two of the plurality of non-financial entities, and estimating the cash flow at risk for the selected one of the at least two of the plurality of non-financial entities based on at least two of the plurality of data elements.",
"In accordance with a first exemplary embodiment of the system of the present invention, there is provided a computer system including means for receiving quarterly data associated with at least two of a plurality of non-financial entities, means for generating a plurality of data elements, each of said plurality of data elements representing a portion of said quarterly data of an associated one of said at least two of said plurality of non-financial entities, means for selecting one of said at least two of said plurality of non-financial entities, and means for estimating said cash flow at risk for said selected one of said at least two of said plurality of non-financial entities based on at least two of said plurality of data elements.",
"BRIEF DESCRIPTION OF THE DRAWINGS Further objects, features, and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the invention, in which: FIG.",
"1 is a flow chart illustrating a process for preparing quarterly income statement data and quarterly balance-sheet data according to the present invention; FIG.",
"2 is a flow chart illustrating a process for generating a cash flow at risk profile for a selected non-financial firm according to the present invention; FIG.",
"3 is a flow chart illustrating a process for dividing the data points stored in the database into standard peer groups according to the present invention; FIG.",
"4 is a flow chart illustrating a process for creating a custom peer group for the selected non-financial firm according to the present invention; and FIG.",
"5 is a flow chart illustrating a process for creating an industry peer group for the selected non-financial firm according to the present invention.",
"Throughout the figures, unless otherwise stated, the same reference numerals and characters are used to denote like features, elements, components, or portions of the illustrated embodiments.",
"Moreover, while the subject invention will now be described in detail with reference to the figures, and in connection with the illustrative embodiments, changes and modifications can be made to the described embodiments without departing from the true scope and spirit of the subject invention as defined by the appended claims.",
"DETAILED DESCRIPTION OF THE INVENTION FIG.",
"1 illustrates a process 100.The process 100 prepares quarterly income statement data and quarterly balance-sheet data for use by a process 200 (shown in FIG.",
"2) whereby a cash flow at risk profile is generated for a specific non-financial firm.",
"The cash flow at risk profile for the specific non-financial firm is used to predict the specific non-financial firm's earnings before interest, taxes, depreciation and amortization (hereinafter “EBITDA”) per assets forecast error over the next quarter and the next year.",
"In a certain embodiment, the cash flow at risk profile predicts a specific non-financial firm's EBITDA per assets forecast error given a five percent tail event over the next quarter or year.",
"In another certain embodiment, the cash flow at risk profile predicts a specific non-financial firm's EBITDA per assets forecast error given a one percent tail event and a five percent tail event over the next quarter or year.",
"The process 100 begins at step 102.At step 102, the process 100 determines whether new data has become available.",
"The process 100 uses quarterly income statement data and quarterly balance-sheet data from a selected group of non-financial firms.",
"In a certain embodiment, the quarterly income statement data and quarterly balance-sheet data is obtained from Compustat Expressfeed, version 2.0, Standard & Poors, 55 Water Street, New York, N.Y. 10041.If new data becomes available, the process 100 advances to step 104.If no new data has become available, the process 100 advances to step 102.At step 104, the process 100 marks out-dated data points stored in a database associated with the process 100 such that the out-dated data point is not used in any analysis.",
"Each data point represents quarterly income statement data and quarterly balance-sheet data for one of the group of selected non-financial firms.",
"A data point is a data structure which may include multiple values indicating the quarter, for example, Q1 1994, EBITDA for the quarter, assets for the quarter, market capitalization for the quarter, average income to assets for the quarter, annualized stock price volatility during the quarter, industry cashflow volatility, industry group identifier, i.e.",
"SIC code, EBITDA per asset forecast error for a quarter ahead, and EBITDA per asset forecast error for a year ahead.",
"The EBITDA per asset forecast error for a quarter ahead and EBITDA per asset forecast error for a year ahead will be described in more detail with reference to step 112.The process 100 marks any data points which are out-dated to keep the population of data points reflective of the current business environment.",
"Preferably, the process 100 marks any data point which is more than five years old.",
"Once the out-dated data points are removed, the process 100 advances to step 106.At step 106, the process 100 retrieves any available quarterly income statement data and quarterly balance-sheet data for the selected group of non-financial firms for quarters which are not currently reflected by the data points stored in the database and for quarterly income statement data and quarterly balance-sheet data that has been updated, generates data points representing that data, and stores the generated data points in the database.",
"When any published quarterly data is not reflected by any of the data points in the database or when any published quarterly data is updated so that it is not accurately reflected by any of the data points in the database, the process 100 preferably retrieves that quarterly data for the non-financial firm.",
"The process 100 extracts the relevant data from the retrieved quarterly income statement data and quarterly balance-sheet data creating a data point reflecting that data, and stores that data point in the database.",
"One data point should be created representing the quarterly data for each of the selected non-financial firms.",
"Retrieving and storing available data points for the selected group of non-financial companies keeps the population of data points stored in the database current.",
"Preferably, the process 100 retrieves data points which are no more than five years old.",
"In a certain embodiment, the selected non-financial firms are those firms for which quarterly income statement data and quarterly balance-sheet data is available to the public.",
"Once the available data points are stored, the process 100 advances to step 108.At step 108, the process 100 marks a first portion of the new data points such that they are not used to generate a cash flow at risk profile.",
"Data points for non-financial firms where the value of book assets for the non-financial firm for a particular quarter fall below a particular predetermined level are marked such that they are not used to generate a cash flow at risk profile.",
"In a certain embodiment, the data points for non-financial firms which fall within the lowest five percent of book assets in any given quarter are so marked.",
"By eliminating the data points for non-financial firms with low values of book assets from the analysis, the ratio of EBITDA per assets does not become unboundedly large.",
"Once the data points for non-financial firms with low values of book assets are marked, the process 100 advances to step 110.At step 110, the process 100 marks a second portion of the data points such that the second portion of data points are not used in the calculation of the cash flow at risk profile.",
"The data points for non-financial firms where the property plant and equipment (hereinafter “PP&E”) for the non-financial firm experience dramatic changes from one quarter to the next are removed from the database.",
"In a certain embodiment, the data points for non-financial firms where the PP&E changes by more than fifty percent are removed.",
"By so marking the data points for non-financial firms with dramatic changes in PP&E, large mergers and other dramatic changes in a company's asset base, which are not surprises from the non-financial firm's point of view, but which induce a great deal of volatility in measured EBITDA per assets, are eliminated from the analysis.",
"Once the data points for non-financial firms with dramatic changes in PP&E are marked, the process 100 advances to step 112.In a certain embodiment, the data points for non-financial firms where the PP&E changes by as little as twenty percent are marked.",
"In step 112, the process 100 generates EBITDA per asset forecast errors for each new data point received.",
"An EBITDA per asset forecast error is the difference between the forecasted EBITDA per asset amount for a given time period and the actual EBITDA per asset amount for the given time period.",
"EBITDA per asset forecast errors are calculated for quarter ahead forecasts and year ahead forecasts.",
"A linear regression algorithm is used to create the coefficients and constant values which are then used in an equation to forecast the quarter ahead forecast and the year ahead forecast.",
"A PROC REG of the SAS/SAT Software, version 8.0, from the SAS Institute, Inc., SAS Campus Drive, Cary, N.C., 27513-2414, is used to perform the linear regression.",
"When the PROC REG performs a quarter ahead linear regression, the quarter-ahead regression used to estimate the parameters used to create the quarter-ahead forecast is the following equation (hereinafter “equation (1)”): ( Income Assets ) i , t = ( Coef Lag1 ) R · ( Income Assets ) i , t - 1 + ( Coef Lag2 ) R · ( Income Assets ) i , t - 2 + ( Coef Lag3 ) R · ( Income Assets ) i , t - 3 + ( Coef Lag4 ) R · ( Income Assets ) i , t - 4 + ( Coef Q1 Dummy ) R · ( Q1 Dummy ) t + ( Coef Q2 Dummy ) R · ( Q2 Dummy ) t + ( Coef Q3 Dummy ) R · ( Q3 Dummy ) t + Constant R + ɛ t , i , R where the income-to-assets ratio for company i in quarter t is defined by the following equation (hereinafter “equation (2)”): ( Income Assests ) i , t = EBITDA i , t Assets i , t - 1 where EBITDAi,t is the EBITDA for company i in quarter t in the five-year period R, and Assetsi,t-1 is the total assets for company i at the end of quarter t−1 (and hence the total assets at the beginning of quarter t).",
"The season dummy variables (Q1 Dummy)t, (Q2 Dummy)t, and (Q3 Dummy)t are defined by the following equation (hereinafter “equation (3)”): (Qq Dummy)l=1 if quarter t equals calendar quarter q, 0 otherwise.",
"The εt,i,R term in Equation 1 is the in-sample error term in the regression for company i in quarter t using the data in the five-year period R Once the coefficients have been calculated for a given five-year period R, the coefficients are used to forecast the income-to-assets ratio for the first quarter beyond this five-year period according to the following equation (hereinafter “equation (4)”): ( Income Assets ) i , Max ( R ) + 1 Forecast = ( Coef Lag1 ) R · ( Income Assets ) i , Max ( R ) + ( Coef Lag2 ) R · ( Income Assets ) i , Max ( R ) - 1 + ( Coef Lag3 ) R · ( Income Assets ) i , Max ( R ) - 2 + ( Coef Lag4 ) R · ( Income Assets ) i , Max ( R ) - 3 + ( Coef Q1 Dummy ) R · ( Q1 Dummy ) Max ( R ) + 1 + ( Coef Q2 Dummy ) R · ( Q2 Dummy ) Max ( R ) + 1 + ( Coef Q3 Dummy ) R · ( Q3 Dummy ) Max ( R ) + 1 + Constant R where the Max(R)+1 notation is used to indicate the first quarter beyond the five-year period R. For example, by using data for the five-year period from 1989Q1 to 1993Q4, we can forecast the income-to-assets ratio for quarter 1994Q1 using Equation 4, data from 1989Q2 to 1994Q1 to forecast the ratio for quarter 1994Q2, etc.",
"By rolling the five-year period forward, forecasted quarter ahead income-to-assets ratios for each company i for each quarter from 1994Q1 to latest quarter for which we have data can be generated.",
"After the quarter ahead forecasts are generated for the income-to-assets ratio, the quarter-ahead forecasted income-to-assets error for company i in quarter t may be calculated by the following equation (hereinafter “equation (5): ( Forecast Error ) i , t Quarter - Ahead = ( Income Assets ) i , t Forecast - ( Income Assets ) i , t .",
"The set of (Forecast Error)i,tQuarter-Ahead is all companies i and all quarters t which are within five years of the quarter in question.",
"Once the EBITDA per asset forecast error for a quarter ahead is calculated it is stored in the associated data point.",
"When the PROC REG performs a year ahead linear regression, the year ahead regression used to estimate the parameters used to create the year ahead forecasts is the following equation (hereinafter “equation (6)”): ( Yearly Income Assets ) i , t = ( Coef Lag1 ) R ′ · ( Income Assets ) i , t - 1 + ( Coef Lag2 ) R ′ · ( Income Assets ) i , t - 2 + ( Coef Lag3 ) R ′ · ( Income Assets ) i , t - 3 + ( Coef Lag4 ) R ′ · ( Income Assets ) i , t - 4 + ( Coef Q1 Dummy ) R ′ · ( Q1 Dummy ) t + ( Coef Q2 Dummy ) R ′ · ( Q2 Dummy ) t + ( Coef Q3 Dummy ) R ′ · ( Q3 Dummy ) t + Constant R ′ + ɛ i , t , R ′ where yearly income to assets ration is defined by the following equation (hereinafter “equation (7)”): ( Yearly Income Assets ) i , t = EBITDA i , t + EBITDA i , t + 1 + EBITDA i , t + 2 + EBITDA it + 3 Assets i , t - 1 The forecasted yearly-income-to-asset ratio for the first quarter after a given five-year period R is then calculated by the following equation (hereinafter “equation (8)”): ( Yearly Income Assets ) i , Max ( R ) + 1 Forecast = ( Coef Lag1 ) R ′ · ( Income Assets ) i , Max ( R ) + ( Coef Lag2 ) R ′ · ( Income Assets ) i , Max ( R ) - ( Coef Lag3 ) R ′ · ( Income Assets ) i , Max ( R ) - 2 + ( Coef Lag4 ) R ′ · ( Income Assets ) i , Max ( R ( Coef Q1 Dummy ) R ′ · ( Q1 Dummy ) Max ( R ) + 1 + ( Coef Q2 Dummy ) R ′ · ( Q2 Dummy ) Max ( R ) + 1 + ( Coef Q3 Dummy ) R ′ · ( Q3 Dummy ) Max ( R ) + 1 + Constant R ′ Finally, the year ahead forecast error is defined by the following equation (hereinafter “Equation (9)”): ( Forecast Error ) i , t Year - Ahead = ( Yearly Income Assets ) i , t Forecast - ( Yearly Income Assets ) i , t .",
"The set of (Forecast Error)i,tYear-Ahead is all companies i and all quarters t which are within five years of the quarter in question.",
"The set of year ahead forecast errors stops three quarters before the set of quarter ahead forecast errors, since four leads of actual data are required for calculating the year ahead forecast error.",
"Once the EBITDA per asset forecast error for a quarter ahead is calculated it is stored in the associated data point.",
"Once the year ahead EBITDA per asset forecast errors and the quarter ahead EBITDA per asset forecast errors are stored in the associated data point, the process 100 advances to step 102.FIG.",
"2 illustrates the process 200 which generates a cash flow at risk profile for a selected non-financial firm.",
"The process 200 starts when it receives an indication of which non-financial firm for which to generate a cash flow at risk profile.",
"Once the process 200 receives an indication of which non-financial firm to generate a cash flow at risk profile for, the process 200 advances to step 204.At step 204, the process 200 waits to receive an indication of which peer group selection criteria are to be used in selecting the peer group for the particular non-financial firm.",
"A peer group can be selected on any characteristic relevant to the non-financial firm's business, but four characteristics of a non-financial firm's business are strongly associated with patterns in forecast-error volatility: market capitalization, average income to assets, industry cashflow volatility, and annualized stock price volatility.",
"Market capitalization is calculated for a particular data point based on the non-financial firm's market capitalization for the particular quarter.",
"Average income to assets is calculated for a particular data point as the average value of EBITDA per assets over the prior four quarters.",
"Annualized stock price volatility is calculated using daily stock price data over the current quarter.",
"Industry cashflow volatility is calculated as the log of squared residuals of in-sample variances, ln(ε2i,t,R), from the quarter ahead regression discussed above in connection with step 112 of process 100, illustrated in FIG.",
"1, and regressing those values, using linear regression, against: the dummy variables for the industry identifiers controlling for market capitalization, EBITDA per assets, and stock volatility.",
"The process 200 uses PROC REG to perform the linear regression.",
"When the PROC REG performs the linear regression, the PROC REG is attempting to generate the coefficient on an industry's dummy (Coef Industry) for the following equation (hereinafter “equation (10)”): ln ( ɛ i , t , R 2 ) = ( Coef Market Cap ) R · ln ( ( Market Cap ) i , t - 1 ) + ( Coef Income - to - Assets ) R · 〈 Income Assets 〉 i , t - 1 + ( Coef Stock Vol ) R · ( Stock Volatility ) i , t - 1 + ∑ all SIC3 ( Coef Industry ) R , SIC3 · ( SIC3 Dummy ) i , SIC3 where (Market Cap)i,t is the market capitalization expressed in units of millions of dollars for company i at the end of quarter t, (Stock Volatility)i,t is the annualized stock volatility for company i for quarter t, and <Income per Assets>i,t is the average EBITDA per assets for the last four quarters.",
"The dummy variables for the three digit SIC codes in Equation (10) are defined by: (SIC3 Dummy)i, SIC3 equals one if company i has a three digit SIC code equal to the industry code for the non-financial firm associated with the particular data point, otherwise (SIC3 Dummy)i, SIC3 equals zero.",
"The higher the coefficient on an industry's dummy (Coef Industry), the riskier that industry is deemed to be.",
"Once the selected peer group selection criteria are received, the process 200 advances to step 206.At step 206, the process 200 determines whether standard peer groups are to be utilized in generating the cash flow at risk profile.",
"If the standard peer groups are to be used, the process 200 advances to step 208.The step 208 generates a cash flow at risk profile using standard peer groups.",
"Once the step 208 is complete, the process 200 exits.",
"The step 208 is shown in more detail in FIG.",
"3.If standard peer groups are not to be used, the process 200 advances to step 210.At step 210, the process 200 determines whether custom peer groups are to be utilized in generating the cash flow at risk profile.",
"If custom peer groups are to be used, the process 200 advances to step 212.The step 212 generates a cash flow at risk profile using custom peer groups.",
"Once the step 212 is complete, the process 200 exits.",
"The step 212 is shown in more detail in FIG.",
"4.If standard peer groups are not to be used, the process 200 advances to step 214.At step 214, the process 200 determines whether industry peer groups are to be utilized in generating the cash flow at risk profile.",
"If industry peer groups are to be used, the process 200 advances to step 216.The step 216 generates a cash flow at risk profile using industry peer groups.",
"Once the step 216 is complete, the process 200 exits.",
"The step 216 is shown in more detail in FIG.",
"5.If industry peer groups are not to be used, the process 200 advances to step 218.At step 218, the process 200 reports an error.",
"The user must specify that standard peer groups, custom peer groups or industry peer groups are to be used to generate the cash flow at risk profile, otherwise an error is reported.",
"Once the error is reported, the process 200 exits.",
"FIG.",
"3 illustrates the process 208, which divides the data points stored in the database into standard peer groups.",
"The process 208 begins at step 302.At step 302, the process 208 divides the data points into three groups based on the first selection criteria.",
"The first group having a third of the data points with the smallest first criterion, the third group having a third of the data points with the largest first criterion, and the second group having the remaining third of the data points.",
"In a certain embodiment, the first criterion is the prior quarter's value of market capitalization, the first group having the third of the data points with small market capitalization, the second group having the third of the data points with medium market capitalization, and the third group having the third of the data points with large market capitalization.",
"A data point may only belong to one of the three groups.",
"Once the data points are divided into three groups, the process 208 advances to step 304.At step 304, the process 208 further divides each of the three groups into three groups based on the second selection criteria, making a total of nine groups.",
"Each of the three groups of data points are divided into three groups, the first group having the third of the data points with the smallest second criterion, the third group having the third of the data points with the largest second criterion, and the second group having the remaining third of the data points.",
"In a certain embodiment, the second criterion is average income to assets, the first group having the third of the data points with the smallest average income to assets, the second group having the third of the data points with medium average income to assets, and the third group having the third of the data points with the largest average income to assets.",
"Once each of the three groups of data points are divided into three additional groups, making a total of nine groups, the process 208 advances to step 306.At step 306, the process 208 further divides each of the nine groups into three groups based on the third selection criteria, making a total of twenty seven groups.",
"Each of the nine groups of data points are divided into three groups, the first group having the third of the data points with the smallest third criterion, the third group having the third of the data points with the largest third criterion, and the second group having the remaining third of the data points.",
"In a certain embodiment, the third criterion is industry cashflow volatility, the first group having the third of the data points with the smallest industry cashflow volatility, the second group having the third of the data points with a medium about of industry cashflow volatility, and the third group having the third of the data points with the largest industry cashflow volatility.",
"Once each of the nine groups of data points are divided into three additional groups, making a total of twenty seven groups, the process 208 advances to step 308.At step 308, the process 208 further divides each of the twenty seven groups into three groups based on the fourth selection criteria, making a total of eighty one groups.",
"Each of the twenty seven groups of data points are divided into three groups, the first group having the third of the data points with the smallest fourth criterion, the third group having the third of the data points with the largest fourth criterion, and the second group having the remaining third of the data points.",
"In a certain embodiment, the fourth criterion is the prior quarter's value of annualized stock price volatility, the first group having the third of the data points with the smallest annualized stock price volatility, the second group having the third of the data points with a medium amount of annualized stock price volatility, and the third group having the third of the data points with the largest annualized stock price volatility.",
"Once each of the twenty seven groups of data points are divided into three additional groups, making a total of eighty one groups, the process 208 advances to step 310.At step 310, the process 208 selects one of the eighty one standard peer groups from which to generate a cash flow at risk profile.",
"The process 208 selects the one of the eighty one standard peer groups within which the selected non-financial firm would be placed given the selection criteria.",
"Once the process 208 selects the one of the eighty one standard peer groups, the process 208 advances to step 312.At step 312, the process 208 generates the values for the five percent and one percent tail events for quarter ahead forecasting error and year ahead forecasting error for the one of the eighty one standard peer groups.",
"A five percent tail event for a quarter ahead forecast error is the quarter ahead forecast error EBITDA per assets for the data point with the highest quarter ahead forecast error EBITDA per assets of the one of the eighty one groups that is within lowest five percent of the data points contained within the one of the eighty one groups.",
"A one percent tail event for a quarter ahead forecast error is a measurement of the quarter ahead forecast error EBITDA per assets for the data point with the highest quarter ahead forecast error EBITDA per assets of the one of the eighty one groups that is within the lowest one percent of the data points contained within the one of the eighty one groups.",
"A five percent tail event for a year ahead forecast error is a measurement of the year ahead forecast error EBITDA per assets for the data point with the highest year ahead forecast error EBITDA per assets of the one of the eighty one groups that is within the lowest five percent of the data points contained within the one of the eighty one groups.",
"A one percent tail event for a year ahead forecast error is a measurement of the year ahead forecast error EBITDA per assets for the data point with the highest year ahead forecast error EBITDA per assets of the one of the eighty one groups that is within the lowest one percent of the data points contained within the one of the eighty one groups.",
"Once the tail events are calculated, the process 208 advances to step 314.At step 314, the process 208 generates a histogram representing the number of data points versus forecast error EBITDA per assets.",
"The number of data points of the one of the eighty one groups having a particular forecast error EBITDA per assets is represented on the y-axis.",
"The forecast error EBITDA per assets is represented on the x-axis.",
"An exemplary histogram 600 is provided as FIG.",
"6.Once the process 208 generates the histogram representing the number of data points versus forecast error EBITDA per assets, the process 208 exits.",
"FIG.",
"4 illustrates the process 212, which creates a custom peer group for the selected non-financial firm.",
"The process 212 begins at step 402.At step 402, the process 212 begins creating a first custom peer group.",
"The process 212 creates the first custom peer group having one third of the data points stored in the database whereby the data points contained within the first custom peer group have first selection criteria values centered around the value of the first selection criteria of the selected non-financial firm.",
"In a certain embodiment, the first criterion is market capitalization.",
"Once the first custom peer group is created, the process 212 advances to step 404.At step 404, the process 212 refines the custom peer group.",
"The process 212 creates a second custom peer group having one third of the data points contained within the first custom peer group whereby the data points contained within the second custom peer group have second selection criteria values centered around the value of the second selection criteria of the selected non-financial firm.",
"In a certain embodiment, the second criterion is average income to assets.",
"Once the second custom peer group is created, the process 212 advances to step 406.At step 406, the process 212 refines the custom peer group further.",
"The process 212 creates a third custom peer group having one third of the data points contained within the second custom peer group whereby the data points contained within the third custom peer group have third selection criteria values centered around the value of the third selection criteria of the selected non-financial firm.",
"In a certain embodiment, the third criterion is industry cashflow volatility.",
"Once the third custom peer group is created, the process 212 advances to step 408.At step 408, the process 212 refines the custom peer group still further.",
"The process 212 creates a fourth custom peer group haying one third of the data points contained within the third custom peer group whereby the data points contained within the fourth custom peer group have fourth selection criteria values centered around the value of the fourth selection criteria of the selected non-financial firm.",
"In a certain embodiment, the fourth criterion is annualized stock price volatility.",
"Once the fourth custom peer group is created, the process 212 advances to step 410.At step 410, the process 212 generates the values for the five percent and one percent tail events for quarter ahead forecasting error and year ahead forecasting error for the fourth custom peer group.",
"A five percent tail event for a quarter ahead forecast error is the quarter ahead forecast error EBITDA per assets for the data point with the highest quarter ahead forecast error EBITDA per assets of the fourth custom peer group that is within the lowest five percent of the data points contained within the fourth custom peer group.",
"A one percent tail event for a quarter ahead forecast error is a measurement of the quarter ahead forecast error EBITDA per assets for the data point with the highest quarter ahead forecast error EBITDA per assets of the fourth custom peer group that is within the lowest one percent of the data points contained within the fourth custom peer group.",
"A five percent tail event for a year ahead forecast error is a measurement of the year ahead forecast error EBITDA per assets for the data point with the highest year ahead forecast EBITDA per assets of the fourth custom peer group that is within the lowest five percent of the data points contained within the fourth custom peer group.",
"A one percent tail event for a year ahead forecast error is a measurement of the year ahead forecast error EBITDA per assets for the data point with the highest year ahead forecast error EBITDA per assets of the fourth custom peer group that is within the lowest one percent of the data points contained within the fourth custom peer group.",
"Once the tail events are calculated, the process 212 advances to step 412.At step 412, the process 212 generates a histogram representing the number of data points versus forecast error EBITDA per assets for the data points of the fourth custom peer group.",
"The number of data points of the fourth custom peer group having a particular forecast error EBITDA per assets is represented on the y-axis.",
"The forecast error EBITDA per assets is represented on the x-axis.",
"Once the process 212 generates the histogram representing the number of data points versus forecast error EBITDA per assets, the process 212 exits.",
"FIG.",
"5 illustrates the process 216, which creates an industry peer group for the selected non-financial firm.",
"The process 216 begins at step 502.At step 502, the process 216 creates the industry peer group by selecting each data point stored within the data base having the same industry group code as the selected non-financial firm.",
"Using an industry peer group is only effective if the industry has a significant number of non-financial firms such that a significant number of forecast errors are available.",
"It may be particularly useful to use an industry peer group if there are specific questions about the industry that cannot be answered if non-financial firms from different industries are included within the peer group.",
"Once the industry peer group is created, the process 216 advances to step 504.At step 504, the process 216 generates the values for the five percent and one percent tail events for quarter ahead forecasting error and year ahead forecasting error for the industry peer group.",
"A five percent tail event for a quarter ahead forecast error is the quarter ahead forecast error EBITDA per assets for the data point with the highest quarter ahead forecast error EBITDA per assets of the industry peer group that is within the lowest five percent of the data points contained within the industry peer group.",
"A one percent tail event for a quarter ahead forecast is a measurement of the quarter ahead forecast error EBITDA per assets for the data point with the highest quarter ahead forecast error EBITDA per assets of the industry peer group that is within the lowest one percent of the data points contained within the industry peer group.",
"A five percent tail event for a year ahead forecast is a measurement of the year ahead forecast error EBITDA per assets for the data point with the highest year ahead forecast error EBITDA per assets of the industry peer group that is within the lowest five percent of the data points contained within the industry peer group.",
"A one percent tail event for a year ahead forecast error is a measurement of the year ahead forecast error EBITDA per assets for the data point with the highest year ahead forecast error EBITDA per assets of the industry peer group that is within the lowest one percent of the data points contained within the industry peer group.",
"Once the tail events are calculated, the process 216 advances to step 506.At step 506, the process 216 generates a histogram representing the number of data points versus forecast error EBITDA per assets for the data points of the industry peer group.",
"The number of data points of the industry peer group having a particular forecast error EBITDA per assets is represented on the y-axis.",
"The forecast error EBITDA per assets is represented on the x-axis.",
"Once the process 212 generates the histogram representing the number of data points versus forecast error EBITDA per assets, the process 216 exits.",
"The invention has been described in connection with certain preferred embodiments thereof.",
"It will be appreciated that those skilled in the art can modify or alter such embodiments without departing from the scope and spirit of the invention which is set forth in the appended claims."
]
] | Patent_10415206 |
[
[
"Non-photochromic, colored, borosilicate inorganic glasses which absorb ultraviolet, and preparations",
"The object of the present invention is non-photochromic, colored, borosilicate inorganic glasses, which absorb ultraviolet and which contain effective amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s).",
"The present invention also relates to the preparation of said glasses.",
"Within the context of said preparation, borosilicate glasses, which absorb ultraviolet and which are of various colors, can be obtained from a single crude borosilicate glass."
],
[
"1.A non-photochromic, colored, borosilicate inorganic glass, which absorbs ultraviolet and which contains effective amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s).",
"2.The glass according to claim 1, characterised in that it contains, per 100 parts by weight of its borosilicate base: from 0.0020 to 1.5 parts by weight of Ag; from 0.15 to 1.5 parts by weight of Cu; and an effective amount of at least one halogen selected from Cl, Br, I and F, which is conjugated to: an effective amount of at least one reducing agent, said effective amounts of halogen(s) and of reducing agent(s) generating sufficient copper halide(s) for the absorption of the ultraviolet; as well as an effective amount of at least one colorant.",
"3.The glass according to claim 1, characterised in that said reducing agent(s) is(are) selected from SnO, SnO2, Sb2O3 and As2O3.4.The glass according to claim 1, characterised in that said effective amount of reducing agent(s) is between 0.1 and 5 parts by weight, per 100 parts by weight of its borosilicate base.",
"5.The glass according to claim 1, characterised in that said effective amount of halogen(s) is between 0.25 and 10 parts by weight, per 100 parts by weight of its borosilicate base; each halogen being advantageously incorporated in an amount less than or equal to 3 parts by weight.",
"6.The glass according to claim 1, characterised in that said colorant(s) is(are) selected from Fe2O3, NiO, CoO, V2O5, MnO, SeO2, Cr2O3 and Nd2O3.7.The glass according to claim 1, characterised in that it contains, per 100 parts by weight of its borosilicate base, up to 7 parts by weight of Fe2O3, and/or up to 2 parts by weight of NiO, and/or up to 3 parts by weight of CoO, and/or up to 10 parts by weight of V2O5, and/or up to 2 parts by weight of SeO2, and/or up to 2 parts by weight of Cr2O3, and/or up to 4 parts by weight of Nd2O3.8.The glass according to claim 1, characterised in that it contains an effective amount of NiO and/or of CoO.",
"9.The glass according to claim 1, characterised in that it contains, per 100 parts by weight of its borosilicate base from 0.0050 to 1 part by weight of Ag; from 0.2 to 1 part by weight of Cu; from 0 to 2 parts by weight of Cl; from 0 to 2 parts by weight of Br, with at least 0.25 parts by weight of Br+Cl; from 0 to 2 parts by weight of I; from 0 to 2 parts by weight of F; from 0.2 to 3 parts by weight of SnO; from 0 to 3 parts by weight of SnO2; from 0 to 3 parts by weight of Sb2O3; from 0 to 3 parts by weight of As2O3; from 0 to 3 parts by weight of Fe2O3; from 0.0100 to 1 part by weight of NiO; from 0.0050 to 1 part by weight of CoO; from 0 to 5 parts by weight of V2O5; from 0 to 0.5 part by weight of MnO; from 0 to 0.5 part by weight of SeO2; from 0 to 0.5 part by weight of Cr2O3; and from 0 to 0.5 part by weight of Nd2O3.10.The glass according to claim 1, characterised in that its borosilicate base has the following composition by weight: SiO2 25-60% B2O3 10-35% Al2O3 3-17% ZrO2 0-13% Li2O 0-15% Na2O 0-15% K2O 0-15% with Li2O + Na2O + K2O > 2% MgO 0-10% CaO 0-15% SrO 0-15% BaO 0-15% with MgO + CaO + SrO + BaO > 1% ZnO 0-15% PbO 0-8% TiO2 0-8% Nb2O5 0-8% La2O3 0-8% Y2O3 0-8%; advantageously the following composition by weight: SiO2 30-55% B2O3 15-28% Al2O3 6-12% ZrO2 3-8% Li2O 1.5-3% Na2O 2-5% K2O 2.5-8% MgO 0-3% CaO 0-5% SrO 0-5% BaO 3-8% ZnO 0-11% PbO 0-5% TiO2 0-5% Nb2O5 0-5% La2O3 0-5% Y2O3 0-5%.",
"11.Sunglasses, which are corrective or non-corrective, made from a glass according to claim 1.12.A method of preparing a glass according to claim 1, characterised in that it comprises: preparing a crude borosilicate glass which contains the suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s); and heat-treating said crude glass in order to generate within it the crystal phase of the halide(s) which is responsible for the absorption of the ultraviolet.",
"13.A method of preparing a glass according to claim to 1, characterised in that it comprises: preparing a crude borosilicate glass which contains the suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s); optionally heat-treating said crude glass in order to generate within it the crystal phase of the halide(s) which is responsible for the absorption of the ultraviolet; polishing the surface of said borosilicate glass which is heat-treated or non-heat-treated; and heat-treating said polished borosilicate glass under a reducing atmosphere; said heat-treatment being carried out under conditions of duration and temperature which are suitable for obtaining the coloration sought after; said heat-treatment also ensuring the generation of the crystal phase of the halide(s) which is responsible for the absorption of the ultraviolet if this crystal phase has not been generated beforehand.",
"14.The method according to claim 12, characterised in that said heat-treatment of the crude glass is carried out, for 10 minutes to 2 hours, at a temperature between 450 and 650° C. 15.The method according to claim 12, characterised in that said heat-treatment under reducing atmosphere is carried out, for 2 minutes to 12 hours, at a temperature between 250 and 650° C. 16.A method for preparing the glasses according to claim 1, which are of various colors, characterised in that it comprises heat-treating, under a reducing atmosphere, under various conditions of duration or(and) of temperature, a polished crude borosilicate glass which has a defined composition containing suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s)."
],
[
"The present invention relates to novel colored borosilicate inorganic glasses, as well as to methods of preparing them.",
"Said novel borosilicate inorganic glasses are both colored and non-photochromic and at the same time absorb ultraviolet and are obtainable in various colors from a single mixture of oxides or a single crude borosilicate glass of defined composition.",
"Said novel glasses of the invention are perfectly suitable as corrective or non-corrective sunglasses.",
"According to prior art, many sunglasses have already been developed, more particularly in the three following basic tints: green, grey and brown.",
"At present, as regards the performances of said glasses, more and more demands are made from the glasses, especially in terms of protection against ultraviolet rays and in terms of recognition of road signals.",
"Conventionally, a given tint is obtained from the presence of an effective amount of colorant(s) in the basic glass composition.",
"A single glass of given color is obtained from said basic composition.",
"Said glass can absorb ultraviolet if it contains iron oxide and/or vanadium oxide.",
"However, UV cut-offs greater than 380 nm, which are compatible with a transmission in the visible which is sufficiently high to render said glass commercially viable, are difficult to obtain according to prior art.",
"In U.S. Pat.",
"No.",
"5,281,562, non-photochromic, borosilicate inorganic glass compositions are described which contain copper halide or copper-cadmium halide crystals, and which have an abrupt optical absorption cut-off at about 400 nm.",
"Within these compositions, the contents of oxides are adjusted in order to maintain a certain basicity (an R value of between 0.15 and 0.45).",
"The glasses in question can contain up to 1% by weight of colorant(s), and can therefore have a particular fixed tint.",
"U.S. Pat.",
"No.",
"5,242,869 also describes non-photochromic borosilicate glasses which, in addition to copper halides for absorbing ultraviolet, contain coloring agents of the iron oxide, nickel oxide, manganese oxide, cobalt oxide, vanadium oxide, chromium oxide, copper oxide, neodymium oxide and palladium oxide type.",
"The (non-photochromic) glass compositions of both these patents do not contain any silver and must be modified in substance in order to obtain various tints.",
"Furthermore, photochromic borosilicate inorganic glasses are known which do not possess any ultraviolet absorbing properties.",
"U.S. Pat.",
"No.",
"3,208,860 describes such photochromic glasses which are not colored.",
"Said glasses contain silver halides and can contain 0.01 to 1% of copper oxide(s).",
"Colored photochromic glasses of this type also exist.",
"They contain suitable colorants.",
"Finally, it has been described in U.S. Pat.",
"No.",
"3,892,582 of the possibility of tinting a photochromic transparent borosilicate inorganic glass (which contains silver halide particles) by heating it under a reducing atmosphere.",
"Said heating permanently confers a tint to said transparent glass without altering its photochromic properties.",
"Within such a context, the inventors thus propose novel non-photochromic, colored glasses which absorb ultraviolet (with abrupt absorption cut-offs of greater than 380 nm), and which are obtainable in various colors from a single mixture of oxides.",
"The presently claimed invention is interesting in both terms of product and in terms of process (of processability).",
"The invention enables non-photochromic, colored glasses, which absorb ultraviolet and which are of various colors, to be obtained from a single mixture of starting materials, even from a single crude borosilicate glass of defined composition (which is non-photochromic despite the presence of silver within it, which absorbs ultraviolet if it has undergone a suitable heat-treatment, and which is colored since it contains at least one colorant within it), by heat-treatments which are carried out under given conditions of duration and/or of temperature.",
"Said starting crude colored borosilicate glass, as well as said colored glasses of various colors which are obtainable by treatment of it, all constitute glasses of the invention, which are first generation glasses and second generation glasses, respectively.",
"According to its first object, the presently claimed invention thus relates to a non-photochromic, colored, borosilicate inorganic glass which absorbs ultraviolet and which contains effective amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s).",
"Said inorganic glass is a glass based on silica (SiO2) and boron oxide (B2O3).",
"Its inorganic matrix can obviously contain other oxides, such as Al2O3, ZrO2, Li2O, Na2O, K2O, MgO, CaO, SrO, BaO, ZnO, PbO, TiO2, Nb2O5, La2O3, Y2O3.Compositions by weight of matrices are specified later on in the present text in an illustrative manner.",
"Said borosilicate inorganic glass is a colored glass.",
"It contains an effective amount of at least one colorant.",
"The following oxides: Fe2O3, NiO, CoO, V2O5, MnO, SeO2, Cr2O3 and Nd2O3 can be cited in a non-limiting manner as suitable colorants which can be incorporated alone or in a mixture within the glasses of the invention.",
"Said colored borosilicate inorganic glass absorbs ultraviolet.",
"It has an abrupt optical absorption cut-off of greater than 380 nm, generally towards or greater than 400 nm (said abrupt UV cut-off, expressed in nm, corresponds to the wavelength at which 1% transmission is observed, below which the glass absorbs at more than 99% and over which it hardly absorbs any more).",
"To this end, it contains suitable amounts of copper, of halogen(s) and of reducing agent(s).",
"The copper is incorporated in the glass of the invention, with reference to the absorption of ultraviolet, in the same way as in the glasses according to U.S. Pat.",
"No.",
"5,281,562 and U.S. Pat.",
"No.",
"5,242,869.It is noted at this juncture that express mention has not been made of the incorporation of said copper in combination with cadmium.",
"The combined presence of copper and cadmium is however not totally excluded, but the person skilled in the art does not ignore the problems of toxicity which are linked to the incorporation of cadmium.",
"Said copper is incorporated in the glass of the invention in amounts which are far greater than in the glasses according to U.S. Pat.",
"No.",
"3,208,860 in which it is incorporated for other purposes.",
"Said copper is generally incorporated in said glasses of the invention at the rate of 0.15 to 1.5 parts by weight per 100 parts by weight of their borosilicate base.",
"Said colored borosilicate inorganic glass which absorbs ultraviolet characteristically further contains silver.",
"In an entirely unexpected way, said silver does not confer photochromic properties to the colored glass of the invention and enables glasses of various colors to be obtained from a single colored base by suitable heat-treatments.",
"Said silver very likely also takes part in the ultraviolet absorption properties of the glasses of the invention.",
"Said silver is in general incorporated in the glasses of the invention at the rate of 0.0020 to 1.5 parts by weight per 100 parts by weight of their borosilicate base.",
"Said silver is present in the first generation and second generation glasses of the invention.",
"It is responsible for the change of color during the heat-treatment of said first generation glasses in order to obtain said second generation glasses.",
"Advantageous variants of the glasses of the invention are now specified below.",
"In general, per 100 parts by weight of their borosilicate base, said glasses of the invention contain: from 0.0020 to 1.5 parts by weight of Ag, as indicated above; from 0.15 to 1.5 parts by weight of Cu, as indicated above; and an effective amount of at least one halogen selected from Cl, Br, I and F, which is conjugated to: an effective amount of at least one reducing agent, said effective amounts of halogen(s) and of reducing agent(s) generating sufficient copper halide(s) for the absorption of the ultraviolet; as well as an effective amount of at least one colorant.",
"The reducing agents which can be incorporated, alone or in a mixture, can notably be selected from SnO, SnO2, Sb2O3 and As2O3.The effective amount of their incorporation is generally between 0.1 and 5 parts by weight per 100 parts by weight of the borosilicate base.",
"As regards the halogens, they are incorporated in general at the rate of at least 0.25 part by weight per 100 parts by weight of the borosilicate base.",
"They are incorporated very rarely at more than 10 parts by weight.",
"Advantageously, each halogen which is incorporated is incorporated in an amount which is less than or equal to 3 parts by weight.",
"Advantageously, chlorine and/or bromine is incorporated at least 0.25 part by weight.",
"The colorants which can be incorporated alone or in a mixture can notably be selected from Fe2O3, NiO, CoO, V2O5, MnO, SeO2, Cr2O3 and Nd2O3.The glasses of the invention generally contain, per 100 parts by weight of its borosilicate base: up to 7 parts by weight of Fe2O3, and/or up to 2 parts by weight of NiO, and/or up to 3 parts by weight of CoO, and/or up to 10 parts by weight of V2O5, and/or up to 2 parts by weight of SeO2, and/or up to 2 parts by weight of Cr2O3, and/or up to 4 parts by weight of Nd2O3.Said glasses of the invention advantageously contain, as colorant(s), nickel oxide (NiO) and/or cobalt oxide (CoO).",
"These two colorants, which are used independently or in a mixture, ensure, within the context of the invention, particularly interesting colorations to the borosilicate glasses.",
"The advantageous amounts of incorporation of each one of the constituents above of the glasses of the invention, which are characteristically added into the borosilicate base, are specified below.",
"Per 100 parts by weight of their borosilicate base, the glasses of the invention thus advantageously contain: from 0.0050 to 1 part by weight of Ag; from 0.2 to 1 part by weight of Cu; from 0 to 2 parts by weight of Cl; from 0 to 2 parts by weight of Br, with at least 0.25 parts by weight of Br+Cl; from 0 to 2 parts by weight of I; from 0 to 2 parts by weight of F; from 0.2 to 3 parts by weight of SnO; from 0 to 3 parts by weight of SnO2; from 0 to 3 parts by weight of Sb2O3; from 0 to 3 parts by weight of As2O3; from 0 to 3 parts by weight of Fe2O3; from 0.0100 to 1 part by weight of NiO; from 0.0050 to 1 part by weight of CoO; from 0 to 5 parts by weight of V2O5; from 0 to 0.5 part by weight of MnO; from 0 to 0.5 part by weight of SeO2; from 0 to 0.5 part by weight of Cr2O3; and from 0 to 0.5 part by weight of Nd2O3.These advantageous ranges of incorporation set forth in combination are also to be considered independently of each other.",
"Thus, for example, the glasses of the invention advantageously contain, per 100 parts by weight of their borosilicate base, from 0.0050 to 1 part by weight of silver and/or from 0.2 to 1 part by weight of copper and/or from 0.2 to 3 parts by weight of SnO and/or .",
".",
".",
".",
"As regards the borosilicate base of the glasses of the invention, to which suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s) are characteristically added, it advantageously has the following composition by weight: SiO2 25-60%, preferably 30-55% B2O3 10-35%, preferably 15-28% Al2O3 3-17%, preferably 6-12% ZrO2 0-13%, preferably 3-8% Li2O 0-15%, preferably 1.5-3% Na2O 0-15%, preferably 2-5% K2O 0-15%, preferably 2.5-8% with Li2O + Na2O + K2O > 2% MgO 0-10%, preferably 0-3% CaO 0-15%, preferably 0-5% SrO 0-15%, preferably 0-5% BaO 0-15%, preferably 3-8% with MgO + CaO + SrO + BaO > 1% ZnO 0-15%, preferably 0-11% PbO 0-8%, preferably 0-5% TiO2 0-8%, preferably 0-5% Nb2O5 0-8%, preferably 0-5% La2O3 0-8%, preferably 0-5% Y2O3 0-8%, preferably 0-5%.",
"The advantageous and preferable incorporation ranges above can also be considered independently of one another.",
"In any case, they define, respectively, taken in combination, an advantageous composition by weight and a preferred composition by weight of the borosilicate base of the glasses of the invention.",
"Said borosilicate base of the glasses of the invention can therefore contain the components listed above, advantageously and preferably in the relative percentages by weight indicated above.",
"It is not excluded that said base contain other components.",
"As regards the optional components, it is indicated that when they are incorporated, it is generally at least 0.25-0.5% (by weight).",
"The first generation glasses and the second generation glasses of the invention, notably the second generation glasses of the invention, as defined above, are more particularly suitable as sunglasses, corrective or not.",
"Such sunglasses constitute the second object of the present invention.",
"Within the first and second objects of the present invention, glasses are found which are particularly interesting and which: have a suitable refractive index (1.523), absorb ultraviolet up to greater than 430 nm, have a transmittance in the UV and visible domains, typically greater than 5% and less-than 25%, even between 8 and 18%, are in accordance to the requirements imposed with reference to the recognition of road signals, such as those defined by the ANSI Z80.3-1986 standard, and have a suitable color in the tones of green, grey and brown.",
"The third object of the present invention has now been arrived at, i.e.",
"the methods of preparation of the inorganic glasses in question.",
"The person skilled in the art will already have understood that the method in question is more or less complex, depending on whether a first generation glass or a second generation glass is prepared.",
"The method in question for obtaining a first generation glass of the invention may be considered to be an analogous method.",
"It comprises: preparing a crude borosilicate glass which contains the suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s); and heat-treating said crude glass in order to generate within it the crystal phase of the halide(s) which is responsible for the absorption of the ultraviolet.",
"The preparation of said colored crude borosilicate glass is classical.",
"It comprises mixing and melting the compounds in question.",
"The heat-treatment of said colored crude glass is of the type of those described in U.S. Pat.",
"No.",
"5,281,562 and U.S. Pat.",
"No.",
"5,242,869.In order to obtain a second generation glass of the invention, the method of the invention comprises: preparing a crude borosilicate glass which contains the suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s); optionally heat-treating said crude glass in order to generate within it the crystal phase of the halide(s) which is responsible for the absorption of the ultraviolet; polishing the surface of said borosilicate glass which is heat-treated or non-heat-treated; and heat-treating said polished borosilicate glass under a reducing atmosphere; said heat-treatment being carried out under conditions of duration and temperature which are suitable for obtaining the coloration sought after; said heat-treatment also ensuring the generation of the crystal phase of the halide(s) which is responsible for the absorption of the ultraviolet if this crystal phase has not been generated beforehand.",
"According to a first variant, the method of the invention can therefore comprise obtaining a first generation glass and then, mainly, its heat-treatment under a reducing atmosphere.",
"According to a second variant, the method of the invention can comprise a single heat-treatment under reducing atmosphere which itself alone ensures the results sought after in terms of coloration and of absorption of ultraviolet (which ensures the direct obtaining of a second generation glass).",
"Said single heat-treatment under reducing atmosphere is, within this context, carried out on a glass, which constitutes a potential precursor of a first generation glass of the invention.",
"The surface treatment—polishing—is carried out before the heat-treatment under reducing atmosphere insofar as said heat-treatment under reducing atmosphere produces effects within the first 100 nm of surface.",
"Said surface heat-treatment is a method known per se.",
"The term <<polishing >> is employed here in an entirely non-limiting manner.",
"In general, it designates any treatment which can confer, to said glass on which it is carried out, the state of the surface sought after for the finished product.",
"With reference to the finishing of the glasses of the invention, a chemical tempering can be added to the steps of the two methods above.",
"Carried out in a classical manner, such a tempering leads to increasing their mechanical resistance.",
"The heat-treatments set forth above are advantageously carried out under the following conditions: the heat-treatment of the crude borosilicate glass intended to generate a first generation glass of the invention: at a temperature between 450 and 650° C., for 10 minutes to 2 hours; the heat-treatment under reducing atmosphere of the polished crude borosilicate glass (potential precursor of a first generation glass of the invention) or of the polished first generation glass of the invention: at a temperature between 250 and 650° C., for 2 minutes to 12 hours.",
"Said reducing atmosphere generally contains hydrogen.",
"Said reducing atmosphere can notably consist of pure hydrogen or of a mixture of nitrogen and hydrogen.",
"The carrying out of this heat-treatment, under reducing atmosphere, on a polished crude borosilicate glass of defined composition containing suitable amounts of silver, of copper, of halogen(s), of reducing agent(s) and of colorant(s) (first generation glass of the invention, if it has undergone the heat-treatment rendering it an ultraviolet-absorber or potential precursor of such a first generation glass, and in any case a precursor of a second generation glass of the invention), in order to confer a different color to said crude glass, is an innovating stepper se.",
"The carrying out of such heat-treatments, under the conditions of different duration or (and) of temperature, on one glass of given suitable composition to generate numerous glasses of various colors, constitutes another object of the present invention.",
"In an entirely surprising way, the inventors have in fact prepared glasses of various colors (which are non-photochromic and which absorb Uv) by heat treating, under a reducing atmosphere, a single crude borosilicate glass of defined, particular composition.",
"Said crude borosilicate glass is a first generation glass of the invention or a potential precursor of such a glass (if it has not undergone the heat-treatment conferring ultraviolet-absorbing properties to it).",
"In any case, it contains suitable amounts of silver, of copper, of halogen(s), reducing agent(s) and colorant(s).",
"The invention will now be illustrated under its different aspects, by the following Examples.",
"Glasses A, B, C (First generation glasses—Table I) The first part of Table I gives the compositions by weight of borosilicate bases of three glasses of the invention.",
"The second part of said Table I indicates the parts by weight of the additives added characteristically to said bases (per 100 parts by weight of said bases).",
"The third part of said Table I gives properties of the three glasses, A, B and C, of the invention (first generation glasses).",
"The glasses in question are obtained in a classical manner, i.e.",
"by successively carrying out the following steps: mixing vitrifiable starting materials in the suitable amounts indicated in the first and second parts of Table I; melting in a crucible between 1,100 and 1,200° C. refining between 1,250 and 1,400° C.; conditioning between 1,200 and 1,250° C. with the view to forming; said forming for obtaining spectacles mouldings; heat-treatment for obtaining the UV cut-off sought after: the mouldings are treated after pressing in an arch at a temperature of 600° C., for 20 minutes.",
"Upon completion of the implementation of these steps, said three glasses A, B and C are obtained.",
"They have a transmission of greater than 25%.",
"Said transmission on the visible spectrum was measured under a thickness of 2 mm.",
"The color of each one of said glasses A, B and C is characterised by its chromatic co-ordinates (x,y) which are determined according to the C.I.E.",
"trichromatic calorimetric system using the D65-illuminant.",
"The UV cut-off, expressed in nm, corresponds to the wavelength at which 1% transmission is observed, lower than which the glass absorbs at more than 99% and greater than which it hardly absorbs any more, given the abrupt character of the optical absorption cut-off.",
"TABLE I A B C Base (% by weight) SiO2 48.7 48.7 39.4 B2O3 21.4 21.4 21.7 Al2O3 8.8 8.8 17.5 ZrO2 4.3 4.3 4.4 Li2O 2.2 2.2 2.2 Na2O 3.6 3.6 3.6 K2O 6 6 6.1 BaO 5 5 5.1 Additives (parts by weight per 100 parts by weight of the base) Ag 0.0300 0.0700 0.1000 CuO 0.450 0.330 0.45 Cl 0.0600 0.0600 0 Br 0.78 0.57 0.80 SnO 0.380 0.290 1.6 Nd2O3 0.0500 0.0500 0 NiO 0.1100 0.1300 0.1000 CoO 0.0750 0.0750 0 Transmission (%) 32.5 33.4 32.0 x 0.2751 0.2961 0.2485 y 0.3043 0.3385 0.2796 UV cut-off (nm) 410 420 400 Refractive index 1.523 1.523 1.523 Glasses 1 to 15 (Second Generation Glasses—Table II).",
"These second generation glasses were obtained from first generation glasses A, B and C. A surfacing of said glasses A, B and C is first of all made according to usual techniques.",
"An optical correction can be given to the piece during this surfacing, which confers an adapted geometry to it.",
"Then, a heat-treatment under hydrogen is carried out under the various conditions of temperature and/or of duration, which are specified in Table II.",
"The glasses were in fact treated in batches, discontinuously, in an adapted static oven under a flow of pure hydrogen.",
"Obtaining the same results can be entirely conceived by a continuous treatment of said glasses, in an industrial arch having a controlled atmosphere which is equipped to this end.",
"The surfaced pieces were thus heat treated under a reducing atmosphere between 400 and 530° C. for 5 to 240 minutes.",
"During this step, the silver atoms and copper atoms present on the surface are reduced.",
"The total UV visible transmission and the tint are also adapted.",
"Six glasses (glasses 1 to 6), six glasses (glasses 7 to 12) and three glasses (glasses 13 to 15), of various colors, were obtained from a same glass A, B or C, respectively.",
"For said glasses 1 to 15, their transmission, their chromatic co-ordinates (x,y), their UV cut-off, as well as their conformity with the tests of road signal recognition, are also given in Table II.",
"TABLE II Examples 1 2 3 4 5 6 Crude glass A A A A A A Heat-treatment temperature under hydrogen (° C.) 400 400 530 450 450 500 Duration of heat-treatment under hydrogen (min) 120 240 240 120 240 120 Transmission (%) 18.3 15.3 14.9 17.5 14.3 11.2 x 0.3139 0.3330 0.3399 0.3453 0.3773 0.3905 y 0.3348 0.3395 0.3415 0.3472 0.3595 0.3689 UV cut-off (nm) 410 410 410 410 410 410 Recognition of road signals (Z80) yes yes yes yes yes yes Examples 7 8 9 10 11 12 Crude glass B B B B B B Heat-treatment temperature under hydrogen (° C.) 400 400 530 450 450 500 Duration of heat-treatment under hydrogen (min) 120 240 240 120 240 120 Transmission (%) 18.8 15.3 14.8 17.6 14.1 16.0 x 0.3500 0.3675 0.3758 0.3725 0.3985 0.3929 y 0.3738 0.3790 0.3751 0.3755 0.3782 0.4049 UV cut-off (nm) 420 420 420 420 420 420 Recognition of road signals (Z80) yes yes yes yes yes yes Examples 13 14 15 Crude glass C C C Heat-treatment temperature under hydrogen (° C.) 530 450 510 Duration of heat-treatment under hydrogen (min) 240 30 5 Transmission (%) 20.3 19.2 24.8 x 0.3550 0.2970 0.2648 y 0.3468 0.3109 0.2882 UV cut-off (nm) 400 400 420 Recognition of road signals (Z80) yes yes yes"
]
] | Patent_10415237 |
[
[
"Preparation of 4-methyl-2, 3, 5, 6-tetrafluorobenzyl alcohol",
"A process for the preparation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene comprising fluorinating 2,3,5,6-tetrachloroterephthalonitrile to obtain 2,3,5,6-tetrafluoroterephthalonitrile, hydrogenating 2,3,5,6-tetrafluoroterephthalonitrile to give 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene and converting 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene to 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene."
],
[
"1.A process for the preparation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene comprising: a) fluorinating 2,3,5,6-tetrachloroterephthalonitrile to obtain 2,3,5,6-tetrafluoroterephthalonitrile; b) hydrogenating 2,3,5,6-tetrafluoroterephthalonitrile to give 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene; and c) converting 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene to 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene.",
"2.A process according to claim 1 wherein step c) is performed by diazotisation of 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene followed by in-situ hydrolytic decomposition of the resulting diazonium salt.",
"3.A process as claimed in claim 1 which comprises the further steps of i) halogenation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene to give a 4-(halomethyl)-2,3,5,6-tetrafluoro-benzyl alcohol where halo is chloro or bromo and ii) hydrogenation of the 4-(halomethyl)-2,3,5,6-tetrafluorobenzyl alcohol to give to 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol.",
"4.A process according to claim 1 which process comprises the further step of reacting 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol with cis-Z 3-(2-chloro-1,1,1-trifluoro-2-propenyl)-2,2-dimethylcyclopropane carbonyl chloride to form tefluthrin.",
"5.A process according to claim 2 in which the step of converting 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene to 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene is followed by a hydrolysis reaction to convert ester by-products to the required 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene.",
"6.A process according to claim 2 wherein the 2,3,5,6-tetrafluoroterephthalonitrile is hydrogenated in a carboxylic acid solvent and the hydrogenation mass is passed straight into the diazotisation stage (step c) after screening, such that no further acid is required.",
"7.A process according to claim 2 wherein the reaction mass containing the 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene is adjusted to pH 7-10 and the 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene is extracted into a solvent.",
"8.A process according to claim 3 wherein 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene from step c) is extracted from the reaction mass in a solvent such as methyl isobutylketone (MiBK), the solvent removed under reduced pressure and a second solvent is added, the second solvent being suitable for the halogenation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene to a 4-(halomethyl)-2,3,5,6-tetrafluoro-benzyl alcohol.",
"9.A process according to claim 3 wherein the 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene from step c) is extracted directly into a solvent that is also used in the halogenation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene to a 4-(halomethyl)-2,3,5,6-tetrafluoro-benzyl alcohol."
],
[
"The present invention relates to a process for making polyfluorinated benzyl alcohols which are useful in the synthesis of pyrethroid pesticides.",
"Esters of cis-3-(haloalkenyl)-2,2-dimethylcyclopropanecarboxylic acid with 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, in particular tefluthrin [2,3,5,6-tetrafluoro-4-methylbenzyl cis-3-((Z)-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropane carboxylate] are important insecticidal and acaricidal products.",
"It is thus desirable to have an industrially acceptable and efficient process for making the necessary intermediates such as 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol.",
"Known processes for making of 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol are described in DE3714602, GB2155464 and EP31199 but the processes involve reduction of the parent benzoic acids or acid halides using expensive reagents such as borohydrides, or lithiation of tetrafluorobenzene at very low temperature followed by methylation.",
"These methods are not suited to industrial application as they involve the use of expensive reagents or require specialised low temperature conditions.",
"The applicants have found that these disadvantages may be avoided by preparing 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol from 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene and have devised a practical and efficient process for the manufacture of both compounds on an industrial scale starting from the readily available compound 2,3,5,6-tetrachloroterephthalonitrile.",
"There is therefore provided a method for producing 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene comprising: a) fluorinating 2,3,5,6-tetrachloroterephthalonitrile to obtain 2,3,5,6-tetrafluoroterephthalonitrile; b) hydrogenating 2,3,5,6-tetrafluoroterephthalonitrile to give 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene; and c) converting 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene to 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene.",
"The fluorination of step a) is suitably carried out using fluoride exchange agents such as alkali metal fluorides, for example potassium fluoride and optionally a phase transfer catalyst, for example tetramethyl ammonium chloride (TMAC).",
"The reaction is suitably carried out in a solvent.",
"Suitable solvents are dipolar aprotic solvents such as dimethylfomamide.",
"The reaction is suitably performed at temperatures between 100-150° C., preferably at 130-150° C. and preferably under anhydrous conditions.",
"The hydrogenation process of step b) may be carried out according to any of the procedures in described in EP99622.A particularly suitable method employs hydrogen and a metal catalyst such as palladium.",
"The reaction may be carried out at from 0° C. to about 60° C. and from ambient pressure to 10 bar of over pressure.",
"Suitable solvents for use in the reaction include alcohols, carboxylic acids, such as formic acid, acetic acid, or mixtures of either class with water.",
"A preferred solvent for the hydrogenation reaction is acetic acid or aqueous acetic acid.",
"The mol ratio of acetic acid is suitably 2-10:1 but is preferably 4-6:1 Step c) is preferably performed by diazotisation and in-situ hydrolytic decomposition of the diazonium salt.",
"The diazotisation process is suitably performed using aqueous sodium nitrite and an acid at a temperature of 0-100° C., preferably 50-80° C. The concentration of the amine in the reaction (prior to nitrite addition) is suitably 5-30%, preferably 5-15%.",
"The mol ratio of nitrite to 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene is suitably 2-5:1 but 2:1 is preferred.",
"After completion of step c), the product may contain some esters (e.g.",
"acetate esters) of the desired benzyl alcohol product.",
"The acetate ester structures are shown below.",
"It has been found advantageous to render the reaction mass alkaline and then heat it to hydrolyse the esters to the desired diol compound.",
"Once the esters have all been hydrolysed, the reaction mass is adjusted to between pH 7-10, preferably to between pH 9-10 prior to extraction of the product into a solvent for purification or for use in subsequent processes.",
"In a further aspect of the invention the 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene from step c) may be converted to 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol by i) halogenation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene to give a 2,3,5,6-tetrafluoro-4-(halomethyl)benzyl alcohol; and ii) hydrogenation of the 2,3,5,6-tetrafluoro-4-halomethyl)benzyl alcohol to give 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol.",
"The halogenation of step i) where the halo group is for example chloro or bromo is suitably carried out using halogenating agents such as aqueous hydrochloric acid or hydrobromic acid.",
"The reaction is suitably carried out in two phases in the presence of a solvent.",
"Suitable solvents are inert and water immiscible solvents, such as aromatic hydrocarbons, for example toluene.",
"The reaction is performed at a temperature between 50-150° C., preferably at 75-100° C. The hydrogenation reaction of step ii) may be performed under process conditions similar to those for outlined for step b) above but in the presence of a base to absorb the liberated hydrogen halide.",
"Suitable bases are alkali or alkaline earth metal hydroxides or carbonates or alkaline earth metal oxides.",
"Suitable solvents are alcohols, esters or aromatic hydrocarbons.",
"In yet a further aspect of the invention, the 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol is reacted with cis-Z 3-(2-chloro-1,1,1-trifluoro-2-propenyl)-2,2-dimethylcyclopropane carbonyl chloride to give tefluthrin.",
"Suitable conditions for performing the reaction are described for example in EP-A-31199.In one preferred embodiment of the invention, the 2,3,5,6-tetrafluoroterephthalonitrile is hydrogenated in a carboxylic acid solvent, for example acetic acid, and the hydrogenation mass is passed straight into the diazotisation stage (step c)) after screening, and no further acid is then required.",
"In another preferred embodiment 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene from step c) is extracted from the reaction mass in a solvent such as methyl isobutylketone (MiBK), the solvent removed under reduced pressure and a second solvent is added, the second solvent being suitable for the halogenation reaction of step i).",
"A suitable second solvent is toluene.",
"In a further preferred embodiment, the 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene from step c) is extracted directly into a solvent that is also used in the halogenation reaction of step i).",
"Suitable solvents are those that are stable to aqueous acid, such as aromatic hydrocarbons (for example toluene, xylene or a halobenzene) or halogenated hydrocarbons (for example ethylene dichloride or perchloroethylene).",
"The following Examples illustrate the processes and the compounds of this invention.",
"1 H NMR was carried out on a Bruker AS 200 (200 MHz 1 H) spectrometer, using TMS as reference standard.",
"Gas chromatography analyses were obtained with a Hewlett Packard 5890 Series II.",
"Hydrogenation reactions under pressure were carried out in a 1 litre stainless steel autoclave, fitted with a gas-inducing turbine agitator at 1000 rpm.",
"Hydrogen feed was through a dip-pipe via a Buchi gas controller type 6002, heating and cooling was controlled by a Jelabo FP40 bath.",
"Palladium on carbon type 58 catalyst was supplied by Johnson Matthey Ltd.",
"EXAMPLE 1 This Example Describes the Preparation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene Step a.",
"Preparation of 2,3,5,6-tetrafluoroterephthalonitrile Potassium fluoride (117.7 g) and DMF (330 g) were charged to a 1 litre jacketed flask fitted with a condenser, nitrogen blanket and external heating cooling coil.",
"The mixture was agitated under a nitrogen atmosphere for 5 minutes after which time a sample (5 ml) was taken and analysed for water content to ensure that the reaction mixture contained <0.2 wt % water.",
"2,3,5,6-Tetrachloroterephthalonitrile (120 g) and tetramethylammonium chloride (1.55 g) were then charged and the reaction mixture heated to 115° C. under agitation.",
"The reaction was maintained at 115° C. under nitrogen for 6 hours after which time a sample was taken to determine if completion of reaction had been achieved (i.e.",
"monochloro impurity was <2%).",
"The reaction mixture was further heated at 115° C. for 30 minutes if necessary.",
"The reaction was cooled to 70° C. and dropped out of the reaction vessel via the bottom run off valve into a stirred 2 litre beaker containing water (670 g) at ambient temperature.",
"The resulting slurry was filtered and the filter cake water washed (3×300 ml).",
"The 2,3,5,6-tetrafluoroterephthalonitrile was partially dried on the filter and discharged into a suitable storage vessel.",
"The reaction produced 97.8 g of water wet paste, GC analysis showed a strength of 78.6% giving a yield of 85.2%.",
"Step b.",
"Preparation of 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene Water (258 g), acetic acid (179 g), 2,3,5,6-tetrafluoroterephthalonitrile paste (90.6 g 100% wt.",
"), wetting agent (Nopco 8050) (0.15 g) and 5% palladium/carbon catalyst, (1.8 g 100% wt.)",
"were charged to the autoclave.",
"The lid was sealed and the autoclave was initially purged with nitrogen to remove residual oxygen and then pressurised to 5 bar g. with hydrogen after which the gas controller was zeroed.",
"The agitator was started and the pressure maintained at 5 bar g. automatically by the Buchi controller.",
"The temperature was monitored manually and the reaction exotherm controlled by manual intervention using the external heating/cooling bath.",
"The reaction temperature was initially at 5° C. rising to 30° C. over the first 120 minutes of hydrogen ingress.",
"The hydrogen consumption was totalled by the Buchi gas controller.",
"The reaction was continued until the consumption of hydrogen ceased (typically 4 hrs.).",
"The residual pressure was released and the autoclave purged with nitrogen.",
"The contents of the autoclave were discharged by the autoclave's bottom run-off valve, followed by a small water wash.",
"The spent catalyst was removed by filtration.",
"The filtrates and filter-cake water washes were combined and analysed for product 1,4-bis(aminomethyl)-2,3,5,6-tetrafluorobenzene.",
"The reaction produced 83.4 g of diamine, this equating to a reaction yield of 88.6%.",
"Step c. Preparation of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene 1,4-Bis(aminomethyl)-2,3,5,6-tetrafluorobenzene (68.6 g), acetic acid (104 g), water (451 g) and a silicone-based defoaming agent (0.2 g) were charged to a 1 litre split neck reaction flask, agitation was maintained throughout.",
"The flask was held at 25° C. and aqueous sodium nitrite solution (36%, 134.2 g) was added over 90 minutes.",
"The reaction temperature was increased to 70° C. over the course of the addition.",
"The reaction was held at 70° C. for a further 30 minutes following completion of the nitrite addition.",
"The reaction mass was sampled and analysed for residual sodium nitrite using starch-iodide paper.",
"Upon confirmation that excess sodium nitrite had been charged (starch-iodide paper turns blue) the reaction was cooled to 60° C. The by-product acetate esters of the product were next hydrolysed by treatment with sodium hydroxide.",
"Sufficient 47% aqueous sodium hydroxide solution was charged to raise the pH to >11.5.The temperature was then raised to 90° C. and held for 30 minutes after which time the pH was re-checked to ensure it was >11.5.If pH was not >11.5, further sodium hydroxide solution was added and agitation maintained for a further 30 minutes.",
"Reaction temperature was lowered to 60° C. and the pH adjusted to 9 by addition of hydrochloric acid.",
"This solution was then extracted with 4-methylpentan-2-one (MiBK, 2×250 ml), the organic extracts were combined and analysed to show a 90% yield of 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene by quantitative GLC analysis.",
"EXAMPLE 2 This Example Describes the Preparation of 4-methyl-2,3,5,6-tetrafluoro-benzyl alcohol Step i.",
"Preparation of 2,3,5,6-tetrafluoro-4-(bromomethyl)benzyl alcohol A solution of 80 gms 1,4-bis(hydroxymethyl)-2,3,5,6-tetrafluorobenzene in MiBK produced as in Example 1 was charged to a 1 litre reaction flask fitted for distillation (both reflux return and total take-off).",
"The solvent was removed by distillation and then steam distillation.",
"Toluene (303 g) was added and residual water removed by azeotropic distillation.",
"The solution was agitated and heated to 60° C. for 30 minutes and Silcolapse (0.2 g) and 48% aqueous hydrobromic acid (109.3 g) were added to the reaction mixture and this was heated to 95° C. The distillation apparatus was set to reflux return for the initial 30 minutes of reaction at 95° C. After this time, the toluene water azeotrope was collected in a separator with the toluene layer being recycled back to the reaction vessel.",
"The reaction was heated for 5.5 hours at 95° C. with toluene recycle.",
"The mixture was cooled to 55° C. with water (150 ml) and 48% aqueous hydrobromic acid (36.6 g) being added.",
"This separation mixture was stirred at 55° C. for 15 minutes and then allowed to settle for 30 minutes prior to the aqueous phase being removed.",
"The remaining toluene/product layer was further washed with a pre-prepared mixture of water (150 ml) and 40% aqueous sodium acetate solution (36 g) this was stirred and settled prior to the aqueous buffer layer being discharged.",
"The toluene layer was analysed for 2,3,5,6-tetrafluoro-4-(bromomethyl)benzyl alcohol product and gave a yield of 96.2%.",
"Step ii.",
"Preparation of 4-methyl-2,3,5,6-tetrafluoro-benzyl alcohol The following procedure used a reducer which was a 1 litre glass autoclave, working volume 350-500 mls fitted with a gas dispersion agitator (1000 rpm), gas feed through a dip-pipe via a Buchi gas controller type 6002, heating and cooling was controlled by a Jelabo FP40 bath.",
"Methanol (362 g), water (6 g), 2,3,5,6-tetrafluoro-4-(bromomethyl)benzyl alcohol (95.1 g 100% wt.",
"), magnesium oxide (18.1 g) and 5% palladium/carbon catalyst, Johnson Matthey Type 58, (0.8 g 100% wt.)",
"were charged to the autoclave.",
"The lid was sealed and the autoclave was initially purged with nitrogen to remove residual oxygen and then pressurised to 2.5 bar g. with hydrogen after which the gas controller was zeroed.",
"The agitator was started and the pressure maintained at 2.5 bar g. automatically by the Buchi controller.",
"The reaction temperature was controlled at 50° C. throughout reaction by means of the external heating/cooling bath.",
"The hydrogen consumption was totalled by the Buchi gas controller.",
"The reaction was continued until the consumption of hydrogen ceased (typically 60 to 90 minutes).",
"The residual pressure was released and the autoclave purged with nitrogen.",
"The contents of the autoclave were discharged by the autoclave's bottom run-off valve, followed by a small methanol wash (30 g).",
"The spent catalyst and inorganic salts was removed by filtration with the filter cake receiving methanol washes (2×30 g).",
"The filtrates and filter-cake washes were combined.",
"On analysis the reaction had produced 60.4 g of 2,3,5,6-tetrafluoro-4-methylbenzyl alcohol, equating to a reaction yield of 89.4%.",
"EXAMPLE 3 This Example Describes the Preparation of 2,3,5,6-tetrafluoro-4-methylbenzyl cis-3-((Z)-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropane carboxylate Cis-Z 3-(2-chloro-1,1,1-trifluoro-2-propenyl)-2,2-dimethylcyclopropane carbonyl chloride (257 g) was charged to a reactor fitted with an agitator, thermometer, sub-surface nitrogen inlet and jacketed dropping funnel.",
"Nitrogen sparging was applied to the acid chloride and maintained throughout subsequent processing.",
"Molten 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol (188.1 g) was charged to the dropping funnel.",
"The temperature in the funnel was maintained at 80° C. The alcohol was charged to the acid chloride mixture over three hours.",
"The maximum reaction temperature allowed during the addition was 45° C. On completion of the addition the temperature of the reaction mass was raised to 95° C. and held for 2 hours.",
"GC analysis was used to determine if full conversion of acid chloride had occurred and that no excess reactants were remaining.",
"The mixture was cooled to 60° C. and the molten reaction mass discharged, weighed and analysed.",
"The reaction yielded 423.3 g of molten 2,3,5,6-tetrafluoro-4-methylbenzyl cis-3-((Z)-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropane carboxylate, analysis against a known standard showed this material to be 92.5 wt % giving an overall reaction yield of 96.5%."
]
] | Patent_10415258 |
[
[
"Medicament dispenser",
"A medicament dispenser comprising: (i) a housing having an outlet; (ii) a medicament container locatable within said housing; (iii) an electronic dose counter associated with said outlet, wherein said dose counter comprises a first sensor for directly detecting a medicament release dispensible from said medicament container through said outlet is disclosed."
],
[
"1.A medicament dispenser comprising: (i) a housing having an outlet; (ii) a medicament container locatable within said housing; (iii) an electronic dose counter associated with said outlet, wherein said dose counter includes a first sensor comprising an emitter and a detector for detecting a medicament release dispensible from said medicament container through said outlet, characterized in that the detector detects electromagnetic radiation in the infra red range.",
"2.A medicament dispenser according to claim 1, wherein said emitter emits electromagnetic radiation in the infrared range.",
"3.A medicament dispenser according to claim 1 wherein the electromagnetic radiation has a wavelength in the range of 0.95 μm to 0.35 μm.",
"4.A medicament dispenser according to claim 3, wherein the electromagnetic radiation has a wavelength of about 0.88 μm.",
"5.A medicament dispenser according to claim 1, wherein the emitter is selected from the group consisting of light emitting diode and laser.",
"6.A medicament dispenser according to claim 1, wherein the emitter further comprises a filter.",
"7.A medicament dispenser according to claim 6, wherein the filter is an optical filter.",
"8.A medicament dispenser according to claim 7, wherein the optical filter is a polarising filter.",
"9.A medicament dispenser according to claim 1, wherein the detector is selected from the group consisting of photodiode, phototransistor, light dependent resistor and bolometer.",
"10.A medicament dispenser according to claim 9, wherein the detector further comprises a filter.",
"11.A medicament dispenser according to claim 10, wherein the filter is an electronic filter.",
"12.A medicament dispenser according to claim 10, wherein the filter is an optical filter.",
"13.A medicament dispenser according to claim 12, wherein the optical filter is a polarising filter.",
"14.A medicament dispenser according to claim 1, wherein the detector is associated with an amplifier.",
"15.A medicament dispenser according to claim 14, wherein the amplifier is positioned close to the detector.",
"16.A medicament dispenser according to claim 14, wherein the amplifier is integrated with the detector.",
"17.A medicament dispenser according to claim 1, wherein the detector detects an increase in radiation compared to the amount of radiation emitted by the emitter.",
"18.A medicament dispenser according to claim 1, wherein the detector detects a decrease in radiation compared to the amount of radiation emitted by the emitter.",
"19.A medicament dispenser according to claim 17, wherein the increase or decrease in detected radiation is due to interference of radiation reaching the detector by the medicament release.",
"20.A medicament dispenser according claim 19, wherein the interference is due to absorption of radiation by the medicament release.",
"21.A medicament dispenser according claim 19, wherein the interference is due to scattering of radiation by the medicament release.",
"22.A medicament dispenser according claim 19, wherein the interference is due to reflection of radiation by the medicament release.",
"23.A medicament dispenser according claim 19, wherein the interference is due to refraction of radiation by the medicament release.",
"24.A medicament dispenser according claim 19, wherein the interference is due to diffraction of radiation by the medicament release.",
"25.A medicament dispenser according to claim 1, wherein the amount of radiation reaching the detector is maintained at a constant level by using an electronic feedback circuit to alter the level of radiation emitted by the emitter.",
"26.A medicament dispenser according to claim 1, wherein the first sensor further comprises a reflector to reflect radiation from the emitter to the detector.",
"27.A medicament dispenser according to claim 1, wherein the emitter emits radiation of more than one wavelength and the detector detects radiation of more than one wavelength.",
"28.A medicament dispenser according to claim 27, wherein the first sensor quantifies the concentration of medicament with the medicament release by measuring radiation at more than one wavelength.",
"29.A medicament dispenser according to claim 1, wherein the dispenser additionally comprises a second sensor for detecting a medicament release.",
"30.A medicament dispenser according to claim 29, wherein the second sensor comprises an emitter and a detector.",
"31.A medicament dispenser according to claim 29, wherein the medicament release passes the second sensor subsequent to passing the first sensor.",
"32.A medicament dispenser according to claim 1, wherein the dispenser further comprises a third sensor.",
"33.A medicament dispenser according to claim 32, wherein the third sensor is sensitive to parameter selected from the group consisting of electromagnetic radiation, magnetic field, light, motion, temperature, pressure, sound, oxygen concentration, carbon dioxide concentration and moisture.",
"34.A medicament dispenser according to claim 32, wherein the third sensor responds to actuation of the dispenser.",
"35.A medicament dispenser according to claim 29, wherein the first and/or second sensors are integral with the outlet.",
"36.A medicament dispenser according to claim 29, wherein the first and/or second sensors are reversibly attachable to the outlet.",
"37.A medicament dispenser according to claim 32, wherein the third sensor is integral with the housing.",
"38.A medicament dispenser according to claim 32, wherein the third sensor is reversibly attachable to the housing.",
"39.A medicament dispenser according to claim 1, wherein the dose counter is reversibly attachable to the housing.",
"40.A medicament dispenser according to claim 1, wherein the first sensor is located on the dose counter.",
"41.A medicament dispenser according to claim 1, wherein the dispenser further comprises one or more optical wave guides.",
"42.A medicament dispenser according to claim 41, wherein the one or more optical wave guides are located on the housing.",
"43.A medicament dispenser according to claim 41, wherein the optical wave guide is composed of an organic polymeric or inorganic glass fibre material.",
"44.A medicament dispenser according to claim 41, wherein the medicament dispenser comprises a first and a second optical wave guide per sensor.",
"45.A medicament dispenser according to claim 44, wherein the first optical wave guide channels radiation from the emitter to the outlet.",
"46.A medicament dispenser according to claim 44, wherein the second optical wave guide channels radiation from the outlet to the detector.",
"47.A medicament dispenser according to claim 1, wherein the emitter and detector are located on the same side of the outlet.",
"48.A medicament dispenser according to claim 47, wherein the radiation emitted from the emitter is reflected back to the detector by a reflective surface on the opposite side of the outlet to the emitter and detector.",
"49.A medicament dispenser according to claim 48, wherein the reflective surface is a surface of the outlet or is an additional component attached thereto.",
"50.A medicament dispenser according to claim 47, wherein the emitter and detector are integrated into a single component.",
"51.A medicament dispenser according to claim 1, wherein the first sensor is controlled by a digital or computational semi-conductor device.",
"52.A medicament dispenser according to claim 51, wherein the digital or computational semi-conductor device energises the first sensor and associated electronic components to detect and respond to a medicament release every 10 to 100 ms. 53.A medicament dispenser according to claim 51, wherein the sensor and associated electronic components are energised for 5 to 30 μs.",
"54.A medicament dispenser according to claim 51, wherein the digital or computational semiconductor device returns the sensor to low power mode after energising the sensor.",
"55.A medicament dispenser according to claim 1, wherein the medicament container is an aerosol container.",
"56.A medicament dispenser according to claim 55, wherein the aerosol container comprises a suspension of medicament in a propellant.",
"57.A medicament container according to claim 56, wherein the propellant is selected from the group consisting of HFA134a, HFA227, carbon dioxide or a mixture thereof.",
"58.A medicament dispenser according to claim 55, wherein the aerosol container comprises a solution of medicament in a solvent.",
"59.A medicament dispenser according to claim 1, wherein the medicament container is a dry-powder container.",
"60.A medicament dispenser according to claim 59, wherein the dry-powder container comprises a medicament and optionally excipient in dry-powder form.",
"61.A medicament dispenser according to claim 1, wherein the dispenser is actuable manually by the patient.",
"62.A medicament dispenser according to claim 1, wherein the dispenser is actuable by the application of non-mechanical energy to a coupling element.",
"63.A medicament dispenser according to claim 1, wherein the dispenser is actuable by the application of mechanical energy to a coupling element.",
"64.A medicament dispenser according to claim 62, wherein the coupling element is one or more shape memory alloy wires.",
"65.A medicament dispenser according to claim 1, wherein the dispenser is actuable by the application of non-mechanical energy to a drive means.",
"66.A medicament dispenser according to claim 1, wherein the dispenser is actuable by the application of mechanical energy to a drive means.",
"67.A medicament dispenser according to claim 1, wherein the medicament container contains a medicament selected from the group consisting of albuterol, salmeterol, flucticasone propionate, beclomethasone dipropionate and salts and solvates and any mixtures thereof.",
"68.A medicament dispenser according to claim 1, wherein the outlet comprises a mouthpiece for inhalation therethrough.",
"69.A medicament dispenser according to claim 1, wherein the dispenser additionally comprises a communicator for communication to enable transfer of data from the dose counter to an electronic data management system.",
"70.A medicament dispenser according to claim 69, wherein the data management system is a local data management system.",
"71.A medicament dispenser according to claim 69, wherein the dispenser further comprises a communicator for wireless communication with a gateway to a network computer system to enable transfer of data between the network computer and the electronic data management system.",
"72.The use of a medicament dispenser as claimed in claim 1, for dispensing medicament to a patient.",
"73.A method of dispensing medicament to a patient, said method comprising the use of a medicament dispenser as claimed in claim 1.74.A housing for receipt of a medicament container, the housing comprising: (i) an outlet for dispensing medicament therethrough; and (ii) an electronic dose counter associated with the outlet, wherein said dose counter includes a first sensor comprising an emitter and a detector for detecting a medicament release dispensible through said outlet, characterized in that the detector detects electromagnetic radiation in the infra red range.",
"75.A housing for receipt of a medicament container, the housing comprising: (i) an outlet for dispensing medicament therethrough; and (ii) a first and second strand of optical wave guide associated therewith, wherein said first and second strand of optical wave guide are associable with an electronic dose counter including a first sensor for detecting medicament release through said outlet."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Medicaments for treating respiratory disorders are frequently administered as dry powder formulations through the mouth and nose.",
"Dry powder inhalation devices, or inhalers, are used in the administration of these drugs, inhalation by the patient resulting in uptake of a specified dosage of medicament through the nose or mouth.",
"The drug may be stored as a dry powder within a reservoir in the body of the inhaler, a metering chamber being utilised to administer a specified dose of medicament.",
"Alternatively, more sophisticated inhalation devices employ medicament carriers, such as individual capsules or blister packs/strips containing defined doses of powdered drug.",
"It is also known to use for such therapy medicaments which are contained in an aerosol and are administered to a patient by means of an inhalation device.",
"The aerosol containers used in such inhalation devices are designed to deliver a predetermined dose of medicament upon each actuation and are known as Metered Dose Inhalers (MDIs); see Peter Byron, Respiratory Drug Delivery, CRC Press, Boca Raton, Fla. (1990) for a general background on this form of therapy.",
"Mechanical and electrical dose indicating devices, which typically count the number of doses delivered from or remaining in the medicament dispenser, are frequently used in dry powder inhalers and MDIs to enable patients to determine how much medicament is available in the dispenser for future use.",
"One problem associated with both mechanical and electrical dose counters is that they may, on occasions, give false readings.",
"Thus the electronic dose counter disclosed in U.S. Pat.",
"No.",
"5,020,527 employs a mechanical trigger, which may be actuated without release of the medicament, thereby giving a false reading.",
"Other forms of triggers are also known, particularly the use of sensing means to detect actuation of the dispenser.",
"U.S. Pat.",
"No.",
"5,544,647 discloses a range of sensors which may be used to detect and record actuation of MDIs.",
"Typical sensors include pressure switches which are sensitive to either physical contact with a movable element of the device or to the inward breath of the patient.",
"Other examples include sensors which are responsive to light, such as reflected or emitted light, or optical sensors which recognise reference points on the container or housing.",
"These sensors are designed to detect movement of the aerosol container following actuation of the device by being aligned to a reference point or a light source, such as a LED.",
"Sensors which are responsive to electromagnetic radiation, such as fluctuations in electromagnetic fields caused by movement of the container, are also disclosed within this document.",
"WO 95/07724 describes a dry powder inhaler with an electronic dose counter which employs a series of sensors to validate actuation of the inhaler.",
"The inhaler uses magnetically responsive proximity reed switches to detect medicament loading within the device, and a thermistor sensor to detect temperature changes due to inhalation by the patient.",
"A general problem encountered with dose counters in the art, such as those described above, is that they are dependent upon sensors which do not directly detect the medicament release from the dispenser.",
"All of the sensors used in the art are arranged to sense some feature associated with actuation of the device, such as movement of the container/housing or pressure/temperature changes due to inhalation.",
"This indirect method of sensing medicament release can lead to false readings being registered on the dose counter, if for example the sensor is activated without release of medicament.",
"Accidental activation or triggering of the sensor may, for example, result from partial movement of the container or from interference with light received by the sensor.",
"Similarly, a blockage or only partial release of the drug from either an MDI or dry powder inhaler would lead to a false reading registering on the dose counter."
],
[
"<SOH> SUMMARY OF INVENTION <EOH>It is an object of the present invention to provide direct sensing means by which release of a medicament can be detected and recorded on a dose counter.",
"It is a further object of the present invention to provide a means of confirming or validating medicament release from a medicament dispenser."
],
[
"This invention relates to a medicament dispenser, particularly dry powder and metered dose inhalation devices, which have dose counters attached thereto.",
"In particular, the invention relates to sensors used with dose counters to detect and validate release of medicament from the dispenser.",
"BACKGROUND OF THE INVENTION Medicaments for treating respiratory disorders are frequently administered as dry powder formulations through the mouth and nose.",
"Dry powder inhalation devices, or inhalers, are used in the administration of these drugs, inhalation by the patient resulting in uptake of a specified dosage of medicament through the nose or mouth.",
"The drug may be stored as a dry powder within a reservoir in the body of the inhaler, a metering chamber being utilised to administer a specified dose of medicament.",
"Alternatively, more sophisticated inhalation devices employ medicament carriers, such as individual capsules or blister packs/strips containing defined doses of powdered drug.",
"It is also known to use for such therapy medicaments which are contained in an aerosol and are administered to a patient by means of an inhalation device.",
"The aerosol containers used in such inhalation devices are designed to deliver a predetermined dose of medicament upon each actuation and are known as Metered Dose Inhalers (MDIs); see Peter Byron, Respiratory Drug Delivery, CRC Press, Boca Raton, Fla. (1990) for a general background on this form of therapy.",
"Mechanical and electrical dose indicating devices, which typically count the number of doses delivered from or remaining in the medicament dispenser, are frequently used in dry powder inhalers and MDIs to enable patients to determine how much medicament is available in the dispenser for future use.",
"One problem associated with both mechanical and electrical dose counters is that they may, on occasions, give false readings.",
"Thus the electronic dose counter disclosed in U.S. Pat.",
"No.",
"5,020,527 employs a mechanical trigger, which may be actuated without release of the medicament, thereby giving a false reading.",
"Other forms of triggers are also known, particularly the use of sensing means to detect actuation of the dispenser.",
"U.S. Pat.",
"No.",
"5,544,647 discloses a range of sensors which may be used to detect and record actuation of MDIs.",
"Typical sensors include pressure switches which are sensitive to either physical contact with a movable element of the device or to the inward breath of the patient.",
"Other examples include sensors which are responsive to light, such as reflected or emitted light, or optical sensors which recognise reference points on the container or housing.",
"These sensors are designed to detect movement of the aerosol container following actuation of the device by being aligned to a reference point or a light source, such as a LED.",
"Sensors which are responsive to electromagnetic radiation, such as fluctuations in electromagnetic fields caused by movement of the container, are also disclosed within this document.",
"WO 95/07724 describes a dry powder inhaler with an electronic dose counter which employs a series of sensors to validate actuation of the inhaler.",
"The inhaler uses magnetically responsive proximity reed switches to detect medicament loading within the device, and a thermistor sensor to detect temperature changes due to inhalation by the patient.",
"A general problem encountered with dose counters in the art, such as those described above, is that they are dependent upon sensors which do not directly detect the medicament release from the dispenser.",
"All of the sensors used in the art are arranged to sense some feature associated with actuation of the device, such as movement of the container/housing or pressure/temperature changes due to inhalation.",
"This indirect method of sensing medicament release can lead to false readings being registered on the dose counter, if for example the sensor is activated without release of medicament.",
"Accidental activation or triggering of the sensor may, for example, result from partial movement of the container or from interference with light received by the sensor.",
"Similarly, a blockage or only partial release of the drug from either an MDI or dry powder inhaler would lead to a false reading registering on the dose counter.",
"SUMMARY OF INVENTION It is an object of the present invention to provide direct sensing means by which release of a medicament can be detected and recorded on a dose counter.",
"It is a further object of the present invention to provide a means of confirming or validating medicament release from a medicament dispenser.",
"DETAILED DESCRIPTION OF INVENTION According to the present invention there is provided a medicament dispenser comprising: (i) a housing having an outlet; (ii) a medicament container locatable within said housing; (iii) an electronic dose counter associated with said outlet; wherein said dose counter comprises a first sensor for directly detecting a medicament release dispensible from said medicament container through said outlet.",
"In one aspect, the first sensor comprises an emitter and a detector.",
"In a second aspect, the sensor comprises only a detector, for example a pyroelectric detector which responds to a decrease in temperature.",
"Suitably, the emitter emits electro magnetic radiation and the detector detects the electromagnetic radiation.",
"The electromagnetic radiation emitted from the emitter may be infrared, visible or ultraviolet radiation.",
"Suitably, radiation in the range 0.95 μm to 0.35 μm is used.",
"More suitably, the radiation is in the infrared range.",
"In particular, infrared radiation with a wavelength of 0.88 μm has been found to be useful.",
"Suitably, the emitter is selected from the group consisting of light emitting diode, laser, incandescent lamp, electroluminescent and fluorescent light sources.",
"Suitably, the emitter emits infra red radiation.",
"In one embodiment, the emitter may include a filter, suitably an optical filter and preferably a polarising filter (particularly if the emitter is an incandescent source) in order to select a particular wavelength, a narrow range or ranges of wavelength.",
"Several advantages may be obtained by selecting a particular wavelength or range/ranges of wavelength, for example a given range of wavelengths may be especially sensitive to a particular drug/propellant combination.",
"Alternatively, one ‘sensitive’ range and one ‘insensitive’ range may be selected—in this case the ratio of the two or more wavelengths reaching the detector would be used to detect the drug, thus making the sensor less prone to errors caused by an overall reduction in intensity due to contamination of the optical path.",
"Suitably, the detector is selected from the group consisting of photodiode, phototransistor, light-dependent resistor and bolometer.",
"Preferably, the detector detects infra red radiation.",
"In one embodiment, the detector additionally comprises a filter, suitably an optical filter and preferably a polarising filter.",
"The use of a filter will enable the wavelength/wavelengths detectable by the detector to be pre-determined giving advantages similar to those described for using a filter with the emitter, for example, the detector could be made sensitive only to the wavelengths chosen for the emitter so the detector could be less sensitive to extraneous light sources, such as room light/sun light.",
"In a further embodiment, the detector is associated with an amplifier, since the output from the detector can be very small (of the order of micro Amps).",
"Suitably, the amplifier is positioned as closed to the detector as possible to avoid amplifying any extraneous noise e.g.",
"any electrical noise picked up in the connecting wires.",
"In one particular embodiment, the amplifier is integrated with the detector, for example the detector and amplifier are positioned on the same integrated circuit or “chip”.",
"The detector may detect either an increase or decrease in radiation, compared to the amount of radiation emitted by the emitter.",
"The increase or decrease may be due to interference of radiation reaching the detector by the medicament release.",
"In one aspect, the interference is due to absorption of radiation by the medicament release.",
"In another aspect, the interference is due to scattering of radiation by the medicament release.",
"In a further aspect, the interference is due to reflection of radiation by the medicament release.",
"In a yet further aspect, the interference is due to refraction of radiation by the medicament release.",
"In a still further aspect, the interference is due to diffraction of radiation by the medicament release.",
"In one aspect, the interference results in a decrease in the amount of radiation reaching the detector, for example due to absorption, scattering, refraction or diffraction, resulting in a decrease in the output signal.",
"Alternatively, the amount of radiation reaching the detector may be maintained at a constant level by increasing the input level to the emitter.",
"For example, an electronic feedback circuit that increases the current flowing through the emitter in order to maintain a constant flux at the detector may be used, resulting in an increase in the current supplied to the emitter as the medicament is released.",
"In a second aspect, the interference results in an increase in the amount of radiation reaching the detector, for example due to reflection by the medicament release, resulting in an increase in the output signal.",
"Alternatively, the amount of radiation reaching the detector may be maintained at a constant level by decreasing the input level to the emitter.",
"For example, an electronic feedback circuit that decreases the current flowing through the emitter in order to maintain a constant flux at the detector may be used, resulting in a decrease in the current supplied to the emitter as the medicament is released.",
"In one aspect, the emitter emits radiation of more than one wavelength and the detector detects radiation of more than one wavelength.",
"Preferably, the first sensor can quantify the concentration of medicament within the medicament release by measuring radiation at more than one wavelength.",
"These data can be processed, for example by a microprocessor, and compared against standardised data for a specified medicament to determine the concentration in the emission.",
"For example, a first wavelength is used as a control to calibrate the system response.",
"Suitably, this wavelength is not affected by the medicament release.",
"A second wavelength is affected by the medicament release, for example due to interference of the radiation by the medicament release.",
"The ratio of the amount of radiation of the first wavelength to the amount of radiation of the second wavelength arriving at the detector will depend on the concentration of medicament in the medicament release.",
"In another aspect, the medicament dispenser additionally comprises a second sensor (suitably having an emitter and a detector) for detecting a medicament release.",
"Suitably, the second sensor is positioned such that the medicament release passes the second sensor subsequent to passing the first sensor.",
"The presence of a second sensor may be used to increase confidence in the detection of the medicament release, for example for a detection to be considered valid, both sensors must be triggered.",
"For example, a single sensor may be “triggered” by a foreign body interrupting the radiation path, but in this case the second sensor would not be “triggered”; thus the detection would not be considered valid and a dose not shown as given.",
"Furthermore, the time lapse between triggering of the first sensor and triggering of the second sensor may be used to determine whether a detection is valid, i.e.",
"the second sensor must be triggered within a specified time of the triggering of the first sensor.",
"In a further aspect, the medicament dispenser additionally comprises a third sensor.",
"Suitably, the third sensor is sensitive to parameters selected from the group consisting of electro magnetic radiation, magnetic field, light, motion, temperature, pressure, sound, oxygen concentration, carbon dioxide concentration and moisture.",
"Preferably the third sensor responds to actuation of the dispenser.",
"In one aspect, the first and/or second sensor is integral with the outlet, for example moulded into the outlet or is attached thereto.",
"In a second aspect, the first and/or second sensor is reversibly attachable to the outlet and may be transferred from one outlet to another.",
"In another aspect, the third sensor is integral with the housing, for example moulded into the housing or attached hereto.",
"Alternatively, the third sensor is reversible attachable to the housing.",
"The dose counter is associated electronically with the sensor(s), such that when the detector detects medicament release in the outlet, a signal is sent to the dose counter to record that a dose has been dispensed.",
"In one aspect, the dose counter comprises a microprocessor.",
"Suitably, the microprocessor performs operations on the data from the first sensor and produces a signal output relating to the data or the outcome of an operation on the data.",
"Preferably, the microprocessor performs operations on the data from the second or third sensor and produces a signal output relating to the data or the outcome of an operation on the data.",
"More preferably, the data from the second and/or third sensor is processed by the microprocessor to validate data from the first sensor.",
"In one aspect, the dose counter additionally comprises a visual display unit for display of the data.",
"Preferably, the visual display unit displays the number of doses of medicament used or remaining within the container.",
"Preferably the doses are displayed numerically, by a series of coloured lights or by a monochrome bargraph.",
"Suitably, the dose counter is reversibly attachable to the housing.",
"In one embodiment, the first and/or second sensors are located on the dose counter (either integral therewith or detachable therefrom).",
"In a further aspect, the medicament dispenser additionally comprises one or more optical wave guides located within the housing.",
"Suitably the one or more optical wave guide is composed of an organic polymeric or inorganic glass fibre material.",
"Suitably, the medicament dispenser comprises two optical wave guides per sensor, i.e.",
"one associated with the emitter and the other associated with the detector.",
"A first optical wave guide channels radiation from the emitter to the outlet and a second optical wave guide channels radiation from the outlet to the detector.",
"Alternatively, the medicament dispenser comprises one optical wave guide per sensor, which may be associated with either the emitter or the detector.",
"In an alternative embodiment the emitter and detector may be located on the same side of the outlet, the radiation emitted from the emitter being reflected back to the detector by a reflective surface on the opposite side of the outlet.",
"The emitter and detector may be separate components or may be integrated into a single component.",
"The reflective surface may be the surface of the outlet or may be an additional component attached thereto.",
"When the medicament dispenser is actuated, the medicament release causes a backscattering of the signal off the particles/droplets in the plume spray, resulting in a reduction in the signal received by the detector.",
"In another aspect, the sensor is controlled by a digital or computational semiconductor device.",
"Suitably, the digital or computational semiconductor device energises the sensor and associated electronic components to detect and respond to a medicament release every 10 to 100 ms. Preferably, the digital or computational semiconductor device energises the sensor and associated electronic components every 40 ms.",
"In one aspect, the sensor and associated electronic components are energised for 5 to 30 μs.",
"Preferably, the sensor and associated electronic components are energised for 15 μs.",
"In a further aspect, the digital or computational semiconductor device returns the sensor to low power mode after energising the sensor.",
"Preferably, the digital or computational semiconductor device returns to low power mode after the sensor has been de-energised.",
"In one aspect, the medicament container is an aerosol container.",
"Preferably, the aerosol container comprises a suspension of a medicament in a propellant; in one embodiment, the propellant comprises liquefied HFA134a, HFA-227, or carbon dioxide; in an alternative embodiment, the propellant comprises a mixture of one or more of liquefied HFA134a, HFA-227, or carbon dioxide.",
"Alternatively, the aerosol container comprises a solution of a medicament in a solvent.",
"In a further aspect, the medicament container is a dry-powder container.",
"Preferably the dry-powder container comprises medicament and optionally excipient in dry-powder form.",
"Medicament dispensers according to the invention may be actuated manually by the patient.",
"Alternatively, the dispenser is actuated on application of mechanical or non-mechanical energy to a coupling element, for example one or more shape memory alloy (SMA) wires, for example a medicament dispenser as disclosed in WO01/41849, which is incorporated herein in its entirety by reference.",
"Alternatively, the dispenser is actuated by the application of mechanical or non-mechanical energy to a drive means, for example as described in UK Patent Application No.",
"0114175.3, which is incorporated herein in its entirety by reference.",
"The sensors of the present invention provide significant advantages for medicament dispensers which are actuated by the application of non-mechanical energy to a SMA coupling element, such advantages including: (i) preventing overheating of the SMA wires by shutting off the power as soon as they have contracted (heating the wires beyond this point does not result in any further contraction but may damage their microstructure, (ii) reducing or eliminating damage to the SMA wires, by preventing the wires driving against a hard stop by switching off the power as soon as the drug is released, (iii) saving energy by reducing the SMA drive pulse to a minimum by shutting the power off as soon as the drug is released thus preventing the wires being heated for longer than necessary, and (iv) giving confirmation of device actuation.",
"For example, the control electronics could be configured to: (i) switch off the drive to the SMA wires as soon as the sensor detects the drug.",
"This would rely on the valve staying open long enough to allow complete release of the dose after power to wires was switched off; (ii) start a timer at the start of the medicament release and then switch off the power to the SMA wires at a pre-set time thereafter; (iii) wait until the sensor detects completion of medicament release and then switch off the power to the SMA wires; (iv) wait until the sensor detects a particular point in the drug profile (such as the peak) and then switch off the power to the SMA wires immediately or after a pre-set time.",
"Preferably, the medicament is selected from the group consisting of albuterol, salmeterol, fluticasone propionate, beclomethasone dipropionate, salts or solvates thereof and any mixtures thereof.",
"In one aspect, the outlet comprises a mouthpiece for inhalation therethrough.",
"In another aspect, the medicament dispenser additionally comprises a communicator for communication to enable transfer of data from the dose counter to an electronic data management system.",
"Preferably, data from the dose counter are transferable onto a local data management system.",
"In one aspect, the medicament dispenser additionally comprises a communicator for wireless communication with a gateway to a network computer system to enable transfer of data between the network computer system and the electronic data management system.",
"In another aspect of the present invention there is provided the use of a medical dispenser according to any of the preceding claims to dispense medicament to a patient.",
"In a further aspect of the present invention there is provided a housing for receipt of a medicament container, the housing comprising (i) an outlet for dispensing medicament therethrough; and (ii) an electronic dose counter associated therewith; wherein the dose counter comprises a sensor for directly detecting a medicament release dispensible through the outlet.",
"In another aspect of the present invention there is provided a housing for receipt of a medicament container, the housing comprising (i) an outlet for dispensing medicament therethrough; and a first and second strand of optical wave guide associated therewith.",
"In a yet further aspect of the invention, there is provided a dose counter comprising a sensor for detecting a medicament release.",
"In yet another aspect of the present invention there is provided a kit of parts comprising a housing according to the present invention and an electronic dose counter comprising a sensor for directly detecting a medicament release.",
"Energy may be conserved by a variety of means to enable the device to operate for longer on a given source of energy, such as a battery.",
"Energy conservation or saving methods have additional advantages in terms of reducing the size requirements of the power source (e.g.",
"battery) and thus the weight and portability of the inhalation device.",
"According to another aspect herein, a variety of energy saving methods are is available which generally involve reducing power consumption.",
"One such method is to use a clock or timer circuit to switch the power on and off at regular or predetermined intervals.",
"In another method the system can selectively switch on/off specific electronic devices, such as visual display units, electronic agitators or sensors, in order to power these devices only when they are required to perform a particular sequence of events.",
"Thus different electronic devices may be switched on and off at varying intervals and for varying periods under control of the system.",
"Sensors, for example, may be energised at predetermined intervals (e.g.",
"every 100 milliseconds) and for predetermined periods (e.g.",
"50 microseconds) thereby conserving power but being active long enough to detect specific stimuli, such as inhalation by the patient.",
"Similarly, emitter-detector pairs may be controlled such that emitters are activated to pulse stimuli (e.g.",
"electro magnetic radiation or sound) at predetermined intervals to appropriate detectors which are only energised to sense these stimuli at corresponding intervals.",
"The power sequencing system may also respond to a sensor, such as a motion or breath sensor, which is activated on use of the device.",
"Low power or “micropower” components should be used within the electronics where possible and if a high power device is required for a particular function this should be put into a low power standby mode or switched off when not required.",
"Similar considerations apply in the selection of transducers.",
"Operation at low voltage is desirable since power dissipation generally increases with voltage.",
"For low power digital applications complementary metal oxide semi-conductor (CMOS) devices are generally preferred and these may be specially selected by screening for low quiescent currents.",
"Clock speeds of processors and other logic circuits should be reduced to the minimum required for computational throughput as power consumption increases with frequency.",
"Supply voltages should also be kept at minimal values consistent with reliable operation because power dissipation in charging internal capacitance's during switching is proportional to the square of the voltage.",
"Where possible, supply voltages should be approximately the same throughout the circuit to prevent current flowing through input protection circuits.",
"Logic inputs should not be left floating and circuits should be arranged so that power consumption is minimised in the most usual logic output state.",
"Slow logic transitions are undesirable because they can result in relatively large class-A currents flowing.",
"Resistors may be incorporated in the power supply to individual devices in order to minimise current in the event of failure.",
"In some control applications, devices that switch between on and off states are preferred to those that allow analog (e.g.",
"linear) control because less power is dissipated in low resistance on states and low current off states.",
"Where linear components are used (e.g.",
"certain types of voltage regulators) then types with low quiescent currents should be selected.",
"In some circuit configurations it is preferable to use appropriate reactive components (i.e.",
"inductors and capacitors) to reduce power dissipation in resistive components.",
"According to another aspect herein, emitter-detector pairs, including those described in the present invention based upon the emission and detection of electro magnetic radiation, may be used as a means of detection in inhalation devices.",
"Other forms of emitter and detector pairs can also be used to sense movement within the inhalation device such as those based upon sound, temperature, and pressure.",
"Thus, for example, actuation of the device in the form of movement of the canister might be registered by interfering with a high frequency sound emanating from an emitter and sensed by an appropriate detector.",
"Similar detection methods could be based upon the emission and detection of a pressure wave (e.g.",
"in the form of an acoustic wave) or of a heat wave.",
"Such emitter-detector pairs can be arranged to detect movement within the inhalation device; typical examples include movement of the mouthpiece, loading, transport and preparation of medicament (e.g.",
"removal of cover strip in a blister pack prior to medicament release) and sample metering (e.g.",
"filling of metering chamber with medicament powder, aerosol or liquid).",
"The position and relative configuration of the emitter-detector pair could also be varied to optimise performance and sensitivity.",
"Thus, for example, the detector might be positioned diametrically opposite, or at any point within a 360° arc around, the emitter for receipt of the appropriate stimulus (e.g.",
"electro magnetic radiation, sound etc.).",
"Alternatively the detector might be attached to the canister, such that an appropriate stimulus was only received when the emitter-detector pair were aligned.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a cross-sectional perspective of a MDI having a single sensor.",
"FIG.",
"2 shows a cross-sectional perspective of a MDI having two sensors.",
"FIGS.",
"3a-d show a cross-sectional, side, front cross-sectional (along the line C-C of FIG.",
"3b) and rear perspective, respectively, of a MDI having two sensors.",
"FIGS.",
"4a & b show side and front-sectional perspective (along the line A-A of FIG.",
"4a) of a MDI having two sensors.",
"FIG.",
"5 is a schematic side perspective of a MDI comprising a detachable dose counter unit having two sensors.",
"FIGS.",
"6a and 6b show front and front-sectional perspective (along line B-B of FIG.",
"6a) of a MDI having a rotatable outlet (mouthpiece).",
"FIG.",
"7 shows a plan sketch view of an outlet having wherein a reflective surface is used to return direct the infra red radiation from the emitter back to the detector.",
"FIG.",
"8 shows a medicament dispenser and an associated system diagram.",
"DETAILED DESCRIPTION OF DRAWINGS For the sake of brevity, each drawing depicts a sensor comprising an emitter/detector pair which emit and are responsive to infra red radiation.",
"It should be understood that other forms of sensors, detailed herein, could equally be described in the drawings and used in the present invention.",
"Furthermore, the drawings could equally apply to a sensor comprising only of a detector and lacking the corresponding emitter.",
"As shown in the cross-sectional perspective of FIG.",
"1, the aerosol canister 20 is located in the housing 10 so that one end protrudes from its open top, the canister being positioned such that the neck 21 and valve ferrule 22 are enclosed within housing 10.Spacer ribs (not shown) may be provided inside the housing to hold the external surface of the canister 20 spaced from the internal surface of the housing 10.A support 30 is provided at the lower end of the housing 10 and has a passage 32 in which the valve stem 40 of the aerosol canister 20 can be located and supported.",
"A second passage 34 is provided in the support 30 and is directed towards the interior of the outlet 50.An emitter 60 is located on outlet 50.Emitter 60 emits an infra red beam (not shown) across outlet 50 onto a detector (not shown) attached to the other side of outlet 50.The emitter 60 may emit a continuous beam of infra red radiation or may emit a pulsed beam at varying intervals and for varying duration.",
"The emitter 60 is under the control of a microprocessor within dose counter unit 70.The detector (not shown) is responsive to change in infra red radiation and generates a signal readable by the microprocessor.",
"When the level of energy falls below a predetermined threshold for a predetermined period (calibrated to respond to an emission of medicament released from canister 20) the microprocessor updates the display on the visual display unit 75 indicating the doses used or remaining within the canister.",
"The detector is calibrated to respond to an emission of medicament released from canister 20.The signal results in a change in the display on the visual display unit 75 indicating the doses used or remaining within the canister.",
"Thus, when the parts are in the positions shown in FIG.",
"1, the protruding portion of the aerosol canister 20 can be depressed to move the canister relative to the valve stem 40 to open the valve and a dose of medicament contained in the canister 20 will be discharged as a medicament release through passage 34 and into the outlet 50 from which it can be inhaled by a patient.",
"One dose will be released from the aerosol canister each time it is fully depressed.",
"The medicament release will interfere with the beam of infra red radiation from emitter 60 resulting in a reduction in radiation reaching the detector.",
"The visual display unit 75 will be updated to display the number of doses used or remaining within the canister 20.This display may consist of a numerical read out, giving the precise number of doses used or remaining.",
"Alternatively, the display may be indicative of the number of doses used or remaining, based upon a series of coloured lights (e.g.",
"green, orange, red) representing an estimated range of the number of doses used or remaining.",
"FIG.",
"2 is another cross-sectional perspective showing a second embodiment of the invention.",
"Two emitters 160, 165 are attached to outlet 150 and emit infra red beams (not shown) onto their respective detectors (not shown) attached to the other side of outlet 150.The infra red beams may be emitted continuously or pulsed (e.g.",
"at 40 millisecond intervals for 15 microseconds) across outlet 150.A dose of medicament is dispensed from canister 120, by depressing and moving the canister relative to valve stem 140, to release a dose of medicament as a medicament release through passage 134 into outlet 150.The infra red beams emitted or pulsed from emitters 160 and 165 are broken by the emission from the canister 120, resulting in a reduction in radiation reaching their respective detectors (not shown).",
"The two electronic signals from the detectors are processed by a microprocessor within the dose counter unit 170.This processing serves both to register that a single dose of medicament has been dispensed from the canister 120 and to validate that the response of each detector is not due to an error (e.g.",
"resulting from dust or dirt in the outlet or on the detector surface).",
"On validation by the processor, the number of doses remaining or used within the canister 120 is displayed on the visual display unit 175.Another embodiment of the present invention is shown in FIGS.",
"3a-d.",
"The cross-sectional perspective diagram of FIG.",
"3a shows a medicament dispenser similar to that of the preceding Figures.",
"Two emitters 260, 267 are attached to the housing 210 and emit a continuous or pulsed beam of infra red energy onto two detectors (not shown) positioned opposite each emitter and attached to the housing 210.On depression of canister 220 a dose of medicament is dispensed as a medicament release through passage 234 into outlet 250.The medicament release breaks the infra red beam emanating from emitter 260 which is sensed by the detector.",
"The depression of the canister 220 also causes ferrule 222 to disrupt the infra red beam emitted from emitter 267, resulting in a reduction in radiation reaching the detector.",
"A microprocessor within the dose counter unit 270 processes the signal from each detector to validate whether or not a single dose of medicament has been dispensed from the canister 220, validation requiring appropriate signals from each detector.",
"If the processor validates that one dose has been emitted, the dose counter displays the number of doses remaining or used within the canister 220 on the visual display unit 275.FIG.",
"3b shows a side elevation of the medicament dispenser of FIG.",
"3a.",
"The dispenser comprises a housing 210 for receipt of aerosol container 220.Medicament within container 220 is dispensed through outlet 250 to the patient.",
"Two emitters 260, 267 are located on the side of the housing and emit infra red radiation across the device as described above.",
"The dose counter unit 270 and visual display unit 275 are attached to the housing.",
"FIG.",
"3c is a cross-sectional view along the line C-C of FIG.",
"3b.",
"The aerosol canister 220 is positioned within housing 210 such that the canister outlet (not shown) is received by support 230.When the dispenser is not in use, emitters 260 and 267 emit or pulse infra red radiation across the interior of the housing onto detectors 261 and 268, respectively.",
"The detectors are calibrated to respond to a reduction in infra red radiation.",
"On depression of canister 220 a dose of medicament in a propellant is released as a medicament release through passage 234 into outlet 250.The movement of ferrule 222 blocks the beam of infra red energy from emitter 267 and this blockage is sensed by detector 268 which sends a signal to the microprocessor within dose counter 270.Simultaneously, the release of medicament and propellant from passage 234 breaks the beam of infra red radiation from emitter 260 and results in detector 261 sending a signal to the microprocessor within the dose counter unit 270.Signals from both detectors 261 and 268 are processed by the microprocessor to validate whether a single dose of medicament has been dispensed.",
"Signal validation results in a change in the number of doses displayed by the visual display unit (not shown).",
"A rear perspective of the medicament dispenser of FIGS.",
"3a-c is shown in FIG.",
"3d.",
"The key feature illustrated in this view is the visual display unit 275 on dose counter unit 270 indicating the number of doses used or remaining within the container 220.FIGS.",
"4a and 4b show another embodiment of the invention.",
"The side perspective (FIG.",
"4a) illustrates a medicament dispenser comprising a housing 310 with outlet 350 and containing an aerosol canister 320.The dispenser has a dose counter 370 with visual display unit 375 for indicating the number of doses used or remaining within the canister 320.An infra red emitter 360 and detector 361 are positioned across the outlet 350 to detect a release of medicament in propellant from passage 334, as discussed above.",
"As can be seen from FIG.",
"4b, a cut away section along the line A-A of FIG.",
"4a, an inductive coil 380 carrying a low electrical current is located on support 330.The inductive coil forms part of an inductive displacement transducer such that depression of canister 320 disturbs the magnetic field created by the flow of current in coil 380.Actuation of the device can therefore be detected as a change in the oscillating frequency of the circuit by the dose counter unit 370.Validation of actuation of the device is achieved by the microprocessor within the dose counter unit 370 is processing signals from both the infra red detector 361 and coil 380.If the processor confirms that actuation and medicament release has occurred then the number of doses used or remaining within the canister 320 is changed accordingly on the visual display unit 375.FIG.",
"5 is a schematic diagram depicting a variant of the present invention.",
"The medicament dispenser of FIG.",
"5 comprises a detachable dose counter unit 470 with emitters 460, 467 and detectors (not shown) attached thereto.",
"The attachment of dose counter 470 to housing 410 is shown by arrows A in FIG.",
"5.The dispenser housing 410 is provided with light piping 490, 494 which is connectable at one end to the emitters 460, 467 on detachable dose counter unit 470.Another pair of light pipes (not shown) are attached to the housing 410 on the opposite side of the dispenser.",
"The other ends 491, 495 of the light piping 490, 494 fit through the body of the housing 410 and are positioned within the housing in a similar manner to the emitters/detectors of FIGS.",
"3a-d.",
"Thus two sets of light tubes are located on both sides of the dispenser and end on the internal surfaces of housing 410, being positioned on either side of the mouthpiece and below the container ferrule (not shown).",
"Infra red radiation emitted or pulsed from emitter 460 will be transmitted through light tube 490 across the interior of outlet 450 and channelled by the corresponding light tube on the opposite side of the mouthpiece to the detector (not shown) on dose counter unit 475.Similarly, infra red radiation from emitter 467 will be channelled through light tube 494 across the interior of the housing, below the ferrule (not shown), and transmitted by the corresponding light tube on the opposite side of the housing 410 to the detector (not shown).",
"On actuation of the device, the release of medicament from the canister 420 will break the beam of infra red radiation transmitted across the interior of outlet 450 from emitter 460.This will result in a reduction in radiation being channelled through the corresponding light pipe to the detector (not shown), on dose indicator 470, and an output signal from the detector.",
"Depression of the canister 420 will push the ferrule (not shown) across the path of the infra red light beam transmitted through and from light pipe 494.The reduction in energy being transferred through the corresponding light pipe on the opposite side of the housing 410 to the detector (not shown) will result in an output signal.",
"The microprocessor within the dose unit 470 will validate these signals to ensure that one signal is not due to an artefact.",
"If satisfied that a dose of medicament has been dispensed, the microprocessor will indicate a change in the number of doses used or remaining within the container 420.FIG.",
"6a shows a perspective view of a medicament dispenser of the invention and FIG.",
"6b shows a cross-sectional cut away view along the line B-B of FIG.",
"6a.",
"The dispenser comprises a housing 510 for receipt of aerosol container 520.Medicament within container 520 is dispensed to the patient through outlet 550 rotatably attached to housing 510.An infra red emitter 560 and detector 561 with connecting wires attached are located on housing 510.Light piping 590, 594 is connectable at one end to emitter 560 and detector 561 respectively.",
"The other ends 591, 595 of light piping 590, 594 fits through the body of the housing 510 and are positioned within the housing in a similar manner to the emitter/detector in FIG.",
"3a-3d.",
"Infra red radiation emitted or pulsed from emitter 560 will be transmitted through light tube 590 across the interior of outlet 550 and channelled by the corresponding light tube 594 on the opposite side of the mouthpiece to the detector 561.On actuation of the device, the release of medicament from the canister 520 will break the beam of infra red radiation transmitted across the interior of outlet 550 from emitter 560.This will result in a reduction in radiation being channelled through the corresponding light pipe 594 to the detector 561 and an output signal from the detector.",
"The reduction in energy being transferred through the corresponding light pipe 594 on the opposite side of the housing 510 to the detector 561 will result in an output signal.",
"The microprocessor within the dose counter (not shown) will validate this signal and if satisfied that a dose of medicament has been dispensed, the microprocessor will indicate a change in the number of doses used or remaining within the container 520.FIG.",
"7a shows a side perspective of a medicament dispenser of the invention comprising housing 610 with outlet 650 and containing an aerosol canister 620.The dispenser has a dose counter 670 with visual display unit 675 for indicating the number of doses used or remaining within the canister 620.FIG.",
"7b and 7c are cross-sectional views along line D-D. A sensor component 665 comprising emitter and detector is located on the housing 610 above outlet 650 and has a clear optical path provided by hole 666 located on the centre line on the upper surface of the mouthpiece.",
"The lower surface of outlet 650 is provided with a reflective surface 655.The infra red signal emitted from sensor 665 is reflected off the reflective surface 655 back to the detector part of sensor 665.During actuation of the device, a medicament release is released from passage 634.The particles/droplets in the medicament release cause a scattering of the infra red beam (FIG.",
"7c) thus causing a reduction of the signal reaching the detector portion of sensor 665.The microprocessor within the dose counter 670 will validate this signal and if satisfied that a dose of medicament has been dispensed, the microprocessor will indicate a change in the number of doses used or remaining within the container 620.FIG.",
"8 shows a schematic representation of a breath-operable medicament dispensing system.",
"The system comprises a metered dose inhaler similar to that shown in more detail in FIG.",
"1 comprising tubular housing 710 having a dispensing outlet 750 in the form of a mouthpiece.",
"Within the housing 710 sits aerosol container 720 which has a valve.",
"Valve stem 740 is supported by valve support 730.Outlet passage 734 is provided in the support 730 to enable passage of dispensed dose to the dispensing outlet 750.An emitter 760 is located in outlet 750.An emitter (not shown) emits an infra red beam (not shown) across outlet 750 on to a detector 761 attached to the other side of outlet 750.It may be seen that the upper part of the aerosol container 720 abuts container seat 780.The container seat 780 comprises an insulating portion 781 which directly contacts the aerosol container 720 and an upper conducting portion 782 (e.g.",
"comprised of aluminium).",
"It may also be seen that the valve support 730 connects with conducting valve seat 736.Plural shape memory alloy wires 799a, 799b connect the conducting portion 782 of the container seat 780 to the conducting valve seat 736.The plural wires 799a, 799b comprise a nickel-titanium alloy which contracts in response to electrical current flow therethrough.",
"It may thus, be appreciated that on passage of electrical current through the plural wires 799a, 799b the container seat 780 and valve seat 736 will be drawn towards each other as the wires 799a, 799b contract.",
"Actuation of the valve will result and a medicament release dispenser through outlet 750.The medicament release will interfere with the beam of infra red radiation from emitter (not shown) resulting in a reduction in radiation reaching the detector 761.Control of electrical current flow to the container seat 780, valve seat 736 and wires 799a, 799b is achievable using the illustrated circuitry.",
"Container seat 780 and valve seat 736 connect to actuation circuit 7100 which includes a power supply 7110 (e.g.",
"a voltaic cell or battery of voltaic cells) and a switch 7115 in the form of a solid state switching device.",
"The switch 7115 itself connects to control circuitry including micro-controller 7120 which has an analogue and digital interface.",
"The power supply for the control circuitry is taken from the power supply 7110 for the wires 799a, 799b after suitable regulation and filtering 7112.The micro-controller 7120 itself connects with pressure transducer 7130 which has an input in the form of a pressure tube 7132 located within the dispensing outlet 750 of the inhaler housing 710.It may be appreciated that current flow to the container seat 780, valve seat 736 and wires 799a, 799b, and hence actuation of the dispenser may be achievable as follows.",
"The patient inhales through the outlet 750 resulting in a change in pressure within the housing 710 and pressure tube 7132.The change in pressure is detected by the pressure transducer 7130 which sends a signal to the micro-controller 7120.The micro-controller 7120, in turn sends a switching signal to the solid state switching device 7115 which results in closing of the actuation circuit and electrical current flow therethrough.",
"The resulting contraction of the shape memory alloy wires 799a, 799b causes actuation of the dispenser and hence, dispensing of medicament to the inhaling patient.",
"Interference of the infra red beam emitted by the emitter (not shown) is detected by the detector 761 and a signal sent to the micro-controller 7120 which can be configured to carry out one or more tasks.",
"For example it may be configured to switch off the actuator circuit as soon as the medicament has been dispensed and to display an error message if the medicament is not dispensed.",
"It may also be seen in FIG.",
"8 that the micro-controller 7120 is connected to a display 7140 for display of information to the patient and also with a computer interface 7142 for exchange of data therewith.",
"Communication with the computer interface 7142 may be via a wired, optical or radio communications link.",
"The micro-controller 7120 is also connected to shake detector 7144 for use in detecting whether the container 720 is shaken prior to actuation of the dispenser and to a clock-calendar module 7146 including a temperature sensor.",
"All circuitry and components thereof including the power supply 7110, display 7140, shake detector 7144, computer interface 7142 and clock-calendar module 7146 may be arranged to be present on the housing 710 such that the system is in the form of a discrete, hand-held device.",
"Whilst the present invention has been described in detail in respect of a metered dose inhaler it will be appreciated that identical sensors could be attached to a dry powder inhalation device in a similar fashion.",
"It may be appreciated that any of the parts of the dispenser which contact the chemical suspension may be coated with materials such as fluoropolymer materials which reduce the tendency of chemical to adhere thereto.",
"Any movable parts may also have coatings applied thereto which enhance their desired movement characteristics.",
"Frictional coatings may therefore be applied to enhance frictional contact and lubricants used to reduce frictional contact as necessary.",
"The medicament dispenser of the invention is suitable for dispensing medicament, particularly for the treatment of respiratory disorders such as asthma and chronic obstructive pulmonary disease.",
"Appropriate medicaments may thus be selected from, for example, analgesics, e.g., codeine, dihydromorphine, ergotamine, fentanyl or morphine; anginal preparations, e.g., diltiazem; antiallergics, e.g., cromoglycate (e.g.",
"s the sodium salt), ketotifen or nedocromil (e.g.",
"as the sodium salt); antiinfectives e.g., cephalosporins, penicillins, streptomycin, sulphonamides, tetracyclines and pentamidine; antihistamines, e.g., methapyrilene; anti-inflammatories, e.g., beclomethasone (e.g.",
"as the dipropionate ester), fluticasone (e.g.",
"as the propionate ester), flunisolide, budesonide, rofleponide, mometasone e.g.",
"as the furoate ester), ciclesonide, triamcinolone (e.g.",
"as the acetonide) or 6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioic acid S-(2-oxo-tetrahydro-furan-3-yl) ester; antitussives, e.g., noscapine; bronchodilators, e.g., albuterol (e.g.",
"as free base or sulphate), salmeterol (e.g.",
"as xinafoate), ephedrine, adrenaline, fenoterol (e.g.",
"as hydrobromide), formoterol (e.g.",
"as fumarate), isoprenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol (e.g.",
"as acetate), reproterol (e.g.",
"as hydrochloride), rimiterol, terbutaline (e.g.",
"as sulphate), isoetharine, tulobuterol or 4-hydroxy-7-[2-[[2-[[3-(2-phenylethoxy)propyl]sulphonyl]ethyl]amino]ethyl-2(3H)-benzothiazolone; adenosine 2a agonists, e.g.",
"2R,3R,4S,5R)-2-[6-Amino-2-(1S-hydroxymethyl-2-phenyl-ethylamino)-purin-9-yl]-5-(2-ethyl-2H-tetrazol-5-yl)-tetrahydro-furan-3,4-diol (e.g.",
"as maleate); α4 integrin inhibitors e.g.",
"(2S)-3-[4-({[4-(aminocarbonyl)-1-piperidinyl]carbonyl}oxy)phenyl]-2-[((2S)-4-methyl-2-{[2-(2-methylphenoxy)acetyl]amino}pentanoyl)amino]propanoic acid (e.g.",
"as free acid or potassium salt), diuretics, e.g., amiloride; anticholinergics, e.g., ipratropium (e.g.",
"as bromide), tiotropium, atropine or oxitropium; hormones, e.g., cortisone, hydrocortisone or prednisolone; xanthines, e.g., aminophylline, choline theophyllinate, lysine theophyllinate or theophylline; therapeutic proteins and peptides, e.g., insulin or glucagon; vaccines, diagnostics and gene therapies.",
"It will be clear to a person skilled in the art that, where appropriate, the medicaments may be used in the form of salts, (e.g., as alkali metal or amine salts or as acid addition salts) or as esters (e.g., lower alkyl esters) or as solvates (e.g., hydrates) to optimise the activity and/or stability of the medicament and/or to minimise the solubility of the medicament in the propellant.",
"Preferred medicaments are selected from albuterol, salmeterol, fluticasone propionate and beclomethasone dipropionate and salts or solvates thereof, e.g., the sulphate of albuterol and the xinafoate of salmeterol.",
"Medicaments can also be delivered in combinations.",
"Preferred formulations containing combinations of active ingredients contain salbutamol (e.g., as the free base or the sulphate salt) or salmeterol (e.g., as the xinafoate salt) or formoterol (e.g.",
"as the fumarate salt) in combination with an antiinflammatory steroid such as a beclomethasone ester (e.g., the dipropionate) or a fluticasone ester (e.g., the propionate) or budesonide.",
"A particularly preferred combination is a combination of fluticasone propionate and salmeterol, or a salt thereof (particularly the xinafoate salt).",
"A further combination of particular interest is budesonide and formoterol (e.g.",
"as the fumarate salt).",
"It will be understood that the present disclosure is for the purpose of illustration only and the invention extends to modifications, variations and improvements thereto."
]
] | Patent_10415279 |
[
[
"Carpet stretcher",
"A sliding hammer apparatus which is attachable to a gripper head part of a carpet stretching device (10), the apparatus having a body portion (14) having an elongate substantially horizontal member (15) which has a front end and a rear end, a hand operable sliding hammer (16) slidably attached to the member, a strike face (17) which is adapted to be struck by the sliding hammer (16) and attachment means to attach the apparatus to a gripper head (12) part of a carpet stretcher."
],
[
"1.A sliding hammer apparatus which is attachable to a gripper head part of a carpet stretching device, the gripper head having a stem, the apparatus having a body portion which has a front end and a rear end, a first member extending upwardly from the body portion and adjacent the front end, a second member extending upwardly from the body portion and adjacent the rear end, a elongate substantially horizontal guide member which extends between the first member and the second member, and adjacent upper ends of the first member and the second member, and which has a front end and a rear end, a hand operable sliding hammer slidably attached to the elongate member, a strike face adjacent the front end of the elongate member, the strike face comprising part of the first member and which is adapted to be struck by the sliding hammer, and attachment means to attach the apparatus to the stem of a gripper head part of a carpet stretcher.",
"2.The apparatus according to claim 1, wherein the hand operable sliding hammer is weighted.",
"3.The apparatus according to claim 2, wherein the substantially horizontal guide member is releasably attached to the body portion of the device, to allow replacement of the sliding hammer with a sliding hammer of the same weight a different weight or the body portion.",
"4.The apparatus according to claim 3, further comprising side attachment, releasably attached to the body portion of the apparatus, adapted to prevent the carpet stretcher from deviating or to be struck using the knee in the manner of a conventional carpet stretching device of the knee kicking type.",
"5.The apparatus according to claim 4, wherein the side attachment is padded.",
"6.The apparatus of claim 1 containing the gripper head part.",
"7.The apparatus according to claim 6, wherein the gripper head part is releasably attached using attachment means to the body portion.",
"8.The apparatus according to claim 7, wherein the attachment means used to attach the grip the head part to the body portion is a pin and slot arrangement.",
"9.The apparatus according to claim 6, wherein the body portion is as guide means to facilitate the attachment of a gripper head portion to the body portion.",
"10.The apparatus of either claim 8, wherein the sliding hammer is adapted to facilitate hand gripping or have a grip enhancing profile."
],
[
"<SOH> BACKGROUND ART <EOH>The most popular carpet stretching tools used are the manual knee kicker stretcher, the lever action stretcher, and a powered stretcher.",
"The invention can be seen as an improvement on the manual knee kicker stretcher.",
"Carpet stretchers are very well known and are used to stretch a carpet towards the perimeter of the floor to eliminate creases and bulges, and to tension the carpet over the smooth edge which is a strip of timber with protruding spikes or nails, and which is attached to the floor around the base of the walls.",
"The carpet is stretched over the spikes which holds the carpet in place.",
"The most popular unit used to stretch the carpet is a knee kicker.",
"The knee kicker is typically formed from two main parts.",
"The first part is the gripper head.",
"The gripper head typically has a rectangular head portion having a bottom face containing protruding spikes which bite into the carpet.",
"A shank or shaft extends behind the gripper head which may be tubular, or channel shaped.",
"The second part of the knee kicker contains a shank or shaft which makes with the shank or shaft on the gripper head.",
"A soft thick pad is fitted to the rear part of the shank or shaft, and the pad is hit by the knee of the carpet layer to cause the gripper head to move forwardly and therefore to stretch or tension the carpet.",
"The knee kicker needs to be kicked by the knee of the carpet layer while the carpet layer is in a kneeling position.",
"This action causes impact which results in serious knee, hip, leg and spinal injuries.",
"Another disadvantage with this type of device is that the carpet layer must be positioned behind or next to the knee kicker.",
"This means that the device cannot be easily used in very confined spaces such as in cupboards or underneath overhangs.",
"Often, the carpet is only partially stretched or is not stretched at all in these confined spaces which results in creases or bulges forming in the carpet over time.",
"Attempts have been made to improve the efficiency of knee kickers.",
"For instance, it is known to provide a knee kicker where a sliding weight slides along the shaft or shank which extends between the gripper head and the knee pad.",
"When the carpet layer kicks the knee pad, the weight is propelled towards the gripper head and it is hoped that this provides some intensification of the force of the blow to the gripper head.",
"In practice, this arrangement increases the weight of the kicker, which is undesirable, and provides no advantage to the problems with confined spaces.",
"Also, the device still needs to be kicked by the persons knee which provides no solution to the injury problem.",
"Another known type of carpet stretcher has a gripper head which is more or less conventional.",
"Behind the gripper head is a rather long tubular member.",
"A long rod slides within the tubular member and is weighted.",
"The rear end of the rod has a hand grip.",
"In practice, the rod is retracted to be almost out of the tubular member, and is then forced back through the tubular member by pushing hard on the hand grip.",
"The front end of the rod extends through the tubular member and strikes a stop in the tubular member.",
"It is hoped that this provides a decent forward force to the gripper head.",
"This arrangement has the advantage that it does not need to be kicked by the persons knee.",
"However, the plunger and rod type assembly is rather long (almost 1 m when in the retracted position) which makes it difficult for use in confined spaces.",
"Also, the rather long stroke of the device when the rod is retracted and pushed forwardly through the tubular member, makes it difficult to provide a quick series of blows to stretch the carpet.",
"The device also has the disadvantage that it is, not very versatile and can only be used as a hand carpet stretcher.",
"Occasionally, there are still advantages in being able to use a knee kicker, and this device does not allow it to be used as a knee kicker when required.",
"Carpet stretchers are known which do not require kicking.",
"For instance, carpet stretchers using a lever action are known, but these stretchers are rather slow and require the device to be pinned to the floor to prevent it from moving backwards as the lever forces the carpet forwards.",
"These devices are also completely unsuitable for use in confined spaces.",
"Powered devices are known and these do not require knee kicking.",
"However such powered devices are expensive and require a source of power which is not always readily available.",
"The devices can be quite large and unsuitable for use in confined spaces."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>An embodiment of the invention will be described with reference to the following drawings in which: FIG.",
"1 illustrates a front perspective view of the carpet stretcher.",
"FIG.",
"2 illustrates a rear perspective view of the carpet stretcher.",
"FIG.",
"3 illustrates a side view of the carpet stretcher.",
"FIG.",
"4 illustrates a top view of the carpet stretcher.",
"FIG.",
"5 illustrates a front view of the carpet stretcher.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF THE INVENTION This invention is directed to a carpet stretcher which does not require knee kicking, and instead uses a hand operated sliding hammer to stretch the carpet.",
"The stretcher can fit to a conventional gripper head, or can be purpose-built.",
"BACKGROUND ART The most popular carpet stretching tools used are the manual knee kicker stretcher, the lever action stretcher, and a powered stretcher.",
"The invention can be seen as an improvement on the manual knee kicker stretcher.",
"Carpet stretchers are very well known and are used to stretch a carpet towards the perimeter of the floor to eliminate creases and bulges, and to tension the carpet over the smooth edge which is a strip of timber with protruding spikes or nails, and which is attached to the floor around the base of the walls.",
"The carpet is stretched over the spikes which holds the carpet in place.",
"The most popular unit used to stretch the carpet is a knee kicker.",
"The knee kicker is typically formed from two main parts.",
"The first part is the gripper head.",
"The gripper head typically has a rectangular head portion having a bottom face containing protruding spikes which bite into the carpet.",
"A shank or shaft extends behind the gripper head which may be tubular, or channel shaped.",
"The second part of the knee kicker contains a shank or shaft which makes with the shank or shaft on the gripper head.",
"A soft thick pad is fitted to the rear part of the shank or shaft, and the pad is hit by the knee of the carpet layer to cause the gripper head to move forwardly and therefore to stretch or tension the carpet.",
"The knee kicker needs to be kicked by the knee of the carpet layer while the carpet layer is in a kneeling position.",
"This action causes impact which results in serious knee, hip, leg and spinal injuries.",
"Another disadvantage with this type of device is that the carpet layer must be positioned behind or next to the knee kicker.",
"This means that the device cannot be easily used in very confined spaces such as in cupboards or underneath overhangs.",
"Often, the carpet is only partially stretched or is not stretched at all in these confined spaces which results in creases or bulges forming in the carpet over time.",
"Attempts have been made to improve the efficiency of knee kickers.",
"For instance, it is known to provide a knee kicker where a sliding weight slides along the shaft or shank which extends between the gripper head and the knee pad.",
"When the carpet layer kicks the knee pad, the weight is propelled towards the gripper head and it is hoped that this provides some intensification of the force of the blow to the gripper head.",
"In practice, this arrangement increases the weight of the kicker, which is undesirable, and provides no advantage to the problems with confined spaces.",
"Also, the device still needs to be kicked by the persons knee which provides no solution to the injury problem.",
"Another known type of carpet stretcher has a gripper head which is more or less conventional.",
"Behind the gripper head is a rather long tubular member.",
"A long rod slides within the tubular member and is weighted.",
"The rear end of the rod has a hand grip.",
"In practice, the rod is retracted to be almost out of the tubular member, and is then forced back through the tubular member by pushing hard on the hand grip.",
"The front end of the rod extends through the tubular member and strikes a stop in the tubular member.",
"It is hoped that this provides a decent forward force to the gripper head.",
"This arrangement has the advantage that it does not need to be kicked by the persons knee.",
"However, the plunger and rod type assembly is rather long (almost 1 m when in the retracted position) which makes it difficult for use in confined spaces.",
"Also, the rather long stroke of the device when the rod is retracted and pushed forwardly through the tubular member, makes it difficult to provide a quick series of blows to stretch the carpet.",
"The device also has the disadvantage that it is, not very versatile and can only be used as a hand carpet stretcher.",
"Occasionally, there are still advantages in being able to use a knee kicker, and this device does not allow it to be used as a knee kicker when required.",
"Carpet stretchers are known which do not require kicking.",
"For instance, carpet stretchers using a lever action are known, but these stretchers are rather slow and require the device to be pinned to the floor to prevent it from moving backwards as the lever forces the carpet forwards.",
"These devices are also completely unsuitable for use in confined spaces.",
"Powered devices are known and these do not require knee kicking.",
"However such powered devices are expensive and require a source of power which is not always readily available.",
"The devices can be quite large and unsuitable for use in confined spaces.",
"OBJECT OF THE INVENTION It is an object of the invention to provide a carpet stretcher which does not require knee kicking, does not require electrical or hydraulic power, does not have a lever action, and which may overcome at least some of the abovementioned disadvantages.",
"In one form, the invention resides in a carpet stretcher which comprises a gripper head and a body portion, the body portion having an elongate member which has a front end and a rear end, a hand operable sliding hammer slidably attached to the member, and a strike face which is adapted to be struck by the sliding hammer.",
"In another form, the invention resides in a sliding hammer apparatus which is attachable to a gripper head part of a carpet stretching device, the apparatus having a body portion having an elongate member which has a front end and a rear end, a hand operable sliding hammer slidably attached to the member, a strike face which is adapted to be struck by the sliding hammer, and attachment means to attach the apparatus to a gripper head part of a carpet stretcher.",
"The carpet stretcher of the invention can be used instead of the conventional knee kicking carpet stretching devices.",
"The size of the carpet stretcher may be similar to conventional knee kicking carpet stretching devices.",
"However, larger or smaller stretchers are envisaged.",
"The gripper head may be of a more or less conventional design, and gripper heads are well-known in the marketplace.",
"The gripper head is typically substantially rectangular or square shaped when viewed in plan and can have a length and width of between 5-20 cm.",
"The gripper head is typically formed of steel.",
"The gripper head typically has a bottom face which is provided with an array of spikes or teeth which bite into the carpet.",
"The length of the spikes or teeth is typically adjustable and can be adjusted by a dial which is on the top face of the gripper head.",
"The gripper head typically has a tapered front nose portion and a substantially abrupt rear portion.",
"This type of gripper head is well-known in the art.",
"Also known in the art is a gripper head having a rearwardly extending shaft or shank to form a first part which is typically called a “gripper head part”.",
"The body portion has an elongate member to support the sliding hammer.",
"The elongate member typically comprises a rod member.",
"The rod member is typically in a substantially horizontal position.",
"The rod member may be formed from steel or other suitable material.",
"The rod member is typically cylindrical, although it is possible for the rod member to have a square cross-section or other type of cross-section shape.",
"The rod member may have a cross-section size, or diameter if the rod member is cylindrical of between 5-20 mm.",
"The rod member may have a length sufficient to allow the sliding hammer to be used effectively.",
"The length will depend on the size of the carpet stretcher, and the size of the sliding hammer.",
"A typical length of the rod member can be between 20-50 cm.",
"It is envisaged that the elongate member may comprise a rail member, other types of guide members, a substantially flat or plate like member and the like, as long as it can support a sliding hammer for sliding movement along the elongate member The body portion may have two extending portions between which the rod member can locate.",
"Suitably, the body portion is plate like and has an upwardly extending rear end portion an upwardly extending front end portion between which the rod member can locate, and a connecting main body portion which connects the two end portions.",
"The body portion has a strike face which is adapted to be struck by the sliding hammer.",
"The strike face the may comprise the upwardly extending front end portion.",
"This front end portion may be formed from fairly thick material (typically metal) to allow it to function efficiently as a strike face without damage.",
"The sliding hammer is slidably attached to the rod member.",
"Typically, the sliding hammer comprises an elongate weighted member and is typically formed from metal.",
"The sliding hammer may be coupled to the rod member by various means.",
"In a preferred form, the sliding hammer has a longitudinal bore through which the rod member passes to sliding the attach the sliding hammer to the rod member.",
"The shape of the bore is typically the same shape of the rod.",
"For instance, the bore may be circular if the rod is cylindrical or may be slot like if the rod is an elongate plate or strip.",
"Other forms of attachment means are envisaged.",
"For instance, the attachment might be via a pin-in-slot arrangement, a stud-in-channel arrangement, and the like.",
"The sliding hammer may be profiled to facilitate hand gripping.",
"The sliding hammer may be substantially cylindrical and have a front face and a rear face.",
"The front face is typically planar and is adapted to strike the strike face of the body portion.",
"In order to minimise metal-metal contact, a washer or bush may be positioned on the front face.",
"The washer or bush may be formed from hard plastic material such as nylon or from other suitable materials.",
"The gripper head may be releasably attachable to the body portion or may be fixed to the body portion.",
"It is preferred that the gripper head is releasably attachable such that a conventional gripper head can be used.",
"Suitably, an attachment means is provided to releasably attach the body portion to the gripper head.",
"The attachment means may comprise fasteners such as screws, bolts, clips and the like.",
"If desired, the body portion may have guide means to facilitate attachment of the gripper head to the body portion.",
"The guide means may comprise an opening in the front end portion through which a shank or shaft of the gripper head may pass.",
"A side attachment may be provided to the body portion.",
"The side attachment may be releasably attachable to the body portion and can be attached to either side of the body portion.",
"If desired, a side attachment may be provided on each side of the body portion.",
"The side attachment can be gripped by a person's hand, or pushed up against by a person's knee or foot to hold the carpet stretcher in place and to prevent the carpet stretcher from moving back.",
"BRIEF DESCRIPTION OF THE DRAWINGS An embodiment of the invention will be described with reference to the following drawings in which: FIG.",
"1 illustrates a front perspective view of the carpet stretcher.",
"FIG.",
"2 illustrates a rear perspective view of the carpet stretcher.",
"FIG.",
"3 illustrates a side view of the carpet stretcher.",
"FIG.",
"4 illustrates a top view of the carpet stretcher.",
"FIG.",
"5 illustrates a front view of the carpet stretcher.",
"BEST MODE Referring to the figures, there is illustrated a carpet stretcher 10.The main components of the carpet stretcher are (1) a gripper head part 11 which is formed from a forward gripper head 12, and a rearwardly extending shank 13, (2) a body portion 14 which comprises a elongate member in the form of a rod 15, a sliding hammer 16, and a front strike face 17.Gripper head 12 is of conventional design and has a substantially block like rectangular configuration with a tapering front nose portion 18, and an abrupt rear portion 19.The gripper head 12 has a bottom face 20 which is formed with an array of extending spikes or teeth 21 which bite into the carpet.",
"A top face 22 has a dial 23 which can be turned to adjust the extent of the spikes or teeth 21 from bottom face 20.The rearwardly extending shank 13 is formed integrally with gripper head 12 to form a single unit.",
"The shank 13 has a C-shaped section which is best illustrated in FIG.",
"2.The gripper head and the shank together form a gripper head part 11.This part 11 is usually conventional and forms one part of the known two part knee kicker device.",
"This illustrates an advantage of the present invention which envisages use of a known gripper head part which means that carpet layers can use part of their existing knee kicker device.",
"The body portion 14 is substantially plate like and comprises a lower profiled but substantially planar connecting main body portion 24.A rear part of main body portion 24 is bent upwardly to form a rear end portion 25.A front part of main body portion 24 contains an upwardly extending front portion 26 which is quite thick as one face of it functions as strike face 17.Rod 15 is cylindrical and extends between rear end portion 25 and front end portion 26, and is supported by each end portion.",
"Rear end portion 25 has an opening 27 through which rod 15 can pass.",
"A lock plate 28 is attached to rod 15 and is locked in place with a fastener 29.Front end portion 26 is provided with a bore (not illustrated) in which the front end of rod 15 can pass.",
"The rod is fixed in the bore by any suitable means which may comprise threads, a fastener such as a grub screw, or a split pin.",
"A sliding hammer 16 slides over rod 15.Sliding hammer 16 is substantially cylindrical but has a profiled outer surface to facilitate hand gripping.",
"Sliding hammer 16 has a longitudinal bore to allow it to slide over rod 15.Sliding hammer 16 has a front end face 30.Attached to front end face 30 is a nylon bush 31 which prevents metal-metal contact as sliding hammer 16 is slammed against strike face 17 to provide a forward thrust to gripper head 12.Front end portion 26 has a guide means which is in the form of an aperture 32 through which shank 13 can pass.",
"Shank 13 is attached to portion 24 and is preferably releasably attached thereto.",
"This can be achieved by fasteners (not illustrated) which connect shank 13 to the body portion.",
"The arrangement may allow shank member 13 to be adjustable relative to the body portion to allow the degree of extension of gripper head 12 to vary.",
"A side attachment 34 is provided.",
"Side attachment 34 has a central steel pin 35 which extends into a bore (not illustrated) on body portion 14.A bore may be provided on each side of body portion 14 to allow the side attachment to be attached to either side of the carpet stretcher.",
"Side attachment 34 is profiled such that it can be gripped by a person's hand to keep a forward force on the carpet stretcher while sliding hammer 16 is being used by the persons other hand.",
"Alternatively, side attachment 34 can be pushed by a person's knee or foot.",
"It can be seen that the carpet stretcher can be operated by hand and that the forward thrust is provided by the hand operated sliding hammer 16.Therefore, knee kicking is no longer required.",
"The sliding hammer can be used quickly and efficiently and can deliver rapid blows to strike face 17.The carpet layer can also use sliding hammer 16 to deliver hard blows or soft blows to strike face 17 to provide a greater precision in the use of the device.",
"The carpet layer is not thrown off-balance as both knees can be used to support the carpet layer and a knee is not required to be repeatedly struck against the device.",
"The device can be used in confined areas as the forward thrust is provided by a hand operated sliding hammer and not by the knee of the carpet layer.",
"Another advantage of the invention is that the main body portion 14 can be removed from the gripper head and shank part which allows the gripper head and shank part be fastened to a conventional kneepad such that the carpet layer can still use this part as a conventional knee kicker.",
"This provides greater versatility to the carpet layer.",
"It should be appreciated that various other changes and modifications may be made to the invention described without departing from the spirit and scope of the invention."
]
] | Patent_10415337 |
[
[
"Latch device for securing cargo containers together and/or to vehicle decks",
"A cargo container hold down device that includes a housing defined by a base which forms a planar surfacing thereabout on which cargo container corner fittings results in the applied relation on the container relative to the supporting structure or platform involved.",
"The hold down device includes a flanged end which engages a cargo container corner casting aperture opening or an appropriate aperture and utilizes the structure for retention of the hold down device so the opposite side of the hold down device is allowed to automatically engage and disengage with a cargo container corner fitting as necessary to achieve proper handling and transport of cargo containers."
],
[
"1.A cargo container hold down arrangement for cargo containers of generally parallelepiped configuration, the four corners of the underside of which are each equipped with a corner fitting for purposes of securing the container to a support that is horizontally disposed, and with such container fittings being disposed in co-planar relation and respectively each defining a similar locking opening and a planar bearing surfacing that are respectively disposed adjacent such openings of the respective fittings, with a container securement device for each of the respective corner fittings for securing the cargo container to a platform, said securement devices each comprising: a shear block defining a front side, a top side, and a back side, with said shear block forming a base portion defining a planar force transmitting surfacing portion that extends to eitherside of the same, and a projecting portion that is generally normal to the planar bearing surfacing; said shear block further defining an internal chamber that in the projecting portion of said shear block is open at said front side of the same; said shear block base portion also defining a second force transmitting surfacing portion for engagement with such support, and including a latch member pivotally mounted in said chamber, for movement therein in a plane that is normally disposed relative to said front and rear sides of said shear block, and that is about a pivot axis that is normal of such plane; said latch member including a nose portion having an upper cam surfacing means for engagement by the bearing surfacing of a correspondingly located container mounted fitting, and an under cam surfacing means for engagement by the bearing surfacing of such correspondingly located container mounting fitting for removal of such container from such platform; resilient means for biasing said latch member to dispose said nose portion thereof exteriorly of said shear block opening in said front side thereof when said member cam surfacing means are not in use, said resilient means having, a resilient member interposed between said latch member and said base portion opposite the latch member tail portion; means for effecting deflection of said latch member about said pivot axis to within a housing on engagement of a container being lowered with said latch member nose upper cam surfacing means, and means for effecting deflection of said latch member within the housing on engagement of a correspondingly located container corner fitting of a container being removed from such platform and about a separate axis spaced frontwise from and parallel to said pivot axis of said latch member, said separate axis is disposed adjacent the level of said second force transmitting surfacing portion.",
"2.The securement device set forth in claim 1, wherein the top and back sides of said shear block are imperforate.",
"3.A container securement device for each respective corner fitting of a lower side of a cargo container of parallelepiped configuration for securing the cargo container to a platform, said securement device comprising: a shear block defining a front side, a top side, and a back side; said shear block forming a base portion defining a planar force transmitting surfacing portion that extends to either side of the same, and a projecting portion that is generally normal to the planar bearing surfacing; said shear block further defining an internal chamber that in the projecting portion of said shear block is open at said front side of the same; said shear block base portion also defining a second force transmitting surfacing portion for engagement with such support, and including a latch member pivotally, but unpinned, mounted in said chamber, for movement therein in a plane that is normally disposed relative to said front and rear sides of said shear block, and that is about a pivot axis that is normal of such plane; said latch member including a nose portion having an upper cam surface; said upper cam surface engaging a first bearing surfacing of a correspondingly located container mounted fitting, said latch member also including an under cam surface; said under cam surface engaging a second bearing surfacing of such correspondingly located container mounting fitting for removal of such container from such platform; a resilient member biasing said latch member to dispose said nose portion thereof exteriorly of said shear block opening in said front side thereof when said member cam surfaces are not in use; said resilient member being interposed between said latch member and said base portion opposite the latch member tail portion; a first deflector associated with said resilient member; said first deflector moving said latch member about said pivot axis to within a housing on engagement of a container being lowered with said latch member nose upper cam surface; a second deflector associated with said resilient member; said second deflector moving said latch member within the housing on engagement of a correspondingly located container corner fitting of a container being removed from such platform and about a separate axis spaced from and parallel to said pivot axis of said latch member.",
"4.A latch device for securing cargo containers comprising: a housing having a cavity opening and an internal pocket, a latch movably carried in the housing; a retainer holding a spring in place so as to urge said latch into a locked position; said latch is pivotally mounted in said chamber in an unpinned manner, for movement about a pivot axis, said latch has a projecting leg having a latch surface such that said latch pivots about said axis through motion within said internal housing pocket imparted through the contacting between latch first and second surfaces and an internal housing pocket at an under surfaces and over surface, respectively.",
"5.The latch device of claim 4 further comprising: said latch member including a nose portion having an upper cam surface; said upper cam surface adapted for engaging a bearing surfacing of a correspondingly located container mounted fitting, and an under cam surface; said under cam surface adapted for engaging a surface of a container mounting fitting for removal of such container from a platform.",
"6.The latch device of claim 5 further comprising: said housing, latch, retainer and spring being held together in relation to one another so that said latch is first inserted into a cavity opening of said housing and then positioned into said internal pocket.",
"7.The latch device of claim 6 further comprising: said spring is mounted in said cavity opening of said housing with a first end of the spring being positioned on a protrusion of said latch and the opposite end of said spring abutting said retainer in said cavity opening.",
"8.The latch device of claim 7 further comprising: said latch device is assembled with an appropriate assembly force is applied to said retainer compressing said spring so as to push retainer into cavity opening past first and second tabs and and four internal nubs; said retainer is then rotated to become aligned with said first and second tabs and said four internal nubs wherein the assembly force that has been applied to said retainer is removed thereby resulting in said retainer backing out of said housing cavity and bearing against said tabs which prevents complete removal of said retainer.",
"9.The latch device of claim 8 further comprising: said four internal nubs trap said retainer in a desired position holding the parts together and preventing said retainer from rotating and inadvertently aligning itself with said housing cavity thereby allowing the parts to come loose.",
"10.The latch device of claim 4 further comprising: an integral attachment adapted to receive a tether to be attached to said housing to secure said latch device to a deck of a vehicle to deter theft or loss of said latch device when it is desired for it to be removed from an appropriate structure and surface from a vehicle deck but yet remain with the vehicle.",
"11.The latch device of claim 4 further comprising: said latch device operates in absence of any pin, bolt or fastener retaining said latch in said housing.",
"12.The latch device of claim 11 further comprising: said latch has a substantially vertical line of action corresponding to the line of clearance of the container corner casting with said nose of said latch; said latch is contoured and functionally matched to fit within the internal contours of said housing and is be restrained therein by the resulting geometry, such that latch surface contacts and pivots in internal housing pocket at said undersurface and said undersurface is displaced as far away and to the left of said line of action thereby minimizing the mechanical force advantage of said spring during the engagement motion of the corner casting, thereby aiding in obtaining low applied forces and smooth engagement motion of latch.",
"13.The latch device of claim 12 further comprising: said device is a deck mountable device.",
"14.The latch device of claim 12 further comprising: said device has a twist lock container engagement.",
"15.The latch device of claim 12 further comprising: said device is one of a class of deck mounted or twist lock devices."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates to container securement devices, and more particularly, to improvements in cargo container securement devices of the type that provides automatic securement and release of a cargo container.",
"The device is mountable and demountable on a deck or frame of a vehicle so that the device can be adapted to different load conditions including a different mix of containers of different length and the like while having unused devices not interfere with the flush mounting of long containers."
],
[
"<SOH> BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT <EOH>A device for securing cargo containers to a vehicle deck and/or two cargo containers together comprising a housing containing a latch mechanism that extends outwardly from the housing to engage a cargo container corner casting.",
"The other side of the housing has two flanges projecting therefrom in a generally “T” shaped plan form.",
"In the case where two cargo containers are to be secured together, the flanges are inserted into the aperture of a cargo containers corner fitting manually and oriented in such a manner as to prevent its removal.",
"The appropriate corner fitting of the other cargo container is brought into contact with the exposed end of the device's latch mechanism that extends outwardly from the housing to engage the cargo container corner casting and secure the two cargo containers together.",
"When appropriate force is exerted to pull the two cargo containers apart, the devices latch mechanism that extends outwardly from the housing will automatically retract into the devices housing allowing the two cargo containers to be separated.",
"In the case where a cargo container is to be secured to a deck or frame of a vehicle, the flanges are inserted into the aperture of a deck or frame of a vehicle manually and oriented in such a manner as to prevent its removal.",
"The appropriate corner fitting of the cargo container is brought into contact with the exposed end of the device latch mechanism that extends outwardly from the housing to engage the cargo container corner casting and secure it to the deck or frame of a vehicle.",
"When appropriate force is exerted to pull the cargo container off of the deck, the devices latch mechanism that extends outwardly from the housing will automatically retract into the devices housing allowing the cargo container to be removed."
],
[
"CLAIM OF PRIORITY This is a non-provisional application claiming priority based on App.",
"No.",
"60/245,965, filed Nov. 3, 2000 and entitled “Latch Device for Securing Cargo Containers Together And/or to Vehicle Decks,” and also claiming priority based on App.",
"No.",
"60/292,505, filed May 21, 2001 also entitled “Latch Device for Securing Cargo Containers Together And/or to Vehicle Decks.” BACKGROUND OF THE INVENTION The invention relates to container securement devices, and more particularly, to improvements in cargo container securement devices of the type that provides automatic securement and release of a cargo container.",
"The device is mountable and demountable on a deck or frame of a vehicle so that the device can be adapted to different load conditions including a different mix of containers of different length and the like while having unused devices not interfere with the flush mounting of long containers.",
"DESCRIPTION OF RELATED ART U.S. Pat.",
"No.",
"3,365,229 teaches a top coupler means for interlocking a pair of opposed container corner brackets to provide for a tandem coupling of said containers, said top coupling means including a pair of first and second severable top coupler elements, each element having a clamp portion for engagement with respective corner bracket and a spacer portion engageable with the spacer portion of the other element attendant to space separation of one corner bracket from the other, said first top coupler element being provided with one coupler element interlock portion and said second top coupler element being provided with another coupler interlock portion for intercoupling with the one interlock portion, and means for pivotally interlocking one element with the other, and bottom coupling means for coupling the bottoms of the containers together, and hoisting means therefore.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"3,603,267 teaches a supporting and securement structure adapted to use on carrier vehicles, including railway flatcars, for the transportation of varied sizes and numbers of box-type containers in which merchandise is shipped; said structure having guide tracks secured to the carrier vehicle structure and one-piece pedestal type supports with integral support portions retained within the guide tracks for movement therealong to predetermined positions of securement and swingable around said support portions between upright and collapsed positions, the support pedestals being constructed and propelled for relative stability in their upright positions and collapsible into relatively small openings in the guide tracks themselves to close said openings when not in use.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"3,604,363 teaches spring-biased latches on a transport carrier for automatically engaging and disengaging bottom corner container fittings are bodily movable to maintain the same latching engagement within limits for various clearances between the container fittings and the housings secured to the transport carrier on which the latches are mounted.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"3,604,364 teaches fittings at the corners of a container that are automatically latched to a railway car when it is lowered thereon and unlatched therefrom when the container is lifted from transport position.",
"Each fitting is received in a housing on the car on which a bellcrank latch is mounted to pivot about a pair of spaced axes under the biasing action of a coil compression spring reacting between the housing and the distal end of one arm of the latch.",
"The distal end of the other arm of the latch has latching engagement with the respective container fitting.",
"The housings are slidable along slots extending lengthwise along opposite sides of the car and can be swung to retracted positions on trunnions extending below the floor or deck of the car.",
"The trunnions are located in spaced relation to the latches to cause them to maintain latching engagement with the container on upward movement of it during transport.",
"The retracted housings are arranged to be bypassed by other housings slidable along the slots.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"3,628,222 teaches a latching mechanism having two pivotally mounted and interacting members.",
"This mechanism provides for automatic locking when moved to the latched position with provisions for unlocking when unlatching is desired.",
"The latching mechanism is particularly adaptable for use in latching shipping containers to the bed of transporting vehicles.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"3,630,155 teaches a railroad car container bracket mounted on transverse sideplate means attached periodically to the sides of the railroad car deck.",
"The bracket is pivoted on an axis transverse to the longitudinal centerline of the car and constructed in such a manner as to prevent longitudinal, transverse, and vertical movement of a container.",
"When the brackets are in position supporting the four bottom corners of a container, the bracket will transmit impact forces to the deck of the railway car in a unique manner which shields the bracket pivot pin from damaging shearing forces.",
"The bracket also contains a spring-loaded pivot latch which prevents dislodgment of the container in a vertical direction, especially when the container is empty and subjected to high wind loading which tends to tip an empty container from the deck of the container car.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"3,774,551 teaches a spring biased latch lever is variably pivoted on the housing of container securing means on a transport carrier to accommodate minimum and maximum clearances between the container securing means and the bottom container fitting mounted thereon.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,236,853 teaches a coating of cadmium applied to a container pedestal latch protuberance which lowers the maximum exit force sufficiently as to be within the 2200 pound maximum in the AAR specification while the minimum exit force of 1600 pounds and the maximum container entry force of 800 pounds were also within the specification.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,277,212 teaches a connector for use in the securement of a first member, such as a cargo container, to a base support includes a base member, a post member extending from the base member in one direction and an attaching means for attaching the connector to the base support extending from the base member in the other direction.",
"Restraining means provides vertical restraint to the container when the post member is positioned to extend into the opening of the web of the corner casting thereof and the container is restrained from movement in at least one horizontal direction.",
"In one form, a restraining surface for the container is located on a cam pivotally mounted by the post member and in a second form, a restraining surface for the container is on the post member itself.",
"The cam of the first form is pivotally mounted so that in response to lifting movement, the cam is rotated about its axis to act upon the web portion to impact a force horizontally whereby the container, in loading, follows a path similar to that in loading.",
"In the second form, a plunger, under a force of compression of a spring, acts on the web portion to provide a similar function.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,372,715 teaches a punch type release lock intended for use primarily in retaining load supports such as pallets in locked position in an aircraft.",
"The lock comprises a detent mechanism which is inserted into a recess preferably at the side of the load support.",
"A preferred form of detent mechanism comprises a pair of relatively movable elements, at least one of which is pivoted.",
"The elements include abutments which are movable apart as the elements are inserted into the recess.",
"When a load is applied to the pivoted detent, as for example, by a parachute extracting system, movement of the element in a direction to withdraw the element to release the load support is prevented by a load cell comprising a fuse plate and punch, in which the punch is prevented from movement by the fuse plate until attainment of a predetermined load on the pivoted detent element.",
"At the pre determined load, the punch penetrates the fuse plate and upon penetration of the fuse plate, the punch is relatively freely movable to effectively permit the load support to move the pivoted detent element to completely release the load support.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,382,734 teaches a container pedestal for supporting and securing a cargo container having a catch opening on a vehicle such as a rail car.",
"The pedestal includes a base defining a platform for supporting the container.",
"A pivotal latch lever is biased by a spring into a latched position wherein a latching nose on the lever registers with a latch recess in the container.",
"The latch nose is contacted for pivoting the latch lever from the latched to a released position when the container is raised or lowered.",
"The latch lever can be manually locked, yet self-entry automatic loading can be carried out in the locked condition.",
"A line contact between the latch lever and the spring provides reliable and consistent latch operation due to a uniform spring lever arm length.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,382,735 teaches a container pedestal for supporting and securing a cargo container having a catch opening on a vehicle such as a rail car.",
"The pedestal includes a base defining a platform for supporting the container.",
"A pivotal latch lever is biased by a spring into a latched position wherein a latching nose on the lever registers with a latch recess in the container.",
"The latch nose is contacted by the container for pivoting the latch lever from the latched to a released position when the container is raised or lowered.",
"The latch lever can be manually locked, yet self-entry automatic loading can be carried out in the locked condition.",
"A line contact between the latch lever and the spring provides reliable and consistent latch operation due to a uniform spring lever arm length.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,430,032 teaches a latch for locking a container to a pedestal on the flat deck of a flat car and particularly containers containing flammable materials.",
"The container is supported on a pedestal at each corner thereof and the pedestals are adjustably mounted in guideways for movement along the deck of the flat car in accordance with the length of the container, to support containers at selected intervals along the car.",
"A spring biased latch is provided to lock the container to the pedestal and a lock is provided for the latch is provided which reacts against the pedestal and includes a biasing spring for the lock to positively hold the latch in a locked position even though the car should be derailed.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"4,626,155 teaches a device for automatically securing a cargo container to a support such as a deck of a vehicle or a second container with the first container is to be stacked.",
"The device includes a base having a projecting shear block received in the locking opening of the container.",
"A head rotates between an unlocked or loading position in which the head moves through the locking opening and a locked position in which the container is secured.",
"Automatic entry and release are provided by a spring within the biasing the head to the locked position but permitting movement to the unlocked position when torque is applied by engagement of the container with a cam surface on the head.",
"Visible indication of the locked position and positive locking of the head in the locked position may be provided.",
"For stacked containers, two aligned shear blocks and two angularly offset heads are provided and the spring may be released for manual locking of the device to one container followed by automatic locking to the second container.",
"The disclosure in this patent is incorporated by reference in the instant application as if filly set forth herein.",
"U.S. Pat.",
"No.",
"5,090,638 teaches a locking mechanism for tying down a piece of freight on a loading floor in an aircraft has a housing recessed in the loading floor.",
"A latch opening member and a latching member are journalled in the housing to tilt toward each other or away from each other.",
"Follower cams of the latching member ride in respective cam guide tracks of the latch operating member.",
"A tension spring tends to bias the latching member and the latch operating member in opposite directions in a freight latching position or into a recessed beyond dead center position.",
"Stop members are so positioned on the latch operating member and on the latching member that the latter cannot be tilted without activating the latch operating member which can be rolled over by a piece of freight in one direction when projecting from the housing and in the other direction when recessed into the housing.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"5,106,247 teaches an automatic hold down and locking as well as automatic load configuration change capability device system, which can be used to hold down and lock either one long container or several shorter containers within the same loading space, regardless of the outside width or width of bottom side rail flange on the container.",
"The locking device system has four fixed non-retractable fully automatic locking devices positioned on the load carrier at the four outer standard locking points of each long container, and at least two retractable fully automatic locking devices positioned at the long side of the load carrier between and in line with the outer locking points.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"5,560,088 teaches a coupling piece includes an abutment and locking member which is shiftable relative to the abutment to allow for an automatic and reliable locking of the containers.",
"The coupling pieces do not jam when the connection is released by means of slightly tilting the upper container.",
"The coupling piece is particularly suitable for automatically locking and releasing tightly stowed containers, especially 20′ containers.",
"In an alternate embodiment, a coupling piece is shaped such that the entire coupling piece is shifted to a locking position when containers are placed on top of one another.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"5,570,981 teaches a cargo container hold down device that includes a shear block defining a base and a housing therefore that is shaped so that the base forms a planar surfacing thereabout on which the cargo container fitting rests in the applied relation of the container relative to the supporting platform involved; the shear block housing pivotally mounts a latch device comprising a latch member that includes a nose portion having an upper cam surfacing for engagement by a correspondingly located container mounted corner fitting, and an under cam surfacing disposed for engagement by such correspondingly located container mounted corner fitting on removal of such container therefrom, the latch member being biased outwardly of the shear block housing to dispose the nose thereof over the container corner fitting supporting surface of the shear block housing, and including an element for withdrawing such latch member within the shear block housing about one pivot axis when the container is applied to the device, and an element for withdrawing such latch member within the shear block housing about a separate axis that is spaced from and parallels the first indicated pivot axis when the container is removed from such device, so as to achieve a smooth and easier loading of the container, and provide for increased force for cam positioning of the device latch member for container removal purposes.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"U.S. Pat.",
"No.",
"5,797,169 teaches a coupling piece for the detachable connection of corner fittings of adjacent containers, especially of containers stacked one above the other on board ships.",
"In order to reduce the manual effort involved in coupling together containers, semi-automatic coupling pieces are known which only need to be manually attached to one container and pre-locked.",
"A full locking after the containers have been placed one on top of the other is effected automatically.",
"Coupling pieces of this type require however, in many respects, a complex automatic actuating mechanism.",
"In order to simplify the automatic actuating mechanism, a plurality of stop faces are provided, which are offset to one another on the locking bolt and which can be brought alternately into a corresponding position to a stop face on a spring-loaded ram.",
"The contact of a stop face of the locking bolt against the stop face of the spring-loaded ram enables the locking bolt to be fixed simply and reliably in the respectively intended position of its crossbolts.",
"The disclosure in this patent is incorporated by reference in the instant application as if fully set forth herein.",
"Canadian Patent No.",
"589031, issued December, 1959, in Class/Subclass 410/80 is believed to generally relate to the subject matter of this invention.",
"A company known as Peck & Hale has offered for sale a model F665 Safe-T-Loc Stacker container lock, believed to be more than one year prior to the filing date of this application.",
"It will be seen that the forgoing prior art teaches certain parameters for container locks and use various complex solutions to meet the needs taught.",
"The instant invention departs from the complex mechanisms and mechanisms of limited functionality in its use of the housing with several camming surfaces, a latch with specific geometry to engage the surfaces in the housing, the corner casting of the container and the spring, moving through the required motion and imparting the required loads and resistance to forces, yet further providing a simple, strong and efficient structure with a minimum of parts, notably without a latch pivot.",
"Additionally, the prior art typically uses either complex spring mounting and seating arrangements or spring mounting and/or seating arrangements that are difficult to work with, particularly when changing broken springs or replacing springs with appropriately calibrated springs.",
"The instant invention utilizes simple, yet high performance spring mounting and seating.",
"BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT A device for securing cargo containers to a vehicle deck and/or two cargo containers together comprising a housing containing a latch mechanism that extends outwardly from the housing to engage a cargo container corner casting.",
"The other side of the housing has two flanges projecting therefrom in a generally “T” shaped plan form.",
"In the case where two cargo containers are to be secured together, the flanges are inserted into the aperture of a cargo containers corner fitting manually and oriented in such a manner as to prevent its removal.",
"The appropriate corner fitting of the other cargo container is brought into contact with the exposed end of the device's latch mechanism that extends outwardly from the housing to engage the cargo container corner casting and secure the two cargo containers together.",
"When appropriate force is exerted to pull the two cargo containers apart, the devices latch mechanism that extends outwardly from the housing will automatically retract into the devices housing allowing the two cargo containers to be separated.",
"In the case where a cargo container is to be secured to a deck or frame of a vehicle, the flanges are inserted into the aperture of a deck or frame of a vehicle manually and oriented in such a manner as to prevent its removal.",
"The appropriate corner fitting of the cargo container is brought into contact with the exposed end of the device latch mechanism that extends outwardly from the housing to engage the cargo container corner casting and secure it to the deck or frame of a vehicle.",
"When appropriate force is exerted to pull the cargo container off of the deck, the devices latch mechanism that extends outwardly from the housing will automatically retract into the devices housing allowing the cargo container to be removed.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a largely schematic perspective view of a support surface to which the indicated pairs of container support or hold down devices have been applied, and a cargo container is to be supported thereon, which support surface may be, for instance, the deck of a railroad car.",
"FIG.",
"2 is an exploded perspective view of one of the cargo container lock or securement devices arranged in accordance with the invention.",
"FIG.",
"3 is a diagrammatic perspective view showing the container lock or securement device of FIG.",
"2 assembled and disposed to receive the conventional lower corner fitting of a cargo container that is being lowered onto same; the securement device of FIG.",
"3 is shown deliberately separated from, for instance, a railroad car deck, that normally supports same, to expose the underside of same for disclosure purposes.",
"FIG.",
"4 is a side elevational view of the latch device for cargo containers.",
"FIG.",
"5 is a frontal elevational view of the latch device for cargo containers.",
"FIG.",
"6 is a back elevational view of the latch device for cargo containers.",
"FIG.",
"7 is a top plan view of the latch device for cargo containers.",
"FIG.",
"8 is a bottom plan view of the latch device for cargo containers.",
"FIG.",
"9 is a side elevational view of the latch device for cargo containers, same as FIG.",
"4.FIG.",
"10 is an alternate frontal elevational view other than FIG.",
"5 of the latch device for cargo containers showing a frontal cavity which is utilized during assembly of the alternate latch that contains two stub protrusions on the end of the leg of the latch.",
"FIG.",
"11 is an alternate bottom plan view other than FIG.",
"8 of the latch device for cargo containers showing a bottom cavity which is utilized during assembly of the alternate latch that contains two stub protrusions on the end of the leg of the latch.",
"FIG.",
"12 is an alternate bottom plan view other than FIGS.",
"8 and 11 of the latch device for cargo containers showing an alternate bottom housing shape which maybe utilized when the said latch device is being applied to a support surface that does not require the housings bottom flanges, such as in the case of welding the housing to a support surface.",
"FIG.",
"13 is a sectional side elevational view of the housing for the latch device.",
"FIG.",
"14 is a sectional side elevational view of the latch for the latch device.",
"FIG.",
"15 is a sectional side elevational view of the housing for the latch device which utilizes an alternate latch that contains two stub protrusions on the end of the leg of the latch.",
"FIG.",
"16 is a side elevational view of the alternate latch for the latch device which contains two stub protrusions on the end of the leg of the latch.",
"FIG.",
"17 is a sectional side elevational view of the housing for the latch device showing the initial insertion technique utilized for assembling the said alternate latch that contains two stub protrusions on the end of the leg.",
"FIG.",
"18 is a sectional side elevational view of the housing for the latch device showing the secondary assembly path utilized for assembling the said alternate latch that contains two stub protrusions on the end of the leg, the two stub protrusions on the end of the leg are being inserted through the frontal cavity shown in FIG.",
"10.FIG.",
"19 is a sectional side elevational view of the housing for the latch device showing the final assembly path utilized for assembling the said alternate latch that contains two stub protrusions on the end of the leg, the two stub protrusions on the end of the leg are being inserted through the bottom cavity shown in FIG.",
"11.FIG.",
"20 is a bottom plan view of the latch device showing the end of a spring in the housings slot cavity and a view of the appropriate retainer.",
"FIG.",
"21 is a bottom plan view of the latch device showing the end of a spring in the housings slot cavity and the retainer placed on top of the spring appropriately.",
"FIG.",
"22 is a bottom plan view of the latch device showing the end of a spring in the housings slot cavity and the retainer turned and secured to captivate the internal spring and latch appropriately.",
"FIG.",
"23 is a sectional side elevational view showing the positions of the latch devices internal components just prior to the cargo containers corner fitting being removed from the device.",
"FIG.",
"24 is a sectional side elevational view showing the positions of the latch devices internal components during partial removal of the cargo containers corner fitting from the device.",
"FIG.",
"25 is a sectional side elevational view showing the positions of the latch devices internal components retracted into the devices housing just after the cargo containers corner fitting has been removed from the device.",
"FIG.",
"26 is a sectional side elevational view showing the positions of the latch devices internal components just prior to the cargo containers corner fitting engaging with the device.",
"FIG.",
"27 is a sectional side elevational view showing the positions of the latch devices internal components fully retracted into the devices housing just after the cargo containers corner fitting has been engaged onto the device.",
"FIG.",
"28 is a sectional side elevational view showing the positions of the latch devices internal components that include the alternate latch that contains two stub protrusions on the end of the leg, just prior to the cargo containers corner fitting being removed from the device.",
"FIG.",
"29 is a sectional side elevational view showing the positions of the latch devices internal components that include the alternate latch that contains two stub protrusions on the end of the leg, during partial removal of the cargo containers corner fitting from the device.",
"FIG.",
"30 is a sectional side elevational view showing the positions of the latch devices internal components that include the alternate latch that contains two stub protrusions on the end of the leg, retracted into the devices housing just after the cargo containers corner fitting has been removed from the device.",
"FIG.",
"31 is a sectional side elevational view showing the positions of the latch devices internal components that include the alternate latch that contains two stub protrusions on the end of the leg, just prior to the cargo containers corner fitting engaging with the device.",
"FIG.",
"32 is a sectional side elevational view showing the positions of the latch devices internal components that include the alternate latch that contains two stub protrusions on the end of the leg, fully retracted into the devices housing just after the cargo containers corner fitting has been engaged onto the device.",
"FIG.",
"33 is a lateral side elevational view of the latch device appropriately rotated so its bottom flanges are orientated with a cargo container corner casting aperture.",
"FIG.",
"34 is a lateral side elevational view of the latch device appropriately rotated so its bottom flanges are orientated with a cargo container corner casting aperture and raised up into the aperture.",
"FIG.",
"35 is a lateral side elevational view of the latch device appropriately rotated so its bottom flanges are orientated to retain the latch device in a cargo container corner casting aperture.",
"FIG.",
"36 is a lateral side elevational view of the latch device appropriately retained in a cargo containers bottom corner casting aligned and ready to be lowered down onto another cargo containers top corner casting.",
"FIG.",
"37 is a longitudinal side elevational view of the latch device appropriately retained in a cargo containers bottom corner casting aligned and ready to be lowered down onto another cargo containers top corner casting.",
"FIG.",
"38 is a sectional horizontal view of the cargo containers bottom corner casting shown in FIG.",
"20.The captivated flanged end of the latch device is clarified.",
"FIG.",
"39 is a lateral side elevational view of the latch device appropriately retained between two cargo containers.",
"FIG.",
"40 is a longitudinal side elevational view of the latch device appropriately retained between two cargo containers.",
"FIG.",
"41 is a lateral side elevational view of the latch device appropriately retained between a cargo container on the top and an appropriate vehicle deck on the bottom.",
"FIG.",
"42 is a longitudinal side elevational view of the latch device appropriately retained between a cargo container on the top and an appropriate vehicle deck on the bottom.",
"FIG.",
"43 is a lateral side elevational view of the latch device appropriately retained between a cargo container on the top and an appropriate vehicle deck on the bottom, said device represents being welded to the vehicle deck.",
"FIG.",
"44 is a longitudinal side elevational view of the latch device appropriately retained between a cargo container on the top and an appropriate vehicle deck on the bottom, said device represents being welded to the vehicle deck.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to FIG.",
"1, there is illustrated in somewhat of a diagrammatic manner a support 22 upon which a cargo container 20 is to be secured by Applicant's improved securement devices 10 that, in this regard, are arranged in accordance with FIGS.",
"2 through 44 of this application, and in accordance with the principles of the present invention herein disclosed.",
"The support 22 may, for instance, be a deck or floor of a railroad flat car or other rail transport vehicle, or support 22 may be another type of vehicle to which the device 10 is applied in multiples of four for the usual application thereof to cargo container corner fittings 21 or the like.",
"The principles of the present invention are applicable to devices for securing various types of containers to various types of supports.",
"In the illustrated embodiment of the invention, the cargo containers 20 are identical and are of the usual parallelepiped configuration that is involved in standard and modular forms of containers of this type as illustrated, each of the four lower corners of each container 20 includes a corner fitting 21 in the nature of a corner casting that may be of the type specified by the standards of the Association of American Railroads.",
"The corner fitting 21 defines an upwardly or downwardly facing horizontal wall 51 (see FIG.",
"3) that defines an opening 24 that is of the familiar quadrilateral configuration.",
"In accordance with the present invention, the securement devices 10 are intended to protrude through the locking opening 24 of the individual container corner fittings to achieve securement and automatic entry and release of the respective containers as hereinafter disclosed.",
"The lock or securement device 10 of the present invention is illustrated in detail in FIGS.",
"2 through 44, which will be described in detail hereinafter.",
"The latch device 10 is comprised of housing 11, a latch 12, a retainer 13 and a spring 14.An exploded perspective view is shown in FIG.",
"2.An assembled side lateral view of device 10 is shown in FIG.",
"4.The housing has flanges 15 and 16 which extend outwardly from the housing which engage a cargo containers corner casting aperture opening 24 or a vehicle decks comparable aperture opening 23.The housing 11 has a base 18 which is spaced between two cargo containers corner fittings 21 or a cargo containers corner fittings 21 and a vehicle decks appropriate structure and surface 25.The illustrations within this document shows that the vehicle decks appropriate structure and surface 25 is identical to the same aperture shape, size, and structure thickness of a standard cargo container corner casting.",
"This is desirable so that the latch device 10 maybe utilized for orientating with latch 12 upwards as shown in FIG.",
"41 or downwards as shown in FIG.",
"39.It is to be noted that the scope of the design of latch device 10 is not to be limited to an appropriate structure and surface 25 being identical to a cargo containers corner fittings 21.Housing 11 and flanges 15 and 16 are allowed to be varied so as to engage an appropriate deck aperture that is defined by the user.",
"The illustration in FIG.",
"12 shows an example of the tailorability of housing 11 where instead of flanges 15 and 16, the bottom end of housing 11 may be formed into a round cylinder shape 52 which is capable of containing retainer 13 and spring 14.FIGS.",
"43 and 44 show a side and frontal view respectively of Device 10 securing a cargo container 20 to the vehicle decks appropriate structure 22.An appropriate surface 54 is to allow device 10 to be supported with proper provisions 53 for shape 52 of housing 11.FIGS.",
"43 and 44 show the example of how housing 11, if made of appropriate materials maybe fastened to surface 54 by welds 55.There are two typical application uses for Latch Device 10.One application of latch device 10 is for securing standard cargo containers 20 (partial side sectional views shown) together by latching their corner castings 21, see FIGS.",
"39 and 40.The other typical application of latch device 10 is for securing a standard cargo container 20 onto a vehicle deck or frame 22 such as shown in FIGS.",
"41, 42, 43 and 44.One unique feature of the latch device 10 is how the device is assembled and its components housing 11, latch 12, retainer 13 and spring 14 are held together in relation to each other.",
"To assemble latch device 10, refer to FIG.",
"26.Leg 29 of latch 12 is first inserted into cavity opening 27 of housing 11 and then positioned into the internal pocket 28 as shown in FIG.",
"26.Secondly, spring 14 which is a typical metal compression or die spring is inserted through cavity opening 26 of housing 11 with one end on the spring being positioned onto the protrusion 30 of latch 12.FIG.",
"20 is a bottom plan view of latch device 10 showing the end of a spring 12 in the housing cavity opening 26 and a view of the appropriate retainer 13.Retainer 13 is orientated and placed into housing cavity opening 26 on top of the end of spring 12 as shown in FIG.",
"21.An appropriate assembly force is applied to retainer 13 compressing spring 12 so as to push retainer 13 into housing cavity opening 26 past tabs 31 and 32 and four internal nubs 33.Retainer 13 is then rotated appropriately as shown in FIG.",
"22 and become aligned with tabs 31 and 32 and the four internal nubs.",
"The assembly force that has been applied to retainer 13 is removed and this results in retainer 13 backing out of the housing cavity 26 and bearing against tabs 31 and 32 which prevents complete removal of retainer 13.The four internal nubs 33 traps the retainer 13 into the desired position holding the parts together and prevents retainer 13 from rotating and inadvertently aligning itself with housing cavity 26 which would allow the parts to come loose.",
"To disassemble latch device 10, the order of these steps are reversed.",
"Another unique feature of the latch device 10 is that it has an integral attachment feature 19.This feature allows an appropriate tether, such as a chain or cable, to be attached to housing 11 which in turn secures latch device 10 to a deck 22 or frame of a vehicle.",
"Attachment feature 19 is typically a through hole and is not unique by itself, but the uniqueness is that this feature is integral with this type of devices housing 11 and has not been represented by any known prior art.",
"The reason for this type of integral attachment feature is to deter theft of latch device 10 when it is desired for it to be removed from an appropriate structure and surface 25 from a vehicle deck 22 but yet remain with the vehicle.",
"Latch device 10 is to be capable of being removed from the appropriate structure and surface 25 and stowed in an appropriate area on the vehicle so the latch device 10 will not be in the way for other types of lading when cargo containers are not being transported.",
"Another unique and novel feature of latch device 10 is that there is no pin, bolt or fastener retaining latch 12 to housing 11.All prior art that utilizes a pivoting latch uses some type of latch/pin arrangement.",
"Latch device 10 utilizes a latch 12 that is contoured and functionally matched to fit within the internal contours of housing 11 and be restrained by the resulting geometry.",
"FIGS.",
"23 through 27 show sectional views of latch device 10 at various operational stages of engaging and disengaging with a corner casting 21 of a standard cargo container 20.FIGS.",
"28 through 32 show sectional views of latch device 10 at various operational stages of engaging and disengaging with a corner casting 21 of a standard cargo container 20, this device 10 utilizes an alternate latch 12 shape that has stub protrusions 45 and 46 on the end of leg 29 (see FIG.",
"2).",
"FIG.",
"23 represents the earliest operational stage of when a corner casting 21 of a standard cargo container 20 is being removed off of the latch device 10 and is starting to make contact with the concave underside 40 of latch 12.It is to be observed that latch 12 is secured in the internal pocket 28 of housing 11 by its leg 29.Latch surfaces 34 and 36 contact internal housing pocket 28 at surfaces 37 and 39 respectively.",
"Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 is secured into proper position.",
"As corner casting 21 of a standard cargo container 20 is being removed off of the latch device 10 and is in contact with the concave underside 40 of latch 12.It is to be observed that latch 12 is pivoting in the internal pocket 28 of housing 11 by its Leg 29.Latch surface 34 is contacting and pivoting about internal housing pocket 28 at surface 37.Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 translates and pivots through the desired motion.",
"The actual location and shape of latch surface 34 and internal housing surface 37 is allowed to be tailored as desired to obtain the desired release action of the latch device 10.FIG.",
"24 represents the operational stage of when a corner casting 21 of a standard cargo container 20 is being removed off of the latch device 10 and is making contact with the concave underside 40 of latch 12.It is to be observed that latch 12 has pivoted and rotated in the internal pocket 28 of housing 11 by its Leg 29.FIG.",
"24 shows that latch surface 35 is now coming into contact and pivoting about internal housing pocket 28 at surface 39.Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 translates and pivots through the desired motion.",
"The actual location and shape of latch surfaces 34 and 35 and internal housing surfaces 37 and 38 is allowed to be tailored as desired to obtain the desired release action of the latch device 10.It is to be observed that this two or more surface pivot and contact areas of latch 12 during the release action of latch device 10 is similar but unique from prior art Brewster U.S. Pat.",
"No.",
"5,570,981.Latch 12 of latch device 10 is not guided and restrained by a pin.",
"Line action 43 defines the travel line which the contact surfaces of corner casting 21 of a standard cargo container 20 travels while it is being removed off of or being placed onto the latch device 10.Typically it is desired that latch surface 34 contacting and pivoting about internal housing pocket 28 at surface 37 is near or to the right of line action 43 which results in minimizing the mechanical force advantage of spring 14.Minimizing the mechanical force advantage of spring 14 during initial release motion of corner casting 21 aids in obtaining low applied forces and smooth startup motion of latch 12.After startup motion of latch 12, it is desired to increase the mechanical force advantage of spring 14 so as to minimize the required size of spring 14.This is accomplished by creating new pivot areas other than surface 34 of latch 12 farther away and to the left of line action 43 as illustrated in FIG.",
"24 and previously described.",
"FIG.",
"25 represents the operational stage further along when a corner casting 21 of a standard cargo container 20 is being removed off of the latch device 10 and clears contact with the concave underside 40 of latch 12.It is to be observed that latch 12 is pivoting in the internal pocket 28 of housing 11 by its leg 29.FIG.",
"25 shows latch surface 35 is contacting and pivoting about internal housing pocket 28 at surface 38.It is allowed for latch surface 42 to be contoured and to contact and pivot about internal housing pocket 28 at surface 38 to obtain the desired release effect of latch device 10.Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 translates and pivots through the desired motion.",
"The actual location and shape of latch surfaces 34, 35 and 42 and internal housing surfaces 37 and 38 are allowed to be tailored as desired to obtain the desired release action of the latch device 10.FIG.",
"26 represents the earliest operational stage of when a corner casting 21 of a standard cargo container 20 is engaging with latch device 10 and is starting to make contact with the convex upper-side 41 of latch 12.It is to be observed that latch 12 is secured in the internal pocket 28 of housing 11 by its Leg 29.Latch surfaces 34 and 36 contact internal housing pocket 28 at surfaces 37 and 39 respectively.",
"Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 is secured into proper position.",
"As corner casting 21 of a standard cargo container 20 is engaging the latch device 10 and is in contact with the convex upper-side 41 of latch 12.It is to be observed that latch 12 is pivoting in the internal pocket 28 of housing 11 by its Leg 29.Latch surface 36 is contacting and pivoting about internal housing pocket 28 at surface 39.Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 translates and pivots through the desired motion.",
"The actual location and shape of latch surface 36 and internal housing surface 39 is allowed to be tailored as desired to obtain the desired engagement action of the latch device 10.It is to be observed that this pivot and contact area 36 of latch 12 during the engagement action of latch device 10 is similar but unique from prior art Brewster U.S. Pat.",
"No.",
"5,570,981.Latch 12 of latch device 10 is not guided and restrained by a pin.",
"Line action 43 defines the travel line which the contact surfaces of corner casting 21 of a standard cargo container 20 travels while it is engaging and being placed onto the latch device 10.Typically it is desired that latch surface 36 contacting and pivoting about internal housing pocket 28 at surface 39 be as far away and to the left of line action 43 which results in minimizing the mechanical force advantage of spring 14 as illustrated in FIG.",
"24.Minimizing the mechanical force advantage of spring 14 during the engagement motion of corner casting 21 aids in obtaining low applied forces and smooth engagement motion of latch 12.FIG.",
"27 represents the operational stage further along when a corner casting 21 of a standard cargo container 20 is being engaged onto latch device 10 and clears contact with the convex upper-side 41 of latch 12.It is to be observed that latch 12 is pivoting in the internal pocket 28 of housing 11 by its leg 29.FIG.",
"27 shows latch surface 36 is contacting and pivoting about internal housing pocket 28 at surface 39.Spring 14 being positioned onto the protrusion 30 of latch 12 assures that latch 12 translates and pivots through the desired motion.",
"The actual location and shape of latch surface 36 and internal housing surface 39 is allowed to be tailored as desired to obtain the desired release action of the latch device 10.An optional unique feature of the latch device 10 is that for double cargo container stacking it may be desirable to include the integral retractable plunger feature 44.FIG.",
"33 is a side elevational view of latch device 10 appropriately rotated so its bottom flanges 15 and 16 are orientated with a cargo container corner casting aperture 24.The integral retractable plunger feature 44 points out from base 18 of housing 11.As latch device 10 is raised into cargo container corner casting aperture 24 as shown in FIG.",
"34 the plunger feature 44 automatically retracts out of the way into the base 18 of housing 11.The latch device 10 is then rotated while in the cargo container corner casting aperture 24 as shown in FIG.",
"35 so flanges 15 and 16 prevent removal of latch device 10 from corner casting 21 of a standard cargo container 20.When latch device 10 has been rotated into the desired position the plunger feature 44 automatically raises out of base 18 of housing 11 into the open area of the cargo container corner casting aperture 24.A horizontal sectional view of corner casting 21 of a standard cargo container 20 in FIG.",
"38 shows a planar view of plunger feature 44 in corner casting aperture 24.Latch device 10 is prevented from inadvertently coming loose and falling out of corner casting 21 while the standard cargo container 20 is being positioned during loading or unloading operations because the plunger feature 44 has been raised out of base 18 and into the open clear area in corner casting aperture 24.To remove latch device 10 from corner casting 21 one has to grasp the extending part of housing 11 of latch device 10 and manually rotate the latch device 10 about it's axis in such a manner to realign flanges 15 and 16 with corner casting aperture 24 as shown in FIG.",
"34.This rotation action results in plunger feature 44 to bear up against the cargo container corner casting aperture 24 cast surfaces and automatically retract back into base 18 of housing 11 no longer acting as a deterrent to removal of latch device 10.Latch device 10 may then be lowered down out of corner casting aperture 24 as shown in FIG.",
"33.Latch device 10 plunger feature 44 performs a similar task as the prior art of the Safe-T-Loc manually operated feature described in the Peck & Hale-F665 Safe-T-Loc Stacker flyer.",
"Plunger feature 44 is unique in that it performs its function automatically and is of an obviously different design.",
"Although the invention is described with respect to a preferred embodiment, modifications thereto will be apparent to those skilled in the art."
]
] | Patent_10415394 |
[
[
"Telephone directory assistane method and telephone directory assistance system",
"This telephone directory assistance system accepts a request for a telephone number of a registrant, and judges whether the registrant is registered in a telephone number data base.",
"When the registrant is registered in the telephone number data base, the telephone directory assistance system judges whether a message from the inquirer is transmitted to the registrant without informing the telephone number to the inquirer based on a telephone number disclosing condition registered in the telephone number data base.",
"Then, in a case of transmission, the inquirer is caused to input a message and the inputted message is informed to the registrant."
],
[
"1.A method for informing a telephone number of a registrant to an inquirer, comprising: accepting a request for a telephone number of the registrant from the inquirer; judging whether the registrant is registered in a telephone number data base; judging whether an information from the inquirer is transmitted to the registrant at least without informing the telephone number of the registrant to the inquirer, corresponding to a disclosing condition registered in the data base when the registrant is registered in the telephone number data base; and causing the inquirer to input information and the registrant to be capable of being informed of the input information when informing the telephone number of the registrant.",
"2.The method according to claim 1, wherein the method includes requesting the inquirer for a message as the information, recording the message and causing the recorded message to be capable of being played back to the registrant.",
"3.The method according to claim 2, wherein the method includes calling the registrant and notifying the recorded message to the registrant when a terminal of the registrant is a conventional telephone set.",
"4.The method according to claim 2, wherein the method includes notifying the registrant that the message is recorded and causing the registrant to access the recorded message when a terminal of the registrant is a cellular phone.",
"5.The method according to claim 1, wherein the method includes requesting the inquirer for a message in a form of a character signal inputted by a push button operation as the information and informing the inputted message to the registrant.",
"6.The method according to claim 5, wherein the inputted message is converted to an audible sound and informing the registrant of the audible sound.",
"7.The method according to claim 1, wherein the method includes requesting the inquirer to transmit an e-mail as the information and causing the registrant to receive the transmitted e-mail.",
"8.The method according to any one of claims 1 to 7, being capable of setting one of a first condition and a second condition for each of the registrants, the first condition always informing the telephone number, the second condition always refusing to inform the telephone number in response to the request for the telephone number from the inquirer, as the disclosing condition registered in the data base.",
"9.The method according to any one of claims 1 to 7, wherein the request for the telephone number of the registrant is accepted by accessing an old telephone number of the registrant.",
"10.A system informing a telephone number of a registrant to an inquirer, comprising: a data base in which at least a telephone number of a registrant and a disclosing condition on whether an information from the inquirer is transmitted to the registrant without informing the telephone number to the inquirer, are corresponding to each other; and means for informing the information from the inquirer to a terminal of the registrant.",
"11.The system according to claim 10, wherein the informing means records a message as the information from the inquirer, and cause the recorded message to be capable of being played back to the registrant.",
"12.The system according to claim 11, wherein the terminal of the registrant is a conventional telephone set, and wherein the informing means calls the registrant and notify the recorded message to the registrant.",
"13.The system according to claim 11, wherein the terminal of the registrant is a cellular phone, and wherein the informing means notifies the registrant that the message is recorded and cause the registrant to access for playback of the recorded message from the cellular phone.",
"14.The system according to claim 10, wherein the informing means informs the registrant of a message in a form of a character signal inputted by a push button operation as the information from the inquirer.",
"15.The system according to claim 14, wherein the inputted message is converted to an audible sound and the audible sound is informed to the registrant.",
"16.The system according to claim 10, the informing means informs the registrant of a message in a form of an e-mail as the information from the inquirer.",
"17.The system according to any one of claims 10 to 16, wherein the system is capable of setting one of a first condition and a second condition for each of the registrants, the first condition always informing the telephone number, the second condition always refusing to inform the telephone number in response to the request for the telephone number from the inquirer, as the disclosing condition registered in the data base.",
"18.The system according to any one of claims 10 to 16, wherein the telephone number of the registrant and the telephone number of the inquirer are corresponds to each other in the data base, and the system further comprising: means for accepting a request for a telephone number of the registrant from the inquirer; means for judging whether the registrant is registered in the telephone number data base; means for judging whether the information from the inquirer is transmitted to the registrant at least without informing the telephone number of the registrant to the inquirer, corresponding to a disclosing condition registered in the data base when the registrant is registered in the telephone number data base; means for causing the inquirer to input information and the registrant to be capable of being informed of the inputted information when informing the telephone number of the registrant.",
"19.The method according to claim 8, wherein the request for the telephone number of the registrant is accepted by accessing an old telephone number of the registrant.",
"20.The system according to claim 17, wherein the telephone number of the registrant and the telephone number of the inquirer are corresponds to each other in the data base, and the system further comprising: means for accepting a request for a telephone number of the registrant from the inquirer; means for judging whether the registrant is registered in the telephone number data base; means for judging whether the information from the inquirer is transmitted to the registrant at least without informing the telephone number of the registrant to the inquirer, corresponding to a disclosing condition registered in the data base when the registrant is registered in the telephone number data base; means for causing the inquirer to input information and the registrant to be capable of being informed of the inputted information when informing the telephone number of the registrant."
],
[
"<SOH> BACKGROUND ART <EOH>In the telephone directory assistance service of this kind, a person, who owns his/her telephone number (registrant), can select disclosure or private.",
"For example, when a certain registrant does not want to make his/her telephone number known to others, the registrant registers his/her telephone number as private and this prevents the telephone number from being known by others via the telephone directory assistance service, so that the registrant's privacy is protected."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a view showing the schematic entire configuration of a first embodiment to which the present invention is applied; FIG.",
"2 is a view showing the configuration of a telephone number data base; FIG.",
"3 is a view showing the configuration of a message box; FIG.",
"4 is a view showing the entire configuration of this system including an information processing unit; FIG.",
"5 is a flowchart (No.1) showing an operation when an inquirer gains access to an old telephone number and automatically receives service from the system; FIG.",
"6 is a flowchart (No.2) showing an operation when an inquirer gains access to an old telephone number and automatically receives service from the system; FIG.",
"7 is a flowchart (No.",
"3) showing an operation when an inquirer gains access to an old telephone number and automatically receives service from the system; FIG.",
"8 is a schematic diagram showing the entire configuration of a second embodiment of the present invention; FIG.",
"9 is a block diagram showing the configuration of a changer data base; FIG.",
"10 is a block diagram showing the configuration of an inquirer data base; FIG.",
"11 is a schematic block diagram showing an address inquiry system; FIG.",
"12 is a flowchart showing the registration process of changer information; FIG.",
"13 shows an example of an interface screen for explaining the registration of the changer information; FIG.",
"14 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"15 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"16 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"17 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"18 is a first flowchart showing the process of registration of inquirer information and inquiry about a new address; FIG.",
"19 shows an example of an interface screen for explaining the registration of inquirer information; FIG.",
"20 shows an example of the interface screen for explaining the registration of the inquirer information; FIG.",
"21 shows an example of the interface screen for explaining the registration of the inquirer information; FIG.",
"22 shows an example of the interface screen for explaining the registration of the inquirer information; FIG.",
"23 is a block diagram showing the configuration of an inquirer terminal in which special-purpose software for inquiry is installed; FIG.",
"24 is a second flowchart showing registration of inquirer information and the process of inquiry about the new address; FIG.",
"25 shows an example of an interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"26 shows an example of the interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"27 shows an example of the interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"28 shows an example of the interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"29 is a third flowchart showing the process of registration of inquirer information and inquiry about the new address; FIG.",
"30 shows an example of an interface screen for explaining an inquiry operation by an address inquiry system; FIG.",
"31 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"32 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"33 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"34 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"35 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"36 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"37 shows an example of an interface screen for explaining another embodiment of the present invention; FIG.",
"38 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; and FIG.",
"39 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to a telephone directory assistance method and telephone directory assistance system that are applicable to, for example, a telephone directory assistance service that informs a telephone number by telling an operator of an address, a name, etc., or service that informs a new telephone number using an old telephone number as a clue.",
"BACKGROUND ART In the telephone directory assistance service of this kind, a person, who owns his/her telephone number (registrant), can select disclosure or private.",
"For example, when a certain registrant does not want to make his/her telephone number known to others, the registrant registers his/her telephone number as private and this prevents the telephone number from being known by others via the telephone directory assistance service, so that the registrant's privacy is protected.",
"DISCLOSURE OF INVENTION However, in such service, there is such a disadvantage that the registrant cannot make his/her telephone number known to even a person whom he/she wants to inform since all persons cannot know the telephone number of the registrant as well when the telephone number is registered as private.",
"Particularly, in recent years, cellular phone replacements are frequently carried out and this extremely increases the opportunities of changing the telephone number, so that the aforementioned disadvantage becomes obvious.",
"As an actual problem, when his/her telephone number is changed, the aforementioned problem can be solved to some degree by individually telling only a person whom he/she wants to inform of his/her telephone number as private in the above directory service.",
"However, the registrant cannot help feeling the annoyance of telling his/her telephone number individually in some cases.",
"Moreover, communication with the person whom he/she has desired to inform originally cannot be carried out afterward by the leakage in some instances.",
"The present invention has been made with consideration given to the aforementioned problems and an object of the present invention is to provide a directory assistance method and telephone directory assistance system that are capable of surely informing his/her telephone number to a person whom he/she wants to inform without requiring time and labor in a directory assistance service.",
"In order to attain the above object, according to a first aspect of the present invention, in a method for informing a telephone number of a registrant to an inquirer, comprising the steps of accepting a request for a telephone number of the registrant from the inquirer, judging whether the registrant is registered in a telephone number data base, judging whether an information from the inquirer is transmitted to the registrant at least without informing the telephone number of the registrant to the inquirer, based on a disclosing condition registered in the data base when the registrant is registered in the telephone number data base, and causing the inquirer to input information and the registrant to be capable of being informed of the input information when informing the telephone number of the registrant.",
"According to a second aspect of the present invention, in a system informing a telephone number of a registrant to an inquirer, comprising a data base in which at least a telephone number of a registrant and a disclosing condition on whether an information from the inquirer is transmitted to the registrant without informing the telephone number to the inquirer, are associated with each other, and means for informing the registrant of the information from the inquirer.",
"The present invention is applicable to a telephone directory assistance service that informs a telephone number by informing an address, a name and the like to an operator or service that informs a new telephone number using an old telephone number as a clue.",
"According to the present invention, once there is a request from the inquirer for a telephone number, whether an information from the inquirer is transmitted to the registrant without informing the telephone number of the registrant to the inquirer is judged, and causing the inquirer to input information and the registrant to be capable of being informed of the inputted information when informing the telephone number of the registrant.",
"The registrant decides whether the telephone number is informed to the inquirer, based on the informed information.",
"In a case of informing, for example, the registrant may inform it to the inquirer via a telephone and the like directly, so that he/she can be sure to inform a person who has informed his/her telephone number without requiring time and labor.",
"Here, the inquirer may be requested to input information and the inputted message may be caused to be capable of being played back to the registrant.",
"In such case, when a terminal of the registrant is a conventional telephone set, the recorded message may be transmitted to the registrant and when a terminal of the registrant is a cellular phone, the registrant is informed that the message is recorded and he/she may be caused to gain access for the playback of the recorded message from the cellular phone.",
"In this way, the voice message is used as information to be transmitted to the registrant from the inquirer, so that burdensome operations may be reduced.",
"Moreover, the inquirer may be requested for a message with a character signal inputted by a push button operation as the information and the inputted message may be informed to the registrant.",
"In such case the inputted message may be converted to a vocal sound and informed to the registrant.",
"In addition, the inquirer may be requested to transmit an e-mail as the information and the registrant may be caused to receive the transmitted e-mail.",
"With this, the telephone number of the inquirer and the like may be known without fail.",
"Furthermore, as the disclosing condition registered in the data base, in response to the request for the telephone number from the inquirer, the condition that always inform the telephone number and the condition that always refuses to inform the telephone number may be able to be set for each registrant and the request for the telephone number of the registrant may be accepted by accessing an old telephone number of the registrant, and the corresponding telephone number may be searched from the data base in which the old telephone number and the new telephone number of the registrant are associated with each other.",
"Other features and advantages of the present invention will become more apparent from the following description and the accompanying drawings.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a view showing the schematic entire configuration of a first embodiment to which the present invention is applied; FIG.",
"2 is a view showing the configuration of a telephone number data base; FIG.",
"3 is a view showing the configuration of a message box; FIG.",
"4 is a view showing the entire configuration of this system including an information processing unit; FIG.",
"5 is a flowchart (No.1) showing an operation when an inquirer gains access to an old telephone number and automatically receives service from the system; FIG.",
"6 is a flowchart (No.2) showing an operation when an inquirer gains access to an old telephone number and automatically receives service from the system; FIG.",
"7 is a flowchart (No.",
"3) showing an operation when an inquirer gains access to an old telephone number and automatically receives service from the system; FIG.",
"8 is a schematic diagram showing the entire configuration of a second embodiment of the present invention; FIG.",
"9 is a block diagram showing the configuration of a changer data base; FIG.",
"10 is a block diagram showing the configuration of an inquirer data base; FIG.",
"11 is a schematic block diagram showing an address inquiry system; FIG.",
"12 is a flowchart showing the registration process of changer information; FIG.",
"13 shows an example of an interface screen for explaining the registration of the changer information; FIG.",
"14 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"15 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"16 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"17 shows an example of the interface screen for explaining the registration of the changer information; FIG.",
"18 is a first flowchart showing the process of registration of inquirer information and inquiry about a new address; FIG.",
"19 shows an example of an interface screen for explaining the registration of inquirer information; FIG.",
"20 shows an example of the interface screen for explaining the registration of the inquirer information; FIG.",
"21 shows an example of the interface screen for explaining the registration of the inquirer information; FIG.",
"22 shows an example of the interface screen for explaining the registration of the inquirer information; FIG.",
"23 is a block diagram showing the configuration of an inquirer terminal in which special-purpose software for inquiry is installed; FIG.",
"24 is a second flowchart showing registration of inquirer information and the process of inquiry about the new address; FIG.",
"25 shows an example of an interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"26 shows an example of the interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"27 shows an example of the interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"28 shows an example of the interface screen for explaining the inquiry process in which the special-purpose software for inquiry is used; FIG.",
"29 is a third flowchart showing the process of registration of inquirer information and inquiry about the new address; FIG.",
"30 shows an example of an interface screen for explaining an inquiry operation by an address inquiry system; FIG.",
"31 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"32 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"33 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"34 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"35 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"36 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; FIG.",
"37 shows an example of an interface screen for explaining another embodiment of the present invention; FIG.",
"38 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system; and FIG.",
"39 shows an example of the interface screen for explaining the inquiry operation by the address inquiry system.",
"BEST MODE FOR CARRYING OUT THE INVENTION Preferred embodiments of the present invention will be explained below with reference to the drawings.",
"(First Embodiment) FIG.",
"1 is a view showing the schematic entire configuration of a first embodiment to which the present invention is applied.",
"In FIG.",
"1, reference numeral 201 indicates a public network, a conventional telephone set 203 is connected to the public network 201 via a station 202.Moreover, a cellular phone 205 is wirelessly connected thereto via a radio base station 204.Furthermore, a telephone directory assistance service center 206 is connected to the public network 201.As the telephone directory assistance service center 206, for instance, in the case of Japan as an example, there is conventionally used a center, which is provided by NTT, offers serve by connection with a telephone number “104.” In addition to this, dedicated centers, which are provided separately therefrom, may be, of course, used.",
"In the telephone directory assistance service center 206, an operator in the center basically informs the telephone number to a person, who desires to be informed about telephone number, (hereinafter referred to as “inquirer” in the first embodiment) by voice.",
"Additionally, the service on telephone number may be automatically executed by access to the old telephone number from the inquirer.",
"In the telephone directory assistance service center 206, there is provided a telephone directory assistance system 207, which is used to provide service on the telephone number.",
"The telephone directory assistance system 207 is mainly composed of an information processing unit 209 for performing processing such as telephone number searching, etc., a telephone number data base 210 in which various kinds of data on the telephone numbers are registered and a message box 211.FIG.",
"2 is a view showing the configuration of telephone number data base 210.As shown in FIG.",
"2, the telephone number data base 210 stores at least registrant information 217, which includes a telephone number 212 of a person, who has received the registration of the telephone number to be subjected to service by the telephone directory assistance service center 206, (hereinafter referred to as “registrant” in the first embodiment) a name or title 213, an address or residence 214, an old telephone number 215, an e-mail address 216, and registrant attribute information 244 including an occupation and others, a telephone number disclosing condition 218, a service refusal list 219 that stores a list of inquirers, who refuse service on the telephone number, and notifying method information 220 that stores notifying methods of information from inquirers to the registrants.",
"In the telephone number disclosing condition 218, there are preregistered operator's response patterns occurred when the operator receives information indicating name of the registrant, address, old telephone number from the inquirer or patterns occurred when the system automatically responses.",
"In this embodiment, the registrant can set by selecting one from the following three kinds of responses (1) to (3): (1) “the telephone number of the registrant is always disclosed to all inquirers.” (221 in FIG.",
"2).",
"This response pattern means that the telephone number of the registrant is always disclosed to all inquirers instead of the point that information from the inquirer is informed to the registrant without informing the inquirer of the telephone number.",
"According to this embodiment, this item is selected, thereby the telephone number of the registrant is always disclosed to all inquirers unconditionally.",
"However, a telephone number disclosure list is prepared so that the telephone number of the registrant may be disclosed to only inquirers that match this list.",
"(2) “The telephone number of the registrant is always refused to be disclosed to all inquirers.” (222 in FIG.",
"2).",
"This response pattern means that the telephone number of the registrant is always rejected to be disclosed to all inquirers instead of the point that information from the inquirer is inform to the registrant without informing the inquirer of the telephone number.",
"Additionally, similar to the aforementioned response, a telephone number non-disclosure list is prepared so that the telephone number of the registrant may be refused to be disclosed to only inquirers matching this list.",
"(3) “Information from the inquirer is notified the registrant without offering telephone directory service to the inquirer.” (223 in FIG.",
"2).",
"This response pattern means that information from the inquirer is informed to the registrant without informing the inquirer of the telephone number.",
"For example, as will be described later, this notification is one in which the inquirer is caused to input a voice message and the input voice message is informed to the registrant.",
"Meanwhile, in this embodiment, the inquirers, who are desirably refused to offer the service, are registered in the service refusal list 219, making it possible to set to refuse the service to the inquirers registered in this service refusal list 219 even if the pattern (1) or (3) is selected.",
"In the service refusal list 219, it is possible to register a person, who is desirably rejected to provide the service, in advance.",
"However, in this embodiment, even if the registrant performs refusal registration by a terminal operation later according to the pattern (2), the inquirer is automatically registered in the service refusal list 219.In the notification method information 220, there are stored notification methods of information when the response pattern is the aforementioned (3) (223 in FIG.",
"2).",
"In this embodiment, the registrant can set a method by selecting one from the following four kinds of notification methods (1) to (4).",
"In addition, after such setting, the registrant can gain access to the telephone directory assistance service center 206 to change the setting.",
"For example, a voice message is first set to be received at a designated telephone number.",
"However, thereafter, the registrant may directly gain access to the telephone directory assistance service center 206 to provide a predetermined operation to the telephone directory assistance system 207 or directly request the operator of the telephone directory assistance service center 206, thereby making it possible to change the setting such that reception is performed via an e-mail at the telephone number or reception is performed at another telephone number.",
"(1) Notification via voice (No.",
"1) (224 in FIG.",
"2) This notification method is particularly useful when the registrant is the general telephone user.",
"The inquirer is requested to record a voice message, and a transmission is given to the registrant, so that the recorded voice message is informed from the message box 211.",
"(2) Notification via voice (No.",
"2) (225 in FIG.",
"2) This notification method is particularly useful when the registrant is the cellular phone user.",
"The inquirer is requested to record a voice message.",
"The registrant is notified that the message has been recorded to cause him/her gain access for playback of the recorded message from the cellular phone.",
"For example, when the message is recorded as mentioned above, the center side sends a signal “presence of message,” to the cellular phone of the registrant, and the registrant gains access to the center side and plays back the message from the message box 211.",
"(3) Notification using character information via a tone signal (226 in FIG.",
"2) This notification method means that the inquirer is requested to make a message by inputting a character signal via a push button operation (tone signal) to inform the registrant of the input message.",
"Regarding the notification method to the registrant, for example, the character signal may be directly transmitted to the terminal of the registrant in the same manner as the short mail and pocket bell (pager) or the character signal may be converted to voice to be transmitted to the terminal of the registrant.",
"(4) Notification via an e-mail (227 in FIG.",
"2) This notification method means that the inquirer is requested to transmit an e-mail to cause the registrant to receive the transmitted e-mail.",
"The e-mail may be once transmitted to the registrant via the center.",
"Or, the e-mail address of the registrant is presented to the inquirer from the center so that the inquirer may directly transmit the e-mail to the registrant.",
"FIG.",
"3 is a view showing the configuration of message box 211.As shown in FIG.",
"3, in the message box 211, there are stored at least a telephone number of the inquirer 228, a message 229 via voice or data inputted by the inquirer, a telephone number of the registrant 230, and a notification method 231 to the registrant.",
"FIG.",
"4 is a view showing the entire configuration of this telephone directory assistance system 207 including the information processing unit 209.In the telephone directory assistance system 207, the information processing unit 209, telephone number data base 210 and message box 211 are connected to a bus 236 to which a CPU 232, a RAM 233, a ROM 234, and various kinds of communication interfaces 235 are connected.",
"As shown in FIG.",
"4, the information processing unit 209 is composed of at least a service request acceptor 237 that accepts a request for the telephone number of the registrant based on the old telephone number informed from the inquirer, a registration judge 238 that judges whether the registrant is registered in the telephone number data base 210 based on the old telephone number, name, address and the like, a disclosure judge 239 that judges whether service on the telephone number is offered or refused to the inquirer or the message from the inquirer is informed to the registrant without informing the inquirer of the telephone number based on the disclosing condition 218 registered in the telephone number data base 210, a message storage processing unit 240 that performs processing for urging the inquirer to input the message to store the input message to the message box 211, a message notification processing unit 241 that informs the registrant of the message stored in the message box 211 according to the notification method 210, a telephone directory service unit 242 that informs the telephone number that is possible to be disclosed to the inquirer, and a service refusal processing unit 243 that performs registration processing and service refusal processing to the service refusal list 219.These elements 237 to 243 and the telephone number data base 210, and the message box 211 are practically composed of computer programs installed in a storage medium of a computer system as shown in FIG.",
"4, and show various functions of this invention by being read and executed in the RAM 223 by means of the CPU 232.The explanation will be next given of the telephone directory service method using the above-configured telephone directory assistance system 207.As a telephone directory service method using the telephone directory assistance system 207, for example, there are one case in which the inquirer gains access to the center 206 to receive serve via the operator and the other case in which the telephone directory assistance system 207 automatically accepts service when the inquirer (a person who simply makes a call) gains access to the old telephone number (this is a mere telephone number to the inquirer).",
"An explanation will be first given of an operation that is performed when the telephone directory assistance system 207 automatically accepts service based on the flowchart of FIG.",
"5.For example, when the inquirer originates a call to the registrant using the old telephone number via the cellular phone 205, this call is transferred to the center 206 via, for example, the public network 201.When the telephone directory assistance system 207 of the center 206 receives the call (step 501), the registration judge 238 searches whether the registrant is registered in the telephone number data base 210 based on the old telephone number included in the call in the telephone directory assistance system 207 (step 502).",
"When it is registered in the telephone number data base 210, the service request acceptor 237 accepts the call from the inquirer as a service request for the telephone number (step 503).",
"On the other hand, when it is not registered in the telephone number data base 210, a message, for example, “This telephone is not available at present.” is sent to the inquirer (step 504) and this process ends.",
"In addition, when the need arises to select a target registration from a plurality of persons in a case in which there are registrations each having a person with the same family and personal name in the same residential area or a case in which there are registrations corresponding to the old telephone number because of time lag before and after the period of service, “registrant attribute information 244 including an occupation and others” relating to each registrant is presented to the inquirer, making it possible to assist the inquirer with his/her selection.",
"In other words, the inquirer may select the target registrant based on the presented “registrant attribute information 244 including an occupation and others.” After acceptation of the service request, the service rejection processing unit 243 judges whether the inquirer is listed based on the service refusal list 219 (step 505).",
"Then, when it is listed, a message, for example, “This telephone is not available at present.” is sent to the inquirer (step 504) and this processing ends.",
"On the other hand, when the inquirer is not listed in the service refusal list 219, the disclosure judge 239 judges the disclosing condition of the registrant corresponding to the old telephone number based on the disclosing condition 218 (step 506).",
"Here, when the disclosing condition 218 is that “the telephone number of the registrant is always disclosed to all inquirers” (step 507), the telephone directory service unit 242 informing the telephone number of the registrant (step 508), and the processing ends.",
"In the service on the telephone number, means such as voice, a tone signal, an e-mail, etc can be used.",
"When the disclosing condition 218 is that “the telephone number of the registrant is always refused to be disclosed to all inquirers” (step 509), a message, for example, “This telephone is not available at present.” is sent to the inquirer (step 504) and this processing ends.",
"When the disclosing condition 218 is that “information from the inquirer is informed to the registrant without informing the inquirer of the telephone number” (step 510), the message storage processing unit 240 sends a message, for example, “This contractor's telephone number has been transferred.",
"A message can be sent to the transfer.",
"Input the message after the tone.” to the inquirer (step 511).",
"Then, a message of the inquirer, for example, “This is Paul.",
"Please tell me John's new telephone number.",
"Contact me at this telephone number: 090-0000-0000.” is stored in the message box 211 (step 512).",
"In the message box 211, this message 229 is stored to be associated with the telephone number of the inquirer 228, telephone number of the registrant 230, and the notification method 231 to the registrant.",
"In addition, the message may be input in the form of dialogue.",
"For example, messages are exchanged between the center and the inquirer in such a form that “First of all, your name, please.” (Center), “This is Taro Shokai.” (Inquirer), “Next, your message, please” (Center), “I had a talk with you at the forum sponsored by .",
".",
".",
", previously.",
"I want to talk with you over business.” (Inquirer), “Finally, your contact telephone number, please.” (Center), “My contact telephone number is 00?1111?2222.You can contact me between nine in the morning and eight in the evening.” (Inquirer).",
"The center sends the registrant the voice message to which the above voice message of the inquirer are combined: “This is Taro Shokai.",
"I had a talk with you at the forum sponsored by .",
".",
".",
", previously.",
"I want to talk with you over business.",
"You can contact me between nine in the morning and eight in the evening.” This obtains an effect that stops the inquirer from forgetting to record the telephone number for return or the name of the inquirer and further an effect that helps to prevent a prank telephone call.",
"When the messages are stored in this way, the message notification processing unit 241 provides notification to the registrant according to the notification method 231 (step 513).",
"FIG.",
"6 is a flowchart showing an operation of message notification to the registrant by the message notification processing unit 241.First of all, the message notification processing unit 241 judges the type of notification method 231 (step 601).",
"When the notification method is “notification via voice (No.",
"1)” (step 602), a transmission is given to the registrant and the recorded message is informed from the message box 211 (step 603).",
"Moreover, when the notification method is “notification via voice (No.",
"2) (step 604), the registrant is informed that the message has been recorded in his/her cellular phone (step 605).",
"After that, as shown in FIG.",
"7, when the registrant gains access to the center 206 (step 701), the message is played back from the message box 211 and sent to the registrant (step 702).",
"Thereafter, when, for example, the registrant presses a specific button on the cellular phone to input registration refusal (step 703), the inquirer is listed in the service refusal list 219 (step 704).",
"As a result, even if the inquirer makes a telephone call using the old telephone number afterward, any message is not informed to the registrant.",
"From the message, the registrant can know that the inquirer has the will to take over via a telephone.",
"Then, when the registrant desires to inform this inquirer of a new telephone number, the registrant may call the inquirer to tell the new telephone number.",
"When the registrant does not desire to inform this inquirer of a new telephone number, the registrant does not have to make contact with the inquirer.",
"Therefore, according to this embodiment, since confirmation is sent to the registrant using the message every time when an inquire is sent from such the inquirer, it is possible to surely inform his/her telephone number to a person who he/she desires to inform without requiring time and labor in the directory assistance service.",
"Additionally, in the aforementioned embodiment, the voice message is informed to the registrant from the inquirer via the center.",
"However, in addition to such the message notification method, notification using character information via a tone signal (226 in FIG.",
"2) may be used and notification via an e-mail (227 in FIG.",
"2) may be used.",
"Moreover, the aforementioned embodiment explained the case in which the telephone directory assistance system 207 automatically accepted service when the inquirer gained access to the old telephone number.",
"However, the present invention is applicable to a case in which, for example, the inquirer gains access to the center 206 and receives service via the operator.",
"In this case, when the inquirer gains access to the center 206, the operator answers to the inquirer and asks him/her to tell an old telephone number, an address, a name and the like that correspond to a telephone number on which he/she desires to receive service, and inputs them to the telephone directory assistance system 207.As a result, the same operations as the aforementioned embodiment (operations including step 502 in FIG.",
"5 and afterward) are carried out as follows.",
"More specifically, according to the aforementioned first embodiment, upon reception of the presentation of the old telephone number from the inquirer, the center informs the new telephone number, refuses to inform, and sends a message to the registrant.",
"However, the present invention is not limited to this.",
"Namely, the center receives the presentation, which is a so-called “clue”, which can specify the registrant, that is, any information relating to the registrant such as an address, a name and the like, from the inquirer as well as the presentation of the old telephone number from the inquirer.",
"Then, the center informs the telephone number of the specified registrant, rejects the service, and sends the message to the registrant.",
"Then, the service on the telephone number, rejection of the service, and notification of message to the registrant are left on the registrant's will.",
"Thereby, the registrant can arbitrarily select whether he/she “tells/does not tell” his/her telephone number at his/her will with respect to the service request for the telephone number from a certain inquirer.",
"Moreover, the present invention is intended to adaptively deal with such a way of selection in accordance with the registrant and inquirer circumstances (for example, the type of terminal they possess).",
"In addition, the aforementioned first embodiment implements the present invention using the public network.",
"However, the present invention can be implemented even in the other network system such as the Internet and the like.",
"In this case, it is possible to use a cellular phone, a personal computer and the like as terminals for the inquirer and registrant.",
"The respective elements of the second embodiment may be adopted to the system of this embodiment as much as possible.",
"For example, a registration method, a changer authentication method and an inquirer authentication method to be described later may be, of course, applied to the system of this embodiment.",
"(Second Embodiment) As shown in FIG.",
"8, an address inquiry system 1 according to this one embodiment is connected to an Internet 2 and structured to be able to bi-directionally communicate with a terminal (a changer terminal 4) of a changer 3 and a terminal (an inquirer terminal 6) of an inquirer 5.The changer 3 is defined here as an individual or a corporation (hereinafter defined as an individual in this embodiment) who has moved, for some reason, from an old address to a new address, has changed his or her own address from the old address to the new address, or has stopped the use of the old address.",
"The inquirer 5 is defined here as an individual or a corporation (hereinafter defined as an individual in this embodiment) which inquires about the new address of the changer 3 since an access by the old address of the changer 3 is refused in transmitting e-mail, requesting data, and the like to the old address.",
"Further, an address is defined here as information for specifying a position of his or her own in communication by the use of existing communication means, and an e-mail address, an IP address, a URL, a telephone number, a mail address, and the like are given as its examples.",
"When these addresses are unknown or incorrect, it is impossible to communicate with the other party through the use of their corresponding communication systems.",
"An object of the present is to provide the address inquiry system 1 capable of flexibly coping with an intention of the changer 3 when the inquirer 5 inquires about the new address based on the old address of the changer 3.Elements of this embodiment will be explained below in detail.",
"This address inquiry system 1 is mainly composed of a changer data base 10 storing information about the changer 3, an inquirer data base 11 storing information about the inquirer 5 who inquires about the new address, and an information processing unit 12 processing the inquiry from the inquirer 5 based on the information stored in these data bases 10 and 11.These elements will be explained below.",
"(Changer Data Base) As shown in FIG.",
"9, the changer data base 10 stores a changer authenticating information 14 including a changer ID 21 and a changer password 22 for specifying the changer 3, change information 15 including one, or two or more old addresses 23 and a new address 24, a changer attribute information 16 storing the property of the changer 3, a changer profile information 33 for characterizing the changer 3, an address disclosing condition 17 storing responses depending on the inquirers 5, an inquiry refusal list 18 storing a list of the inquirers 5 who are refused an inquiry, a confirmation form format 19 which is sent to the changer 3 to confirm whether the new address 24 is disclosed or not, and a changer reference address 20 (an e-mail address) provisionally assigned to the changer 3.The changer authenticating information 14 such as the changer ID 21 and the changer password 22 is information required for security when information is registered or changed in the changer data base 10 and issued to the changer 3 who has registered the new address 24 and the like in the address inquiry system 1.The change information 15 is composed of a disclosure rule 25 in addition to the old address 23 and the new address 24 described above.",
"The sorts of addresses which can be registered in this embodiment are four sorts such as an e-mail address, a home page URL, a telephone number, and an address, and the changer 3 is requested to register the old address 23 and the new address 24 concerning at least one or more sorts out of the aforesaid sorts.",
"In this embodiment, after the old address 23 and the new address 24, codes 26 and 27 denoting the sorts of these addresses are given (FIG.",
"9).",
"Incidentally, two or more old addresses 23 or new addresses 24 may be registered per one sort.",
"Moreover, by setting the disclosure rule 25, the changer 3 can freely set the sort of the new address 24 to be disclosed when receiving an inquiry with the presentation of each old address 23.Namely, when any of the aforesaid four sorts of old addresses 23 is presented, the new address 24 different from the presented old address 23 in sort can be disclosed or a plurality of new addresses 24 can be disclosed.",
"For example, such setting that a new e-mail address is disclosed when an inquiry with the presentation of an old telephone number is received, or a new e-mail address and a new home page URL are disclosed when an inquiry with the presentation of an old mail address is received can be performed.",
"The changer attribute information 16 is one in which the changer 3 previously registers information other than the old address 23 such as a name and an address capable of distinguishing and specifying the changer 3 himself or herself.",
"The changer profile information 33 is one in which the changer 3 previously registers a profile other than the changer attribute information 16 such as a self-introduction capable of characterizing the changer 3.For example, in many cases, an e-mail address is composed by a combination of alphabets, numbers and symbols, and the like, and the owner thereof cannot be specified by this e-mail address only.",
"Therefore, the changer attribute information 16 and the changer profile information 33 are useful information for confirming whether the owner of the address is the changer 3 of whom the inquirer 5 wishes to inquire.",
"Response patterns (the numerals 28 to 30 in FIG.",
"9) of this address inquiry system 1 in the case where an inquiry with the presentation of the old address 23 is received from the inquirer 5 are previously registered in the address disclosing condition 17.In this embodiment, the changer 3 can set a response by selecting one from the following three sorts of responses (1) to (3).",
"(1) “Disclose a new address instantly.” (the numeral 28 in FIG.",
"9) This response pattern means that the new address 24 is disclosed to the specific inquirer 5 or the inquirer 5 which fulfills a fixed condition without making the changer 3 confirm whether the new address 24 is disclosed or not.",
"In this embodiment, the new address 24 is disclosed to all the inquirers 5 unconditionally by the selection of this item.",
"Incidentally, it is suitable that a new address disclosure list is prepared and that the new address 4 is disclosed only to the inquirers 5 included in this list without the confirmation of the changer 3.",
"(2) “Make the changer 3 judge whether to disclose a new address by transmitting information about the inquirer 5 without disclosing the new address Instantly.” (the numeral 29 in FIG.",
"9) This response pattern means that when the inquirer 5 which inquires about an address fulfills or does not fulfill a specific condition, the information of this inquirer 5 is transmitted to the changer 3 for confirmation.",
"In this case, the changer 5 can judge whether to give permission to disclose the new address 24 after confirming who the inquirer 5 is, for what purpose the inquirer 5 requests the disclosure of the new address 24, and the like from the information about the inquirer 5.",
"(3) “Refuse an inquiry about a new address instantly.” (the numeral 30 in FIG.",
"9) This response pattern means that when the inquirer 5 which is going to inquire about an address fulfills or does not fulfill a specific condition, the inquiry about the address is automatically refused.",
"Incidentally, in this embodiment, when this item is selected, inquiries from all the inquirers 5 are refused unconditionally.",
"Meanwhile, in this embodiment, by registering the inquirers 5, inquiries from whom the changer 3 wishes to refuse, in the inquiry refusal list 18, setting such that inquiries from the inquires 5 registered in this inquiry refusal list 18 are refused even when the pattern (1) or the pattern (2) is selected is possible.",
"It is possible to previously register the other parties, inquiries from whom the changer 3 wishes to refuse, in the inquiry refusal list 18.In this embodiment, however, also when the response of “Do not disclose new address, and refuse inquiry from this inquirer hereafter” (See the hyperlink 112 in FIG.",
"30) is selected when the changer 3 makes a judgment in the pattern (2), an e-mail address of this inquirer 5 is automatically registered in the inquiry refusal list 18.In this embodiment, the respective responses of the patterns (1) to (3) can be set in each of the registered old addresses 23.For example, it is possible that when an inquiry about the new address 24 with the presentation of an old home page URL is received, “Disclose a new address instantly” (the pattern (1)) is selected, and that when an inquiry with the presentation of an old telephone number is received, the response of “Make a changer judge whether to disclose a new address by transmitting information about an inquirer without disclosing the new address instantly.” (the pattern (2)) is sent through the address inquiry system 1.Meanwhile, in the confirmation form format 19, the format of the confirmation form for the permission of disclosure sent from the address inquiry system 1 to the changer 3 can be set in the case of the response pattern (2).",
"Namely, in this embodiment, as will be explained later, the inquirer 5 first makes out an inquiry message for the address inquiry system 1, and the address inquiry system 1 draws up the confirmation form for the changer 3 based on this inquiry message.",
"It is possible to include an important matter (a message text and the like) from the inquirer 5 in this confirmation form.",
"In the confirmation form format 19, “with message text” 31 or “without message text” 32, which means that a message text included in an inquiry message from the inquirer 5 is included or not included in the confirmation form for the changer 3, can be selected.",
"As will be explained later, even if the “without message text” 32 is set in the confirmation form format 19, the changer 3 can obtain the message text from the inquirer 5 by e-mail later.",
"The changer reference address 20 is an e-mail address issued by the address inquiry system 1 to exchange communications including the confirmation form with the changer 3, and issued when the changer 3 has no e-mail address or does not want to use its own e-mail address even if he or she has the address, or issued for ensuring security.",
"(Inquirer Data Base) Meanwhile, as shown in FIG.",
"10, the inquirer data base 11 stores an inquirer authenticating information 35 composed of an inquirer ID 41 and an inquirer password 42 for authenticating the inquirer 5, an inquiry log 36, an inquirer attribute information 37, an inquirer profile information 38, an inquirer reference address 39, and the like.",
"The inquirer authenticating information 35 such as the inquirer ID 41 and the inquirer password 42 is necessary information in terms of security when a demand for the search for the new address 24 from the inquirer 5 is executed and its result is confirmed.",
"The inquiry log 36 is information for monitoring an inquiry process by the inquirer 5, and for example, utilized for confirming whether a response from the changer 3 to the confirmation is within a predetermined period.",
"The inquirer attribute information 37 is information about the inquirer 5 presented to the changer 3 for making the changer 3 confirm whether the new address 24 is disclosed to the inquirer 5.In this embodiment, this inquirer attribute information 37 is composed of a name, an address (an e-mail address, a telephone number) and the like of the inquirer 5.The inquirer profile information 38 is a brief composition of introduction for characterizing the inquirer 5 and registered to characterize the inquirer 5 in making the changer 3 confirm whether to disclose the new address 24.The registration of the inquirer profile information 38 is left to the discretion of the inquirer 5, but it is desirable to persuade the inquirer 5 to register it as much as possible since there is a case where the changer 3 refuses the disclosure of the new address 24 since the inquirer 5 is not identified only by an e-mail address and the like.",
"The inquirer reference address 39 is an e-mail address issued by the address inquiry system 1 to exchange communications including the inquiry message with the inquirer 5, and issued when the inquirer 5 has no e-mail address or does not want to use its own e-mail address even if he or she has the address, or issued for ensuring security.",
"(Information Processing Unit) Next, the information processing unit 12 will be explained.",
"FIG.",
"11 is a diagram showing the entire configuration of the address inquiry system 1 including this information processing unit 12.The information processing unit 12, the changer data base 10, and the inquirer data base 11 are connected to a bus 59 to which a CPU 55, a RAM 56, a ROM 56, and communication interfaces 58 of various kinds are connected.",
"As shown in FIG.",
"11, the information processing unit 12 of the address inquiry system 1 is composed of a changer authenticator 45 authenticating the changer 3, a changer register 46 receiving the change information 15 such as the new address 24 inputted by the changer 3 and storing it in the changer data base 10, an inquirer authenticator 47 authenticating the inquirer 5, an inquirer register 48 registering information about the inquirer 5 in the inquirer data base 11, a changer searcher 49 searching the changer data base 10 with the old address 23 of the changer 3 when the inquirer 5 inquires about the new address 24 of the changer 3, a changer selector 50 making the inquirer 5 select from the changers 3 on whom the inquiry about the new address 24 is executed when there exist plural changers 3 who fulfill the condition in the search, a new address disclosure judge 51 judging whether to disclose the new address 24 to the inquirer 5 based on the address disclosing condition 17 for the new address 24 of the changer 3, a changer confirmer 52 making the changer 3 confirm whether the new address 24 is disclosed or not, an inquiry process monitor 53 administering the period of each inquiry process, and a discloser 54 disclosing an inquiry result to the inquirer 5.These elements 45 to 54, the changer data base 10, and the inquirer data base 11 are practically composed of computer programs installed in a storage medium of a computer system as shown in FIG.",
"11 and show various functions of the present invention by being read and executed in the RAM 56 by means of the CPU 55.The detailed configurations and functions of the elements (45 to 54) will be explained below with reference to flowcharts and interface examples.",
"(Registration by Changer) FIG.",
"12 is a flowchart showing the registration process of changer information by the changer register 46 and the like.",
"The changer register 46 first judges whether the changer 3 concerned in an access has already registered changer information or not by inquiring of the changer data base 10 about it (step S1).",
"When the changer information is registered for the first time, the changer register 46 receives the information, registers it in the changer data base 10 (step S2), and thereafter issues the changer ID 21 and the changer password 22 to the changer 3 (step S3).",
"Meanwhile, when the changer 3 wishes to changes information which has been already registered, the changer authenticator 45 authenticates an access through the use of the changer ID 21 and the changer password 22 by making inquiries at the changer data base 10 (step S4), and thus a change and a renewal of information in the changer data base 10 are permitted (step S5).",
"FIG.",
"13 to FIG.",
"17 show examples of an interface screen when changer information (See FIG.",
"9) in the changer data base 10 is registered.",
"Although a series of registration processes are executed in connection with a Web site offered by the address inquiry system 1 in this example, registration may be performed by transmitting and receiving necessary information to/from the address inquiry system 1 by e-mail.",
"FIG.",
"13 is an example of an interface screen for registering the changer attribute information 16.The changer 3 inputs basic information such as a name 60, a present address 61, a telephone number 62, a FAX number 63, on this screen.",
"The changer register 46 registers this information as the changer attribute information 16 (See FIG.",
"9).",
"If the changer 3 wishes, the changer reference address 20 for receiving notice from the address inquiry system 1 is set and acquired through this screen.",
"Incidentally, in registering basic information about the changer 3, individual registration or corporate registration can be selected, and an example of the individual registration is shown here.",
"When the registration of the basic information is completed, the changer register 46 displays an interface screen shown in FIG.",
"14.This screen is an interface for registering the old address 23 and the new address 24 to be disclosed based on the old address 23 from the change information 15, and FIG.",
"14 shows an example of a screen for an e-mail address.",
"The changer 3 registers an address before change (the old address 23) necessary for receiving an inquiry and an address after change (the new address 24) which is disclosed when there is an inquiry on this screen.",
"In this embodiment, a plurality of (three in this embodiment) old addresses 23 can be registered and renewed at any time by the aforesaid process.",
"When the new address 24 to be disclosed is the same as that in the basic information (the changer attribute information 16) registered in the interface in FIG.",
"13, a button of “Same as basic information” 67 is pressed, whereby the basic information is transferred and repeated input can be avoided.",
"The changer register 46 also accepts the changer profile information 33 in an input section shown by the numeral 68 in this screen.",
"This changer profile information 33 is information given to the inquirer 5 to confirm and specify the other party (the changer 3) about whose address the inquirer 5 wishes to inquire as described above.",
"Subsequently, the changer register 46 displays an interface screen shown in FIG.",
"15.This interface screen is to register and set the address disclosing condition 17, the disclosure rule 25, and the confirmation form format 19 from the information in the changer data base 10 shown in FIG.",
"9.Namely, on this screen, the changer 3 registers the sort of the new address 24 to be disclosed to the inquirer 5 out of the registered new addresses 24 such as a new e-mail address and a new address, a condition of disclosure (including unconditional disclosure and unconditional non-disclosure) in the case where an inquiry is sent from the inquirer 5.The example in FIG.",
"8 shows the setting of the address disclosing condition 17, the disclosure rule 25, and the like when an inquiry with the presentation of an e-mail address as the old address 23 is sent.",
"In this example, “Make changer judge whether to disclose address to inquirer after acquisition of inquiry information (information about the inquirer 5).” (the pattern (2) shown by the numeral 29 in FIG.",
"9) is registered as the address disclosing condition 17, “New e-mail address” is registered as the sort of the new address 24 to be disclosed based on the e-mail address (the disclosure rule 25), and “Exclude message text (from the inquirer 5) from inquiry information (information about the inquirer 5)” is registered as the confirmation form format 19.The above information is registered in the changer data base 10 by the changer register 46.Meanwhile, FIG.",
"16 is an example of a registration screen of the inquiry refusal list 18 (See FIG.",
"9).",
"On this screen, the changer 3 inputs conditions such as e-mail addresses of the inquirers from whom the changer 3 wishes to refuse inquiries.",
"By pressing a “Registration” button 69 on this screen, these addresses are registered in the inquiry refusal list 18, and stored in the changer data base 10.Incidentally, this inquiry refusal list 18 can be renewed (added, deleted) at any time by the aforesaid process.",
"When the changer 3 finishes inputting changer information on each of the above screens, the changer register 46 displays a confirmation screen such as shown in FIG.",
"17.Incidentally, in order to confirm that the e-mail address registered by the changer 3 is owned by the changer himself or herself, it is suitable that the issued changer ID 21 is not displayed on this screen and that an e-mail notifying the completion of changer registration is separately transmitted from the address inquiry system 1 to the changer 3 to notify the changer ID 21 by this e-mail.",
"Moreover, an e-mail in which the same contents are described may be transmitted to the changer 3 without displaying this confirmation screen (FIG.",
"17).",
"(Registration by Inquirer) Next, the registration of inquirer information by the inquirer register 48 will be explained with reference to a flowchart shown in FIG.",
"18 and examples of an interface screen shown in FIG.",
"19 to FIG.",
"22.This registration of inquirer information is executed as a part of the process of an inquiry about the new address 24 from the inquirer 5.Namely, the inquiry from this inquirer 5 is executed when the inquirer 5 cannot get access to the changer 3 when he or she sends an e-mail to the old address 23 without knowing the change thereof or tries to refer to a home page by an old URL address without knowing the change thereof.",
"The inquirer register 48 first judges whether an inquiry from the said inquirer 5 is his or her initial inquiry by inquiring of the inquirer data base 11 about it (step S6).",
"When the inquirer 5 uses the address inquiry system 1 for the first time, that is, when the inquirer 5 inquires about an address for the first time, the inquirer register 48 accepts the registration of this inquirer 5 (step S7).",
"FIG.",
"19 and FIG.",
"20 are examples of screens for accepting the registration of information about the inquirer 5.FIG.",
"19 is an example of a screen in which the inquirer register 48 accepts the registration of attribute information of the inquirer 5, and the inquirer 5 registers basic information such as a name 70, an address 71, a telephone number 72, a FAX number 73 and sets and acquires the inquirer reference address 39 for receiving an inquiry result if he or she wishes.",
"Incidentally, in registering the basic information about the inquirer 5, individual registration or corporate registration can be selected, and an example of the individual registration is shown here.",
"The information inputted on this screen is registered as the inquirer attribute information 37 and the inquirer reference address 39 in the inquirer data base 11 (See FIG.",
"10).",
"Then, the changer selector 50 performs setting on receipt of a response to a search result on a screen shown in FIG.",
"20.Specifically, when the matching changers 3 are searched as a result of the search of the change information, it is selected that the inquiry is immediately made for (all) the matching changers 3 (a pattern shown by the numeral 76 in FIG.",
"20), that an inquiry is made after the selection of the changer 3 when a plurality of matching changers 3 are searched (a pattern shown by the numeral 77 in FIG.",
"20), or that the inquiry is made after a memo, a profile, or the like which characterizes the changer 3 is confirmed (a pattern shown by the numeral 78 in FIG.",
"20).",
"Thereafter, the inquirer register 48 displays a screen shown in FIG.",
"21 for the inquirer 5 which has acquired the inquirer reference address 39 on the registration screen of inquirer information in FIG.",
"19, and notifies him or her of account information.",
"After the completion of the aforesaid registration operations, the inquirer register 48 displays a screen of the completion of inquirer registration shown in FIG.",
"22.This screen shows a list of information contents inputted or selected by the inquirer 5 in order that the inquirer 5 confirms registration contents.",
"Incidentally, an e-mail in which the same contents are described may be sent to the inquirer 5.Thereafter, the address inquiry system 1 makes the inquirer 5 judge whether to download special-purpose software for assisting the inquiry about the address (step S8-1).",
"When this special-purpose software is downloaded, the download and set-up thereof is executed in step S8-2.Namely, after this special-purpose software is downloaded in the computer system of the inquirer 5, an installer is automatically started, and this software is installed in the inquirer terminal 6 at the request of the inquirer 5 (step S8-2).",
"This special-purpose software is automatically started by recognizing that e-mail is returned because its destination is unknown or that a URL address is not found as will be explained later (step S9), and has a function of assisting an access to the address inquiry system 1 and the search for the new address 24.Even if the inquirer 5 is not a person who sends an inquiry to the address inquiry system 1 for the first time (No in step S6), the special-purpose software is downloaded when it has not been downloaded yet (step S10).",
"When the special software is not downloaded in the aforesaid step S8-1, subsequent processes are executed on Web page provided by the address inquiry system 1 (step S8-3).",
"The inquiry process on this Web page is almost the same as the under-mentioned inquiry process by the use of the special-purpose software, and thus the explanation thereof is omitted.",
"(Inquiry by Inquirer) Next, the process of an inquiry about the new address 24 by the use of the special-purpose software will be explained.",
"FIG.",
"23 is a schematic block diagram showing the inquirer terminal 6 in which the special-purpose software is installed in a storage medium such as a hard disk.",
"FIG.",
"23 shows the configuration of only a software program related to the present invention, and the illustration of hardware such as a CPU and basic software such as an OS is omitted.",
"In the inquirer terminal 6 in which this software is installed, a browser 80 for browsing an Internet home page and a mailer 81 for transmitting and receiving e-mail are set up in advance.",
"The aforesaid software has e-mail account setter 82 setting e-mail account of the inquirer reference address 39 in the mailer 81, a starter 83 starting this software based on the receipt of a notice that an address is unknown by the browser 80 and the mailer 81, an inquiry message maker 84 making out an inquiry message for the address inquiry system 1 based on information from the browser 80 and the mailer 81, an inquiry executor 85 logging in the address inquiry system 1 and registering the inquiry message, and a new location displayer 86 displaying a new location (for example, a home page) based on the acquisition of the new address 24 of the new location as the result of the inquiry.",
"The starter 83 and the inquiry message maker 84 operate based on starting conditions stored in a starting condition storage shown by the numeral 87 in FIG.",
"23.Namely, in the starting condition storage 87, starting conditions such that the browser 80 and the mailer 81 automatically starts this software based on the receipt of a notice that an access with presentation of the old address 23 is refused are stored depending on the sort of the browser 80 and the mailer 81 as described above.",
"Moreover, this software has an inquirer authenticating information storage 88 storing the inquirer authenticating information 35.The inquiry message maker 84 automatically takes in the inquirer authenticating information 35 from this inquirer authenticating information storage 88, and the inquiry executor 85 logs in the address inquiry system 1 by using the inquirer authenticating information 35.The inquiry process of the new address 24 will be explained below by means of flowcharts in FIG.",
"24 and FIG.",
"29 and interface screen examples in FIG.",
"25 and the following figures.",
"When the aforesaid special-purpose software is used, the starter 83 starts the inquiry message maker 84 based on the receipt of an e-mail of a non-delivery notice, in which a message that a destination is unknown is described, by the mailer 81 (steps S11-1 and S11-2 in FIG.",
"17).",
"Incidentally, manual starting is also possible by changing starting conditions stored in the starting condition storage 87.FIG.",
"25 is a conceptual diagram showing operations at the time of the starting of inquiry software.",
"The inquiry message maker 84 has functions of displaying an input screen for an inquiry message such as shown by the numeral 90 in FIG.",
"25 in the inquirer terminal 6 and transferring a sender 93, a destination 94, a subject 95, a message text 96, and the like out of the contents of original e-mail 92 attached to the non-delivery notice e-mail to the input screen in response to the click of a “Incorporate e-mail information” button 91 on this screen (step S12).",
"It is possible to directly amend the inputted matters on this screen 90 when the inquirer 5 wants to supplement the information.",
"Thus, the inquiry message to be sent to the address inquiry system 1 is made out.",
"If the making of the inquiry message is completed, the inquiry executor 85 is started by pressing a “Search change information” button 97 on the screen 90, and gets access to the address inquiry system 1, and logs in the system 1 using the inquirer authenticating information 35.Authentication on this occasion is executed by the inquirer authenticator 47 (See FIG.",
"11) of the system.",
"When the authentication by the inquirer authenticator 47 is completed, the destination 94, that is, the old address 23 of the changer 3 out of the inquiry information is sent to the changer searcher 49.The changer searcher 49 searches the changer data base 10 by means of the received old address 23 (step S14).",
"FIG.",
"26 to FIG.",
"28 show examples of interface screens for showing search results by the changer searcher 49 on the inquirer terminal 6.FIG.",
"26 shows an example of a screen of a Web page for informing the inquirer 5 that as the result of a search of the changer data base 10 in the address inquiry system 1 for the old address 23 of the changer 3, the said address is not found (step S15).",
"In this case, the inquiry about the corresponding new address 24 comes to an end at this stage (step S16).",
"Meanwhile, FIG.",
"27 shows an example of a screen of a Web page for informing the inquirer 5 that only one matching address is found as the result of the search of the change information 15.The changer searcher 49 displays the changer profile information 33 together with the old address 23 of the changer 3, whereby the inquirer 5 can judge whether the owner of this old address 23 is the changer 3 about whose new address 24 the inquirer 5 wishes to inquire.",
"When he wishes an inquiry, a “Start inquiry” button 100 is pressed (steps S17 and S19).",
"FIG.",
"28 shows an example of a screen of a Web page of the address inquiry system 1 informing the inquirer 5 that a plurality of matching addresses are found as the result of the search of the change information 15.In this case, the inquirer 5 can select the changer 3 about whose new address 24 the inquirer 5 wishes to inquire with reference to the changer profile information 33 (the changer attribute information 16) of the changer 3.By pressing a “Start inquiry” button 103 after the selection of the changer 3 by the use of check boxes 101 and 102, an inquiry about the new address 24 of the changer 3 can be started (step S17 to S19).",
"Incidentally, the communication between the changer 3 and the inquirer 5, and the address inquiry system 1 can be performed by e-mail, in which case it is possible to use the changer reference address 20 and the inquirer reference address 39 which are assigned by the address inquiry system 1.Based on the press of the “Start inquiry” buttons 100 and 103 by the inquirer 5, the new address disclosure judge 51 takes the address disclosing condition 17 out of the changer data base 10 and confirms which pattern out of the numerals 28 to 30 shown in FIG.",
"9 the address disclosing condition 17 of the changer 3 imposed on the inquiry about the new address 24 is (step S20 in FIG.",
"29).",
"When the address disclosing condition 17 is “Disclose new address instantly” (the numeral 28 in FIG.",
"9), the inquiry refusal list 18 is then checked in this embodiment, and only when the inquirers 5 are not registered in the inquiry refusal list 18, the new address 24 is disclosed unconditionally to all the inquirers 5 (steps S21, S22-1, and S22-2).",
"In this case, the address disclosure judge 51 and the discloser 54 in the address inquiry system 1 display a screen shown in FIG.",
"26 in the inquirer terminal 6 and disclose the new address 24 of the changer 3 to the inquirer 5.In this example, only an e-mail address is disclosed as the new address 24, but other sorts of new addresses 24, that is, a URL address, an address, a telephone number, and the like can be displayed according to the disclosure rule 25.When the address disclosing condition 17 registered by the changer 3 is “Make changer judge whether to disclose new address without disclosing new address instantly” (the numeral 29 in FIG.",
"9), the inquiry refusal list 18 is then checked in this embodiment, and only when the inquirer 5 is not registered in the inquiry refusal list 18, the process advances to the next step (steps S23 and S24).",
"FIG.",
"34 shows an example of a notice screen in the inquirer terminal 6 by the new address disclosure judge 51 and the discloser 54 when the inquirer 5 is not registered in the inquiry refusal list 18.This example shows a notice of the inquiry result when the plural changers 3 (a changer A and a changer B) are selected on the screen shown in FIG.",
"28, and the new address 24 regarding the changer A is disclosed by the pattern shown by the numeral 28 in FIG.",
"9.Meanwhile, regarding the changer B, the inquirer 5 is informed that the changer 3 judges whether to disclose the new address 24 according to the pattern shown by the numeral 29 in FIG.",
"9.Incidentally, even when this pattern is selected, if the inquirer 5 is registered in the inquiry refusal list 18, his or her inquiry is refused (step S25).",
"In this case, the screen shown in FIG.",
"32 is displayed in the inquirer terminal 6.Further, when the address disclosing condition 17 registered by the changer 3 is “Refuse inquiry about new address instantly” (the numeral 30 in FIG.",
"9), inquiries from all the inquirers 5 are refused unconditionally in this embodiment (steps S26 and S25).",
"Also in this case, the same screen shown in FIG.",
"32 is displayed in the inquirer terminal 6.Next, the process of the confirmation of the changer 3 in step S27 and the following steps will be explained.",
"In this case, the new address disclosure judge 51 sends an e-mail of a confirmation form 105 with contents shown in FIG.",
"30 to the changer 3 to confirm whether the new address 24 is disclosed or not (step S27 in FIG.",
"29).",
"In the confirmation form 105, a notice 106 that an inquiry about the new address 24 is sent, a date and time of the inquiry 107, an address 108 of the inquirer 5, a profile 109 of the inquirer 5, and a subject 110 are displayed.",
"When “without message text” (the pattern shown by 32 in FIG.",
"9) is set in the confirmation form format 19, the message text in the inquiry message 90 shown in FIG.",
"25 received from the inquirer 5 is not displayed as described above.",
"However, by selecting a link 111 for obtaining and displaying the message text, an e-mail in which the message text is described can be received as shown in FIG.",
"31 (step S28).",
"Meanwhile, in the confirmation form 105, as shown in FIG.",
"30, the changer 3 who receives it can select a response to this inquiry among hyperlinks shown by the numeral 112 to 114 in FIG.",
"30, that is, in this embodiment, can select a response from “Do not disclose new address, and refuse inquiry from this inquirer hereafter” (the hyperlink shown by the numeral 112 in FIG.",
"30), “Disclose new address” (the hyperlink shown by the numeral 113 in FIG.",
"30), or “Do not disclose new address this time, but receive inquiries from this inquirer hereafter” (the hyperlink shown by the numeral 114 in FIG.",
"30).",
"FIG.",
"38 is an example of a notice to the inquirer 5 when “Do not disclose new address, and refuse inquiry from this inquirer hereafter” (the hyperlink shown by the numeral 112 in FIG.",
"30) is selected.",
"In this case, the inquirer 5 is registered to the inquiry refusal list 18, and hereafter inquiries from the said inquirer 5 are automatically refused until the changer 3 changes the setting (steps S30 and S31).",
"FIG.",
"39 is an example of a notice to the inquirer 5 when “Disclose new address” (the hyperlink shown by the numeral 113 in FIG.",
"30) is selected (step S32).",
"Via this screen, the inquirer 5 can know the new address 24 of the changer 3 and get access to the said changer 3 by e-mail or the like.",
"FIG.",
"35 shows an example of the contents of an e-mail received when the changer 3 selects “Do not disclose new address this time, but receive inquiries from this inquirer hereafter” (the hyperlink shown by the numeral 114 in FIG.",
"30) in the confirmation form 105 shown in FIG.",
"30.In this example, the changer 3 refuses an inquiry about the new address 24 from the inquirer 5 but does not register this inquirer in the inquiry refusal list 18, and thereby a notice that the similar inquiries can be made hereafter is given (step S33).",
"Moreover, when the changer 3 selects nothing within a predetermined period, an e-mail shown in FIG.",
"36 is transmitted, the inquiry is refused, and the processing is completed (steps S34 and S35).",
"Incidentally, the display screens in FIGS.",
"35, 36, 38, and 39 show examples in the case where the changer 3 who discloses the new address is singular.",
"When an inquiry is made about a plurality of changers 3 at the same time, a list of results of responses to the permission of disclosure of new addresses of the plurality of changers 3 may be displayed all at once by the same screens.",
"Further, the display screens in FIGS.",
"35, 36, 38, and 39 are notice screens when a notice from the address inquiry system 1 is received by e-mail.",
"Meanwhile, the inquirer 5 can confirm this notice through a Web page by being authenticated by the address inquiry system 1 and logging therein.",
"A notice of the inquiry result to the inquirer 5 is executed by the discloser 54, and the monitoring of the aforesaid predetermined period is executed by the inquiry process monitor 53.If the new address 24 obtained as the result of the inquiry is a home page address, the new location displayer 86 operates to make the browser 80 display a home page of the new location automatically.",
"According to the configuration explained above, the following effects can be obtained.",
"(1) In most of conventional services, the inquirer 5 is informed about the new address of the changer 3 unconditionally if the inquirer 5 inquires about the old address 23 and the said old address is registered.",
"Namely, in such services, it is impossible to selectively inform the inquirers 5 about the new address 24 depending on the inquirers 5.Contrary to this, according to the aforesaid embodiment, the changer 3 can freely set the address disclosing condition 17 of the new address 24 and judge whether to disclose the new address 24 after checking the profile of the inquirer 5, thereby producing an effect that flexible responses to meet the situation of the changer 3 can be made with regard to the disclosure of the new address 24.",
"(2) In conventional services, the changer 3 side cannot know when or to whom the new address 24 is disclosed.",
"Contrary to this, according to the aforesaid embodiment, the changer 3 can receive a confirmation form to confirm whether the new address 24 is disclosed to the inquirer 5, and the changer 3 can determine when and to whom the new address 24 is disclosed by controlling his or her response to the confirmation form.",
"(3) Conventionally, to notify a third party of the change information 15 of a URL of a home page, it is necessary to notify him or her of a URL (the new address 24) of a new home page on the old home page.",
"Therefore, a domain name of the old home page and a contract with an Internet service provider need to be maintained, which causes a problem that the cost thereof needs to be borne during the notification of the change information 15.Contrary to this, according to the aforesaid embodiment, the address inquiry system 1 can provide an inquiry about the URL (the new address 24) of the new home page, so that a domain server of the old home page or the contract with the Internet service provider does not need to be maintained.",
"Thus, to the changer 3, server administration becomes easier, and the cost of maintaining servers can be reduced.",
"(4) When the change information 15 about a telephone number or a mail address is given, there is a limit in terms of a period to the present service of a telephone office or a post office.",
"Contrary to this, according to the aforesaid embodiment, the new address 24 can be disclosed regardless of the service of the telephone office or the post office.",
"(5) In the case of mail service, the receiver needs to inform a sender of mail delivered to the old address about the new address on another occasion by himself or herself as described above.",
"Contrary to this, according to the aforesaid embodiment, the system can respond to an inquiry about a new mail address (the new address 24) in place of the receiver of the mail, thereby eliminating such a trouble.",
"(6) Meanwhile, in transport by the majority of private companies, it is impracticable to make a forwarding request to all of transport companies, and moreover, some private companies do not accept such a request.",
"Contrary to this, if the address inquiry system 1 is used, a private transporter can be used after the new address 24 of the changer 3 is known, which makes it possible to ensure the delivery of a load and the like by such transporter.",
"(7) Addresses sometimes cease to be used for some reason.",
"Namely, the use of the addresses themselves is sometimes stopped without the telephone number being changed.",
"In this case, since the conventional service handles only addresses of the same sort, some who know only the telephone number cannot know other addresses of the changer 3.Contrary to this, according to this embodiment, by setting the disclosure rule 25, inquiries about the new addresses 24 of different sorts can be made, for example, a new telephone number (the new address 24) is detected based on the e-mail address (the old address 23), whereby the aforesaid trouble is eliminated.",
"It should be mentioned that the present invention is not limited to the aforesaid embodiment, and various changes may be made therein without changing the spirit of the present invention.",
"For example, it is already described that processing can be executed by the same process as above also in the case of an inquiry about a new home page URL as the new address 24.The home page, however, aims at being opened to the public, and hence the permission of disclosure of a URL at a new location is often given unconditionally.",
"In this case, it is imagined that it is troublesome to follow the same steps as that of the e-mail address, which is sometimes unbecoming for the purpose of the home page.",
"To cope with this, in the aforesaid special-purpose software, based on the detection of “An access destination is unknown” by the browser 80, the inquiry message maker 84 and the inquiry executor 85 may automatically make out an inquiry message and transmit it to the address inquiry system 1 without obtaining confirmation from the inquirer 5.If the changer searcher 49 finds the new address 24 (new location URL), the discloser 54 or the new location displayer 86 may give a command to the inquirer terminal 6 to display a Web page concerned in the new address 24.Such a configuration enables the automatic display of a new home page 116 at the new location in the inquirer terminal 6 based on the detection of “An access destination to a desired home page is unknown” by the browser 80 as shown in FIG.",
"37.In this case, it is preferable to display also a screen 117 for persuading the inquirer 5 to renew the access destination in order to call the attention of the inquirer 5.Further, it is more preferable to display the new home page 116 at the new location without displaying an error display screen of “An access destination is unknown” by the browser 80.Furthermore, in the aforesaid embodiment, the confirmation form (the screen shown by the numeral 105 in FIG.",
"30) from the address inquiry system 1 to the changer 3 is sent only when the response pattern (2) is selected (step S23) as shown in step S27 in FIG.",
"29, but the present invention is not limited to this.",
"For example, even when the response pattern (1) or (3) is selected (step S21 or S26), it is possible to send the changer 3 this form as an general notice in which information such that about which address, from what sort of the inquirer 5, and on what sort of matter an address inquiry is received is described when the inquiry about the new address 24 is received from the inquirer 5.Moreover, it is possible to include a message text to the changer in this notice.",
"In this case, however, it is preferable that only in the notice when the response pattern (2) is selected, the hyperlinks (the hyperlinks shown by the numeral 112 to 114 in FIG.",
"30) for confirming whether to disclosed the new address to the inquirer exist and that a response to the permission of disclosure of the new address is given with reference to the information about the inquirer 5 displayed in this notice.",
"Moreover, in the aforesaid embodiment, a registrant is explained with the changer 3 who has changed his or her own address from the old address 23 to the new address 24 as an example, but the registrant in the present invention is not limited to the changer 3 like this.",
"For example, the registrant may be one who registers his or her own e-mail address or the like only to disclose it to others (the inquirer 5).",
"Furthermore, based on the old address 23 of the changer 3, the new address 24 thereof is searched and disclosed in the aforesaid embodiment, but the present invention is not limited to this.",
"The new address 24 may be searched by using information except the old address 23, for example, some attribute information of the changer 3.INDUSTRIAL APPLICABILITY As explained above, according to the present invention, it is possible to surely inform his/her telephone number to a person who he/she desires to inform surely without requiring time and labor in the directory assistance service."
]
] | Patent_10415439 |
[
[
"Software development",
"A method of developing software capable of performing a task is disclosed.",
"The task comprises at least one task step required to perform the task.",
"The task step has a resultant effect.",
"The method comprises: recording a use case comprising one or more scenarios, the or each scenario comprising one or more further steps; recording said scenario or one of said scenarios as a main success scenario such that the resultant effect of said main success scenario is the same as the resultant effect of said task step.",
"Also disclose is the re-use of use cases which have been stripped of context-specific information.",
"Such information may be included in a reference to the re-used use case."
],
[
"1.A method of developing software capable of performing a task, said task comprising at least one task step required to perform said task, the task step having a resultant effect, wherein the method comprises: recording a use case comprising one or more scenarios, the or each scenario comprising one or more further steps; recording said scenario or one of said scenarios as a main success scenario such that the resultant effect of said main success scenario is the same as the resultant effect of said task step.",
"2.-63.",
"(canceled)"
],
[
"This invention relates to development of software, and more particularly to methods, software and tools to facilitate such development.",
"Software development projects in many cases involve as much manpower resources as architectural or civil engineering projects, however, the progress and the results are not as easy to visualise.",
"Thus software development involving more than one person has always been difficult to control effectively, and for this reason it has been the consensus for some time that a defined process is required.",
"Development without a process gives rise to the following problems: over-reliance on having the right people.",
"Difficulties in co-ordination and communication between people.",
"Inaccurate planning.",
"Lack of code/module re-use.",
"Lack of predictability of the software.",
"Processes have been developed in recent years, however, for various reasons they have not been widely used.",
"The following are some of the problems: The process is not suitable for Object-Oriented (O) development.",
"There is a perception that implementation of a process involves too much overhead and additional developer time.",
"There is a perception that projects below a certain size are too small for implementation of a process.",
"The available processes do not strike a good balance between the traditional “waterfall” approach in which every stage must be completely finished before proceeding to the next stage, and the other extreme of constantly iterating in apparently endless cycles.",
"It is an aim of the present invention at least partially to alleviate these problems.",
"When viewed from a first aspect, the present invention provides a method of developing software capable of performing a task, said task comprising at least one task step required to perform said task, the step having a resultant effect, wherein the method comprises: recording a use case comprising one or more scenarios, the or each scenario comprising one or more further steps; and recording said scenario or one of said scenarios as a main success scenario such that the resultant effect of said main success scenario is the same as the resultant effect of said step.",
"Thus it will be seen by those skilled in the art that in accordance with the present invention a software development method is provided whereby a task step to be carried out by the software may be broken down by means of a main success scenario which has further steps which add up to the same net effect as the overall task step.",
"In other words detail may be added to each step in an organised and straightforward way.",
"This process may, and typically will, be continued for several layers.",
"In other words, for some or all of the ‘further’ steps in the main success scenario, there may be recorded use cases which themselves have main success scenarios and a number of yet further steps.",
"Detail may continue to be added until such time as there is sufficient detail to translate into code.",
"Thus the same use case structure may be applied at all levels of abstraction throughout the software development life cycle.",
"Such an organised structure is beneficial in the development of software as it ensures openness during the development process and, for example, allows easy evaluation of the extent of development which has been achieved at any given point in the software development life cycle.",
"The relationship between a task step and its more detailed use case is known as a refinement relationship and in preferred embodiments, the method comprises recording a refinement relationship which links the task step to the use case.",
"By recording the relationship, it is clear how further detail has been added.",
"Preferably the method comprises recording a justification for said refinement relationship, said justification demonstrating how the resultant effect of the main success scenario within the more detailed use case equals the resultant effect of the task step.",
"Use cases in accordance with the invention may be allowed only a main success scenario.",
"Preferably however the use case may also have one or more alternative scenarios.",
"The alternative scenario(s) may not have a resultant effect which is the same as that of the task step which the use case refines.",
"In some preferred embodiments, the use case has a required post-condition.",
"That is, a condition which must be satisfied once the use case has been executed.",
"A post-condition may be visually represented as applying to the use case as a whole, but preferably is represented in a meta model as applying to the main success scenario—since an alternative scenario may not be able to meet the post-condition.",
"In some preferred embodiments, the use case has a required pre-condition.",
"That is, a condition that must be satisfied before the use case is executed.",
"A pre-condition may be applied to the use case as a whole since it does not depend on whether the main success scenario or an alternative scenario is followed.",
"It will be appreciated by those skilled in the art that the methods set out hereinabove in accordance with the invention may benefit any single software development project or part of a project by giving enhanced structure and visibility to the project.",
"This is true even if all of the task steps and use cases are built from scratch.",
"Preferably, however, the method comprises recording a reference to one or more use cases stored elsewhere—i.e.",
"that have been previously built.",
"This is extremely beneficial in allowing use cases to be reused.",
"Not only does this save the time that would otherwise have been taken to build the use case, but also the time associated with checking, testing, validating etc.",
"The use case referred to may previously have been built in an earlier project or in a different part of the same project.",
"It will be appreciated that uses cases at any level of detail may be reused.",
"Where a higher level use case is reused, the further steps comprised in its main success or alternative scenarios will be reused.",
"These further steps may themselves be refined into lower level use cases which might, although need not necessarily, also be reused.",
"In other words, if a user reuses a use case he/she may utilise the refinements within it, or equally may define new refinements, subject to any pre- or post-conditions.",
"It will be appreciated that this is an advantage conferred by the fact that the resultant effect of the task step and the main success scenario of the refining use case are ensured to be the same.",
"The benefits of reusing use cases may be achieved by reusing the same use case in the same context in which it was built.",
"In these circumstances, the use case could be specific to that context.",
"It is preferred however that use cases do not contain any context-specific information.",
"For example any context-specific information required for the use case could be recorded separately, e.g.",
"in a separate reference object rather than in the use case itself.",
"The term context, when applied to use cases, should be understood as referring to the ability to specify additional attributes of that use case which are specific to the particular environment in which the use case is used, without affecting the pre- and/or post-conditions (where provided for) of the use case itself.",
"For example the context of a use case may be the project in which the use case is used.",
"In this case the context specific information could be attributes such as the priority of that use case in that project, or the delivery increment in which it is due to be delivered.",
"Alternatively the context of a use case may be a subset of a whole project.",
"Effectively this means that the structure of the use case, i.e.",
"the collection of steps and optional pre- and/or post-conditions, may be maintained while allowing the use case to show context-specific attributes.",
"By separating a Use Case into two types: a pure use case and a use case reference, and relating these two types by means of a reference relationship, reuse of use cases across different contexts, e.g.",
"different projects, may be achieved.",
"This apparently simple feature in fact greatly enhances the power and value of reusability in accordance with the invention.",
"By stripping use cases of their context, the opportunity for reusing them becomes significantly greater and the avoidance of repeated effort may be optimised.",
"For example, the task step “Make Payment” may be used in a business model to pay for received goods, issue a refund or award a prize.",
"Regardless of the business context that the use case is used in, its resultant effect is still the same: money passes from payer to payee.",
"Such an arrangement is considered to be novel and inventive in it's own right and thus when viewed from a further aspect the invention provides a method of developing software capable of performing a task, said task comprising at least one main step required to perform said task, the main step having a resultant effect, wherein the method comprises recording a reference to a use case comprising one or more further steps such that said further steps have a resultant effect which is the same as the resultant effect of the main step; wherein said use case is stored elsewhere and has no context-specific information, thereby allowing re-use of said use case.",
"At least some use cases may be stored in the part of the project where they are first built.",
"This is most applicable in the circumstances of reuse within a given software development project.",
"Additionally or alternatively, use cases may be stored in a repository.",
"This is most applicable in the circumstances of reuse between separate software development projects.",
"Indeed as well as storing use cases which are built in the context of a particular project, such an approach allows use cases to be proactively built outside of any particular project for employment in subsequent projects.",
"The use cases in a repository of the type set out above may simply be stored there once they have been built.",
"Preferably however the repository comprises a separate section for storing use cases which have been ‘qualified’—i.e.",
"those which have been tested, checked for lack of context-specific information, or had context-specific information removed.",
"In the following, the term “collaboration” may be taken to be interchangeable with the term “use case”.",
"Furthermore the term “action” may be taken to be interchangeable with the term “step” or “task step”.",
"No inference is to be drawn by use of the former term rather than the latter or vice versa.",
"When viewed from a yet further aspect the present invention provides a software development method comprising the steps of defining process workflows for use cases and roles, wherein: the use cases are represented as actions, each having an associated effect defining a required post-condition for the action; and use cases are stored in an asset repository according to a meta model which allows retrieval of use cases for re-use.",
"In one embodiment, at least some use cases are represented in the meta model as a collaboration linked to actions, in which each action and the collaboration is linked to an associated effect, and the collaboration is a more detailed representation of the actions.",
"In one embodiment, each effect states both a pre-condition and a post-condition.",
"In one embodiment, a collaboration is linked to an action by a refinement relationship.",
"In one embodiment, the refinement relationship states what stage of the project life-cycle the project is at.",
"In one embodiment, there is a justification associated with each refinement relationship, stating how the action is realised by the refining collaboration.",
"In one embodiment, the method uses a predicate justification stating how each clause in an action's pre-condition expression is realised by a clause in a collaboration pre-condition.",
"In one embodiment, the method uses a diagrammatic justification defining how entities of the action are realised in the refining collaboration.",
"In one embodiment, a model for a project is generated from the meta model by re-use of parts of the meta model in the asset repository.",
"In one embodiment, the model is generated by use of an include pattern for inclusion of actions and collaborations of the meta model.",
"In one embodiment, the model comprises only a reference to a collaboration or action, the actual action or collaboration being stored in the meta model.",
"In one embodiment, the model is extended by inheriting behaviour of reference types and refinement of included collaborations.",
"In one embodiment, the meta model models a business context for a business process.",
"In one embodiment, the method uses a proactive content group that proactively creates re-useable artefacts.",
"In one embodiment, the method comprises the step of mapping requirements to software components for technical mapping.",
"In one embodiment, the meta model represents use cases at different levels of abstraction and in different contexts.",
"In one embodiment, the meta model comprises refinement relationships linking views of processes at different levels of detail to provide traceability.",
"In one embodiment, a refinement relationship links an action to a collaboration, in which a collaboration is a more detailed representation of an action.",
"In one embodiment, the meta model represents use cases independent of context, and allows a context to be applied without change to a base use case.",
"According to another aspect, the invention provides a software development system comprising means for defining process workflows for use cases and roles in a method as defined above.",
"When viewed from further aspects the present invention provides respectively a software development tool, a computer program product and complementary client and sever software for carrying out the methods set out hereinabove.",
"Certain preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: FIG.",
"1 is a diagram showing refinement using a meta model in accordance with an embodiment of the present invention; FIG.",
"2 is a diagram showing refinement in the meta model; FIG.",
"3 is a diagram showing Include and Exclude patterns using a meta model; FIG.",
"4 is a diagram showing Include and Extend patterns in the meta model; FIG.",
"5 is a diagram showing the Include pattern; FIG.",
"6 is a diagram showing an Extend pattern; FIG.",
"7 is a diagram showing action re-use in the meta model; FIG.",
"8 is a diagram showing action re-use using the meta model; FIG.",
"9 is a diagram illustrating a “Procure Goods” Action; FIG.",
"10 is a diagram illustrating a “Procure Goods” Collaboration; FIG.",
"11 is a diagram showing a Predicate Justification snapshot; FIG.",
"12 is a diagram showing a Justification snapshot; FIG.",
"13 is a diagram showing a Verbal Justification snapshot; FIG.",
"14 is a diagram showing Justification generally in the meta model; FIG.",
"15 is a class diagram of a meta model in accordance with another embodiment of the invention; FIGS.",
"16 to 18 are more detailed views of the mea model of FIG.",
"15; and FIG.",
"19 is a schematic diagram demonstrating the use of a qualified asset pool.",
"An embodiment of the invention is described in detail below with reference to FIGS.",
"1 to 14.In at least the preferred embodiments, the invention provides a software development process which allows iteration, but does so in a well-controlled manner.",
"A use case may, for example, be “Purchase Goods” in a shopping portal.",
"Although a use case may be nothing more than a textual description of functionality, when used correctly it is a very effective tool for “fencing in” the project scope and allows for excellent segmentation of deliverables within the project plan.",
"Also, use cases define a set of requirements for a project, allowing a project manager to implement change control in a well-defined and transparent way.",
"Thus if a customer decides to change direction during a project then there is a verifiable “audit trail” of easily understood use cases that allow the changes to be quantified for planning purposes.",
"A problem with the known Rational RUP approach is that iterations are difficult to control in many projects.",
"In some embodiments of the present invention, this is overcome by defining work-flows which collectively describe the project life-cycle.",
"Each workflow corresponds to a set of activities, the outputs (artefacts) of which are verifiable and facilitate a clear progression within the project life-cycle.",
"At the end of a work-flow all outputs are clearly defined and verifiable.",
"Each workflow has a predecessor and a successor, with the exception of the first and the last.",
"Each iteration corresponds to a set of completed functional groups, a functional group comprising one or more related use cases.",
"Iterations are controlled and may be run in parallel, sequentially, or a hybrid of both depending on the inter-dependencies between functional groups themselves and between the functional groups and shared packages.",
"The project manager and senior technical people decide on the dependencies.",
"A preferred embodiment of the invention also provides an asset repository, in which an asset is a reusable artefact developed during the project life-cycle.",
"The asset repository comprises a meta model and content.",
"Content is knowledge related to the delivery of systems and is embodied in reusable artefacts.",
"The meta model is a schema for storage of the content.",
"The problem of demonstrating that certain software components actually deliver on their requirements is referred to as “traceability”.",
"For example, the following are two use cases; “Procure Goods”, a highly abstract representation of a procurement process, and “Order Goods”, a representation of one of the sub-actions of the Procure Goods use case.",
"Use Case 1, Procure Goods Characteristic Information To procure goods using one Description/Goal of use of several procurement case: techniques Primary Actor: Buyer Precondition: None Success End Condition: Buyer owns procured goods and has paid for them.",
"Main Success Scenario 1.The buyer orders goods from a supplier.",
"2.The buyer pays the supplier for the goods.",
"3.The supplier fulfils the order.",
"4.The use case ends.",
"Use Case 2: Order Goods (expansion of step 1 in the main success scenario of use case 1) Characteristic Information Description/Goal of use To formally request goods case: from a supplier Primary Actor: Buyer Precondition: None Success End Condition: An order for the buyer's required goods is placed with a supplier.",
"Main Success Scenario 1.The buyer requests the goods by submitting a purchase request to the purchasing department.",
"2.The purchasing department approves the request.",
"3.The purchasing department creates a purchase order and sends it to the supplier.",
"4.The use case ends.",
"Traceability is achieved by a mechanism known as “Refinement”.",
"Refinement is the linking of two views of a business process, where one view is a more detailed representation of the other.",
"Noting that progressing through the stages of a software life-cycle essentially boils down to adding detail, it becomes clear that refinement is the means by which the project manager can trace from requirements to code.",
"The diagram of FIG.",
"1 shows how the two use cases can be represented using an object diagram.",
"Use case 1 is represented by the Collaboration “Procure Goods”.",
"It contains three Actions (steps)—“Order Goods”, “Fulfill Order” and “Make Payment”.",
"These actions (steps) represent the steps in the “Main Success Scenario” of the use case.",
"All Actions (steps) have an “Effect” that states its precondition and post-condition (but only the Effect for “Order Goods” is shown in the diagram).",
"The second use case “Order Goods”, is represented by the Collaboration (use case) of the same name.",
"It too contains three actions (steps) that represent the steps of the main success scenario.",
"The traceability link is modelled using the refinement relationship.",
"It links an Action (step) to a Collaboration (use case), with the meaning that the Collaboration (use case) is a more detailed representation of the Action (step).",
"Applying this relationship repeatedly allows one to navigate any depth of abstraction, including from business process models to software code.",
"This mechanism is generalised in the representative implementation illustrated in FIG.",
"2, illustrating the section of the meta model which enables refinement.",
"The Collaboration (use case) is a, representation of a business process at some level of abstraction.",
"It has a single Effect, which is an optional Pre-condition and a required Post-condition.",
"The Effect defines the changes the business process has on business entities.",
"A Collaboration (use case) consists of a number of actions (steps) that represent the steps in the business process.",
"Each Action (steps also has an Effect that describes what changes it causes in business entities.",
"It is important to note therefore that Actions (steps) and Collaborations (use cases) are very similar—they both define changes in business entities.",
"The only difference is that Collaborations (use cases) describe the steps involved in the changes and Actions (steps) do not.",
"This allows one to say that Actions (steps) are abstract representations of Collaborations (use cases) (or alternatively Collaborations (use cases) are Actions (steps) that “expose their innards”).",
"Now, if Actions (steps) are abstract representations of Collaborations (use cases), it is a relatively simple step to formally represent this relationship.",
"This is achieved via the Refinement type.",
"It links an Action (step) (via the ActionRef type) and a Collaboration (use case) and has the semantic that the Collaboration (use case) is a more detailed view of the Action (step).",
"The Refinement type has an attribute called “type”, which states what stage of the project life-cycle that the associated Collaboration (use case) is at (business analysis, system analysis, or design for example).",
"So, an Action (step) and a Collaboration (use case) can be associated via Refinement, and the semantic of the association is that the Collaboration (use case) is a more detailed view of the Action (step).",
"In other words, the Collaboration (use case) has exactly the same Effect as the Action (step), but is modelled at a greater level of detail.",
"This is what enables the traceability from business to code.",
"Every refining Collaboration (use case) has to be a plug-in replacement for its abstract Action (step).",
"This requirement is so important in fact that it needs to be enforced.",
"This is achieved by the Justification type, described below.",
"In summary, traceability is applied via refinement in the following ways: Manage detail during Business Analysis—a refinement object of type “business” linking an Action (step) and a Collaboration (use case) states that the Collaboration (use case) is a more detailed view of the Action (step) but that both Action (step) and Collaboration (use case) express Business Analysis level detail.",
"Bridge Business Analysis and Systems Analysis—a refinement object of type “system” linking an Action (step) at Business Analysis level to a Collaboration (use case) states that the Collaboration (use case) is a System Analysis level representation of the Action (step).",
"Manage detail during System Analysis—a refinement object of type “system” linking an Action (step) at System Analysis level and a Collaboration (use case) states that the Collaboration (use case) is a more detailed view of the Action (step) but that both Action (step) and Collaboration (use case) express System Analysis level detail.",
"Bridge System Analysis and Design—a refinement object of type “design” linking an Action (step) at System Analysis level to a Collaboration (use case) states that the Collaboration (use case) is a Design level representation of the Action (step).",
"Refinement is also used to trace into code, but that is treated as a separate pattern.",
"Reuse This section presents the pattern that deals with the software problem of reuse.",
"A number of reuse goals, as described below, are realised: 1.Reuse across the software life-cycle.",
"2.Intra-model reuse.",
"3.“Proactive” reuse.",
"Each type of reuse is dealt with below.",
"Reuse Across the Software Life-Cycle To take the example of a customer that is looking for a procurement site.",
"The developers have completed a project that has a requirement in common with this new one.",
"The common requirement is “Procure Goods”.",
"Additionally the customer has a requirement for a business process called “Special Procurement” and other functionality called “Customer Specific”.",
"Re-use applies to the earlier life-cycle stages as well as it does to construction.",
"Every Internet site requires authentication and session management.",
"Every B2C site requires Catalog Management, even if not obviously.",
"Every B2B site, using the strict definition of B2B, requires exchange of business documents between businesses.",
"Reuse also applies to the requirements stage of the life-cycle, to system requirements, software and network architecture and navigation mechanisms—the entire software life-cycle infact.",
"In accordance with at least preferred embodiments of the invention, the goal of a life-cycle is regarded as a process by which business requirements are continuously and traceably refined to a more software-like form until they actually are software.",
"This interpretation allows one to combine the notions of process and reusable assets in a very interesting and powerful way; any given reusable asset can be a more software-like representation of another reusable asset and as such represents a step along the software life cycle.",
"This means that reusable assets linked in this way form a map that begins at business requirements and ends in code.",
"Enabling reuse across the life-cycle makes use of two mechanisms; refinement to provide traceability through the life cycle (as described above) and the asset repository.",
"The asset repository is a repository of reusable content that spans the software life cycle.",
"It includes business models that are refined into system models that are refined into design models that are refined into software components.",
"Every element in the asset repository is made as generic as possible so it can be included in many customer-specific models.",
"A user can nominate elements from the asset repository for inclusion in his project.",
"He can also extend the content he has included from the asset repository.",
"This mechanism is known as the “Include and Extend Pattern”.",
"The diagram of FIG.",
"3 helps to illustrate.",
"At the top of this diagram is an asset that contains a single Collaboration (use case) Package “Procurement”.",
"That package contains two collaborations (use cases): “Procure Goods” and “Make Payment”.",
"“Procure Goods” is the reusable Collaboration (use case) mentioned in the example above.",
"There is a model towards the bottom of the diagram.",
"A model represents a project for a particular customer.",
"This particular model needs to reference the reusable “Procure Goods” Collaboration (use case) in the Asset repository 100.It does so by first of all including a reference to the package that contains the reusable collaboration (use case).",
"This is achieved by the CollaborationPackageRef called “refl” which points to the required Collaboration (use case) Package in the asset repository 100.“ref1” contains a ModelCollaborationRef called “mrefl” that refers to the required reusable Collaboration (use case) in the Asset repository 100.This has included the reusable Asset repository content in the Model.",
"The content can be extended since it is referred to indirectly by the ModelCollaborationRef instance.",
"This instance can have other instances and content associated with it.",
"In this case it has a PhaseInstance instance associated with it.",
"Additionally, the “Procurement” package included in the Model from the Asset repository 100 can be extended, since it is included by reference.",
"In this example, the referring object, “refl”, has a second Collaboration (use case) added to it—“Special Procurement”.",
"This addition is made without affecting the content in the asset repository 100.In a sense, this content is “layered” over the asset repository 100 content.",
"As a further illustration of the independence of the Model from the asset repository 100, the Model has a second CollaborationPackage, “Customer Specific” that is purely local to it.",
"It can be shown that the refinement mechanism described above applies to content included from the asset repository 100.This allows model builders to include a Collaboration (use case) from the Asset repository and make it more specific via Refinement.",
"For example, the model builder might include a Collaboration (use case) called “Make Payment” that includes an Action (step) called “Transfer Funds”.",
"The model builder can add a refinement to the “Transfer Funds” Action (step) to support customer specific transfer funds mechanisms—“Transfer Funds by EFT” for example.",
"The diagram of FIG.",
"4 shows the section of the meta model that enables the “Include and Extend Pattern”.",
"The include and extend pattern is best explained-by looking at the mechanism of inclusion and extension separately.",
"Any content that has to be included in a model from an asset repository 100 is represented by two types.",
"One is the content itself, the other is a reference to the content (in terms of FIG.",
"4, the content is represented by the type called “CollaborationPackage”, which is a package of pure use cases and the reference type is “CollaborationPackageRef”, which is a reference to such a package and contains the context attributes).",
"References inherit from real types—they are the real types and more.",
"The “more” bit is that they may refer to a real type and delegate toit while still retaining the ability to act exactly like the real type.",
"For example, a CollaborationPackageRef will refer to a single CollaborationPackage (use case package).",
"However, it can also contain Collaborations (use cases), as if it was the real type itself.",
"Real content is always navigated to via the reference types.",
"This enables one to ignore where real content is located—the real content might share the same parent as the reference type, or it might be in some totally different parent.",
"So inclusion is supported.",
"The only question left to be answered is where to locate the real content.",
"The rule here is to always locate it where it is first created.",
"For example, the real collaboration (use case) “Procure Goods” is located in the “Procurement” package in the Asset repository.",
"This is because the Asset repository editor first created the collaboration (use case) in the content of that package.",
"Additionally, the CollaborationPackage “Customer Specific” is located in the Model, because this is where the Model builder first created it.",
"Therefore, inclusion is supported using the pattern shown in FIG.",
"5.In this diagram, the types that represent real content are known as “Real” and the types that refer to content, and contain contexts attributes, are known as “Reference”.",
"Extension builds on the Include pattern by introducing special cases of reference types.",
"In terms of the diagram of FIG.",
"4, ModelCollaborationRef is a special case of a reference type.",
"It inherits from a Reference type and defines new behaviour to achieve extension.",
"In this case it adds a PhaseInstance object.",
"So this is the rule for extension of included content: create a type that inherits from the relevant Reference type and define the extending behaviour.",
"Such extending types are known as “ExtendingReferences”.",
"So, extension is supported using the pattern illustrated in FIG.",
"6: The Include and Extend Pattern in the meta model is used for the following applications: Inclusion of Collaboration (use case) Packages in Models Inclusion of Collaborations (use cases) in Models Inclusion of Actions (steps) in Models Inclusion of Actors and Types in Models Extension of included Collaboration (use case) Packages (by addition of Collaborations (use cases)) Refinement of included Collaborations (use cases) Sharing of Types and Actors—in the asset repository 100, in models and between the asset repository 100 and models.",
"Intra-Model Reuse A business model uses business processes and entities in numerous contexts.",
"For example, the business process “Make Payment” may be used in a business model to pay for received goods, issue a refund or award a prize.",
"Regardless of the business context that the process is used in, its effect is still the same: money passes from payer to payee.",
"So, the problem is how to realise this reuse opportunity.",
"The meta model realises this reuse opportunity by separating the effect of a business process and its business context.",
"The effect is modelled using an Action (step), which captures the business entities and what happens to them during the business process.",
"The business context is modelled using a Business Description, which captures what the process means to the business.",
"To enable inclusion of an Action (step) in many Collaborations (use cases), Actions (steps) are always navigated to via ActionRef objects.",
"An ActionRef may refer to an Action (step) that is contained by its parent Collaboration (use case) or it may refer to an Action (step) that is contained in some other Collaboration (use case).",
"The question then is where to locate Actions (steps).",
"The rule is that Actions (steps) are always placed in the Collaboration (use case) in which they are created and referred to via an ActionRef.",
"This mechanism is called the include and extend Pattern and is applied through the meta model to allow intra-model reuse of Actions (steps), as described here, and also Types and Actors.",
"The diagram of FIG.",
"7 shows the section of the meta model that enables intra-model reuse.",
"The Collaboration (use case) is a business process that has a purpose in a given business.",
"This purpose is represented using a BusinessDesc object.",
"The Collaboration (use case) describes the steps involved in the business process it represents by referring, via ActionRef objects, to any number of Action (step) objects that describes a discrete change of state in a number of business entities.",
"The sum of these changes of state realise the Collaboration (use case) change of state (Effect).",
"However, every ActionRef adds a business reason for the Action (step) that it refers to.",
"This business reason is independent of Action (step), thereby enabling reuse of Actions (steps) in many business contexts.",
"The diagram of FIG.",
"8 illustrates.",
"FIG.",
"8 shows one Collaboration (se case) “Procure Goods” that contains an Action (step) called “Make Payment”.",
"That Action (step) is used for the business reason for paying a supplier for goods received.",
"There is a second Collaboration (use case) “Make Refund” that also uses the “Make Payment” action (step) in the first Collaboration (use case).",
"But this second Collaboration (use case) uses it for the different business reason to refund a customer for substandard services.",
"Thus the embodiment described hereinabove provides for Sharing of Actions (steps)—in the asset repository 100, in models and between the asset repository 100 and models.",
"Proactive Reuse The typical approach to acquiring reusable assets is to retrospectively harvest them from completed projects.",
"There is a problem with this approach in that it is applicable only on projects that are quite similar to previous projects.",
"This makes it difficult to move into a new business area and retain the benefits of a reuse strategy.",
"The ability to quickly move into new business areas is critical for any software organisation, especially those involved in the dynamic business of e-business solution provision.",
"The present embodiment provides a division, called the Content Group, that proactively creates reusable artifacts for given businesses and disseminates them through the asset repository 100.The Content Group can be instructed to create assets in selected businesses.",
"The group will actively pursue that task while the sales force is winning business.",
"The Content Group will create new assets and may re-factor existing assets to make them more suitable for the given business domain.",
"When a contract is won and a project becomes live, project team members can leverage the assets that the Content Group has already generated.",
"Thus the embodiment described hereinabove provides for proactive reuse—proactively generating high quality models for business domains that are strategic.",
"Technology Mapping Once the customer has signed off requirements, technology must be selected to realise them.",
"Some of this technology will be off-the-shelf software; and some of it will be customised development.",
"The problem is choosing the right technology for the customer's requirements.",
"An important thing to note about the meta model is that it uses a uniform modelling methodology from business to code.",
"Business processes are represented as changes in the state of business entities via their Effect object.",
"The same mechanism is used to capture business processes that are modelled at the system level.",
"And the same process can be used to model software components.",
"A software component has an effect in the same way as the highly abstract process has, except that the former speaks in terms of software entities.",
"This enables one to leverage the refinement mechanism to map requirements to software components.",
"The components can be Sun™, Microsoft™ or any other component type.",
"Alternatively they can be notional components that capture the effect of non-componentised software entity.",
"What this means is that once a customer has selected their requirements, one can look at the technologies that implement their requirements and make recommendations accordingly.",
"In other words, the system advises on what technologies one needs to implement the system.",
"Thus the embodiment described hereinabove achieves Technology Mapping—by leveraging the mechanism of refinement, business requirements can be associated with the technologies that implement them.",
"Justification The refinement mechanism realises traceability, as described above.",
"It enables association of an Action (step) and a Collaboration (use case) with the semantic that the Collaboration (use case) is a more detailed representation of the Action (step), and as such represents a step along the software life-cycle.",
"But how does one prove that this statement is correct?",
"Therefore, it is important to apply rigor to the refinement relationship such that the refining Collaboration (use case) is demonstrably equivalent to the abstract Action (step).",
"The present embodiment addresses this problem as described with reference to FIG.",
"9.In this situation, there are no pre-conditions.",
"The post-condition is that the buyer owns the Goods and the Supplier has been paid for them.",
"This is equivalent to a more detailed Collaboration (use case) illustrated in FIG.",
"10.In this situation, there are no pre-conditions and the post-condition is that the Buying Company owns the Goods and the Supplying Company has been paid for them.",
"The solution to this problem is enabled by the introduction of the mechanism of Justification.",
"Every refinement has an associated Justification that states how the abstract Action (step) is realised by the refining Collaboration (use case).",
"There are three types of justification.",
"1.The first, Predicate Justification, is the most rigorous as it can be made subject to machine validation.",
"It states how each clause in the Action's (step's) precondition expression is realized by a clause (or a set of clauses) in the Collaboration (use case) precondition.",
"The same applies for the post-condition.",
"2.Diagrammatic Justification contains UML relationships that show how entities involved in the abstract Action (step) are realised in the refining Collaboration (use case).",
"3.Verbal Justification is the least rigorous justification mechanism as it consists purely of an explanation in English (or whatever language) how the abstract entities are realized in the refinement.",
"Each justification type is illustrated by snapshot diagrams, and referring to FIG.",
"11 a Predicate Justification diagram is shown.",
"Predicates based on Object Constraint Language (OCL) can be justified using Predicate Justifications.",
"Assume there is an Action (step) whose post-condition is “buyer inventory +=goods” and there is a refinement of that Action (step) that has the post-condition “goodsReceiver.employer.inventory +=goods”.",
"The snapshot diagram for this case is illustrated in FIG.",
"11.In this diagram, the Predicate Justification links two predicates and explains verbally how they are equivalent.",
"Assume there is an Action (step) that involves an Actor called “buyer” and that this Action (step) is refined by a Collaboration (use case) that replaces the notion of “buyer” with an Actor called “goodsReceiver” and a type called “Company” and the goodsReceiver is employed by the Company.",
"The snapshot diagram for this case is presented in FIG.",
"12.This snapshot states that the Actor called “buyer” is the same as an Actor called “goodsReceiver” that is employed by a company.",
"This is achieved through the Diagrammatic Justification instance—bottomcentre.",
"It has three associations.",
"The leftmost one states that the “buyer” Actor has an aggregate relationship with the “goodsReceiver” Actor.",
"This means that the “buyer” consists of the “goodsReceiver”.",
"The middle association states that the “buyer” Actor has an aggregate relationship with the Company instance.",
"The rightmost association states that there is a relationship between the “goodsReceiver” Actor and the Company and that this association is known as “employment”.",
"A Verbal Justification is a series of paragraphs of text that explain in English how the entities in the abstract Action (step) involved in a refinement are realised in the refining Collaboration (use case).",
"The snapshot diagram of FIG.",
"13 illustrates this, and the diagram of FIG.",
"14 shows the section of the meta model that enables justification.",
"A Justification consists of any number of DiagrammaticJustifications, VerbalJustifications and PredicateJustifications.",
"Thus, the embodiment described hereinabove applies rigor to traceability—by requiring that refinements; are justified, the justification processes forces the developer to think about and make explicit the relationships between abstract Actions (steps) and the refining Collaborations (use cases).",
"Additionally, the presence of this information guides the reader through the levels of abstraction and makes traceability explicit.",
"A further embodiment is described with reference to FIGS.",
"15 to 19.As in the foregoing description, the term “Collaboration” may be taken to be interchangeable with the term “use case”.",
"Furthermore the term “Action” may be taken to be interchangeable with the term “step” or “task step”.",
"No inference is to be drawn by use of the latter term rather than the former.",
"Any features not described in relation to FIGS.",
"15 to 19 should be taken to be the same as the corresponding features described with reference to FIGS.",
"1 to 14.FIG.",
"15 shows the meta model 2 of a further embodiment of the invention in the form of a mete model class diagram.",
"The meta model 2 is the schema by which the content of the model is structured.",
"The various parts thereof are described with reference to FIGS.",
"16 to 18 from which they may be seen more clearly and thus are not described with reference to FIG.",
"15.The relationship between a project 4 and use cases 6 is shown in FIG.",
"16.The project 4 contains all the components that model a real system—i.e.",
"it represents software which has been developed or is being developed in accordance with a method and tool embodying the invention.",
"The use cases 6 are an arrangement of related steps to achieve some business goal.",
"Use case packages 8 are the named containers of use cases 6.They may in turn contain other use case packages 8.Use case packages 8 allow for the logical or conceptual grouping of associated use cases.",
"The root node of the meta model is a qualified asset pool (QAP) 10.The QAP 10 comprises use case packages 8, which in turn contain use cases 6, which have been modified or purified and approved by an asset purifier team.",
"Use case pointers 12 are pointers to real use cases 6, which may be created in the current project 4 or a ‘read only’ link to a reusable use case in another project.",
"Similarly, use case package pointers 14 are pointers to real use cases packages 8, which may be created in the current project 4 or a ‘read only’ link to a reusable use case package in another project.",
"The project 4 has a collection 16 of goal levels 18.The goal levels 18 are a process specific representation of what >level=a use case 6 can be demoted as.",
"By default the described embodiment specifies five such goal levels: “very high summary”; “summary”; “user goal”; “sub-function”; and “too low”.",
"A list 20 of actors, i.e.",
"entities with which the project 4 may interact, is stored with the project.",
"Also stored with the project 4 are freeform notes 22, a list 24 of links between the project and external systems, a glossary 26 of the terms used in the project and a list 28 of non-functional requirements.",
"These are requirements that must be met by the project as a whole but which do not translate into functionality.",
"Examples of non-functional requirements are “we must be able to process a hundred transactions a second”, or “the user interface must be usable”.",
"Although not shown in FIG.",
"16, non-functional requirements may also be associated with individual use cases.",
"Further details of the meta model 2 may be seen in FIG.",
"17.It will be seen that each use case 6 has one main success scenario 30 and may also have one or more alternative scenarios 32.Additionally a use case 6 May have one or more exceptions 34.An exception(s) 34 handle(s) the case(s) in which the effect of the associated Step will not happen.",
"An exception 34 has a single pre-condition object that states the condition under which the exception 34 will execute.",
"The exception 34 will also have an associated use case that shows how to deal with the exceptional condition.",
"A scenario 30, 32, 34 (represented collectively by the scenario object 36) has, in general one or more steps 38 within the scenario 36.The steps are linked to use case 6 by means of a refinement 40.This is the nature of the recursive structure in accordance with the invention: a step 38 may be refined by a refinement 40 to give a use case 6, a more detailed but equivalent description of the step 38; the use case has scenarios 36; scenarios contain, in general, a number of steps 38; each of these steps 38 may themselves be refined to a yet more detailed use case 6 etc.",
"Each refinement 40 has a justification 42.The justification 42 demonstrates how the resultant effect of a use case 6 (or more accurately the main success scenario 30 thereof) is equivalent to that of the parent step 38.The justification 42 thus provides traceability between the abstract step 38 and the use case 6 that is a more concrete representation of it.",
"The justification may be formal or informal and may have an optional verification 44.Verification 44 proves that the resultant-effect of the abstract and refined views are the same.",
"Verification 44 could be casual, rigorous, mathematical etc.",
"An asset 46 is a piece of work in a business context which is capable of fulfilling the resultant effect of a step 38 exactly.",
"Typically assets 46 are only associated with detailed refined use cases 6.Assets can be soft 46a, e.g.",
"documents, html pages etc., or hard 46b, e.g.",
"computer code.",
"As described above, actors 48 are entities that participate in one or more steps 38.Finally, a business description 50, that is a textual explanation of a business action being carried out by a use case 6, may be recorded.",
"FIG.",
"18 describes the role of pre- and post-conditions.",
"A pre-condition 52 defines the situation in which a step 38 (FIG.",
"17) can start—i.e.",
"it must be true before the step may take place.",
"A post-condition 54 must be true when the step has been performed i.e.",
"it must equal the resultant effect of the further steps of the main success scenario of the use case which is a refinement of the step.",
"The predicate object 54 fully supports predicate logic usage by implementing Object Constraint Language (OCL) which is part of the Unified Modelling Language specification of the Object Management Group; Sets and Predicate basics.",
"This forms the basis for OCL used in pre and post-conditions.",
"The Set interface 58 defines the mathematical Set logic.",
"The Predicate interface 60 defines the mathematical predicate logic.",
"The OCL interface 62 defines the OCL exactly as specified in the Unified Modelling Language specification of the Object Management Group.",
"Finally FIG.",
"19 is a schematic view of the asset repository.",
"This diagram demonstrates that the software development tool comprises an asset repository 64 which includes a separate Qualified Asset Pool (QAP) 66.The asset repository 64 in general contains unqualified assets 68, whilst the QAP 66 contains qualified assets 70.These are assets which have been ‘purified’ by removing any context or project specific information to allow their reuse across other projects.",
"It will be appreciated by those skilled in the art that whilst certain preferred embodiments of the present invention have ben described, many modifications and variations may be made within the scope of the present invention."
]
] | Patent_10415466 |
[
[
"Bridge for attaching auxiliary lenses",
"An auxiliary spectacle apparatus is provided wherein auxiliary lenses have a mechanical bridge fastener for releasably engaging the bridge portion of the primary frames in a manner which provides security of attachment in all directions."
],
[
"1-21.",
"(canceled) 22.An auxiliary spectacle apparatus, comprising: (a) two auxiliary lenses; (b) a hole in each lens; (c) an auxiliary bridge extending between and connecting said auxiliary lenses to each other; (d) an attachment for attaching said auxiliary bridge to a primary spectacle; and (e) lens rivets extending from said auxiliary bridge through said holes to secure said lenses to said bridge; wherein said lens rivets extend rearward from said auxiliary bridge.",
"23.An auxiliary spectacle apparatus as claimed in claims 22, wherein said auxiliary bridge is Polyflex™ plastic or memory plastic.",
"24.An auxiliary spectacle apparatus as claimed in claim 22, wherein said lens rivets terminate in a bulb.",
"25.An auxiliary spectacle apparatus as claimed in claim 22, further comprising at least two holes in each lens and at least two lens rivets extending from each side of said auxiliary bridge through said holes to secure said lenses to said bridge.",
"26.An auxiliary spectacle bridge as claimed in claim 22, wherein said lens rivets terminate in a cap.",
"27.An auxiliary spectacle bridge, comprising a bridge having: (a) lateral ends; (b) an attachment for attaching said auxiliary spectacle to a primary spectacle; and (c) lens rivets extending horizontally from said lateral ends; wherein said lens rivets extend rearward from said auxiliary bridge.",
"28.An auxiliary spectacle bridge as claimed in claim 27, wherein said auxiliary bridge is Polyflex™ or memory plastic.",
"29.An auxiliary spectacle bridge as claimed in claim 27 wherein said lens rivets terminate in a bulb.",
"30.An auxiliary spectacle bridge as claimed in claim 27, wherein said lens rivets terminate in a cap.",
"31.A spectacle bridge and lenses comprising: (a) two lenses; (b) a bridge having: (i) lateral ends; (ii) lens rivets extending horizontally from said lateral ends; wherein said lens rivets have a distal end of said lens rivet spread against said lenses.",
"32.An spectacle bridge as claimed in claim 31, wherein said lens rivets extend rearward from said bridge.",
"33.An spectacle bridge as claimed in claim 31, wherein said bridge is a bridge for auxiliary spectacles.",
"34.A spectacle apparatus, comprising: (a) a primary frame adapted for fitting on a wearer's head, said primary frame having a primary bridge medial in said primary frame; (b) a ridge extending forward from said primary bridge; (c) two auxiliary lenses; (d) an auxiliary bridge extending between and connecting said auxiliary lenses to each other; (e) an upper projection extending rearward from said auxiliary bridge and a lower projection extending rearward from said auxiliary bridge; wherein said upper projection and said lower projection of said auxiliary bridge are configured such that there is an upper face, a lower face, and lateral faces, collectively configured to form a snug interference with said ridge which is correspondingly configured.",
"35.A spectacle apparatus as claimed in claim 34, comprising said protrusions on both said projections, said protrusions configured for releasably engaging said primary bridge.",
"36.A spectacle apparatus as claimed in claim 34 wherein said ridge has a ridge face which extends laterally and said auxiliary bridge has an rearward auxiliary bridge face which extends laterally and said faces are configured such that an interference fit between said faces prevents rotation between said primary spectacle and said auxiliary bridge.",
"37.A spectacle apparatus as claimed claim 34, wherein said protrusion is beveled.",
"38.A spectacle apparatus, comprising: (a) a primary frame adapted for fitting on a wearer's head, said primary frame having a primary bridge medial in said primary frame; (b) a ridge extending forward from said primary bridge; (c) two auxiliary lenses; (d) an auxiliary bridge extending between and connecting said auxiliary lenses to each other; (e) an upper projection extending rearward from said auxiliary bridge and a lower projection extending rearward from said auxiliary bridge and at least one said projection is elastic; and (f) at least one protrusion on at least one of said projections; wherein the lateral shape of said projections and the corresponding lateral shape of said ridge prevents horizontal movement of the apparatus when assembled.",
"39.A spectacle apparatus as claimed in claim 38, comprising said protrusions on both said projections, said protrusions configured for releasably engaging said primary bridge.",
"40.A spectacle apparatus as claimed in claim 38 wherein said ridge has a ridge face which extends laterally and said auxiliary bridge has an rearward auxiliary bridge face which extends laterally and said faces are configured such that an interference fit between said faces prevents rotation between said primary spectacle and said auxiliary bridge.",
"41.A spectacle apparatus as claimed in claim 38, wherein said protrusion is beveled."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>A spectacle frame having an attachable one-piece slide-on rim is disclosed in U.S. Pat.",
"No.",
"4,070,103 to Meeker.",
"In Meeker, a spectacle frame includes a magnetic material secured to the front face of the frame for facilitating attachment of an auxiliary frame.",
"The lens rim cover also includes a magnetic strip for engaging with the magnetic material of the spectacle frame.",
"Other typical auxiliary eyeglass attachments are disclosed in U.S. Pat.",
"No.",
"5,416,537 to Sadler, U.S. Pat.",
"No.",
"5,737,054 to Chao, U.S. Pat.",
"No.",
"5,975,961 to Ku, Canadian patent application No.",
"2,180,714 to Chao, Canadian patent application No.",
"2,235,897 to Chao, Canadian patent application No.",
"2,236,025 to Ku, Canadian patent application No.",
"2,223,088 to Madison, U.S. Pat.",
"No.",
"5,568,207 to Chao, Canadian patent No.",
"2,223,295 to Chao, German patent application No.",
"DE 43 16 698 to Karp, PCT application No.",
"WO 026718 to Zelman, Canadian patent application No.",
"2,235,917 to Chao, U.S. Pat.",
"No.",
"6,053,611 to Ku, U.S. Pat.",
"No.",
"6,542,177 to Nishioka, and PCT application no.",
"WO 9009611 to Stemme.",
"In all cases, the auxiliary lenses are attached to the frames by magnetic materials.",
"The use of magnets to attach auxiliary lenses has several drawbacks.",
"The strength of magnets weakens over time.",
"In addition, magnetic attachment means are only effective in association with selected metal frames, unless magnets are embedded in the frames of the eyeglasses.",
"This requires cavities in the frames for engaging with the magnetic members such that the strength of the frames comprised.",
"Also, there is some literature which suggests that magnets placed near the body may alter body chemistry or physiology.",
"U.S. Pat.",
"No.",
"6,053,611 to Ku teaches auxiliary glasses with magnets located on upper and lower flanges which extend backwardly to the primary bridge, which is also equipped with upper and lower magnets corresponding to the auxiliary glasses magnets.",
"The insertion of four magnets into this confined space is difficult and therefor costly.",
"Also, as discussed above, there are several other drawbacks associated with the use of magnets in eyeglasses.",
"As the strength of the magnets weakens, the design taught by Ku will become ineffective.",
"One magnet-free method of attaching auxiliary lenses that has attempted to address these problems is the ColorClip™ system.",
"This system provides for holes drilled at various places around the outer perimeter of the auxiliary lenses.",
"Soft plastic clips are then inserted into the holes.",
"A similar system, ECLIPS, requires that the lenses be scored, coated with primer and glue, and rely on metal clips on the auxiliary spectacles.",
"In both systems, the clips engage the primary spectacles.",
"The use of these clip systems creates the risk that drilling, scoring, gluing or attaching the clips will damage the auxiliary lenses in positions, such as the upper, lower and lateral portions of the lenses, that tend to impair vision.",
"Furthermore, each auxiliary lens must be separately attached to and removed from the primary frames.",
"In use, the presence of the clips on the lenses may obscure vision.",
"Also, many wearers consider the clips to be unsightly or unappealing.",
"Thus, it would be desirable to have a means for attaching auxiliary lenses to primary spectacles which did not rely on magnets, and did not require placement of clips on the lenses."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a spectacle apparatus having: (a) a primary frame adapted for fitting on a wearer's head, the primary frame having a primary bridge medial in the primary frame; (b) a ridge extending forward from the primary bridge; (c) an indentation on a horizontal surface on the ridge; (d) two auxiliary lenses; (e) an auxiliary bridge extending between and connecting the auxiliary lenses to each other; and (f) an upper projection extending rearward from the auxiliary bridge and a lower projection extending rearward from the auxiliary bridge and (g) at least one protrusion on at least one of the projections, the protrusion configured for releasably inserting into the indentation, and the at least one the projection is elastic.",
"In one embodiment the spectacle apparatus has protrusions on both the projections, and the protrusions are configured for releasably inserting into at least one indentation on the ridge.",
"In another embodiment, there are protrusions on both the projections, and the protrusions extend laterally in correspondence with at least one the indentation for releasably inserting into the indentation.",
"In a further embodiment, the at least one protrusion extends laterally in correspondence with the indentation for releasably inserting into the indentation.",
"In another embodiment, the indentation on the ridge extends laterally.",
"The indentation on the ridge can be a hole through the ridge.",
"In an embodiment, the ridge has a ridge vertical face which extends laterally and the auxiliary bridge has a rearward vertical auxiliary bridge face which extends laterally and the faces are configured such that an interference fit between the faces prevents horizontal rotation between the primary spectacle and the auxiliary bridge.",
"In another embodiment, the ridge has a horizontal ridge face which extends laterally and at least one projection has a projection face which extends laterally and the faces are configured such that an interference fit between the faces prevents vertical rotation between the primary spectacle and the auxiliary bridge.",
"In a further embodiment, the ridge has a horizontal ridge face which extends laterally and at least one projection has a projection face which extends laterally and the faces are configured such that an interference fit between the faces prevents vertical rotation between the primary spectacle and the auxiliary bridge.",
"The protrusion may be beveled.",
"The auxiliary bridge may be made from Polyflex™ plastic or memory plastic.",
"The invention also provides an auxiliary spectacle apparatus, comprising: (a) two auxiliary lenses; (b) a hole in each lens; (c) an auxiliary bridge extending between and connecting the auxiliary lens to each other; (d) an attachment for attaching the auxiliary spectacle to a primary spectacle; and (e) lens rivets extending from the auxiliary bridges through the holes to secure the lenses to the bridge.",
"In a further embodiment, the lens rivets extend rearward from the auxiliary bridge.",
"The lens rivets may terminate in a bulb.",
"The lens rivets may terminate in a cap.",
"The invention also provides an auxiliary spectacle bridge, comprising a bridge having: (a) lateral ends; (b) an attachment for attaching the auxiliary spectacle to a primary spectacle; and (c) lens rivets extending horizontally from the lateral ends.",
"In a further embodiment, the lens rivets extend rearward from the auxiliary bridge."
],
[
"FIELD OF THE INVENTION The invention relates to mechanical means by which auxiliary lenses are attached to spectacles at the bridge.",
"BACKGROUND OF THE INVENTION A spectacle frame having an attachable one-piece slide-on rim is disclosed in U.S. Pat.",
"No.",
"4,070,103 to Meeker.",
"In Meeker, a spectacle frame includes a magnetic material secured to the front face of the frame for facilitating attachment of an auxiliary frame.",
"The lens rim cover also includes a magnetic strip for engaging with the magnetic material of the spectacle frame.",
"Other typical auxiliary eyeglass attachments are disclosed in U.S. Pat.",
"No.",
"5,416,537 to Sadler, U.S. Pat.",
"No.",
"5,737,054 to Chao, U.S. Pat.",
"No.",
"5,975,961 to Ku, Canadian patent application No.",
"2,180,714 to Chao, Canadian patent application No.",
"2,235,897 to Chao, Canadian patent application No.",
"2,236,025 to Ku, Canadian patent application No.",
"2,223,088 to Madison, U.S. Pat.",
"No.",
"5,568,207 to Chao, Canadian patent No.",
"2,223,295 to Chao, German patent application No.",
"DE 43 16 698 to Karp, PCT application No.",
"WO 026718 to Zelman, Canadian patent application No.",
"2,235,917 to Chao, U.S. Pat.",
"No.",
"6,053,611 to Ku, U.S. Pat.",
"No.",
"6,542,177 to Nishioka, and PCT application no.",
"WO 9009611 to Stemme.",
"In all cases, the auxiliary lenses are attached to the frames by magnetic materials.",
"The use of magnets to attach auxiliary lenses has several drawbacks.",
"The strength of magnets weakens over time.",
"In addition, magnetic attachment means are only effective in association with selected metal frames, unless magnets are embedded in the frames of the eyeglasses.",
"This requires cavities in the frames for engaging with the magnetic members such that the strength of the frames comprised.",
"Also, there is some literature which suggests that magnets placed near the body may alter body chemistry or physiology.",
"U.S. Pat.",
"No.",
"6,053,611 to Ku teaches auxiliary glasses with magnets located on upper and lower flanges which extend backwardly to the primary bridge, which is also equipped with upper and lower magnets corresponding to the auxiliary glasses magnets.",
"The insertion of four magnets into this confined space is difficult and therefor costly.",
"Also, as discussed above, there are several other drawbacks associated with the use of magnets in eyeglasses.",
"As the strength of the magnets weakens, the design taught by Ku will become ineffective.",
"One magnet-free method of attaching auxiliary lenses that has attempted to address these problems is the ColorClip™ system.",
"This system provides for holes drilled at various places around the outer perimeter of the auxiliary lenses.",
"Soft plastic clips are then inserted into the holes.",
"A similar system, ECLIPS, requires that the lenses be scored, coated with primer and glue, and rely on metal clips on the auxiliary spectacles.",
"In both systems, the clips engage the primary spectacles.",
"The use of these clip systems creates the risk that drilling, scoring, gluing or attaching the clips will damage the auxiliary lenses in positions, such as the upper, lower and lateral portions of the lenses, that tend to impair vision.",
"Furthermore, each auxiliary lens must be separately attached to and removed from the primary frames.",
"In use, the presence of the clips on the lenses may obscure vision.",
"Also, many wearers consider the clips to be unsightly or unappealing.",
"Thus, it would be desirable to have a means for attaching auxiliary lenses to primary spectacles which did not rely on magnets, and did not require placement of clips on the lenses.",
"SUMMARY OF THE INVENTION The present invention provides a spectacle apparatus having: (a) a primary frame adapted for fitting on a wearer's head, the primary frame having a primary bridge medial in the primary frame; (b) a ridge extending forward from the primary bridge; (c) an indentation on a horizontal surface on the ridge; (d) two auxiliary lenses; (e) an auxiliary bridge extending between and connecting the auxiliary lenses to each other; and (f) an upper projection extending rearward from the auxiliary bridge and a lower projection extending rearward from the auxiliary bridge and (g) at least one protrusion on at least one of the projections, the protrusion configured for releasably inserting into the indentation, and the at least one the projection is elastic.",
"In one embodiment the spectacle apparatus has protrusions on both the projections, and the protrusions are configured for releasably inserting into at least one indentation on the ridge.",
"In another embodiment, there are protrusions on both the projections, and the protrusions extend laterally in correspondence with at least one the indentation for releasably inserting into the indentation.",
"In a further embodiment, the at least one protrusion extends laterally in correspondence with the indentation for releasably inserting into the indentation.",
"In another embodiment, the indentation on the ridge extends laterally.",
"The indentation on the ridge can be a hole through the ridge.",
"In an embodiment, the ridge has a ridge vertical face which extends laterally and the auxiliary bridge has a rearward vertical auxiliary bridge face which extends laterally and the faces are configured such that an interference fit between the faces prevents horizontal rotation between the primary spectacle and the auxiliary bridge.",
"In another embodiment, the ridge has a horizontal ridge face which extends laterally and at least one projection has a projection face which extends laterally and the faces are configured such that an interference fit between the faces prevents vertical rotation between the primary spectacle and the auxiliary bridge.",
"In a further embodiment, the ridge has a horizontal ridge face which extends laterally and at least one projection has a projection face which extends laterally and the faces are configured such that an interference fit between the faces prevents vertical rotation between the primary spectacle and the auxiliary bridge.",
"The protrusion may be beveled.",
"The auxiliary bridge may be made from Polyflex™ plastic or memory plastic.",
"The invention also provides an auxiliary spectacle apparatus, comprising: (a) two auxiliary lenses; (b) a hole in each lens; (c) an auxiliary bridge extending between and connecting the auxiliary lens to each other; (d) an attachment for attaching the auxiliary spectacle to a primary spectacle; and (e) lens rivets extending from the auxiliary bridges through the holes to secure the lenses to the bridge.",
"In a further embodiment, the lens rivets extend rearward from the auxiliary bridge.",
"The lens rivets may terminate in a bulb.",
"The lens rivets may terminate in a cap.",
"The invention also provides an auxiliary spectacle bridge, comprising a bridge having: (a) lateral ends; (b) an attachment for attaching the auxiliary spectacle to a primary spectacle; and (c) lens rivets extending horizontally from the lateral ends.",
"In a further embodiment, the lens rivets extend rearward from the auxiliary bridge.",
"BRIEF DESCRIPTION OF THE DRAWINGS A preferred embodiment of the invention will be described with reference to the accompanying drawings, in which: FIG.",
"1 is a side view of a disassembled primary spectacle and auxiliary bridge according to the present invention.",
"FIG.",
"2 is a top view of a disassembled spectacle apparatus according to the present invention.",
"FIG.",
"3 is a rear view of an auxiliary bridge according to the present invention.",
"FIG.",
"4 is a rear view of auxiliary spectacles according to the present invention.",
"FIG.",
"5 is a front view of a primary bridge according to the present invention.",
"FIG.",
"6 is a top view of a primary and auxiliary bridges (assembled) and the rivet caps (disassembled).",
"FIG.",
"7 is a rear view of an auxiliary bridge according to the present invention.",
"FIG.",
"8 is a side view of an auxiliary bridge according to the present invention.",
"FIG.",
"9 is a top view of an assembled auxiliary spectacle apparatus according to the present invention.",
"FIG.",
"10 is a front view of a spectacle apparatus according to the present invention.",
"DETAILED DESCRIPTION OF EMBODIMENTS Preferred embodiments of the present invention will now be described in detail with reference to the appended drawings, in which like elements are denoted by like reference numerals.",
"As shown in FIG.",
"1, the spectacle apparatus of the present invention has an auxiliary bridge 60 for releasably attaching to a primary frame 32.FIG.",
"1 shows the apparatus in a dissembled form, with the auxiliary lenses removed in order to better show detail on auxiliary bridge 60.The primary frame 32 is configured to be worn on the head of the wearer by means of the stems 44, which fit over a wearer ears to support the spectacles.",
"Primary frame 32 includes a primary bridge 34 which has a ridge 36.Ridge 36 has a ridge front face 38 and indentations 40.At least one indentation 40 must be present for the effective operation of the apparatus.",
"In the embodiment shown, two indentations 40 are present, one at the top and one at the bottom of the ridge.",
"The ridge 36 and indentation 40 are configured to releasably engage with the auxiliary bridge 60.Auxiliary bridge 60 has an upper projection 62 and a lower projection 64.Projections 62 and 64 have a protrusion 66 which is configured to releasably fit into indentation 40.In the embodiment shown, two protrusions 66 are present, one at the top and one at the bottom of the ridge.",
"Turning to FIG.",
"2, primary face 32 includes a primary bridge 34 which is medial within the frame and which extends between and connects primary lenses 18.The primary frame 32 provides retaining structure for primary lenses 18.Primary lenses 18 may be clear or slightly tinted glass or plastic material.",
"One or both primary lenses 18 may be shaped to the wearer's visual deficiencies.",
"The auxiliary spectacle 20, when assembled, is a one-piece unit.",
"The auxiliary lenses 50 are configured to cover both of the primary lenses 18.The external shape of the auxiliary lenses 50 generally correspond with the external shape of the primary lenses.",
"One use for the auxiliary lenses 50 is to provide protection to the wearer from sunlight and other vision-damaging elements.",
"Therefore, the auxiliary lenses 50 may be tinted, and may also include a material therein or a coating thereon to filter ultraviolet light or other specific wavelengths of light.",
"Auxiliary spectacles 20 comprise an auxiliary bridge 60 which provides a retaining means for auxiliary lenses 50.As shown in the embodiment of FIG.",
"1, auxiliary bridge 60 has lens rivets 72 which terminate in bulb 74.When assembled, lens rivets 72 extend horizontally through holes in lenses 50.Lens rivets 72 terminate in bulb 74.Bulb 74 provides security of attachment of lenses 50 to auxiliary bridge 60.Bulb 74 may be present during assembly of lenses 50 to auxiliary bridge 60, or it may be formed or partially deformed upon assembly, to increase the security of attachment.",
"Auxiliary bridge 60 may be made of any flexible material with a plastic memory.",
"In embodiments the invention, auxiliary bridge 60 is made with Polyflex™ (Hyundai Optical Co., Inchon City, Korea) or memory plastic.",
"Memory plastic and Polyflex™ are lightweight plastics that can be bent or stretched without losing their original shape.",
"Polyflex™ is particularly resilient in this regard.",
"B use of such materials, the clipping apparatus between the auxiliary bridge 60 and primary bridge 34 may be used repeatedly without any deformation of shape or degradation of security of attachment.",
"Such materials are also particularly suitable for the mechanical assembly of auxiliary bridge 60 and auxiliary lenses 50.Upon assembly of auxiliary bridge 60 and auxiliary lenses 50, lens rivet 72 is inserted through a hol 52 in each lens.",
"By the use of Polyflex or memory plastic this insertion may be carried out without risk of damage to the lenses.",
"Bulb 74 of FIG.",
"1 is configured such that it is greater in diameter than hole 52 when not deformed.",
"The use of such materials also allows for the insertion of bulb 74 through hole 52, by temporary deformation of bulb 74.Upon being inserted through the hole 52, bulb 74 reestablishes, by plastic memory, its original diameter, thus securing lens 50 to auxiliary bridge 60, in an interference fit.",
"The security of attachment may be increased by treating bulb 74 with a glue or other fixative after assembly, or by treating bulb 74 to compression forces which are great enough to permanently deform bulb 74, partially spreading it against the rear surface or lens 50 immediately around hole 52.In another embodiment, seen in FIG.",
"2, FIG.",
"6 and FIG.",
"9, upon assembly of auxiliary bridge 60 and auxiliary lenses 50, lens rivet 72 is inserted through a hole 52 in each lens.",
"Cap 78 is configured such that projections 73 of the cap are received in corresponding orifices 81 extending lengthwise in lens rivet 72.Upon being inserted into the orifices 81, projections 73 secure cap 78 to auxiliary bridge 60, in an interference fit, thus securing auxiliary lenses 50 to auxiliary bridge 60, as best seen in FIG.",
"9 and FIG.",
"4.The security of attachment may be increased by treating projections 73 with a glue, cement, or other fixative during assembly, or by treating cap 78 to compression forces which are great enough to deform projections 73, spreading them outwards within the orifices.",
"Part 80 facilitates the fit between primary bridge 34 and the rest of primary frame 32.As best seen in FIG.",
"1, primary bridge 34 has a ridge front face 38 which forms a tight fit with auxiliary bridge face 70 when the auxiliary bridge is assembled with the primary spectacles.",
"This tight fit prevents rotation of the bridge off its vertical axis, thus preventing rattling, particularly whe the assembled apparatus of the invention is being used by a wearer during vigorous activity.",
"As best seen in FIG.",
"2, ridge front face 38 extends parallel, and forms a tight fit with auxiliary bridg face 70 which also extends parallel, when the auxiliary bridge is assembled with the primary spectacles.",
"Again, this tight fit prevents rotation of the bridge off its vertical axis, thus preventing rattling.",
"As best seen in FIG.",
"1 and FIG.",
"2, primary bridge 34 has a ridge vertical face 38 which forms a tight fit with auxiliary bridge face 70 when the auxiliary bridge is assembled with the primary spectacles.",
"This tight fit prevents rotation of the bridge off its horizontal axis, thus preventing rattling.",
"As best seen in FIG.",
"3, FIG.",
"5 and FIG.",
"7, a curvature exists in the shape of primary bridge 34 which corresponds to a curvature in the shape of auxiliary bridge 60.When assembled, these corresponding curvatures form a tight fit which further prevents rotation of the bridge off its horizontal axis.",
"As seen in FIG.",
"6 and FIG.",
"7, the upper projection 62 and lower projection 64 of auxiliary bridge 60 can be configured such that there is an upper face 82, a lower face 84, and lateral faces 86, collectively configured to form a snug interference with a correspondingly configured ridge 36 of th primary bridge 34.This prevents movement in any direction.",
"As best seen in FIG.",
"2, protrusion 66 (shown using a dotted line, as it is underneath protrusion 62) extends lengthwise in correspondence with indentation 40.Thus, when assembled, protrusion 66 forms a tight fit with indentation 40 when the auxiliary bridge is assembled with the primary spectacles.",
"This tight fit prevents rotation of the bridge off its vertical axis.",
"Thus it can be seen tha the mechanical clamping of the protrusion/indentation system of the present invention provides an exceptional security of fit on all axis that is not provided by the prior art.",
"As seen in FIG.",
"1, FIG.",
"2 and FIG.",
"5, primary bridge 34 is constructed such that there is physical obstruction created by the shape of primary bridge 32 corresponding to auxiliary bridge 60.This obstruction is not only horizontal, i.e., the interference between primary bridge 34 and auxiliary bridge 60 prevents the auxiliary spectacles from moving downward, but also vertical.",
"This ensures a more secure attachment between the auxiliary spectacles 20 and the primary frame 32.By using the multiple interference fits described herein between the auxiliary and primary bridges, auxiliary spectacles 20 are less likely to collide with primary lenses 18 during physical activity.",
"Thi prevents an annoying rattling noise between the auxiliary spectacles 20 and primary spectacles 42 and also reduces the risk of scratching or otherwise damaging the lenses by reducing contact between primary lenses 18 and auxiliary spectacles 20.The risk of accidental detachment of the auxiliary spectacles is also greatly reduced.",
"A bump or collision which dislodges other, known apparatus will not give rise to the present auxiliary spectacles detaching from the primary spectacle because of the multiple interference fits described herein.",
"When the fastening apparatus of the invention is engaged, auxiliary lenses 50 are located proximal primary lenses 18, so that a minimum amount of light may enter the between the respective lenses.",
"Because projections 62, 64 and ridge 36 project outwardly from the respective frames, no recesses are required in the frames, which would weaken the frames.",
"Furthermore, by avoiding the use of recesses in the fastening apparatus, one is provided with an auxiliary spectacle which can be easily removed and replaced on the primary spectacles, without any flexing or bending of the frames or bridges and without engaging auxiliary spectacle 20 at difficult angles in relation to primary frame 32.In the embodiment as shown in FIG.",
"4 and FIG.",
"8, protrusions 66 are beveled.",
"Beveling the protrusions facilitates the ease of attaching and removing the auxiliary spectacles from the primary spectacles.",
"It will thus be appreciated that the present invention provides a secure method of attaching auxiliary spectacles to primary spectacles without sacrificing the ease with which one can remove and replace the auxiliary spectacles.",
"Removal and replacement can be done with one hand, without the use of awkward angles of engagement and disengagement.",
"The use of a mechanical means of securing attachment, as opposed to a magnetic means, provides significant benefits.",
"First, in producing the apparatus, the auxiliary bridge and the primary frame ma be cast from a desired material, and is then ready for assembly.",
"No drilling or puncturing is required to produce holes for insertion of magnets.",
"No magnets or other additional pieces are required, and no fixative is required to install the means for securing attachment.",
"As the apparatus for securing attachment of the present invention is a “unibody” design, the apparatus will not become loose or fall out with the passage of time and the weathering effect of changes in temperature and humidity.",
"Another benefit of the present means for attaching the auxiliary bridge to the auxiliary lenses is that no lens frames are required for the auxiliary lenses, thus decreasing the weight of the auxiliary spectacles and decreasing the cost of producing the auxiliary spectacles.",
"The above-detailed description with reference to the illustrations is considered to be illustrative and not restrictive in character.",
"Modifications and variations on the embodiments described may be made within the scope of the invention.",
"For example, the auxiliary lenses 50 may also be used for magnification of vision.",
"In this case, the auxiliary lenses 50 would be fashioned in the same manne as a magnifying glass and fit onto the primary lenses 18, which may or may not be prescription lenses.",
"The auxiliary lenses 30 would then enlarge small or intricate details to make them more readily observable, so as to reduce the strain on the eyesight of the wearer.",
"The auxiliary lenses 30 may also have decorations thereon to be viewed by observers of the wearer, to enhance the appearance of the lenses or for amusement purposes.",
"In another embodiment, the present invention also contemplates primary spectacles 42 which contain no primary lens 18.This embodiment would be useful in the circumstances wherein the use desires to remove and replace the auxiliary lenses 50, but does not require the primary lenses 18 for assisting vision.",
"For example, this embodiment would be useful in the context of welding, wherein very dark lenses are required during the welding, but between welding, it may be desirable to have no lenses on.",
"As another example, where an individual suffers from a visual defect which makes reading difficult, it may be desirable to have no lenses except when reading.",
"For this use, the embodiment shown in FIG.",
"10 will be particularly useful, wherein the lower portion 90 of the auxiliary lens 50 is adapted to assist reading and the upper portion 88 is not.",
"In the embodiment shown, the lateral shape of projections 62 and 64 and corresponding lateral shape of ridge 36 prevents horizontal movement of the assembled apparatus.",
"However, in other embodiments (not shown) horizontal movement could be prevented by supplying two of each of protrusions 62 and 64 set laterally apart and corresponding to ridge 36.As best seen in FIG.",
"1, FIG.",
"2 and FIG.",
"5, primary bridge 34 has at least one indentation 40 on ridge 36.In the embodiment shown, indentations 40 are found at the top and bottom of ridge 36.In other embodiments (not shown), only one indentation 40 may be present.",
"In a further embodiment (not shown), a single indentation 40 may comprise a hole extending vertically through ridge 36.It is to be understood that only the preferred embodiments have been shown, and that modifications thereof would be readily apparent to one skilled in the art.",
"Therefore, the true scope and spirit of the invention resides in the appended claims and their legal equivalents, rather than by the given examples."
]
] | Patent_10415467 |
[
[
"Means for detecting pathological transformation of the app protein and their uses",
"A method of intra-articular drug delivery may include selecting an attachment zone in a synovial joint; affixing a drug release device in the attachment zone, the drug release device comprising a base affixable in the attachment zone, a sustained-release drug carrier, and a drug, the device positioned so that the device releases the drug into the synovial fluid of the synovial joint, and so that agitation of the synovial fluid facilitates elution of the drug from the drug release device."
],
[
"1.A method of intra-articular drug delivery, comprising: selecting an attachment zone in a synovial joint; and affixing a drug release device in the attachment zone, the drug release device comprising a base affixable in the attachment zone, a sustained-release drug carrier, and a drug, the device positioned so that the device releases the drug into the synovial fluid of the synovial joint, and so that agitation of the synovial fluid facilitates elution of the drug from the drug release device.",
"2.The method of claim 1, wherein the attachment zone comprises a non-articulating portion of bone and/or cartilage within the synovial joint.",
"3.The method of claim 2, further comprising removing the bone and/or cartilage in the attachment zone to create a void, and so inserting the drug release device into the void that at least one surface of the drug release device is in communication with the synovial fluid.",
"4.The method of claim 3, wherein the drug release device is so inserted that its surface in communication with the synovial fluid is about flush with surrounding bone and/or cartilage.",
"5.The method of claim 2, wherein the attachment zone comprises a band of bone and/or cartilage adjacent to an articulating surface within the synovial joint.",
"6.The method of claim 5, wherein the band extends from about 0.5 millimeters to about 1 centimeter away from the articulating surface.",
"7.The method of claim 5, further comprising removing the bone and/or cartilage in the attachment zone to create a void, and so inserting the drug release device into the void that at least one surface of the drug release device is in communication with the synovial fluid.",
"8.The method of claim 7, wherein the drug release device is so inserted that its surface in communication with the synovial fluid is about flush with surrounding bone and/or cartilage.",
"9.The method of claim 1, wherein the synovial joint is a hip joint, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the hip.",
"10.The method of claim 9, wherein the attachment zone comprises a band of bone and/or cartilage adjacent to at least one of a femoral head, and an acetabulum.",
"11.The method of claim 1, wherein the synovial joint is a knee joint, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the knee.",
"12.The method of claim 11, wherein the attachment zone comprises a band of bone and/or cartilage adjacent to at least one of a tibial plateau, a femoral condyle, a patellofemoral area, the medial rim of a femoral trochlea, the lateral rim of a femoral trochlea, and the periphery of an intercondylar notch.",
"13.The method of claim 1, wherein the synovial joint is a shoulder joint, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the shoulder.",
"14.The method of claim 13, wherein the attachment zone comprises a band of bone and/or cartilage adjacent to at least one of the anatomical neck of a humerus, a glenoid cavity, and a glenoid neck.",
"15.The method of claim 1, wherein the synovial joint is an arthroplastic joint comprising at least one prosthesis, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the joint.",
"16.The method of claim 15, wherein the attachment zone comprises a band of bone and/or cartilage adjacent to the at least one prosthesis.",
"17.The method of claim 1, wherein the drug release device is forcefully injected by gun.",
"18.The method of claim 1, wherein the drug release device comprises threads on its outer surface, and the drug release device is affixed by drilling a hole in the attachment zone and screwing the drug release device into the hole.",
"19.A method of intra-articular drug delivery, comprising: step for selecting a para-articular attachment zone in a synovial joint; step for creating a void in the para-articular attachment zone; and step for implanting in the void a drug-release means for sustainedly releasing a drug into the synovial fluid of the synovial joint.",
"20.A sustained-release intra-articular drug delivery device, comprising: a base, so sized and shaped as to be affixable in an attachment zone of a synovial joint; and a sustained-release drug carrier coupled to the base, the carrier including a drug, the carrier so formed as to elute the drug into synovial fluid, upon implantation of the device in a joint, sufficient to sustain a therapeutically effective concentration of the drug in the synovial fluid for at least 8 hours."
],
[
"<SOH> INTRODUCTION <EOH>Pain and disability from arthritis, joint degeneration, and surgery have been treated by a combination of oral medications or intra-articular injections of steroid compounds designed to reduce inflammation.",
"In addition, other devices, such as hyaluronic acid products, have been injected to provide visco-supplementation.",
"Unfortunately, these approaches have significant systemic side effects or are not effective for extended periods of time.",
"In order to provide local or regional blockade for extended periods, clinicians currently use local anesthetics administered through a catheter or syringe to a site where the pain is to be blocked.",
"This requires repeated administration where the pain is to be blocked over a period of greater than one day, either as a bolus or through an indwelling catheter connected to an infusion pump.",
"These methods have the disadvantage of potentially causing irreversible damage to nerves or surrounding tissues due to fluctuations in concentration and high levels of anesthetic.",
"In addition, anesthetic administered by these methods are generally neither confined to the target area, nor delivered in a linear, continuous manner.",
"In all cases, analgesia rarely lasts for longer than six to twelve hours, more typically four to six hours.",
"In the case of a pump, the infusion lines are difficult to position and secure, the patient has limited, encumbered mobility and, when the patient is a small child or mentally impaired, they may accidentally disengage the pump.",
"In part, this disclosure describes implantable devices that may be used to deliver drugs to a joint."
],
[
"<SOH> SUMMARY <EOH>In one aspect, this disclosure describes devices and methods for delivering drugs to the synovial fluid of a joint by locally implanting a drug delivery device.",
"In certain embodiments, the device is positioned in such a way that agitation of synovial fluid facilitates elution of the drug from the device.",
"In one aspect, a method of intra-articular drug delivery includes selecting an attachment zone in a synovial joint and affixing a drug release device in the attachment zone.",
"Exemplary suitable attachment zones include intra-articular regions of the synovial joint where there is no interfacing articular cartilage.",
"In certain instances, an attachment zone may include intra-articular regions of bone that are non-load-bearing and optionally removed from the articulation surface.",
"In certain embodiments, the drug release device includes a base affixable in the attachment zone, a sustained-release drug carrier, and a drug.",
"The device may be positioned, in certain applications, so that the device releases the drug into the synovial fluid of the synovial joint, and further, so that agitation of the synovial fluid may facilitate elution of the drug from the drug release device.",
"In a further aspect, a method of providing a therapeutic to a skeletal articulation includes identifying a safe zone of the articulation and coupling a therapeutic elution apparatus in the safe zone.",
"Exemplary safe zones include non-load-bearing regions in or around the articulation.",
"In certain embodiments, the therapeutic elution apparatus includes a body couplable in the safe zone, and a therapeutic dispersed in a controlled-release binder.",
"The apparatus may be positioned, in certain applications, so that it releases the therapeutic into the articulation environment.",
"In another aspect, a drug delivery device includes a base and a sustained-release drug carrier coupled to the base.",
"In certain embodiments, the base may be so sized and shaped as to be capable of affixation in an attachment zone of a synovial joint.",
"Typically, the carrier includes a drug to be eluted in vivo, often into the synovial fluid upon implantation of the device in a joint.",
"In certain applications, the carrier may be so formed as to elute the drug into synovial fluid sufficient to sustain a therapeutically effective concentration of the drug in the synovial fluid for at least 8 hours.",
"In yet another aspect, a wide range of therapeutic drugs are contemplated, including but limited to antiinflammatories, antiinfectives, analgesics, and anesthetics.",
"A wide range of drug carrier materials are contemplated, including but not limited to polymers, such as polytetrafluoroethylene, polyfluorinated ethylenepropylene, polylactic acid, polyglycolic acid, silicone, and mixtures thereof.",
"In still another aspect, a drug delivery device may be delivered by a wide variety of methods, such as by placement into a drill site, or forceful injection by gun.",
"In an embodiment, a method of intra-articular drug delivery may include selecting an attachment zone in a synovial joint, and affixing a drug release device in the attachment zone, the drug release device comprising a base affixable in the attachment zone, a sustained-release drug carrier, and a drug, the device positioned so that the device releases the drug into the synovial fluid of the synovial joint, and so that agitation of the synovial fluid facilitates elution of the drug from the drug release device.",
"In any preceding embodiment, the attachment zone may include a non-articulating portion of bone and/or cartilage within the synovial joint.",
"Any preceding embodiment may further include removing the bone and/or cartilage in the attachment zone to create a void, and so inserting the drug release device into the void that at least one surface of the drug release device is in communication with the synovial fluid.",
"In any preceding embodiment, the drug release device may be so inserted that its surface in communication with the synovial fluid is about flush with surrounding bone and/or cartilage.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to an articulating surface within the synovial joint.",
"In any preceding embodiment, the band may extend from about 0.5 millimeters to about 1 centimeter away from the articulating surface.",
"In any preceding embodiment, the synovial joint may be a hip joint, and the attachment zone may include a non-articulating portion of bone and/or cartilage within the hip.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to at least one of a femoral head, and an acetabulum.",
"In any preceding embodiment, the synovial joint may be a knee joint, and the attachment zone may include a non-articulating portion of bone and/or cartilage within the knee.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to at least one of a tibial plateau, a femoral condyle, a patellofemoral area, the medial rim of a femoral trochlea, the lateral rim of a femoral trochlea, and the periphery of an intercondylar notch.",
"In any preceding embodiment, the synovial joint may be a shoulder joint, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the shoulder.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to at least one of the anatomical neck of a humerus, a glenoid cavitym and a glenoid neck.",
"In any preceding embodiment, the synovial joint may be an arthroplastic joint comprising at least one prosthesis, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the joint.",
"In any preceding embodiment, wherein the attachment zone may include a band of bone and/or cartilage adjacent to the at least one prosthesis.",
"In any preceding embodiment, the drug release device may be forcefully injected by gun.",
"In any preceding embodiment, the drug release device may include threads on its outer surface, and the drug release device may be affixed by drilling a hole in the attachment zone and screwing the drug release device into the hole.",
"In any preceding embodiment, the drug release device may include a base, so sized and shaped as to be affixable in an attachment zone of a synovial joint, and a sustained-release drug carrier coupled to the base, the carrier including a drug, the carrier so formed as to elute the drug into synovial fluid, upon implantation of the device in a joint, sufficient to sustain a therapeutically effective concentration of the drug in the synovial fluid for at least 8 hours."
],
[
"CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S.",
"Provisional Application Ser.",
"No.",
"60/536,135, filed Jan. 13, 2004, and also claims the benefit of U.S.",
"Provisional Application Ser.",
"No.",
"60/566,737, filed Apr.",
"29, 2004.The aforementioned patent applications are hereby incorporated herein by this reference.",
"INTRODUCTION Pain and disability from arthritis, joint degeneration, and surgery have been treated by a combination of oral medications or intra-articular injections of steroid compounds designed to reduce inflammation.",
"In addition, other devices, such as hyaluronic acid products, have been injected to provide visco-supplementation.",
"Unfortunately, these approaches have significant systemic side effects or are not effective for extended periods of time.",
"In order to provide local or regional blockade for extended periods, clinicians currently use local anesthetics administered through a catheter or syringe to a site where the pain is to be blocked.",
"This requires repeated administration where the pain is to be blocked over a period of greater than one day, either as a bolus or through an indwelling catheter connected to an infusion pump.",
"These methods have the disadvantage of potentially causing irreversible damage to nerves or surrounding tissues due to fluctuations in concentration and high levels of anesthetic.",
"In addition, anesthetic administered by these methods are generally neither confined to the target area, nor delivered in a linear, continuous manner.",
"In all cases, analgesia rarely lasts for longer than six to twelve hours, more typically four to six hours.",
"In the case of a pump, the infusion lines are difficult to position and secure, the patient has limited, encumbered mobility and, when the patient is a small child or mentally impaired, they may accidentally disengage the pump.",
"In part, this disclosure describes implantable devices that may be used to deliver drugs to a joint.",
"SUMMARY In one aspect, this disclosure describes devices and methods for delivering drugs to the synovial fluid of a joint by locally implanting a drug delivery device.",
"In certain embodiments, the device is positioned in such a way that agitation of synovial fluid facilitates elution of the drug from the device.",
"In one aspect, a method of intra-articular drug delivery includes selecting an attachment zone in a synovial joint and affixing a drug release device in the attachment zone.",
"Exemplary suitable attachment zones include intra-articular regions of the synovial joint where there is no interfacing articular cartilage.",
"In certain instances, an attachment zone may include intra-articular regions of bone that are non-load-bearing and optionally removed from the articulation surface.",
"In certain embodiments, the drug release device includes a base affixable in the attachment zone, a sustained-release drug carrier, and a drug.",
"The device may be positioned, in certain applications, so that the device releases the drug into the synovial fluid of the synovial joint, and further, so that agitation of the synovial fluid may facilitate elution of the drug from the drug release device.",
"In a further aspect, a method of providing a therapeutic to a skeletal articulation includes identifying a safe zone of the articulation and coupling a therapeutic elution apparatus in the safe zone.",
"Exemplary safe zones include non-load-bearing regions in or around the articulation.",
"In certain embodiments, the therapeutic elution apparatus includes a body couplable in the safe zone, and a therapeutic dispersed in a controlled-release binder.",
"The apparatus may be positioned, in certain applications, so that it releases the therapeutic into the articulation environment.",
"In another aspect, a drug delivery device includes a base and a sustained-release drug carrier coupled to the base.",
"In certain embodiments, the base may be so sized and shaped as to be capable of affixation in an attachment zone of a synovial joint.",
"Typically, the carrier includes a drug to be eluted in vivo, often into the synovial fluid upon implantation of the device in a joint.",
"In certain applications, the carrier may be so formed as to elute the drug into synovial fluid sufficient to sustain a therapeutically effective concentration of the drug in the synovial fluid for at least 8 hours.",
"In yet another aspect, a wide range of therapeutic drugs are contemplated, including but limited to antiinflammatories, antiinfectives, analgesics, and anesthetics.",
"A wide range of drug carrier materials are contemplated, including but not limited to polymers, such as polytetrafluoroethylene, polyfluorinated ethylenepropylene, polylactic acid, polyglycolic acid, silicone, and mixtures thereof.",
"In still another aspect, a drug delivery device may be delivered by a wide variety of methods, such as by placement into a drill site, or forceful injection by gun.",
"In an embodiment, a method of intra-articular drug delivery may include selecting an attachment zone in a synovial joint, and affixing a drug release device in the attachment zone, the drug release device comprising a base affixable in the attachment zone, a sustained-release drug carrier, and a drug, the device positioned so that the device releases the drug into the synovial fluid of the synovial joint, and so that agitation of the synovial fluid facilitates elution of the drug from the drug release device.",
"In any preceding embodiment, the attachment zone may include a non-articulating portion of bone and/or cartilage within the synovial joint.",
"Any preceding embodiment may further include removing the bone and/or cartilage in the attachment zone to create a void, and so inserting the drug release device into the void that at least one surface of the drug release device is in communication with the synovial fluid.",
"In any preceding embodiment, the drug release device may be so inserted that its surface in communication with the synovial fluid is about flush with surrounding bone and/or cartilage.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to an articulating surface within the synovial joint.",
"In any preceding embodiment, the band may extend from about 0.5 millimeters to about 1 centimeter away from the articulating surface.",
"In any preceding embodiment, the synovial joint may be a hip joint, and the attachment zone may include a non-articulating portion of bone and/or cartilage within the hip.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to at least one of a femoral head, and an acetabulum.",
"In any preceding embodiment, the synovial joint may be a knee joint, and the attachment zone may include a non-articulating portion of bone and/or cartilage within the knee.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to at least one of a tibial plateau, a femoral condyle, a patellofemoral area, the medial rim of a femoral trochlea, the lateral rim of a femoral trochlea, and the periphery of an intercondylar notch.",
"In any preceding embodiment, the synovial joint may be a shoulder joint, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the shoulder.",
"In any preceding embodiment, the attachment zone may include a band of bone and/or cartilage adjacent to at least one of the anatomical neck of a humerus, a glenoid cavitym and a glenoid neck.",
"In any preceding embodiment, the synovial joint may be an arthroplastic joint comprising at least one prosthesis, and the attachment zone comprises a non-articulating portion of bone and/or cartilage within the joint.",
"In any preceding embodiment, wherein the attachment zone may include a band of bone and/or cartilage adjacent to the at least one prosthesis.",
"In any preceding embodiment, the drug release device may be forcefully injected by gun.",
"In any preceding embodiment, the drug release device may include threads on its outer surface, and the drug release device may be affixed by drilling a hole in the attachment zone and screwing the drug release device into the hole.",
"In any preceding embodiment, the drug release device may include a base, so sized and shaped as to be affixable in an attachment zone of a synovial joint, and a sustained-release drug carrier coupled to the base, the carrier including a drug, the carrier so formed as to elute the drug into synovial fluid, upon implantation of the device in a joint, sufficient to sustain a therapeutically effective concentration of the drug in the synovial fluid for at least 8 hours.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1 depicts a cross section of an exemplary intra-articular drug delivery device.",
"FIG.",
"2 depicts an exemplary positioning of an intra-articular drug delivery device.",
"FIG.",
"3 depicts a cross section of a generalized synovial joint.",
"FIG.",
"4 depicts a cross section of a knee joint showing exemplary placements of an intra-articular drug delivery device.",
"FIGS.",
"5-8 depict various synovial joints and exemplary placements of an intra-articular drug delivery device therein.",
"FIGS.",
"9-11A depict exemplary drug delivery devices and components.",
"FIGS.",
"12-13 depict exemplary placements of drug delivery devices.",
"FIG.",
"14 depicts an exemplary insertion device for a drug delivery device.",
"FIGS.",
"15-17 depict various synovial joints and exemplary placements of an intra-articular drug delivery device therein.",
"FIG.",
"18 depicts an exemplary placements of an intra-articular drug delivery device in the context of arthroplasty.",
"DETAILED DESCRIPTION 1.Overview The present disclosure provides devices and methods for delivering drugs to joints, particularly synovial joints.",
"In part, this disclosure provides controlled-release devices that are capable of being implanted in the joint to deliver therapeutic agents, often to the synovial fluid.",
"In certain examples, the devices, when implanted, are capable of providing sustained drug release of therapeutic agents into the synovial fluid of the affected joint for the relief of pain, reduction of inflammation, the enhancement of joint lubrication, the treatment of infection, and/or the treatment or prevention of other diseases or conditions.",
"In a preferred embodiment, a device is implanted within a joint into bone in an area that is substantially exposed to synovial fluid flow, preferably allowing sustained elution of the therapeutic compound into the synovial fluid and a substantially even distribution of the desired therapeutic within the joint.",
"In certain instances, the implantation site is constantly exposed to synovial fluid flow.",
"Each joint contains specified areas defined hereafter as “attachment zones” that permit drilling, anchoring, or other types of affixing of a device.",
"Certain attachment zones will not cause substantial damage to the load-bearing or articulating cartilage or other surfaces of the specified joint.",
"In certain embodiments, the placement and location of the device does not cause material damage to the cartilage surface, as it may placed and secured using an anchoring device that fixes the device to bone, in the joint cavity, but not into or on the articulating surface of the joint in a manner that could cause such damage while the device is in place.",
"The controlled release devices may be biocompatible and may be capable of being implanted or otherwise placed into a joint.",
"In an exemplary embodiment, a device may have a core including a drug/polymer composition and an outer layer along the long axis of the device that is substantially impermeable to the entrance of an environmental fluid and substantially impermeable to the release of the drug during a delivery period, with drug release possible across a short axis plane of the device, which axis or end of the device may be exposed to synovial fluid upon placement.",
"In certain instances, the end of the device may have a semipermeable membrane or alternatively it may expose the core directly.",
"In certain instances, the short axis plane of a device may account for a minority, such as no more than about 10%, no more than about 20%, no more than about 30%, no more than about 40%, or less than 50%, of the total surface area of the device.",
"The device can be so sized and shaped, and formed of such materials, as to facilitate delivering a variety of drugs with varying degrees of solubility and molecular weight.",
"Methods are also provided for using these drug delivery devices.",
"In certain aspects, the devices may be implanted in regions of a joint that are non-load-bearing and/or that do not form part of the cartilaginous articulation surfaces of bones.",
"In further examples, the devices may be anchored to and recessed within bone either fully or in part.",
"In certain instances, usually when recessed fully, the portion of the device's surface exposed to the synovial fluid is substantially flush with the bone surface.",
"In still further embodiments, the devices may be positioned in a joint so that agitation of synovial fluid past or through the exposed surface facilitates elution of a therapeutic incorporated in the device.",
"In other examples, the one or more therapeutics may be formulated for sustained release, such as by impregnating them in a matrix, or mixing them with a suitable composition, such as a polymer.",
"2.Definitions For convenience, before further description of exemplary embodiments, certain terms employed in the specification, examples, and appended claims are collected here.",
"These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art.",
"The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.",
"By way of example, “an element” means one element or more than one element.",
"The term “access device” is an art-recognized term and includes any medical device adapted for gaining or maintaining access to an anatomic area Such devices are familiar to artisans in the medical and surgical fields.",
"An access device may be a needle, a catheter, a cannula, a trocar, a tubing, a shunt, a drain, or an endoscope such as an otoscope, nasopharyngoscope, bronchoscope, or any other endoscope adapted for use in the joint area, or any other medical device suitable for entering or remaining positioned within the preselected anatomic area.",
"The terms “biocompatible polymer” and “biocompatibility” when used in relation to polymers are art-recognized.",
"For example, biocompatible polymers include polymers that are generally neither themselves toxic to the host, nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host.",
"In certain embodiments, biodegradation generally involves degradation of the polymer in a host, e.g., into its monomeric subunits, which may be known to be effectively non-toxic.",
"Intermediate oligomeric products resulting from such degradation may have different toxicological properties, however, or biodegradation may involve oxidation or other biochemical reactions that generate molecules other than monomeric subunits of the polymer.",
"Consequently, in certain embodiments, toxicology of a biodegradable polymer intended for in vivo use, such as implantation or injection into a patient, may be determined after one or more toxicity analyses.",
"It is not necessary that any subject composition have a purity of 100% to be deemed biocompatible; indeed, it is only necessary that the subject compositions be biocompatible as set forth above.",
"Hence, a subject composition may comprise polymers comprising 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or even less of biocompatible polymers, e.g., including polymers and other materials and excipients described herein, and still be biocompatible.",
"To determine whether a polymer or other material is biocompatible, it may be necessary to conduct a toxicity analysis.",
"Such assays are well known in the art.",
"One example of such an assay may be performed with live carcinoma cells, such as GT3TKB tumor cells, in the following manner: the sample is degraded in 1M NaOH at 37° C. until complete degradation is observed.",
"The solution is then neutralized with 1M HCl.",
"About 200 μL of various concentrations of the degraded sample products are placed in 96-well tissue culture plates and seeded with human gastric carcinoma cells (GT3TKB) at 104/well density.",
"The degraded sample products are incubated with the GT3TKB cells for 48 hours.",
"The results of the assay may be plotted as % relative growth vs. concentration of degraded sample in the tissue-culture well.",
"In addition, polymers and formulations may also be evaluated by well-known in vivo tests, such as subcutaneous implantations in rats to confirm that they do not cause significant levels of irritation or inflammation at the subcutaneous implantation sites.",
"The term “biodegradable” is art-recognized, and includes polymers, compositions and formulations, such as those described herein, that are intended to degrade during use.",
"Biodegradable polymers typically differ from non-biodegradable polymers in that the former may be degraded during use.",
"In certain embodiments, such use involves in vivo use, such as in vivo therapy, and in other certain embodiments, such use involves in vitro use.",
"In general, degradation attributable to biodegradability involves the degradation of a biodegradable polymer into its component subunits, or digestion, e.g., by a biochemical process, of the polymer into smaller, non-polymeric subunits.",
"In certain embodiments, two different types of biodegradation may generally be identified.",
"For example, one type of biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone.",
"In such biodegradation, monomers and oligomers typically result, and even more typically, such biodegradation occurs by cleavage of a bond connecting one or more of subunits of a polymer.",
"In contrast, another type of biodegradation may involve cleavage of a bond (whether covalent or otherwise) internal to side chain or that connects a side chain to the polymer backbone.",
"For example, a therapeutic agent or other chemical moiety attached as a side chain to the polymer backbone may be released by biodegradation.",
"In certain embodiments, one or the other or both generally types of biodegradation may occur during use of a polymer.",
"As used herein, the term “biodegradation” encompasses both general types of biodegradation.",
"The degradation rate of a biodegradable polymer often depends in part on a variety of factors, including the chemical identity of the linkage responsible for any degradation, the molecular weight, crystallinity, biostability, and degree of cross-linking of such polymer, the physical characteristics of the implant, shape and size, and the mode and location of administration.",
"For example, the greater the molecular weight, the higher the degree of crystallinity, and/or the greater the biostability, the biodegradation of any biodegradable polymer is usually slower.",
"The term “biodegradable” is intended to cover materials and processes also termed “bioerodible”.",
"In certain embodiments, if the biodegradable polymer also has a therapeutic agent or other material associated with it, the biodegradation rate of such polymer may be characterized by a release rate of such materials.",
"In such circumstances, the biodegradation rate may depend on not only the chemical identity and physical characteristics of the polymer, but also on the identity of any such material incorporated therein.",
"In certain embodiments, polymeric formulations biodegrade within a period that is acceptable in the desired application.",
"In certain embodiments, such as in vivo therapy, such degradation occurs in a period usually less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or even one day on exposure to a physiological solution with a pH between 6 and 8 having a temperature of between 25 and 37° C. In other embodiments, the polymer degrades in a period of between about one hour and several weeks, depending on the desired application.",
"The terms “comprise,” “comprising,” “include,” “including,” “have,” and “having” are used in the inclusive, open sense, meaning that additional elements may be included.",
"The terms “such as”, “e.g.”, as used herein are non-limiting and are for illustrative purposes only.",
"“Including” and “including but not limited to” are used interchangeably.",
"The term “drug delivery device” is an art-recognized term and refers to any medical device suitable for the application of a drug to a targeted organ or anatomic region.",
"The term includes those devices that transport or accomplish the instillation of the compositions towards the targeted organ or anatomic area, even if the device itself is not formulated to include the composition.",
"As an example, a needle or a catheter through which the composition is inserted into an anatomic area or into a blood vessel or other structure related to the anatomic area is understood to be a drug delivery device.",
"As a further example, a stent or a shunt or a catheter that has the composition included in its substance or coated on its surface is understood to be a drug delivery device.",
"A drug delivery device can include a rigid or flexible container.",
"It may include a semi-solid composition that release drug by dissolution of the device or by leaching of drug from the device.",
"We should also be clear that “implant” covers attaching to the joint in any way, e.g., by implanting into a cavity in bone or cartilage or by suturing or otherwise adhering the device to the surface of bone, tendon, or cartilage.",
"When used with respect to a therapeutic agent or other material, the term “sustained release” is art-recognized.",
"For example, a subject composition that releases a substance over time may exhibit sustained release characteristics, in contrast to a bolus type administration in which the entire amount of the substance is made biologically available at one time.",
"For example, in particular embodiments, upon contact with body fluids including blood, tissue fluid, lymph or the like, the polymer matrices (formulated as provided herein and otherwise as known to one of skill in the art) may undergo gradual degradation (e.g., through hydrolysis) with concomitant release of any material incorporated therein, for a sustained or extended period (as compared to the release from a bolus).",
"This release may result in prolonged delivery of therapeutically effective amounts of any incorporated a therapeutic agent.",
"Sustained release will vary in certain embodiments as described in greater detail below.",
"The term “delivery agent” is an art-recognized term, and includes molecules that facilitate the intracellular delivery of a therapeutic agent or other material.",
"Examples of delivery agents include: sterols (e.g., cholesterol) and lipids (e.g., a cationic lipid, virosome or liposome).",
"The term “or” as used herein should be understood to mean “and/or”, unless the context clearly indicates otherwise.",
"The phrases “parenteral administration” and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.",
"The term “treating” is art-recognized and includes preventing a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition.",
"Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.",
"The term “fluid” is art-recognized to refer to a non-solid state of matter in which the atoms or molecules are free to move in relation to each other, as in a gas or liquid.",
"If unconstrained upon application, a fluid material may flow to assume the shape of the space available to it, covering for example, the surfaces of an excisional site or the dead space left under a flap.",
"A fluid material may be inserted or injected into a limited portion of a space and then may flow to enter a larger portion of the space or its entirety.",
"Such a material may be termed “flowable.” This term is art-recognized and includes, for example, liquid compositions that are capable of being sprayed into a site; injected with a manually operated syringe fitted with, for example, a 23-gauge needle; or delivered through a catheter.",
"Also included in the term “flowable” are those highly viscous, “gel-like” materials at room temperature that may be delivered to the desired site by pouring, squeezing from a tube, or being injected with any one of the commercially available injection devices that provide injection pressures sufficient to propel highly viscous materials through a delivery system such as a needle or a catheter.",
"When the polymer used is itself flowable, a composition comprising it need not include a biocompatible solvent to allow its dispersion within a body cavity.",
"Rather, the flowable polymer may be delivered into the body cavity using a delivery system that relies upon the native flowability of the material for its application to the desired tissue surfaces.",
"For example, if flowable, a composition comprising polymers can be injected to form, after injection, a temporary biomechanical barrier to coat or encapsulate internal organs or tissues, or it can be used to produce coatings for solid implantable devices.",
"In certain instances, flowable subject compositions have the ability to assume, over time, the shape of the space containing it at body temperature.",
"Viscosity is understood herein as it is recognized in the art to be the internal friction of a fluid or the resistance to flow exhibited by a fluid material when subjected to deformation.",
"The degree of viscosity of the polymer may be adjusted by the molecular weight of the polymer and other methods for altering the physical characteristics of a specific polymer will be evident to practitioners of ordinary skill with no more than routine experimentation.",
"The molecular weight of the polymer used may vary widely, depending on whether a rigid solid state (higher molecular weights) desirable, or whether a fluid state (lower molecular weights) is desired.",
"The phrase “pharmaceutically acceptable” is art-recognized.",
"In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.",
"The phrase “pharmaceutically acceptable carrier” is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body.",
"Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.",
"In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic.",
"Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.",
"The term “pharmaceutically acceptable salts” is art-recognized, and includes relatively non-toxic, inorganic and organic acid addition salts of compositions, including without limitation, therapeutic agents, excipients, other materials and the like.",
"Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.",
"Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and the like.",
"Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.",
"For purposes of illustration, the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; (trihydroxymethyl)aminoethane; and the like.",
"See, for example, J. Pharm.",
"Sci., 66:1-19 (1977).",
"A “patient,” “subject,” or “host” to be treated by the subject method may mean either a human or non-human animal, such as primates, mammals, and vertebrates.",
"The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions.",
"If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).",
"The term “synovial joint” refers to a moveable articulation of two or more bones.",
"The articulation is defined by a synovial cavity, which contains a volume of synovial fluid, is lined with a synovial membrane, and is surrounded by a fibrous capsule.",
"The opposing bone surfaces are each covered with a layer of cartilage.",
"The cartilage and synovial fluid reduce friction between the articulating bone surfaces and enable smooth movements.",
"Synovial joints can be further distinguished by their shape, which controls the movements they allow.",
"For example, hinge joints act like the hinge on a door, allowing flexion and extension in just one plane.",
"An example is the elbow between the humerus and the ulna.",
"Ball and socket joints, such as the hip, allow movement in several planes simultaneously.",
"Condyloid (or ellipsoid) joints, such as the knee, permit motion in more than one plane in some positions but not others.",
"For example, no rotation is possible in the extended knee, but some rotation is possible when the knee is flexed.",
"Pivot joints, such as the elbow (between the radius and the ulna), allow one bone to rotate around another.",
"Saddle joints, such as at the thumb (between the metacarpal and carpal) are so named because of their saddle shape, and allow movement in a variety of directions.",
"Finally, gliding joints, such as in the carpals of the wrist, allow a wide variety of movement, but not much distance.",
"Synovial joints include but are not limited to shoulder (glenohumeral and acromioclavicular), elbow (ulno-humeral, radio-capitellar and proximal radioulnar), forearm (radioulnar, radiocarpal, ulnocarpal), wrist (distal radioulnar, radio-carpal, ulno-carpal, mid carpal), hand (carpo-metacarpal, metocarpophalangeal, interphalangeal), spine (intervertebral), hip, knee, ankle (tibiotalar, tibiofibular), and foot (talocalcaneal, talonavicular, intertarsal, tarso-metatarsal, metatarsal-phalangeal, interphalangeal).",
"The terms “therapeutic agent”, “drug”, “medicament” and “bioactive substance” are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition, such as joint pain, degeneration, inflammation, or infection.",
"The terms include without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.",
"Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides and the like that are biologically activated when administered into a patient or subject.",
"The phrase “therapeutically effective amount” is an art-recognized term.",
"In certain embodiments, the term refers to an amount of a therapeutic agent that, when incorporated into a polymer, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment.",
"In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain (e.g., prevent the spread of) a tumor or other target of a particular therapeutic regimen.",
"The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition.",
"One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.",
"The term “preventing”, when used in relation to a condition, such as a local recurrence, a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.",
"Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.",
"Prevention of an infection includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.",
"“Radiosensitizer” is defined as a therapeutic agent that, upon administration in a therapeutically effective amount, promotes the treatment of one or more diseases or conditions that are treatable with electromagnetic radiation.",
"In general, radiosensitizers are intended to be used in conjunction with electromagnetic radiation as part of a prophylactic or therapeutic treatment.",
"Appropriate radiosensitizers to use in conjunction with treatment with the subject compositions will be known to those of skill in the art.",
"“Electromagnetic radiation” as used in this specification includes, but is not limited to, radiation having the wavelength of 10−20 to 10 meters.",
"Particular embodiments of electromagnetic radiation employ the electromagnetic radiation of: gamma-radiation (10−20 to 10−13 m), x-ray radiation (10−11 to 10−9 m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to 30 cm).",
"The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” are art-recognized, and include the administration of a subject composition or other material at a site remote from the disease being treated.",
"Administration of an agent directly into, onto or in the vicinity of a lesion of the disease being treated, even if the agent is subsequently distributed systemically, may be termed “local” or “topical” or “regional” administration, other than directly into the central nervous system, e.g., by subcutaneous administration, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.",
"In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on a number of factors, including: the rate of release of the agent from the polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent.",
"The term “ED50” is art-recognized.",
"In certain embodiments, ED50 means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations.",
"The term “LD50” is art-recognized.",
"In certain embodiments, LD50 means the dose of a drug which is lethal in 50% of test subjects.",
"The term “therapeutic index” is an art-recognized term which refers to the therapeutic index of a drug, defined as LD50/ED50.The terms “incorporated” and “encapsulated” are art-recognized when used in reference to a therapeutic agent and a polymeric composition, such as a composition disclosed herein.",
"In certain embodiments, these terms include incorporating, formulating or otherwise including such agent into a composition which allows for sustained release of such agent in the desired application.",
"The terms may contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including for example: attached to a monomer of such polymer (by covalent or other binding interaction) and having such monomer be part of the polymerization to give a polymeric formulation, distributed throughout the polymeric matrix, appended to the surface of the polymeric matrix (by covalent or other binding interactions), encapsulated inside the polymeric matrix, etc.",
"The term “co-incorporation” or “co-encapsulation” refers to the incorporation of a therapeutic agent or other material and at least one other a therapeutic agent or other material in a subject composition.",
"More specifically, the physical form in which a therapeutic agent or other material is encapsulated in polymers may vary with the particular embodiment.",
"For example, a therapeutic agent or other material may be first encapsulated in a microsphere and then combined with the polymer in such a way that at least a portion of the microsphere structure is maintained.",
"Alternatively, a therapeutic agent or other material may be sufficiently immiscible in a controlled-release polymer that it is dispersed as small droplets, rather than being dissolved, in the polymer.",
"Any form of encapsulation or incorporation is contemplated by the present disclosure, in so much as the sustained release of any encapsulated therapeutic agent or other material determines whether the form of encapsulation is sufficiently acceptable for any particular use.",
"The term “biocompatible plasticizer” is art-recognized, and includes materials which are soluble or dispersible in the controlled-release compositions described herein, which increase the flexibility of the polymer matrix, and which, in the amounts employed, are biocompatible.",
"Suitable plasticizers are well known in the art and include those disclosed in U.S. Pat.",
"Nos.",
"2,784,127 and 4,444,933.Specific plasticizers include, by way of example, acetyl tri-n-butyl citrate (c. 20 weight percent or less), acetyl trihexyl citrate (c. 20 weight percent or less), butyl benzyl phthalate, dibutyl phthalate, dioctylphthalate, n-butyryl tri-n-hexyl citrate, diethylene glycol dibenzoate (c. 20 weight percent or less) and the like.",
"“Small molecule” is an art-recognized term and refers to a molecule which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu.",
"3.Implant Structure A wide variety of structures may be employed for providing drug delivery to synovial joints.",
"Typically, a drug delivery device may include a base that is so sized and shaped as to be affixable in the synovial joint, and a drug carrier coupled to the base that includes a therapeutic drug.",
"An exemplary implantable intra-articular drug delivery device is depicted in FIG.",
"1.As shown in this exemplary embodiment, the base of the device may include a housing (5), and the drug carrier may include a mass (4).",
"The mass (4) may be disposed inside the housing.",
"In some instances, the mass (4) may be a cartridge.",
"For example, the cartridge may be manufactured separately from the housing and later inserted into the housing.",
"The cartridge or mass (4) may be replaceable, so that the drug carrier may be removed from the device without disturbing the housing's affixation in a joint The outer layer of the mass (4) may be formed at least in part by a material substantially impermeable to the drug and/or environmental fluids (such as synovial fluid in a joint).",
"The material may include a polymer.",
"Examples of polymers include polytetrafluoroethylene, polyfluorinated ethylenepropylene, polylactic acid, polyglycolic acid, silicone, and mixtures thereof.",
"The mass (4) may have a surface (1) exposed to the environment, such as synovial fluid.",
"The surface (1) may be covered at least in part by a membrane, such as a semi-permeable membrane.",
"The membrane may be formed to prevent particulate materials, such as biodegradable polymer, from passing out of the drug carrier and into the synovial fluid, while permitting the drug released from the carrier (2) to pass out of the cartridge and in to the joint.",
"This filter may take consist of a semi-permeable, osmotic membrane or a porous cellulose filter such as a Millipore filter.",
"In certain embodiments, the housing (5) may be made at least in part of a biocompatible material.",
"Furthermore, in some embodiments, the housing (5) may be made of an implantable material, such as a material suitable for implantation in bone, implantation in cartilage, and/or implantation in other biomaterials in a joint.",
"In certain embodiments, the housing is formed at least in part of a material of sufficient strength to be implanted into bone without damage.",
"In particular, the housing may be formed at least in part of a material that can maintain the housing's integrity during implantation.",
"This may help prevent leakage of a drug in the carrier through a crack or fissure in the housing.",
"In some embodiments, the reservoir housing may be constructed from a metal, such as titanium, nickel titanium, stainless steel, anodized aluminum, or tantalum, or a plastic, such as polyethylene, nylon, or polyurethane.",
"Alternatively a composite or ceramic may be used.",
"The housing may also include a material or modified material to allow for osseous integration of the implant—i.e., bone ingrowth.",
"Other suitable materials will be apparent to one of ordinary skill in the art.",
"Moreover, combinations of materials may be used.",
"In certain embodiments, the device may be affixable in an attachment zone of a joint.",
"As depicted in FIG.",
"1, the housing (5) may include one or more barbs (3).",
"The barb or barbs (3) may lodge in and/or against, for example, a bony surface, and thereby minimize the device's motion relative to the bone.",
"In some embodiments, barbs (3) may be axially aligned.",
"They may be circumferentially spaced in relation to each other about the base.",
"In some embodiments, the barb or barbs may be able to adopt different states; in some states, the barbs may be retracted or otherwise disposed to facilitate mobility and positioning of the device; in other states, the barbs may be expanded or otherwise disposed to facilitate lodgment and immobility of the device.",
"The barb or barbs may be transitionable between one or more such states.",
"For example, a barb may have a first, or “retracted” state, in which the barb's span lies close enough to the device as not to impede positioning the device.",
"The barb may have a second, or “expanded” state, in which the barb's free end so protrudes from the device as to impinge surrounding anatomy, thereby promoting affixation of the device.",
"In some embodiments, the barb may be deformable among various states.",
"For example, a barb may be biased toward a particular state.",
"A barb may be constrained to an unbiased state and allowed to assume a biased state when the constraint is removed.",
"In some embodiments, a constraint may be mechanical, such as a sleeve or an adhesive.",
"In some embodiments, a constraint may be chemical, so that the barb changes configuration in response to a chemical reaction.",
"In some embodiments, the barb may have a shape memory.",
"A shape memory may be dependent, for example, on temperature.",
"In one embodiment, a barb may assume a “retracted” state below attachment zone temperature and an “expanded” state at attachment zone temperature, so that disposition of a device in an attachment zone causes the barb to transition from the retracted state to the expanded state.",
"A barb may be elastically or plastically deformable; a barb may be reversibly or irreversible deformable.",
"In the embodiment depicted in FIG.",
"1, the barbs extend rearwardly and radially outwardly from the body.",
"In some embodiments, the barb end(s) may extend to positions outside a longitudinal projection of the largest geometric cross-section of the body transverse to its longitudinal axis.",
"Barbs (3) may be formed at least in part by a wide variety of materials.",
"Examples include materials disclosed in U.S. Pat.",
"No.",
"4,665,906 entitled “Medical Devices Incorporating SIM Alloy Elements”, issued May 19, 1987 to Jervis.",
"Other exemplary materials are nickel-titanium alloys, such as nitinol.",
"The combination of the material and orientation of the barbs on the reservoir housing can help the barbs to “spring back” toward their normal, unstressed condition after the insertion process of the device into bone, for example, is complete.",
"The device may include other features to make it affixable in an attachment zone of a joint.",
"For example, the base may have a variable diameter.",
"The device may adopt a first diameter to facilitate insertion, and a second diameter to facilitate affixation.",
"The device may be provided with an adhesive, such as bone cement, that promotes adhesion of the device to the material of the attachment zone.",
"The device may be provided with cells, growth factors, cytokines, or other biomaterials to promote infiltration and anchoring of the device by host tissue.",
"The device of this exemplary embodiment may be embedded in a bone such that the device's surface is flush with the surrounding bone, and so that the semipermeable membrane faces the synovial cavity.",
"A schematic depiction of this orientation is shown in FIG.",
"2.The device may be thus positioned so as not to present an obstruction for potential interference with joint motion.",
"Furthermore, when so positioned, the device may be exposed to synovial fluid through the semipermeable membrane.",
"Thus, as synovial fluid flows over the membrane, diffusion of therapeutic agent may occur.",
"In addition, synovial fluid can infiltrate the device through the membrane and thus provide fluid communication for the agent to reach the synovial fluid.",
"In preferred embodiments, a sustained drug delivery device for intraarticular use may have a cross-sectional diameter in the range of about 0.5 mm to about 5 mm.",
"It may have a length in the range of about 3 mm to about 20 mm.",
"Other structures are contemplated.",
"In one exemplary embodiment, the device includes a base coated with the drug carrier.",
"In another exemplary embodiment, the device may be so sized and shaped to be a prosthetic replacement for all or a portion of a bone.",
"For example, the device base may be a portion of a femur used in a total knee replacement.",
"Many other examples of bone prosthetics will be readily apparent to one of ordinary skill in the art.",
"The drug carrier is coupled to the base as described above.",
"Alternatively, the prosthetic base can define a recess into which a drug carrier, such as a modular cartridge, may be placed.",
"FIGS.",
"9-11 depict other embodiments of drug delivery devices.",
"In one embodiment, the device 10 may include a stage/housing 11.The stage 11 may be externally threaded.",
"The external threads can help keep the stage in position.",
"For example, the threaded stage could be screwed into a pre-drilled hole in an attachment zone.",
"Alternatively, the device could be inserted in a self boring/self tapping manner, in the recipient attachment zone.",
"The stage may include a tip, such as a sharpened tip 15, suitable for this purpose.",
"FIG.",
"11a shows another embodiment of a drug delivery device.",
"The housing may include an outer threaded surface and inner threaded or smooth surface.",
"The housing may define an inner cavity.",
"A plug of drug/polymer mixture may be placed the cavity.",
"The inner threads on the housing facilitate anchoring the plug and may also facilitate removal and exchange of a cartridge with the drug polymer mixture.",
"FIG.",
"12 shows some exemplary placements of threaded devices, similar to those shown in FIG.",
"4.The stage 11 may also include a central socket 12.The central socket may be threaded.",
"A drug implant 13 may be disposed in the central socket 12.The implant 13 may have the same or similar shape as the socket 12 to help it remain in position.",
"For example, the implant can be molded with threads on its body to match the recipient threads of the socket of the stage.",
"The threads may be machined to have a thread interval or “pitch” of about 0.25 mm to 1.5 mm and can vary to about 0.1 mm to 0.25 mm at the lower end of the implant.",
"The threads may have flat top lands with a nominal width of about 0.10 mm to 0.15 mm.",
"The implant 13 can be removed and/or replaced when it has degraded, when the drug is degraded, exhausted, or being delivered in subtherapeutic concentration, or when the need or desirability has passed for the particular drug being eluted.",
"The insert may be held in the stage by the aforementioned threading.",
"In one embodiment, a differential thread pitch can be provided to “lock” the insert in place, to preclude loosening and escape into the joint space In certain embodiments, the device can have an outer diameter in the range of about 1 mm to about 10 mm.",
"In certain embodiments, the device can have a length of about 3 mm to about 2 cm.",
"As shown in FIG.",
"13, The cap of the drug/polymer insert may include a rounded surface 14.This surface can reduce physical interaction with surrounding soft tissue.",
"This surface can also expose an adequate surface for drug elution and desired pharmacokinetic release.",
"4.Attachment Zones As discussed above, each joint contains specified areas defined hereafter as “attachment zones” that permit drilling, anchoring, or other types of affixing of a device.",
"Certain attachment zones will not cause substantial damage to the load-bearing or articulating cartilage or other surfaces of the specified joint.",
"In certain embodiments, the placement and location of the device does not cause material damage to the cartilage surface, as it may be placed and secured using an anchoring device that fixes the device to bone, in the joint cavity, but not into or on the articulating surface of the joint in a manner that could cause such damage while the device is in place.",
"In some embodiments, an attachment zone may be an intra-articular region of a synovial joint where there is no interfacing articular cartilage.",
"It may be located, for example, in a bone portion that is non-load-bearing and removed from the articulation surface of the synovial joint.",
"The device may be attached at an attachment zone within the synovial joint, allowing for continuous exposure to synovial fluid flow and resulting release of therapeutic, without damaging the articular surface that is in apposition during range of motion of the given joint.",
"FIG.",
"3 shows one example of an attachment zone in a synovial joint.",
"The joint depicted is an idealized synovial joint but roughly approximates the femur-tibia articulation at the knee.",
"An exemplary attachment zone is indicated by the bracket.",
"In this example, the indicated attachment zone is located in the joint and is remote from the load-bearing portions of the bone and also from the articulating surfaces of the bones.",
"Although the zone may include portions of the bone with cartilage, the cartilage is not interfacing cartilage, i.e., does not form part of the articulation surface of the joint.",
"FIG.",
"4 shows exemplary placements of drug delivery devices in the depicted attachment zone.",
"In accordance with the attachment zone example shown in FIG.",
"3, the device may be placed in a non-load-bearing and non-articulating portion of the bone.",
"As suggested in FIG.",
"4, more than one device may be implanted in a single joint.",
"Attachment zones exist in every synovial joint.",
"A joint may have more than one attachment zone, and attachment zones may noncontiguous (i.e., they may be regions of the joint isolated from each other).",
"FIGS.",
"5-8 depict examples of attachment zones in the shoulder, elbow, hip, and wrist/thumb joints, respectively.",
"As shown in FIG.",
"5, attachment zones in the shoulder joint can be in the area of redundant capsule medially and inferiorly.",
"As shown in FIG.",
"6, attachment zones in the elbow can be at the distal aspect of the medial or lateral epicondyle superior to the trochlea and capitellum respectively, or just distal to the radial head on the neck of the radius.",
"As shown in FIG.",
"7, a hip attachment zone exists just distal to the femoral head in the femoral neck.",
"In FIG.",
"8, wrist and thumb (carpometacarpal) attachment zones are shown on the ulna at the distal aspect of the distal radioulnar joint, at the distal aspect of the radial styloid, and at the base of the first metacarpal.",
"Other joints, such as various interfaces in the ankle, also have suitable attachment zones.",
"FIGS.",
"15-17 depict additional exemplary placements for drug delivery devices.",
"The attachment zones indicated in the figures including nonarticulating regions of articular cartilage or bone.",
"They may be said to be “para-articular” because they are located just outside the portions of articular cartilage that receive the articulating load.",
"For example, FIG.",
"15, like FIG.",
"3, shows an idealized synovial joint that roughly approximates the knee articulation.",
"The indicated attachment zones are identified as those portions of the articular cartilage that do not bear weight.",
"Many synovial joints include articular cartilage that is non-load-bearing.",
"A portion of non-load-bearing cartilage, then, can be removed in order to place a drug delivery device without harming the joint's load-bearing capacity, overall function, or health.",
"Portions of bone just beyond the non-load-bearing articulating cartilage can also be selected as attachment zones.",
"An advantage of selecting an attachment zone illustrated in FIGS.",
"15-17 is that these attachment zones are as close as possible to the articulating surfaces of the joint without interfering with articulation.",
"As a result, they are exposed to relatively robust synovial fluid circulation compared to the recesses of the joint where the synovial membrane folds back on itself.",
"They are also more accessible by various surgical and minimally-invasive techniques for implantation, exchange, and/or removal.",
"Another advantage is that regions of bone or cartilage nearer the articulation are less likely to become scarred or otherwise inaccessible following trauma or arthritic episodes in the joint.",
"Nonarticulating articular cartilage, as it might be called, can be found in the knee joint at both the tibial plateau and the femoral condyles, as well as in the patellofemoral area, the medial rim of the femoral trochlea, the lateral rim of the femoral trochlea, and the periphery of the intercondylar notch.",
"FIG.",
"16 depicts an exemplary shoulder joint and indicates some para-articular attachment zones.",
"An attachment zone on the humerus may be found in a band just inferior to the anatomical neck, while an attachment zone on the scapula may be found around the ridge of the glenoid cavity or the glenoid neck.",
"FIG.",
"17 depicts an exemplary hip joint and indicates some para-articular attachment zones.",
"An attachment zone on the femur is a band just inferior to the femoral head, while a para-articular attachment zone on the hip bone may be found in a band just around the rim of the acetabulum.",
"This figure shows that articulating cartilage extends just past the margin of the acetabulum.",
"The cartilage extending past the margin is para-articular because it does not touch the head of the femur and so does not bear a load.",
"FIG.",
"18 depicts exemplary attachment zones in a joint that has been subjected to arthroplasty.",
"When the natural para-articular cartilage and/or bone is removed, new attachment zones may be defined in non-articulating areas adjacent the prosthetic surfaces.",
"As shown in FIG.",
"18 for a hip arthroplasty, attachments zones are defined as rims surrounding the prostheses.",
"For example, a hip attachment zone is located in band adjacent the outer edge of the prosthetic acetabular cup, while a femoral attachment zone is located in a band around the prosthetic femoral stem.",
"In an arthroplastic shoulder joint, attachment zones may be defined on the humerus as a band surrounding the humeral stem, and on the scapula in a band adjacent the outer edge of a glenoid component.",
"In an arthroplastic knee, attachment zones may be defined on the femur as a band surrounding the femoral component of the knee replacement, and on the tibia as a band surrounding the tibial tray of the replacement.",
"When a bone is spared in an arthroplasty, the attachment zone may be defined as that of the native bone.",
"In some cases, however, such a partial arthroplasty may change which portions of articulating cartilage bear weight.",
"In that case, the para-articular attachment zone will be defined in a band around the articulating surface of the implant.",
"A drug delivery device may be implanted contemporaneously with arthroplasty.",
"Benefits of contemporaneous fitting may be the forestalling, diminishing, or prevention of inflammation, infection, pain, etc., depending on which drugs are included in the drug delivery device.",
"In one embodiment, a drug delivery device can be seated in the bone cement used to affix the prosthesis.",
"Such seating would place the device in a very close but still para-articular position and would also eliminate the need for drilling a hole in bone.",
"The para-articular bands may vary widely in size and extent, depending on the joint and upon the anatomy and shape of the subject.",
"A para-articular attachment zone may extend away from an articulating surface as much as about 1 cm, or as little as 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, or about 0.5 mm.",
"An attachment zone can be selected in a variety of ways.",
"In one embodiment, an image may be obtained of a bone or joint, such as a radiograph, a CT scan, an MRI, or other modality.",
"The articulating surfaces can be identified by observing which portions of the subject bones are apposed.",
"An attachment zone, such as one depicted in FIGS.",
"3-8, or a para-articular attachment zone, as depicted in FIGS.",
"15-18, may then be selected.",
"The boundaries of a para-articular attachment zone can be identified by measuring an appropriate distance from the edge of an articulating surface.",
"An attachment zone may be selected during arthroscopy or an open procedure by direct visualization of the articulating and non-articulating surfaces.",
"5.Therapeutic Agents Possible biologically active agents include without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances that affect the structure or function of the body.",
"The therapeutic agents are used in amounts that are therapeutically effective, which varies widely depending largely on the particular agent being used.",
"The amount of agent incorporated into the composition also depends upon the desired release profile, the concentration of the agent required for a biological effect, and the length of time that the biologically active substance has to be released for treatment.",
"In certain embodiments, the biologically active substance may be blended with a polymer matrix at different loading levels, in one embodiment at room temperature and without the need for an organic solvent.",
"In other embodiments, the compositions may be formulated as microspheres.",
"There is no critical upper limit on the amount of therapeutic agent incorporated except for that of an acceptable solution or dispersion viscosity to maintain the physical characteristics desired for the composition.",
"The lower limit of the agent incorporated into the polymer system is dependent upon the activity of the drug and the length of time needed for treatment.",
"Thus, the amount of the agent should not be so small that it fails to produce the desired physiological effect, nor so large that the agent is released in an uncontrollable manner.",
"Typically, within these limits, amounts of the therapeutic agents from about 1% up to about 60% may be incorporated into the present delivery systems.",
"However, lesser amounts may be used to achieve efficacious levels of treatment for agent that are particularly potent.",
"Specific types of biologically active agents include, either directly or after appropriate modification, without limitation: anti-angiogenesis factors, antiinfectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antihelmintics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antidiuretic agents; antidiarrheals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antiproliferatives; antimitotics; antimetabolite compounds; angiostatics; angiostatic steroids; antipruritics; antipsychotics; antipyretics, antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including calcium channel blockers and beta-blockers such as pindolol and antiarrhythmics; antihypertensives; catecholamines; diuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; cough and cold preparations, including decongestants; growth factors, hormones such as estradiol and other steroids, including corticosteroids; hypnotics; immunosuppressives; steroids; corticosteroids; glucocorticoids; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers; and naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, lipoproteins, interferons, cytokines, chemotherapeutic agents and other anti-neoplastics, antibiotics, anti-virals, anti-fungals, anti-inflammatories, anticoagulants, lymphokines, or antigenic materials.",
"To illustrate further, other types of biologically active agents that may be used, either directly or after appropriate modification, include peptide, proteins or other biopolymers, e.g., interferons, interleukins, tumor necrosis factor, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), cholinergic differentiation factor/Leukemia inhibitory factor (CDF/LIF), epidermal growth factor (EGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), erythropoietin, growth hormone, Substance-P, neurotensin, insulin, erythropoietin, albumin, transferrin, and other protein biological response modifiers.",
"Other examples of biologically active agents that may be used either directly or after appropriate modification include acebutolol, acetaminophen, acetohydoxamic acid, acetophenazine, acyclovir, adrenocorticoids, allopurinol, alprazolam, aluminum hydroxide, amantadine, ambenonium, amiloride, aminobenzoate potassium, amobarbital, amoxicillin, amphetamine, ampicillin, androgens, anesthetics, anticoagulants, anticonvulsants-dione type, antithyroid medicine, appetite suppressants, aspirin, atenolol, atropine, azatadine, bacampicillin, baclofen, beclomethasone, belladonna, bendroflumethiazide, benzoyl peroxide, benzthiazide, benztropine, betamethasone, betha nechol, biperiden, bisacodyl, bromocriptine, bromodiphenhydramine, brompheniramine, buclizine, bumetanide, busulfan, butabarbital, butaperazine, caffeine, calcium carbonate, captopril, carbamazepine, carbenicillin, carbidopa & levodopa, carbinoxamine inhibitors, carbonic anhydsase, carisoprodol, carphenazine, cascara, cefaclor, cefadroxil, cephalexin, cephradine, chlophedianol, chloral hydrate, chlorambucil, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chlorotrianisene, chlorpheniramine, 6× chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cholestyramine, cimetidine, cinoxacin, clemastine, clidinium, clindamycin, clofibrate, clomiphere, clonidine, clorazepate, cloxacillin, colochicine, coloestipol, conjugated estrogen, contraceptives, cortisone, cromolyn, cyclacillin, cyclandelate, cyclizine, cyclobenzaprine, cyclophosphamide, cyclothiazide, cycrimine, cyproheptadine, danazol, danthron, dantrolene, dapsone, dextroamphetamine, dexamethasone, dexchlorpheniramine, dextromethorphan, diazepan, dicloxacillin, dicyclomine, diethylstilbestrol, diflunisal, digitalis, diltiazen, dimenhydrinate, dimethindene, diphenhydramine, diphenidol, diphenoxylate & atrophive, diphenylopyraline, dipyradamole, disopyramide, disulfiram, divalporex, docusate calcium, docusate potassium, docusate sodium, doxyloamine, dronabinol ephedrine, epinephrine, ergoloidmesylates, ergonovine, ergotamine, erythromycins, esterified estrogens, estradiol, estrogen, estrone, estropipute, etharynic acid, ethchlorvynol, ethinyl estradiol, ethopropazine, ethosaximide, ethotoin, fenoprofen, ferrous fumarate, ferrous gluconate, ferrous sulfate, flavoxate, flecainide, fluphenazine, fluprednisolone, flurazepam, folic acid, furosemide, gemfibrozil, glipizide, glyburide, glycopyrrolate, gold compounds, griseofuwin, guaifenesin, guanabenz, guanadrel, guanethidine, halazepam, haloperidol, hetacillin, hexobarbital, hydralazine, hydrochlorothiazide, hydrocortisone (cortisol), hydroflunethiazide, hydroxychloroquine, hydroxyzine, hyoscyamine, ibuprofen, indapamide, indomethacin, insulin, iofoquinol, iron-polysaccharide, isoetharine, isoniazid, isopropamide isoproterenol, isotretinoin, isoxsuprine, kaolin & pectin, ketoconazole, lactulose, levodopa, lincomycin liothyronine, liotrix, lithium, loperamide, lorazepam, magnesium hydroxide, magnesium sulfate, magnesium trisilicate, maprotiline, meclizine, meclofenamate, medroxyproyesterone, melenamic acid, melphalan, mephenyloin, mephobarbital, meprobamate, mercaptopurine, mesoridazine, metaproterenol, metaxalone, methamphetamine, methaqualone, metharbital, methenamine, methicillin, methocarbamol, methotrexate, methsuximide, methyclothinzide, methylcellulos, methyldopa, methylergonovine, methylphenidate, methylprednisolone, methysergide, metoclopramide, metolazone, metoprolol, metronidazole, minoxidil, mitotane, monamine oxidase inhibitors, nadolol, nafcillin, nalidixic acid, naproxen, narcotic analgesics, neomycin, neostigmine, niacin, nicotine, nifedipine, nitrates, nitrofurantoin, nomifensine, norethindrone, norethindrone acetate, norgestrel, nylidrin, nystatin, orphenadrine, oxacillin, oxazepam, oxprenolol, oxymetazoline, oxyphenbutazone, pancrelipase, pantothenic acid, papaverine, para-aminosalicylic acid, paramethasone, paregoric, pemoline, penicillamine, penicillin, penicillin-v, pentobarbital, perphenazine, phenacetin, phenazopyridine, pheniramine, phenobarbital, phenolphthalein, phenprocoumon, phensuximide, phenylbutazone, phenylephrine, phenylpropanolamine, phenyl toloxamine, phenytoin, pilocarpine, pindolol, piper acetazine, piroxicam, poloxamer, polycarbophil calcium, polythiazide, potassium supplements, pruzepam, prazosin, prednisolone, prednisone, primidone, probenecid, probucol, procainamide, procarbazine, prochlorperazine, procyclidine, promazine, promethazine, propantheline, propranolol, pseudoephedrine, psoralens, psyllium, pyridostigmine, pyrodoxine, pyrilamine, pyrvinium, quinestrol, quinethazone, quinidine, quinine, ranitidine, rauwolfia alkaloids, riboflavin, rifampin, ritodrine, salicylates, scopolamine, secobarbital, senna, sannosides a & b, simethicone, sodium bicarbonate, sodium phosphate, sodium fluoride, spironolactone, sucrulfate, sulfacytine, sulfamethoxazole, sulfasalazine, sulfinpyrazone, sulfisoxazole, sulindac, talbutal, tamazepam, terbutaline, terfenadine, terphinhydrate, teracyclines, thiabendazole, thiamine, thioridazine, thiothixene, thyroblobulin, thyroid, thyroxine, ticarcillin, timolol, tocainide, tolazamide, tolbutamide, tolmetin trozodone, tretinoin, triamcinolone, trianterene, triazolam, trichloromethiazide, tricyclic antidepressants, tridhexethyl, trifluoperazine, triflupromazine, trihexyphenidyl, trimeprazine, trimethobenzamine, trimethoprim, tripclennamine, triprolidine, valproic acid, verapamil, vitamin A, vitamin B-12, vitamin C, vitamin D, vitamin E, vitamin K, xanthine, parathyroid hormone, enkephalins, and endorphins.",
"To illustrate further, antimetabolites may be used as upon appropriate modification if necessary, including without limitation methotrexate, 5-fluorouracil, cytosine arabinoside (ara-C), 5-azacytidine, 6-mercaptopurine, 6-thioguanine, and fludarabine phosphate.",
"Antitumor antibiotics may include but are not limited to doxorubicin, daunorubicin, dactinomycin, bleomycin, mitomycin C, plicamycin, idarubicin, and mitoxantrone.",
"Vinca alkaloids and epipodophyllotoxins may include, but are not limited to vincristine, vinblastine, vindesine, etoposide, and teniposide.",
"Nitrosoureas, including carmustine, lomustine, semustine and streptozocin, may also be prodrugs, upon appropriate modification if necessary.",
"Hormonal therapeutics may also be prodrugs, upon appropriate modification if necessary, such as corticosteriods (cortisone acetate, hydrocortisone, prednisone, prednisolone, methyl prednisolone dexamethasone, and fluocinolone acetonide), estrogens, (diethylstibesterol, estradiol, esterified estrogens, conjugated estrogen, chlorotiasnene), progestins (medroxyprogesterone acetate, hydroxy progesterone caproate, megestrol acetate), antiestrogens (tamoxifen), aromastase inhibitors (aminoglutethimide), androgens (testosterone propionate, methyltestosterone, fluoxymesterone, testolactone), antiandrogens (flutamide), LHRH analogues (leuprolide acetate), and endocrines for prostate cancer (ketoconazole).",
"Antitumor drugs that are radiation enhancers may also be used as prodrugs, upon appropriate modification if necessary.",
"Examples of such biologically active agents include, for example, the chemotherapeutic agents 5′-fluorouracil, mitomycin, cisplatin and its derivatives, taxol, bleomycins, daunomycins, and methamycins.",
"Antibiotics may be used as prodrugs as well, upon appropriate modification if necessary, and they are well known to those of skill in the art, and include, for example, penicillins, cephalosporins, tetracyclines, ampicillin, aureothicin, bacitracin, chloramphenicol, cycloserine, erythromycin, gentamicin, gramacidins, kanamycins, neomycins, streptomycins, tobramycin, and vancomycin.",
"Other agents, upon appropriate modification if necessary, which may be used include those presently classified as investigational drugs, and can include, but are not limited to alkylating agents such as Nimustine AZQ, BZQ, cyclodisone, DADAG, CB10-227, CY233, DABIS maleate, EDMN, Fotemustine, Hepsulfam, Hexamethylmelamine, Mafosamide, MDMS, PCNU, Spiromustine, TA-077, TCNU and Temozolomide; antimetabolites, such as acivicin, Azacytidine, 5-aza-deoxycytidine, A-TDA, Benzylidene glucose, Carbetimer, CB3717, Deazaguanine mesylate, DODOX, Doxifluridine, DUP-785, 10-EDAM, Fazarabine, Fludarabine, MZPES, MMPR, PALA, PLAC, TCAR, TMQ, TNC-P and Piritrexim; antitumor antibodies, such as AMPAS, BWA770U, BWA773U, BWA502U, Amonafide, m-AMSA, CI-921, Datelliptium, Mitonafide, Piroxantrone, Aclarubicin, Cytorhodin, Epirubicin, esorubicin, Idarubicin, Iodo-doxorubicin, Marcellomycin, Menaril, Morpholino anthracyclines, Pirarubicin, and SM-5887; microtubule spindle inhibitors, such as Amphethinile, Navelbine, and Taxol; the alkyl-lysophospholipids, such as BM41-440, ET-18-OCH3, and Hexacyclophosphocholine; metallic compounds, such as Gallium Nitrate, CL286558, CL287110, Cycloplatam, DWA2114R, NK121, Iproplatin, Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium compounds; and novel compounds such as, for example, Aphidoicolin glycinate, Ambazone, BSO, Caracemide, DSG, Didemnin, B, DMFO, Elsamicin, Espertatrucin, Flavone acetic acid, HMBA, HHT, ICRF-187, Iododeoxyuridine, Ipomeanol, Liblomycin, Lonidamine, LY186641, MAP, MTQ, Merabarone SK&F104864, Suramin, Tallysomycin, Teniposide, THU and WR2721; and Toremifene, Trilosane, and zindoxifene.",
"6.Controlled-Release or Sustained Release Compositions In certain aspects, controlled-release compositions, upon contact with synovial fluid, release the joint therapeutic over a sustained or extended period (as compared to the release from an isotonic saline solution).",
"Such a system may result in prolonged delivery (over, for example, 2 to 4,000 hours, even 4 to 1500 hours) of effective amounts (e.g., 0.00001 mg/kg/hour to 10 mg/kg/hour) of the drug.",
"This dosage form may be administered as is necessary depending on the subject being treated, the severity of the affliction, the judgment of the prescribing physician, and the like.",
"For treatment of joints, controlled-release compositions are adapted for application to the joint.",
"As used herein, the term “anatomic area” refers to an area of synovial joint anatomy, i.e., a joint that facilitates movement of the bones it articulates.",
"In certain embodiments, the pharmaceutical compositions are understood to exert their effect in part by contact with a portion of the anatomic area being treated.",
"Contact refers to a physical touching, either directly with the subject composition being applied without intervening barrier to the anatomic area being treated, or indirectly, where the subject composition is applied to or is formed on a surface of an interposed material, passing through to come into direct contact with the anatomic area being treated.",
"Contact, as used herein, includes those situations where the pharmaceutical compounds are initially positioned to contact the anatomic area being treated, and those situations where the controlled-release compositions are initially positioned in proximity to the anatomic area being treated without contacting it, and subsequently move, migrate, flow, spread, or are transported to enter into contact with the anatomic area being treated.",
"Contact may include partial contacts, wherein the pharmaceutical compounds only contact a portion of the anatomic area being treated, or the edge or periphery or margin of the anatomic area being treated.",
"Contact of the pharmaceutical compounds with the anatomic area being treated occurs from a local rather than systemic administration of said compounds, as these terms are defined hereinafter.",
"The composition may be formed as a flowable material, insertable into the anatomic area.",
"A variety of devices and methods for inserting the composition into the preselected anatomic area will be familiar to practitioners of ordinary skill in the art, for example infusion, injection, topical application, spraying, painting, coating, formed gel placement, and others.",
"The composition, alternatively, may be formed as a solid object implantable in the anatomic area, or as a film or mesh that may be used to cover a segment of the area.",
"A variety of techniques for implanting solid objects in relevant anatomic areas will be likewise familiar to practitioners of ordinary skill in the art.",
"Some examples of sustained release devices and compositions are described in U.S. Pat.",
"Nos.",
"5,618,563, 5,792,753, 5,942,241, 5,985,850, 6,096,728, 6,214,387, 6,217,911, 6,248,345, 6,335,035, 6,346,519, 6,426,339, 6,428,804, 6,451,335, 6,511,958, 6,514,514, 6,514,516, 6,521,259, 6,524,606, 6,524,607, 6,527,760, 6,528,097, 6,528,107, 6,534,081, 6,565,534, 6,582,715, 6,590,059, and 6,699,471; and in U.S. Patent Application Publication Nos.",
"US 2003/0139811 A1 and US 2003/0093157 A1; and in PCT Publication No.",
"WO/0061152 A1.All of these documents are hereby incorporated herein by this reference.",
"In some embodiments, the polymer composition may be a flexible or flowable material.",
"When the polymer used is itself flowable, the polymer composition, even when viscous, need not include a biocompatible solvent to be flowable, although trace or residual amounts of biocompatible solvents may still be present.",
"In certain embodiments, a fluid polymer may be especially suitable for the treatment of joint problems.",
"A fluid material may be adapted for injection or instillation into a tissue mass or into an actual or potential space.",
"Certain types of fluid polymers may be termed flowable.",
"A flowable material, often capable of assuming the shape of the contours of an irregular space, may be delivered to a portion of an actual or potential space to flow therefrom into a larger portion of the space.",
"In this way, the flowable material may come to coat an entire post-operative surgical site after being inserted through an edge of an incision or after being instilled through a drain or catheter left in the surgical bed.",
"Alternatively, if the flowable material is inserted under pressure through a device such as a needle or a catheter, it may perform hydrodissection, thus opening up a potential space and simultaneously coating the space with polymer.",
"Such potential spaces suitable for hydrodissection may be found in various identifiable anatomic areas in joints.",
"A flowable polymer may be particularly adapted for instillation through a needle, catheter or other delivery device such as an endoscope, since its flowable characteristics allow it to reach surfaces that extend beyond the immediate reach of the delivery device.",
"A flowable polymer in a highly fluid state may be suitable for injection through needles or catheters into tissue masses, such as tumors or margins of resection sites.",
"Physical properties of polymers may be adjusted to achieve a desirable state of fluidity or flowability by modification of their chemical components and crosslinking, using methods familiar to practitioners of ordinary skill in the art.",
"A flexible polymer may be used in the fabrication of a solid article.",
"Flexibility involves having the capacity to be repeatedly bent and restored to its original shape.",
"Solid articles made from flexible polymers are adapted for placement in anatomic areas where they will encounter the motion of adjacent organs or body walls.",
"Certain areas of motion are familiar to practitioners dealing with joint problems.",
"A flexible solid article can thus be sufficiently deformed by those moving tissues that it does not cause tissue damage.",
"Flexibility is particularly advantageous where a solid article might be dislodged from its original position and thereby encounter an unanticipated moving structure; flexibility may allow the solid article to bend out of the way of the moving structure instead of injuring it Solid articles may be formed as films, meshes, sheets, tubes, or any other shape appropriate to the dimensions and functional requirements of the particular anatomic area.",
"Physical properties of polymers may be adjusted to attain a desirable degree of flexibility by modification of the chemical components and crosslinking thereof, using methods familiar to practitioners of ordinary skill in the art.",
"While it is possible that the biocompatible polymer or the biologically active agent may be dissolved in a small quantity of a solvent that is non-toxic to more efficiently produce an amorphous, monolithic distribution or a fine dispersion of the biologically active agent in the flexible or flowable composition, it is an advantage that, in an embodiment, no solvent is needed to form a flowable composition.",
"Moreover, the use of solvents may be avoided because, once a polymer composition containing solvent is placed totally or partially within the body, the solvent dissipates or diffuses away from the polymer and must be processed and eliminated by the body, placing an extra burden on the body's clearance ability at a time when the illness (and/or other treatments for the illness) may have already deleteriously affected it.",
"However, when a solvent is used to facilitate mixing or to maintain the flowability of the polymer composition, it should be non-toxic, otherwise biocompatible, and should be used in relatively small amounts.",
"Solvents that are toxic clearly should not be used in any material to be placed even partially within a living body.",
"Such a solvent also must not cause substantial tissue irritation or necrosis at the site of administration.",
"Examples of suitable biocompatible solvents, when used, include N-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol, acetone, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, caprolactam, dimethyl-sulfoxide, oleic acid, or 1-dodecylazacycloheptan-2-one.",
"In one embodiment, solvents include N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, and acetone because of their solvating ability and their biocompatibility.",
"The microspheres may be manufactured by incorporating the drug into the polymer matrix by either dissolving or suspending the drug into polymer solution and the mixture will be subsequently dried by techniques familiar to those skill in the arts to form microspheres.",
"These techniques include but not limited to spray drying, coating, various emulsion methods and supercritical fluid processing.",
"The microspheres may be mixed with a pharmaceutically acceptable diluent prior to the administration for injection.",
"They may also be directly applied to the desired site, such as a surgical wound or cavity, by various delivery systems including pouring and spraying.",
"The microspheres may also be mixed with pharmaceutically acceptable ingredients to create ointment or cream for topical applications.",
"In certain embodiments, the subject polymers are soluble in one or more common organic solvents for ease of fabrication and processing.",
"Common organic solvents include such solvents as chloroform, dichloromethane, dichloroethane, 2-butanone, butyl acetate, ethyl butyrate, acetone, ethyl acetate, dimethylacetamide, N-methylpyrrolidone, dimethylformamide, and dimethylsulfoxide.",
"In addition, the polymer compositions may comprise blends of the polymer with other biocompatible polymers or copolymers, so long as the additional polymers or copolymers do not interfere undesirably with the biocompatible, biodegradable and/or mechanical characteristics of the composition.",
"Blends of the polymer with such other polymers may offer even greater flexibility in designing the precise release profile desired for targeted drug delivery or the precise rate of biodegradability desired.",
"Examples of such additional biocompatible polymers include other poly(phosphoesters), poly(carbonates), poly(esters), poly(orthoesters), poly(amides), poly(urethanes), poly(imino-carbonates), and poly(anhydrides).",
"Pharmaceutically acceptable polymeric carriers may also comprise a wide range of additional materials.",
"Without being limited thereto, such materials may include diluents, binders and adhesives, lubricants, disintegrants, colorants, bulking agents, flavorings, sweeteners, and miscellaneous materials such as buffers and adsorbents, in order to prepare a particular medicated composition, with the condition that none of these additional materials will interfere with the intended purpose of the subject composition.",
"Plasticizers and stabilizing agents known in the art may be incorporated in polymers.",
"In certain embodiments, additives such as plasticizers and stabilizing agents are selected for their biocompatibility.",
"A composition may further contain one or more adjuvant substances, such as fillers, thickening agents or the like.",
"In other embodiments, materials that serve as adjuvants may be associated with the polymer matrix.",
"Such additional materials may affect the characteristics of the polymer matrix that results.",
"For example, fillers, such as bovine serum albumin (BSA) or mouse serum albumin (MSA), may be associated with the polymer matrix.",
"In certain embodiments, the amount of filler may range from about 0.1 to about 50% or more by weight of the polymer matrix, or about 2.5, 5, 10, 25, 40 percent.",
"Incorporation of such fillers may affect the biodegradation of the polymeric material and/or the sustained release rate of any encapsulated substance.",
"Other fillers known to those of skill in the art, such as carbohydrates, sugars, starches, saccharides, celluloses and polysaccharides, including mannitose and sucrose, may be used in certain embodiments.",
"In other embodiments, spheronization enhancers facilitate the production of subject polymeric matrices that are generally spherical in shape.",
"Substances such as zein, microcrystalline cellulose or microcrystalline cellulose co-processed with sodium carboxymethyl cellulose may confer plasticity to the subject compositions as well as implant strength and integrity.",
"In particular embodiments, during spheronization, extrudates that are rigid, but not plastic, result in the formation of dumbbell shaped implants and/or a high proportion of fines, and extrudates that are plastic, but not rigid, tend to agglomerate and form excessively large implants.",
"In such embodiments, a balance between rigidity and plasticity is desirable.",
"The percent of spheronization enhancer in a formulation depends on the other excipient characteristics and is typically in the range of 10-90% (w/w).",
"Buffers, acids and bases may be incorporated in the subject compositions to adjust their pH.",
"Agents to increase the diffusion distance of agents released from the polymer matrix may also be included.",
"Disintegrants are substances which, in the presence of liquid, promote the disruption of the subject compositions.",
"Disintegrants are most often used in implants, in which the function of the disintegrant is to counteract or neutralize the effect of any binding materials used in the subject formulation.",
"In general, the mechanism of disintegration involves moisture absorption and swelling by an insoluble material.",
"Examples of disintegrants include croscarmellose sodium and crospovidone that, in certain embodiments, may be incorporated into the polymeric matrices in the range of about 1-20% of total matrix weight.",
"In other cases, soluble fillers such as sugars (mannitol and lactose) may also be added to facilitate disintegration of the subject compositions upon use.",
"Other materials may be used to advantage to control the desired release rate of a therapeutic agent for a particular treatment protocol.",
"For example, if the sustained release is too slow for a particular application, a pore-forming agent may be added to generate additional pores in the matrix.",
"Any biocompatible water-soluble material may be used as the pore-forming agent.",
"They may be capable of dissolving, diffusing or dispersing out of the formed polymer system whereupon pores and microporous channels are generated in the system.",
"The amount of pore-forming agent (and size of dispersed particles of such pore-forming agent, if appropriate) within the composition should affect the size and number of the pores in the polymer system.",
"Pore-forming agents include any pharmaceutically acceptable organic or inorganic substance that is substantially miscible in water and body fluids and will dissipate from the forming and formed matrix into aqueous medium or body fluids or water-immiscible substances that rapidly degrade to water-soluble substances.",
"Suitable pore-forming agents include, for example, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, and polymers such as hydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone.",
"The size and extent of the pores may be varied over a wide range by changing the molecular weight and percentage of pore-forming agent incorporated into the polymer system.",
"The charge, lipophilicity or hydrophilicity of any subject polymeric matrix may be modified by attaching in some fashion an appropriate compound to the surface of the matrix.",
"For example, surfactants may be used to enhance wettability of poorly soluble or hydrophobic compositions.",
"Examples of suitable surfactants include dextran, polysorbates and sodium lauryl sulfate.",
"In general, surfactants are used in low concentrations, generally less than about 5%.",
"Binders are adhesive materials that may be incorporated in polymeric formulations to bind and maintain matrix integrity.",
"Binders may be added as dry powder or as solution.",
"Sugars and natural and synthetic polymers may act as binders.",
"Materials added specifically as binders are generally included in the range of about 0.5%-15% w/w of the matrix formulation.",
"Certain materials, such as microcrystalline cellulose, also used as a spheronization enhancer, also have additional binding properties.",
"Various coatings may be applied to modify the properties of the matrices.",
"Three exemplary types of coatings are seal, gloss and enteric coatings.",
"Other types of coatings having various dissolution or erosion properties may be used to further modify subject matrices behavior, and such coatings are readily known to one of ordinary skill in the art.",
"The seal coat may prevent excess moisture uptake by the matrices during the application of aqueous based enteric coatings.",
"The gloss coat generally improves the handling of the finished matrices.",
"Water-soluble materials such as hydroxypropyl cellulose may be used to seal coat and gloss coat implants.",
"The seal coat and gloss coat are generally sprayed onto the matrices until an increase in weight between about 0.5% and about 5%, often about 1% for a seal coat and about 3% for a gloss coat, has been obtained.",
"Enteric coatings consist of polymers which are insoluble in the low pH (less than 3.0) of the stomach, but are soluble in the elevated pH (greater than 4.0) of the small intestine.",
"Polymers such as EUDRAGIT, RohmTech, Inc., Malden, Mass., and AQUATERIC, FMC Corp., Philadelphia, Pa., may be used and are layered as thin membranes onto the implants from aqueous solution or suspension or by a spray drying method.",
"The enteric coat is generally sprayed to a weight increase of about one to about 30%, or about 10 to about 15% and may contain coating adjuvants such as plasticizers, surfactants, separating agents that reduce the tackiness of the implants during coating, and coating permeability adjusters.",
"The present compositions may additionally contain one or more optional additives such as fibrous reinforcement, colorants, perfumes, rubber modifiers, modifying agents, etc.",
"In practice, each of these optional additives should be compatible with the resulting polymer and its intended use.",
"Examples of suitable fibrous reinforcement include PGA microfibrils, collagen microfibrils, cellulosic microfibrils, and olefinic microfibrils.",
"The amount of each of these optional additives employed in the composition is an amount necessary to achieve the desired effect.",
"The subject polymers may be formed in a variety of shapes.",
"For example, in certain embodiments, subject polymer matrices may be presented in the form of microparticles or nanoparticles.",
"Such particles may be prepared by a variety of methods known in the art, including for example, solvent evaporation, spray-drying or double emulsion methods.",
"The shape of microparticles and nanoparticles may be determined by scanning electron microscopy.",
"Spherically shaped nanoparticles are used in certain embodiments for circulation through the bloodstream.",
"If desired, the particles may be fabricated using known techniques into other shapes that are more useful for a specific application.",
"In addition to intracellular delivery of a therapeutic agent, it also possible that particles of the subject compositions, such as microparticles or nanoparticles, may undergo endocytosis, thereby obtaining access to the cell.",
"The frequency of such an endocytosis process will likely depend on the size of any particle.",
"In certain embodiments, solid articles useful in defining shape and providing rigidity and structural strength to the polymeric matrices may be used.",
"For example, a polymer may be formed on a mesh or other weave for implantation.",
"A polymer may also be fabricated as a stent or as a shunt, adapted for holding open areas within body tissues or for draining fluid from one body cavity or body lumen into another.",
"Further, a polymer may be fabricated as a drain or a tube suitable for removing fluid from a post-operative site, and in some embodiments adaptable for use with closed section drainage systems such as Jackson-Pratt drains and the like familiar in the art.",
"The mechanical properties of the polymer may be important for the processability of making molded or pressed articles for implantation.",
"For example, the glass transition temperature may vary widely but must be sufficiently lower than the temperature of decomposition to accommodate conventional fabrication techniques, such as compression molding, extrusion or injection molding.",
"In certain embodiments, the polymers and blends, upon contact with body fluids, undergo gradual degradation.",
"The life of a biodegradable polymer in vivo depends, among other things, upon its molecular weight, crystallinity, biostability, and the degree of crosslinking.",
"In general, the greater the molecular weight, the higher the degree of crystallinity, and the greater the biostability, the slower biodegradation will be.",
"If a subject polymer matrix is formulated with a therapeutic agent, release of such an agent for a sustained or extended period as compared to the release from an isotonic saline solution generally results.",
"Such release profile may result in prolonged delivery (over, say 1 to about 4,000 hours, or alternatively about 4 to about 1500 hours) of effective amounts (e.g., about 0.00001 mg/kg/hour to about 10 mg/kg/hour) of the agent associated with the polymer.",
"A variety of factors may affect the desired rate of hydrolysis of polymers, the desired softness and flexibility of the resulting solid matrix, rate and extent of bioactive material release.",
"Some of such factors include: the selection of the various substituent groups, such as the phosphate group making up the linkage in the polymer backbone (or analogs thereof), the enantiomeric or diastereomeric purity of the monomeric subunits, homogeneity of subunits found in the polymer, and the length of the polymer.",
"For instance, the present disclosure contemplates heteropolymers with varying linkages, and/or the inclusion of other monomeric elements in the polymer, in order to control, for example, the rate of biodegradation of the matrix.",
"To illustrate further, a wide range of degradation rates may be obtained by adjusting the hydrophobicities of the backbones or side chains of the polymers while still maintaining sufficient biodegradability for the use intended for any such polymer.",
"Such a result may be achieved by varying the various functional groups of the polymer.",
"For example, the combination of a hydrophobic backbone and a hydrophilic linkage produces heterogeneous degradation because cleavage is encouraged whereas water penetration is resisted.",
"In another example, it is expected that use of substituent on phosphate in the polymers that is lipophilic, hydrophobic or bulky group would slow the rate of degradation.",
"For example, it is expected that conversion of the phosphate side chain to a more lipophilic, more hydrophobic or more sterically bulky group would slow down the rate of biodegradation.",
"Thus, release is usually faster from polymer compositions with a small aliphatic group side chain than with a bulky aromatic side chain.",
"One protocol generally accepted in the field that may be used to determine the release rate of any therapeutic agent or other material loaded in the polymer matrices involves degradation of any such matrix in a 0.1 M PBS solution (pH 7.4) at 37° C., an assay known in the art.",
"For purposes of the present disclosure, the term “PBS protocol” is used herein to refer to such protocol.",
"In certain instances, the release rates of different polymer systems may be compared by subjecting them to such a protocol.",
"In certain instances, it may be necessary to process polymeric systems in the same fashion to allow direct and relatively accurate comparisons of different systems to be made.",
"Such comparisons may indicate that any one polymeric system releases incorporated material at a rate from about 2 or less to about 1000 or more times faster than another polymeric system.",
"Alternatively, a comparison may reveal a rate difference of about 3, 5, 7, 10, 25, 50, 100, 250, 500 or 750.Even higher rate differences are contemplated by the present disclosure and release rate protocols.",
"In certain embodiments, when formulated in a certain manner, the release rate for polymer systems may present as mono- or bi-phasic.",
"Release of any material incorporated into the polymer matrix, which is often provided as a microsphere, may be characterized in certain instances by an initial increased release rate, which may release from about 5 to about 50% or more of any incorporated material, or alternatively about 10, 15, 20, 25, 30 or 40%, followed by a release rate of lesser magnitude.",
"The release rate of any incorporated material may also be characterized by the amount of such material released per day per mg of polymer matrix.",
"For example, in certain embodiments, the release rate may vary from about 1 ng or less of any incorporated material per day per mg of polymeric system to about 5000 or more ng/day·mg.",
"Alternatively, the release rate may be about 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or 900 ng/day·mg.",
"In still other embodiments, the release rate of any incorporated material may be 10,000 ng/day·mg or even higher.",
"In certain instances, materials incorporated and characterized by such release rate protocols may include therapeutic agents, fillers, and other substances.",
"In another aspect, the rate of release of any material from any polymer matrix may be presented as the half-life of such material in the such matrix.",
"In addition to the embodiment involving protocols for in vitro determination of release rates, in vivo protocols, whereby in certain instances release rates for polymeric systems may be determined in vivo, are also contemplated by the present disclosure.",
"Other assays useful for determining the release of any material from the polymers of the present system are known in the art.",
"7.Combinations of Controlled-Release Composition and Therapeutic Agent In some embodiments, for delivery of a therapeutic agent, the agent is added to the polymer composition.",
"A variety of methods are known in the art for encapsulating a biologically active substance in a polymer.",
"For example, the agent or substance may be dissolved to form a homogeneous solution of reasonably constant concentration in the polymer composition, or it may be dispersed to form a suspension or dispersion within the polymer composition at a desired level of “loading” (grams of biologically active substance per grams of total composition including the biologically active substance, usually expressed as a percentage).",
"In part, a polymer composition useful in the treatment of joint pain, inflammation, infection, or other problems, includes both: (a) a therapeutic agent, and (b) a biocompatible and optionally biodegradable polymer, such as one having the recurring monomeric units shown in one of the foregoing formulas, or any other biocompatible polymer mentioned above or known in the art.",
"In certain embodiments in which the subject composition will be used to treat pain, the agent is an analgesic or anesthetic; for inflammation, a steroidal or non-steroidal antiinflammatory agent; and for infection, an antimicrobial effective against the pathogen(s) of concern, such as an antibiotic, antifungal, antimycotic, antimalarial, antimycobacterial, antiparasitic, or antiviral.",
"In some embodiments, the subject compositions encapsulate more than one agent for treatment of one or more joint problems.",
"8.Delivery Systems In its simplest form, a delivery system for a therapeutic agent for treatment of a joint problem consists of a dispersion of such an agent into one of the polymers described above.",
"In other embodiments, an article is used for implantation, injection, or otherwise placed totally or partially within the body, the article comprising a composition for treatment of a joint problem.",
"It may be particularly important that such an article result in minimal tissue irritation when applied to, implanted in or injected into vascularized tissue, hypovascularized post-operative tissue or tissue exposed to previous radiation that is part of a joint.",
"In certain embodiments, a solid, flowable or fluid article is inserted within an anatomic area by implantation, injection, endoscopy or otherwise being placed within an anatomic area of the subject being treated for a joint problem.",
"As a structural medical device, the polymer compositions provide a wide variety of physical forms having specific chemical, physical and mechanical properties suitable for insertion into an anatomic area Biocompatible delivery systems and articles thereof, may be prepared in a variety of ways known in the art.",
"The subject polymer may be melt processed using conventional extrusion or injection molding techniques, or these products may be prepared by dissolving in an appropriate solvent, followed by formation of the device, and subsequent removal of the solvent by evaporation or extraction, e.g., by spray drying.",
"By these methods, the polymers may be formed into articles of almost any size or shape desired, for example, implantable solid discs or wafers or injectable rods, microspheres, or other microparticles.",
"Typical medical articles also include such as implants as laminates for degradable fabric or coatings to be placed on other implant devices.",
"In one embodiment, certain polymer compositions may be used to form a soft, drug-delivery “depot” that can be administered as a liquid, for example, by injection, but which remains sufficiently viscous to maintain the drug within the localized area around the injection site.",
"By using a polymer composition in flowable form, even the need to make an incision can be eliminated.",
"In any event, the flexible or flowable delivery “depot” will adjust to the shape of the space it occupies within the body with a minimum of trauma to surrounding tissues.",
"When the polymer composition is flexible or flowable, it may be placed anywhere within the body, including into an anatomic area of a joint.",
"It may be inserted into the anatomic area either through an open surgical wound, under direct or indirect vision, or through any of the access devices routinely used in the art to enter such areas, for example, indwelling or acutely-inserted catheters, needles, drains, superselective angiography means and the like.",
"A flowable or fluid polymer may be adapted for mixing with the transudate or exudate found within or expected to gather within the anatomic area A flowable or fluid polymer may be instilled in an anatomic area during surgery on organs or structures therein to decrease the likelihood of recurrent disease when there is a high risk for its development.",
"In certain embodiments, a polymer composition may also be incorporated in access devices so that a therapeutic agent is released into the anatomic area within which the access device resides.",
"The polymer composition may also be used to produce coatings for other solid implantable devices for treatment of joint problems.",
"Once a system or implant article is in place, it should remain in at least partial contact with a biological fluid, such as blood, tissue fluid, lymph, or secretions from organ surfaces or mucous membranes, and the like to allow for sustained release of any encapsulated therapeutic agent, e.g., a therapeutic agent.",
"These examples of the clinical utility of the disclosed devices and methods have been provided for illustrative purposes only.",
"Other exemplary utilizations will be apparent to practitioners of ordinary skill in the art using no more than routine experimentation.",
"EXAMPLES Example 1 In one example, the drug carrier is provided as a cartridge.",
"The drug may be loaded into the cartridge as a powder, compressed solid, or as granules without a polymer mixture.",
"Alternative the drug may be combined with any suitable biocompatible, biodegradable polymer.",
"Examples of such biocompatible biodegradable polymers useful include: hydroxyaliphatic carboxylic acids, either homo- or copolymers, such as polylactic acid, polyglycolic acid, polylactic glycolic acid; polysaccharides such as cellulose or cellulose derivatives such as ethyl cellulose, cross-linked or uncross-linked sodium carboxymethyl cellulose, sodium carboxymethylcellulose starch, cellulose ethers, cellulose esters such as cellulose acetate, cellulose acetate phthallate, hydroxypropylmethyl cellulose phthallate and calcium alginate, polypropylene, polybutyrates, polycarbonate, acrylate polymers such as polymethacrylates, polyanhydrides, polyvalerates, polycaprolactones such as poly-.epsilon.-caprolactone, polydimethylsiloxane, polyamides, polyvinylpyrollidone, polyvinylalcohol phthallate, waxes such as paraffin wax and white beeswax, natural oils, shellac, zein, hyaluronic acid, or a mixture thereof.",
"Example 2 In one exemplary embodiment, a sustained release device includes a polymeric matrix or liposome from which drug is released by diffusion and/or degradation of the matrix.",
"The release pattern is usually principally determined by the matrix material, as well as by the percent loading, method of manufacture, type of drug being administered and type of device, for example, microsphere.",
"A major advantage of a biodegradable controlled release system over others is that it does not require the surgical removal of the drug depleted device, which is slowly degraded and absorbed by the patient's body, and ultimately cleared along with other soluble metabolic waste products.",
"Systemic anesthetics such as methoxyflurane, have been incorporated into liposomes and lecithin microdroplets, for example, as described by Haynes, et al., Anesthesiology 63:490-499 (1985).",
"To date, the liposome and lecithin preparations have not been widely applied in clinical or laboratory practice, because of their inability to provide dense blockade for a prolonged period of time (i.e., three or more days) in a safe and controlled manner.",
"The lecithin microdroplets and liposomes degrade or are phagocytized too rapidly, in a matter of hours.",
"Other lipid based devices, formed in combination with polymer, for release of local anesthetics are described by U.S. Pat.",
"No.",
"5,188,837 to Domb.",
"Researchers have also explored the use of polymer microspheres constructed of poly-lactic-glycolic acid combinations for the release of local anesthetics and antiinflammatories such as bupivicaine and dexamethasone with some initial promise in nerve blockade (Drager et.",
"al., Anesthesiology 89(4):969-979 (1998)).",
"Example 3 A biocompatible intra-articular device size for implantation within a joint to continuously deliver a drug within the joint for a period of at least several weeks may include an outer bone anchor construct and an inner core consisting of a drug/polymer reservoir.",
"The drug/polymer reservoir elutes a drug that dissolves in joint fluid through a semipermeable membrane at the synovial fluid interface.",
"The device is implanted in a non-loaded but intra-articular portion of the joint where synovial fluid agitation across the semi-permeable membrane promotes drug elution.",
"Example 4 Exemplary sustained release compositions include poly-glycolic acid, polylactic acid, polyester, collagen, a hydrogel, hyaluronic acid, and combinations of these.",
"Example 5 Exemplary therapeutic agents include bupivicaine, lidocaine, dexamethasone, a non-steroidal antiinflammatory agent, an antibiotic, an immunomodulator, a bone morphogenic protein, a cytokine, a growth factor, a vascular endothelial growth factor, and combinations of these.",
"Example 6 An exemplary device may be affixed in the joint implantation into a pre-drilled bone tunnel.",
"In such an embodiment, the outer shell of the device includes a bone anchor design with axially aligned and concentrically spaced barbs.",
"Example 7 In some embodiments, a sustained release device for a joint is deployed in an ambulatory setting, such as an office-based ambulatory surgical suite.",
"The specific point of insertion will be identified using orthogonal radiography or stereotactic CT scanning.",
"An injector gun will be used to pre-drill and then insert the device into the bone tunnel.",
"Example 8 In a typical procedure, the device may be inserted in a minor procedure or ambulatory surgical setting via a percutaneous delivery gun, using an insertion device (such as depicted in FIG.",
"14).",
"The specific target attachment zone can be identified by palpation of anatomic landmarks and/or radiographically by orthogonal plain radiographs, computerized tomographic imaging, or three dimensional computerized tomographic imaging and localization, or by other localization techniques.",
"Local anesthetic can be administered into the skin and subcutaneous tissues and into the synovium.",
"A stab incision may be made in the skin.",
"Blunt dissection may then be carried down through subcutaneous tissue.",
"A cannulated trochar may then be used to penetrate the joint capsule.",
"The trochar may be advanced to the target delivery site identified previously.",
"The delivery gun or insertion device may be placed into the cannula of the trochar.",
"A bone tunnel may be pre-drilled or the implant directly inserted into the bone of the attachment zone.",
"If the device has outer threads, such as shown in FIG.",
"11, it may be seated by using those self tapping and self boring outer threads of the stage.",
"The surgical wound may then be closed using conventional techniques.",
"Example 9 A drug insert may be replaced by a minor surgical procedure.",
"Surgical access may be gained to the device—i.e., anesthesia, incision, dissection, and capsule penetration using a cannulated trocar.",
"A drug insert retrieval device may be placed in the trocar and advanced to the device.",
"The drug insert may be impaled or otherwise attached to the retrieval device, which may then be withdrawn to remove the drug insert.",
"A replacement drug insert, mounted on a drug insert placement device (which may be the same as the drug insert retrieval device), may then be placed in the cannula and advanced to the attached drug delivery device.",
"Example 10 A drug insert can include a polymer solution/drug mixture having a polymer and a co-dissolved or suspended drug.",
"This drug/polymer insert may be constructed by forming a polymer solution/drug mixture including a polymer-dissolved in an organic solvent and a co-dissolved or suspended drug and then removing the solvent from the polymer solution/drug mixture to form the solid polymer/drug matrix.",
"The polymer can be any biocompatible polymer, such as poly(lactic acid) or a poly(lactic acid-co-glycolic acid) copolymer.",
"The drug can be a therapeutic, prophylactic or diagnostic agent, such as a protein, nucleic acid or small organic molecule.",
"Example 11 A drug insert may include dexamethasone and polylactic glycolic acid.",
"Example 12 A drug insert may include gentamycin and polylactic glycolic acid.",
"EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and practices described herein.",
"Such equivalents are intended to be encompassed by the following claims."
]
] | Patent_10415479 |
[
[
"Food grade transglutaminase inhibitor and uses thereof",
"Transglutaminase inhibitor containing composition obtainable from milk, in particular from a whey fraction obtainable from skimmed milk by forming a curd from the milk, separating the curd from the whey in a centrifugation step followed by subjecting the whey to an ultrafiltration step or, alternatively, by subjecting the skimmed milk to a diafiltration step, a method for the production of a transglutaminase inhibitor and the use of the transglutaminase inhibitor in the production of food or pharmaceutical compositions and to the use in the preparation of a medicament for the treatment of pathologies."
],
[
"1.Transglutaminase inhibitor containing composition obtainable from milk.",
"2.Transglutaminase inhibitor containing composition according to claim 1 obtainable from a whey fraction obtainable from skimmed milk by forming a curd from the milk, separating the curd from the whey in a centrifugation step followed by subjecting the whey to an ultrafiltration step.",
"3.Transglutaminase inhibitor containing composition according to claim 1, obtainable by subjecting skimmed milk to a diafiltration step.",
"4.Transglutaminase inhibitor according to claim 26, wherein the filtrate obtained from the ultrafiltration step or from the diafiltration step is further subjected to a concentration step.",
"5.Transglutaminase inhibitor according to claim 2, wherein the concentration step comprises lyophilisation.",
"6.Transglutaminase inhibitor according to claim 26, wherein the whey fraction is at least partially further purified in a purification step, comprising gel filtration and/or ion exchange chromatography.",
"7.Transglutaminase inhibitor according to claim 5, wherein the purification comprises the removal of at least part of the lactose.",
"8.Transglutaminase inhibitor according to claim 2, wherein curd is formed by the acidification of the milk with food grade acid or by the addition of acid generating micro-organisms.",
"9.Transglutaminase inhibitor according to claim 2, wherein the milk is acidified to a pH of 2.8-5.2, preferably 3-5, more preferably 4.0-4.6.10.Transglutaminase inhibitor according to claim 26, whereby the milk is derived from domestic animals, preferably cow, goat, sheep, deer, ass, reindeer, or humans.",
"11.Transglutaminase inhibitor according to claim 10, whereby the milk has been heat treated at a temperature lower than 80° C. 12.Transglutaminase inhibitor according to claim 1, having a molecular weight in the order of magnitude of about 200 Dalton.",
"13.Method for obtaining a transglutaminase inhibitor containing composition from milk, in particular from a whey fraction, comprising the steps of forming a curd from the milk, separating the curd from the whey in a centrifugation step.",
"14.Method for obtaining a transglutaminase inhibitor containing composition comprising subjecting skimmed milk to a diafiltration step.",
"15.canceled.",
"16.canceled.",
"17.canceled.",
"18.Method of preparing a medicament, comprising the step of utilizing a transglutaminase inhibitor containing composition according to claim 1 in the preparation of a medicament for the treatment of Alzheimer's disease, haemophilia, apoptosis, celiac disease, Huntington's disease, dermatological afflictions, cataract, spinobulbar atrophy (Kennedy's disease), (spino) cerebellar ataxia, dentatorubral-pallidoluysian atrophy, inflammatory diseases of the central nervous system, including multiple sclerosis, rheumatoid arthritis, diabetes such as insulin dependent diabetes mellitus, tetanus and other Clostridium related pathologies, Rett's syndrom, HIV infections and inflammatory processes.",
"19.Method of preparing a medicament for the treatment of Alzheimer's disease, haemophilia, apoptosis, celiac disease, Huntington's disease, dermatological afflictions, cataract, spinobulbar atrophy (Kennedy's disease), (spino) cerebellar ataxia, dentatorubral-pallidoluysian atrophy, inflammatory diseases of the central nervous system, including multiple sclerosis, rheumatoid arthritis, diabetes such as insulin dependent diabetes mellitus, tetanus and other Clostridium related pathologies, Rett's syndrom, HIV infections and inflammatory processes, comprising the step of utilizing a transglutaminase inhibitor containing a composition obtainable by a method of subjecting skimmed milk to a diafiltration step, or controlling the activity of transglutaminase in the cross-linking of proteins.",
"20.Method of controlling the activity of transglutaminase in the cross-linking of proteins, comprising the step of utilizing a transglutaminase inhibitor containing composition according to claims 1.21.Method of controlling the activity of transglutaminase in the cross-linking of proteins, comprising the step of utilizing a transglutaminase inhibitor containing composition obtainable by a method of subjecting skimmed milk to a diafiltration step, or by controlling the activity of transglutaminase in the cross-linking of proteins.",
"22.Method for the production of food, comprising the step of utilizing a transglutaminase inhibitor containing composition according to claim 1.23.Method for the production of food, comprising the step of utilizing a transglutaminase inhibitor containing composition obtainable by a method of subjecting skimmed milk to a diafiltration step, or by controlling the activity of transglutaminase in the cross-linking of proteins.",
"24.Pharmaceutical composition comprising the transglutaminase inhibitor containing composition according to claim 1.25.Pharmaceutical composition comprising the transglutaminase inhibitor containing composition obtainable by a method of subjecting skimmed milk to a diafiltration step, or by controlling the activity of transglutaminase in the cross-linking of proteins.",
"26.Transglutaminase inhibitor containing composition according to claim 1, obtainable: from a whey fraction obtainable from skimmed milk by forming a curd from the milk, separating the curd from the whey in a centrifugation step followed by subjecting the whey to an ultrafiltration step; or by subjecting skimmed milk to a diafiltration step.",
"27.Transglutaminase inhibitor according to claim 6, wherein the purification comprises the removal of at least part of the lactose."
],
[
"The present invention is directed to a transglutaminase inhibitor that is obtainable from milk, a method for obtaining said transglutaminase inhibitor from milk, the use of said transglutaminase inhibitor in regulating the cross-linking of proteins and its use in food products or in pharmaceutical compositions and to the use of said transglutaminase inhibitor in the preparation of a medicament for the treatment of certain diseases.",
"Transglutaminase (protein-glutamine-γ-glutamyltransferase, EC 2.3.2.13) catalyses acyl transfer reactions introducing covalent cross-linkages between proteins, thereby creating high molecular weight molecules.",
"Transglutaminases are a group enzymes, some of which being Ca2+ dependent, that catalyse the formation of isopeptide bonds between the side chains of glutamine and lysine residues.",
"When a lysine bound to a protein serves as the primary amine donor, the reaction results in the formation of an ε-(γ-glutamyl)-lys isopeptide bond that serves to cross-link the proteins.",
"The resulting bond is covalent, stable and is considered relatively resistant to proteolysis.",
"The enzymes have been isolated and purified from a variety of sources, among which bacteria, fungi, plants and mammals (Zhu, Y., Rinzema, A., Tramper, J., J. Appl.",
"Microbial Biotechnol (1995) 44: 277-282).",
"The biological role and structural features of the transglutaminases derived from different sources differ enormously, but they all contain a cystein in the active site responsible for the cross-linking reaction and therefore the cross-linking of proteins is one feature that all types of transglutaminase have in common.",
"Among the transglutaminases, plasma glutaminase (Factor XIIIa) from mammals and humans is one of the best studied enzymes.",
"This enzyme plays a crucial role in the blood clotting pathway as the enzyme cross-links fibrin molecules to enhance the strength of the fibrin networks in bloodclots.",
"Another type of transglutaminase, also from mammals and humans, is generally depicted as tissue transglutaminase.",
"This type of transglutaminase has been shown to be involved in a number of processes like Huntington's disease, apoptosis, celiac disease and Alzheimer's disease.",
"Other processes in which transglutaminases play a role are dermatological afflictions, cataract, spinobulbar atrophy (Kennedy's disease), (spino)cerebellar ataxia, dentatorubral-pallidoluysian atrophy, inflammatory diseases of the central nervous system, including multiple sclerosis, rheumatoid arthritis, diabetes such as insulin dependent diabetes mellitus, tetanus and other Clostridium related pathologies, Rett's syndrom, HIV infections and inflammatory processes.",
"The discovery of a Ca2+-independent bacterial transglutaminase from a Streptovertillium strain has given the research on transglutaminase a big boost, because of the possibility of producing the enzyme in large amounts.",
"As the enzyme has become available in large amounts there is currently an increasing interest in the application of the enzyme in various fields of technology.",
"Some of the applications of transglutaminase are in the preparation of food and protein ingredients for food.",
"The protein cross-linking activity of the enzyme can be used to improve the texture and structure of food products and the functional properties of proteins.",
"EP-A-0 610 649 discloses the use of transglutaminase in the production of yoghurt.",
"WO 93/19610 describes the use of transglutaminase in milk, thereby providing a product with improved consistency.",
"Because of the large number of biological processes in which transglutaminases are involved and the increased use of transglutaminase, there is a widespread interest in compounds that modify or inhibit the activity of transglutaminases.",
"Several inhibitors, among which monodansyl cadaverine, mono-and diamines such as cystamine, putrescine, GABA (gamma-amino benzoic acid), N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl ester, CuSO4 and tolbutamide have been described in WO 99/65516 for use in the treatment of various pathologies.",
"Amines such as spermine and spermidine have been referred to as substrates for transglutaminase.",
"Inhibitors such as monodansyl cadaverine, putrescine, spermine, spermidine and glycine methylester are primary amines that inhibit the protein cross-linking activity of transglutaminase through competition reactions, they do not block the transglutaminase activity.",
"Other well-known transglutaminase inhibitors, such as N-ethylmaleimide, iodoacetate and parachloromercuribenzoic acid, are known to work by covalently blocking the active site cystein of transglutaminase.",
"However, these compounds are known to be toxic and hence not suitable for application in food and food ingredients.",
"Other transglutaminase inhibitors described in the patent literature are isoxazoles, imidazoles (U.S. Pat.",
"No.",
"5,098,707), and thiadiazoles (WO-A-99/45027).",
"Natural inhibitors of transglutaminase are scarce.",
"Certain specific peptides are known to inhibit transglutaminase (WO-A 95/17426).",
"An antimicrobial agent produced by a fungus, cerulinin, is reported as a natural non-peptide inhibitor of plasma transglutaminase (Tymiak, A.",
"A., Tuttle, J. G., Kimball, S. D., Wang, T. Lee, V. G. (1993) J. Antiobiot.",
"46:204-206).",
"Alutacenoic acids, isolated from fungi have also shown activity as potent inhibitors of plasma transglutaminase (Kogen, H., Kiho, T., Tago., K., Miyamoto, S., Fujioka, T., Otsuka, N., Suzuki-konagai, K., Ogita, T., 2000.J.",
"Am.",
"Chem.",
"Soc.",
"122:18421-18430.)",
"One of the disadvantages of the use of transglutaminases in foods is that the products in which the enzyme remains present in an active form may have a negative health effect.",
"It is therefore preferred that the activity of the enzyme is controlled prior to the consumption of the product.",
"Preferably the control of the enzyme occurs to the extent that the enzyme is completely inhibited or inactivated.",
"The inactivation of the enzyme can be achieved by thermal treatment of the product.",
"However, thermal treatment of products in many cases may lead to unwanted changes in the characteristics of the products.",
"In the case of foods, this may lead to a product that is less attractive for the consumer.",
"The transglutaminase inhibitors that are presently known in the art are in general compounds or compositions that, mostly due to their toxicity, are not allowed, or otherwise regarded as unsuitable for application in foods.",
"Accordingly, a need exists for a transglutaminase inhibitor that is suitable for application in foods (i.e.",
"food-grade) and at the same time provides for an effective inhibition of the activity of the transglutaminase while adverse effects of the inhibitor on either the product or the consumer are at least substantially reduced or absent.",
"The present inventors have now found that milk contains an inhibitor of transglutaminase.",
"They found in cross-linking experiments of skimmed milk with transglutaminase only a very small degree of formation of higher molecular weight casein polymers.",
"Intensive heat treatment of the skimmed milk resulted in a much higher degree of cross-linking as was observed by the formation of large polymers and the disappearance of casein monomers.",
"Removal of casein and whey proteins from the milk resulted in a fraction in which the transglutaminase inhibiting activity was retained.",
"They also found that this inhibitor not only inhibits bacterial transglutaminase but is also effective in inhibiting the activity of two transglutaminases that are present in blood.",
"By a combination of isolation and purification steps, the inhibitor can be partly purified while the inhibiting activity remains and the fraction thus obtained can be used in the inhibition of transglutaminase.",
"The invention, in a first aspect, provides for a transglutaminase inhibitor containing composition obtainable from milk, in particular from a whey fraction obtainable from milk, preferably from skimmed milk.",
"In a first embodiment, the inhibitor is obtainable from whey obtained by (i) subjecting milk to an acid treatment and subsequent removing the precipitated proteins by the acid treatment leaving a whey fraction, or (ii) by forming a curd from the milk, for instance by use of micro-organisms or proteases, separating the curd from the whey in a centrifugation step, followed by subjecting the whey obtained to an ultrafiltration step.",
"In an alternative embodiment, the inhibitor is obtainable by subjecting milk to a diafiltration step.",
"By subjecting the whey fraction obtainable from milk to an ultrafiltration step, the desired fraction containing the transglutaminase inhibitor is obtained in the permeate which is separated from the whey proteins.",
"Preferably, the whey fraction is ultrafiltrated using a membrane with a cut-off of 10 kDa.",
"The limit of the cut-off of the membrane is determined by the capability to remove two of the major whey proteins α-lactalbumin (14.1 kDa) and β-lactoglobulin (18 kDa).",
"As the inhibitor according to the present invention is thought to have a molecular weight in the order of about 200 Da, a suitable cut-off will be from 1 kDa, but cut-offs of 2, 3 4 or 5-10 kDa are also suitable.",
"In case the desired fraction containing the transglutaminase inhibitor is obtained by diafiltration, it is preferred that the diafiltration membrane has a cut-off of about 3 kDa.",
"However, a suitable membrane for diafiltration will have a cut-off in the same range as the membrane for ultrafiltration, ranging from 1-10 kDa.",
"The invention, in a second aspect, provides for a method for obtaining a transglutaminase inhibitor containing composition from milk, in particular from a whey fraction, comprising the steps of forming a curd from the milk, separating the curd from the whey in a centrifugation step followed by subjecting the whey to an ultrafiltration step.",
"Preferably the curd is formed by acidifying the milk with a food-grade acid such as hydrochlorid acid, malic acid, tartaric acid, citric acid, lactic acid or glucone (delta glucone), or as an alternative by addition of acid generating microorganisms.",
"Alternatively, the process encompasses subjecting the skimmed milk to a diafiltration step.",
"In U.S. Pat.",
"No.",
"5,198,213, it is described that the top fraction of whey, which top fraction contains the higher molecular weight proteins contains a measurable but low level of immunologically active immunoglobulin plus other pathogen specific antibodies.",
"No reference is made to any useful applications of the low molecular weight compounds containing whey fractions associated with transglutaminase.",
"WO-A-98/48640 discloses an insulin-free protein fraction, which consists of larger proteins.",
"These fractions are used in infant formulae and other nutritive preparations.",
"In WO-A-98/09717 a particular separation technique is described.",
"In examples 3 and 4 proteins are recovered from milk and whey, respectively.",
"No particular uses in accordance with the present invention of the said products are given, especially not for the low molecular weight fractions.",
"The transglutaminase inhibitor containing composition, which will also be referred to as transglutaminase inhibitor, according to the invention is thought to be a low molecular weight material, preferably with a molecular weight in the order of magnitude of about 200 Dalton.",
"With respect to the characteristics of the transglutaminase inhibitor according to the invention, it has been found that the inhibitor is not a free metal ion.",
"The inhibitor according to the invention inhibits the activity of transglutaminase in the cross-linking of casein and other proteins and in several activity assays of transglutaminase.",
"The inhibitor may be used in the form of the whey fraction.",
"It is preferred to subject the fraction containing the inhibitor to a concentration step.",
"A suitable concentration step is lyophilisation or spray drying.",
"In this embodiment of the invention, the inhibitor is obtained in a concentrated form while the inhibiting capability of the inhibitor is not substantially affected.",
"Re-dissolving the dried product from the lyophilisation step and/or the spray drying step results in the inhibiting effect on transglutaminase being largely maintained.",
"In a further embodiment of the invention the inhibitor may be further purified.",
"Suitable purification steps are gel filtration and/or ion exchange techniques.",
"When purifying the inhibitor, preferably at least part of the lactose is removed.",
"The inhibitor according to the invention is stable over a wide temperature range.",
"The isolation and purification and concentration steps by which the inhibitor is obtained can be carried out at temperatures up to about 80° C. Care should be taken not to exceed the upper limit of this temperature range.",
"Exceeding the upper temperature limit, in general, results in loss or undesirable reduction of the activity of the transglutaminase inhibitor.",
"Preferably the inhibitor is not heated to temperatures of above 70° C., more preferably of above 60° C. The inhibitor according to the present invention may be isolated from milk.",
"Milk from which a fraction containing the inhibitor according to the invention can be obtained comes from animals and preferably domestic animals such as cow, goat, sheep, horse, camel, buffalo, deer, ass, reindeer; it is in principle also possible to use human milk as a suitable source.",
"In a preferred embodiment the inhibitor is obtained from cow's milk.",
"Preferably the milk is untreated or at least not extensively heat treated.",
"The fatty fraction is preferably removed, e.g.",
"by centrifugation.",
"Proteins are preferably removed through ultrafiltration, precipitation and/or diafiltration or a combination of these techniques; the inhibitor composition is in the permeate of the ultrafiltration and diafiltration step.",
"The inhibitor according to the invention binds in a substantially covalent fashion to the transglutaminase as has been shown by MALDI-TOF analysis.",
"Furthermore it has been found by titration experiments that the inhibitor binds in a approximately equimolar amount to transglutaminase.",
"Without wishing to be bound to any particular theorty, it is thought that the inhibitor according to the invention is bound to transglutaminase cystein residues.",
"The invention finds application in the production of food and food products.",
"When transglutaminase is used in the cross-linking of proteins to improve the structure of protein containing foods, for instance foods such as dairy (yoghurt), meat or fish, the transglutaminase activity can be stopped or reduced by the addition of the inhibitor according to the invention.",
"In one aspect, the invention relates to the use of the transglutaminase inhibitor in the production of food.",
"An important aspect of the inhibitor according to the invention is the use of the inhibitor in the regulation of the degree of cross-linking.",
"It has been found that partial cross-linking of β-casein results in the formation of a gel when heated, whereas uncross-linked β-casein or extensively cross-linked β-casein does not form a gel on heating.",
"Regulation of the degree of cross-linking is easily achieved by the addition of the inhibitor according to the invention whereas other means for activation, for instance through heating, may very well destroy protein functionality.",
"Furthermore the inhibitor according to the invention may be used in a form that allows for controlled release, such as encapsulation.",
"It can easily be envisaged that the addition of transglutaminase in combination with (micro)encapsulated inhibitor to a substrate will result in the homogenous distribution of both transglutaminase and inhibitor.",
"Cross-linking of the proteins can then be governed by the release profile of the inhibitor.",
"This is advantageous when the product after cross-linking is viscous or gelated.",
"The invention pertains in a further aspect to a pharmaceutical composition comprising the transglutaminase inhibitor according to the invention.",
"The pharmaceutical composition can be advantageously used in the treatment of diseases that are in any way associated with or related to transglutaminase activity.",
"The pharmaceutical composition according to the invention can comprise at least one transglutaminase inhibitor in a pharmaceutically acceptable form, optionally combined with a pharmaceutical acceptable carrier.",
"These compositions can be administered by any means that achieve their intended purposes.",
"Amounts and regimens for the administration of a transglutaminase inhibitor according to the invention for the treatment of diseases mediated by transglutaminase activity can readily be determined by those with ordinary skill in the art of treating these diseases.",
"The invention further relates to the use of the transglutaminase inhibitor in the preparation of a medicament for the treatment of diseases in which transglutaminase plays a role such as for example Alzheimer's disease, haemophilia, apoptosis, celiac disease, Huntington's disease, dermatological afflictions, cataract, spinobulbar atrophy (Kennedy's disease), (spino)cerebellar ataxia, dentatorubral-pallidoluysian atrophy, inflammatory diseases of the central nervous system, including multiple sclerosis, rheumatoid arthritis, diabetes such as insulin dependent diabetes mellitus, tetanus and other Clostridium related pathologies, Rett's syndrom, HIV infections and inflammatory processes.",
"DESCRIPTION OF THE FIGURES FIG.",
"1: Cross-linking of skimmed milk with bacterial transglutaminase (30 μg/ml, before and after heat treatment (3 h, 80° C.).",
"Lane 1 Molecular weight markers Lane 2 untreated skimmed milk+transglutaminase, t=0 min; Lane 3 untreated skimmed milk+transglutaminase, t=10 min; Lane 4 untreated skimmed milk+transglutaminase, t=30 min; Lane 5 untreated skimmed milk+transglutaminase, t=120 min; Lane 6 heated skimmed milk+transglutaminase, t=0 min; Lane 7 heated skimmed milk+transglutaminase, t=10 min; Lane 8 heated skimmed milk+transglutaminase, t=30 min; Lane 9 heated skimmed milk+transglutaminase, t=120 min.",
"FIG.",
"2: Activity assay of bacterial transglutaminase after incubation with the inhibitor fraction.",
"A: bacterial transglutaminase after incubation with the inhibitor fraction obtained through ultrafiltration of the whey fraction.",
"B: bacterial transglutaminase after incubation with the inhibitor fraction obtained through gelfiltration of the whey fraction.",
"FIG.",
"3: Separation of protein free inhibitor fraction on Biogel P2.The inhibitor fraction was obtained by procedure 1.The position where the inhibitor eluted is depicted in the figure.",
"FIG.",
"4 Cross-linking of casein with untreated bacterial transglutaminase and with bacterial transglutaminase preincubated with a protein free inhibitor fraction.",
"A: Cross-linking of casein with untreated transglutaminase Lane 1 molecular weight marker Lane 2 casein after cross-linking with transglutaminase t=0 min; Lane 3 casein after cross-linking with transglutaminase t=10 min; Lane 4 casein after cross-linking with transglutaminase t=20 min; Lane 5 casein after cross-linking with transglutaminase t=40 min.",
"B: Cross-linking of casein with transglutaminase after preincubation with inhibitor.",
"Lane 1 molecular weight marker Lane 2 casein after cross-linking with transglutaminase t=0 min; Lane 3 casein after cross-linking with transglutaminase t=10 min; Lane 4 casein after cross-linking with transglutaminase t=20 min; Lane 5 casein after cross-linking with transglutaminase t=40 min.",
"EXAMPLES Materials and Methods Materials.",
"Source Q, Source S and Superdex peptide 30 were purchased from Pharmacia (Uppsala, Sweden).",
"Biogel P2 was obtained from BioRad.",
"N,N,-Dimethyl casein, casein, Cbz-glutaminylglycine, hydroxylamine and guinea pig liver transglutaminase were obtained from Sigma.",
"All other reagents were of analytical grade.",
"Skimmed milk was obtained from a local shop, fresh milk directly from a farm.",
"Streptoverticillium mobaraense was grown as described [1] and purification of transglutaminase was performed according to [2].",
"Human plasma transglutaminase was obtained from Behring Werke.",
"Cross-Linking of Skimmed Milk Proteins Skimmed milk proteins were cross-linked by the addition of 30 μg bacterial transglutaminase per ml skimmed milk.",
"As a control, skimmed milk was incubated at 80° C. for 3 hours prior to the addition of transglutaminase.",
"Incubations were performed at 37° C., and samples were taken at time intervals.",
"The cross-linking reaction was terminated by incubation at 80° C. for 5 min.",
"Analyses of the cross-linking reactions were performed by gel electrophoresis.",
"Measurement of Transglutaminase Inhibition Using Activity Assays.",
"Quantitative analysis of inhibition of bacterial and guinea pig transglutaminase activity was determined with the hydroxamate assay [3].",
"30 μl (1 mg/ml) transglutaminase was incubated with variable amounts of inhibitor fraction (0 to 30 μl).",
"The mixture was incubated at room temperature for 30 min, to ensure coupling of the inhibitor to the transglutaminase.",
"Activity measurements were performed with the inhibited fractions.",
"Quantitative analysis of inhibition of plasma transglutaminase activity was determined by the incorporation of monodansylcadaverine into dimethylated casein according to Lorand [4].",
"Plasma transglutaminase was measured in this assay, because this enzyme does not show any activity in the hydroxamate assay.",
"Dithiotreitol was omitted from the reaction mixture in order to prevent possible removal of the inhibitor from the transglutaminase through reduction.",
"Inhibition was calculated from the relative decrease in activity.",
"Measurement of Transglutaminase Inhibition Using Cross-Linking Experiments.",
"Qualitative analyses of inhibition of bacterial transglutaminase activity was determined with cross-linking of casein.",
"20 μg transglutaminase was incubated with 200 μl of the ultrafiltrated inhibitor fraction.",
"The mixture was incubated at room temperature for 30 min, to ensure coupling of the inhibitor to the transglutaminase.",
"The standard reaction mixture (total volume of 2 ml) contained 50 mM sodium acetate pH 6, 10 mg/ml casein, and 10 μg pre-incubated transglutaminase.",
"Incubations were performed at 40° C., and samples were taken at time intervals.",
"The reaction was terminated by incubation at 80° C. for 5 min.",
"Analyses of the cross-linking reactions were performed by gel electrophoresis.",
"Polyacrylamide-gel electrophoresis (SDS-PAGE).",
"Subunit molecular masses were determined under denaturing conditions by SDS-PAGE according to Laemmli (5) using ready gels and Mini Protean equipment (Bio-Rad).",
"Enzyme or reaction mixture samples were denatured by incubation for 5 min.",
"at 100 C in 2% SDS and 1% dithiotreitol.",
"Gels were stained for protein with Coomassie Brilliant Blue G250.A high molecular weight calibration kit (Pharmacia) was used to derive the molecular masses.",
"Preparation of a Substantially Protein-Free Inhibitor Fraction Procedure 1 Skimmed milk was brought to pH 4.4 by addition of hydrochloric acid.",
"Precipitated casein was removed by centrifugation (20 min 25000 g).",
"The clear supernatant was ultrafiltrated using a 10 kDa membrane (Amicon).",
"The concentrated whey proteins were removed and the solution that has passed the membrane was frozen or lyophilised The inhibitor could be further purified by gel filtration on a Superdex 30 peptide column or Biogel P2 to remove lactose and other non inhibitory components.",
"Procedure 2 1 litre of skimmed milk was dialysed against 5 liter of demineralized water using a dialysis tube with a cut off of 3500 Da.",
"After 24 hours dialysis, the dialysis tube containing the protein fraction was removed.",
"The solution containing the molecules of lesser size than 3500 contained the transglutaminase inhibitor.",
"This fraction can be lyophilised in order to concentrate the inhibitor.",
"Solutions of the inhibitor can be prepared by dissolving the lyophilised sample in water.",
"Residual non dissolving compounds were removed by centrifugation.",
"Maldi TOF Analysis of Transglutaminase Before and after Inhibition Bacterial transglutaminase (1 mg) was incubated with 10 ml of inhibition fraction obtained by procedure 1.After 30 min of incubation at room temperature the inhibition of transglutaminase was tested in the hydroxamate assay.",
"The inhibited transglutaminase was ultrafiltrated using a 30 kDa Amicon filter to remove unbound components and concentrate the bacterial transglutaminase.",
"Molecular masses of the inhibited transglutaminase and of the blank were measured using Maldi TOF.",
"Results Cross-Linking of Skimmed Milk Proteins SDS/PAGE analysis after cross-linking of skimmed milk showed only little indication of cross-linking (FIG.",
"1).",
"The skimmed milk after heat treatment at 80° C. for 3 hours, however, showed normal cross-linking of the caseins.",
"Extra addition of transglutaminase to the unheated skimmed milk resulted in cross-linking, comparable to the heated skimmed milk (data not shown).",
"Measurement of Transglutaminase Inhibition Using Activity Assays Activities of bacterial transglutaminase after incubation with the inhibitor fraction were lowered as could be observed in the hydroxamate assay (FIG.",
"2a).",
"Depending on the type of inhibitor fraction used, components present in the fraction were responsible for disturbances in the activity measurements.",
"These components caused colour formation that interfered with the absorbance measurements of the transglutaminase activity.",
"Further purification of the inhibitor on gel filtration removed the interfering compounds which results in an linear inhibition curve (FIG.",
"2b).",
"Guinea pig liver transglutaminase gave similar results (data not shown).",
"Activities of plasma transglutaminase after incubation with the inhibitor fraction were lowered as could be observed in the fluorescence assay (data not shown).",
"This result clearly shows that it is possible to completely inactivate plasma transglutaminase by the inhibitor.",
"Measurement of Transglutaminase Inhibition Using Cross-Linking Experiments.",
"Cross-linking of casein was not observed with bacterial transglutaminase after inhibition with the fraction obtained after procedure 1 (FIG.",
"3).",
"The control with untreated transglutaminase showed normal cross-linking.",
"Preparation of a Protein Free Inhibitor Fraction Procedure 1 Caseins were removed by centrifugation after precipitation at pH 4.4.The whey protein fraction was subsequently ultrafiltrated using a 10 kDa ultrafiltration unit (Amicon).",
"The low molecular weight fraction that passed the membrane showed transglutaminase inhibition as can be seen in FIG.",
"2a.",
"Concentration of this fraction was achieved by lyophilisation of the solution and dissolving the solids in a small volume of water.",
"The concentrated fraction was further purified on a Biogel P2 (FIG.",
"4).",
"Procedure 2 Caseins and whey protein were separated from the inhibitor fraction through dialysis of the skimmed milk.",
"The proteins remained in the dialysis tube, whereas the inhibitor together with other low molecular weight components passed the dialysis membrane.",
"The dialysis procedure caused dilution of the inhibitor, therefore the solution was lyophilised and the solids dissolved in a small volume of water.",
"Maldi TOF Analysis of Trans Glutaminase The molecular weight of transglutaminase as determined with Maldi TOF was 37.900 Da, the molecular weight of the inhibited transglutaminase was 38.064 Da.",
"Because it is possible that only a part of the inhibitor is coupled to the transglutaminase, the mass obtained does not have to correspond to the mass of the inhibitor.",
"REFERENCES 1 Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H. and Motoki, M. (1989) Agric.Biol.",
"Chem 53, 2613-2617 2 Zhu, Y., Rinzema, A., Tramper, J., Bruin, E.de.",
"and Bol, J.",
"(1998) Appl.",
"Micr.Biotech.",
"49, 251-257 3 Folk, J. E. and Cole, P. W (1965).",
"J. Biol.",
"Chem.",
"241, 2951 4 Lorand, L., Lockridge, O. M., Campbell, L. K., Myhrman, R. and Bruner-Lorand, J.",
"(1972) Anal.",
"Biochem.",
"44, 221-231 5 Laemuli, U. K. (1970) Nature 227, 680-685"
]
] | Patent_10415584 |
[
[
"Aminoglycosides as antibiotics",
"The present invention provides aminoglycosides and pharmaceutical compositions that include the aminoglycosides.",
"The aminogylcosides are useful to treat or prevent infectious diseases (e.g., bacterial infections) in a mammal (e.g., human)."
],
[
"1.A compound of formula (I): wherein: R1 is thio, sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is O or S; R2 is (C1-C12)alkyl, (C6-C10)aryl (C1-C12)alkyl, heteroaryl (C1-C12)alkyl, or (C3-C8)cycloalkyl (C1-C12)alkyl; wherein any alkyl, aryl, heteroaryl, or cycloalkyl is substituted with one or more amino or hydroxy; R3 is a direct bond or C═X, wherein X is O or S; R4 is (C1-C12)alkyl optionally substituted with one or more NRaRb, wherein Ra and Rb are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; wherein any alkyl or aryl of R4, Ra or Rb is optionally substituted with one or more amino, hydroxy, or guanidinyl; R5 is hydroxy, halo, or hydrogen and R6 is a direct bond; or R5 is (C1-C12)alkyl optionally substituted with one or more NRcRd, wherein Rc and Rd are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl of R5, Rc or Rd is optionally substituted with one or more amino, hydroxy, or guanidinyl, and R6 is oxy, thio, or C═X, wherein X is O or S; R7 is hydrogen, halo, or hydroxy; R8 is hydrogen or hydroxy; and R9 is hydrogen or hydroxy; wherein any alkyl is optionally interrupted with one or more oxy, thio, sulfinyl, or sulfonyl; and wherein any alkyl, aryl, heteroaryl, or cycloalkyl is optionally substituted on carbon with one or more halo, cyano, nitro, trifluoromethyl, hydroxy, (C1-C6)alkoxy, mercapto, oxo, thioxo, or NReRf, wherein Re and Rf are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; or a pharmaceutically acceptable salt thereof.",
"2.The compound of claim 1 wherein R1 is sulfonyl, a direct bond, or C═X, wherein X is O or S. 3.The compound of claim 1 wherein R2 is hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl is substituted with one or more amino and wherein any alkyl is optionally substituted with one or more hydroxy or mercapto.",
"4.The compound of claim 3 wherein the (C1-C8)alkyl is terminally substituted with an amino or the aryl of (C6-C10)aryl (C1-C12)alkyl is substituted with an amino.",
"5.The compound of claim 1 wherein R1R2 is hydrogen or a group of the formula: wherein n is 0 to about 6; R10 is sulfinyl, sulfonyl, a direct bond, or C═X wherein X is O or S; and R11 is hydroxy, mercapto, or NRgRh, wherein Rg and Rh are each independently hydrogen, (C1-C8)alkyl, or (C6-C10)aryl (C1-C8)alkyl.",
"6.The compound of claim 1 wherein R3 is a direct bond.",
"7.The compound of claim 1 wherein R3 is C═X, wherein X is S or O.",
"8.The compound of claim 1 wherein R4 is (C1-C12)alkyl substituted with one or more NRaRb, wherein Ra and Rb are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; wherein any alkyl is optionally substituted with one or more hydroxy or mercapto.",
"9.The compound of claim 1 wherein R4 is (C1-C12)alkyl substituted with one or more NRaRb, wherein Ra and Rb are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; wherein any alkyl or aryl of R4, Ra or Rb is substituted with one or more amino or guanidinyl.",
"10.The compound of claim 1 wherein R3R4 is a group of the formula: wherein Ri is hydrogen, Rj is wherein R12 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R13 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R14 is hydrogen, hydroxy, mercapto, or NRiRj, wherein Ri and Rj are each independently hydrogen, (C1-C8)alkyl, or (C6-C10)aryl(C1-C8)alkyl; and m is 0 to about 8.11.The compound of claim 10 wherein Ri is (CH2)n—NH2, wherein n is 2 to about 8; or Ri is guanidinyl.",
"12.The compound of claim 10 wherein Rj is hydrogen or a group of the formula: wherein R15 is C═X, wherein X is O or S; R16 is hydrogen, hydroxy or amino; and q is 2.13.The compound of claim 1 wherein R5 is hydroxy, halo, or hydrogen and R6 is a direct bond.",
"14.The compound of claim 1 wherein R5 is (C1-C12)alkyl optionally substituted with one or more oxo or NRmRn, wherein Rm and Rn are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl of R5, Rm or Rn is optionally substituted with one or more amino or guanidinyl, and R6 is oxy, thio, or C═X, wherein X is O or S. 15.The compound of claim 1 wherein R5R6 is hydrogen, hydroxy, flouro, or a group of the formula: wherein Ro is hydrogen, Rp is wherein R17 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R18 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R19 is hydrogen, hydroxy, mercapto, or NRqRs, wherein Rq and Rs are each independently hydrogen, (C1-C8)alkyl, or (C6-C10)aryl(C1-C8)alkyl; and p is 0 to about 6.16.The compound of claim 1 wherein R5R6 is hydroxy.",
"17.The compound of claim 1 wherein R7 is hydrogen, fluoro, or hydroxy.",
"18.The compound of claim 1 wherein R7 is hydrogen.",
"19.The compound of claim 1 wherein R8 is hydroxy or hydrogen.",
"20.The compound of claim 1 wherein R8 is hydroxy.",
"21.The compound of claim 1 wherein R9 is hydroxy or hydrogen.",
"22.The compound of claim 1 wherein R9 is hydroxy.",
"23.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"24.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"25.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"26.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof 27.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"28.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"29.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"30.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"31.The compound of claim 1 which is or a pharmaceutically acceptable salt thereof.",
"32.A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier or diluent.",
"33.The pharmaceutical composition of claim 32 further comprising one or more additional antibiotic agents.",
"34.The pharmaceutical composition of claim 33 wherein the additional antibiotic agent kills or effectively inhibits the growth of one or more of the following bacterium: Escherichia coli; Pseudomonas spp.",
"; Proteus spp.",
"; Bacteroides spp.",
"; Haemophilus influenzae; Klebsiella spp.",
"; Enterobacter spp.",
"; Neisseria gonorrhoeae; Acinetobacter; Citrobacter spp.",
"; Serratia marcescens; Branhamella (Moraxella) catarrhalis; Morganella morganii; Providencia stuartii; Salmonella spp.",
"; Shigella spp.",
"; Campilobacter spp.",
"; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Streptococcus pyogenes; Streptococcus (alpha-hemolytic); Streptococcus pneumoniae; and Enterococcus faecium.",
"35.The pharmaceutical composition of claim 33 wherein the additional antibiotic agent is an aminoglycoside, β-lactam antibiotic, cephalosporin, macrolide, miscellaneous antibiotic, penicillin, tetracycline, antifungal, antimalarial agent, antituberculosis agent, antiviral, leprostatic, miscellaneous anti-infective, quinolone, sulfonamide, urinary anti-infective, nasal antibiotic, ophthalmic antibiotic, ophthalmic antiviral, ophthalmic quinalone, ophthalmic sulfonamide, skin and mucous membrane antibiotic, skin and mucous membrane antifungal, skin and mucous membrane antiviral, skin and mucous membrane miscellaneous anti-infective, skin and mucous membrane scabicide and pedulicide, skin and mucous membrane antineoplast, nitrofuran, or oxazolidinone.",
"36-41.",
"(canceled) 42.A method for preventing or treating a bacterial infection in a mammal comprising administering to the mammal in need of such prevention or treatment a compound of claim 1.43.The method of claim 42 wherein the bacterial infection is caused by one or more of the following bacterium: Escherichia coli; Pseudomonas spp.",
"; Proteus spp.",
"; Bacteroides spp.",
"; Haemophilus influenzae; Klebsiella spp.",
"; Enterobacter spp.",
"; Neisseria gonorrhoeae; Acinetobacter; Citrobacter spp.",
"; Serratia marcescens; Branhamella (Moraxella) catarrhalis; Morganella morganii; Providencia stuartii; Salmonella spp.",
"; Shigella spp.",
"; Campilobacter spp.",
"; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Streptococcus pyogenes; Streptococcus (alpha-hemolytic); Streptococcus pneumoniae; and Enterococcus faecium.",
"44.The method of claim 42 further comprising administering one or more additional antibiotic agents.",
"45.The method of claim 44 wherein the additional antibiotic agent effectively kills or effectively inhibits the growth of one or more of the following bacterium: Escherichia coli; Pseudomonas spp.",
"; Proteus spp.",
"; Bacteroides spp.",
"; Haemophilus influenzae; Klebsiella spp.",
"; Enterobacter spp.",
"; Neisseria gonorrhoeae; Acinetobacter; Citrobacter spp.",
"; Serratia marcescens; Branhamella (Moraxella) catarrhalis; Morganella morganii, Providencia stuartii; Salmonella spp.",
"; Shigella spp.",
"; Campilobacter spp.",
"; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Streptococcus pyogenes; Streptococcus (alpha-hemolytic); Streptococcus pneumoniae; and Enterococcus faecium.",
"46.The method of claim 44 wherein the additional antibiotic agent is an aminoglycoside, β-lactam antibiotic, cephalosporin, macrolide, miscellaneous antibiotic, penicillin, tetracycline, antifungal, antimalarial agent, antituberculosis agent, antiviral, leprostatic, miscellaneous anti-infective, quinolone, sulfonamide, urinary anti-infective, nasal antibiotic, ophthalmic antibiotic, ophthalmic antiviral, ophthalmic quinalone, ophthalmic sulfonamide, skin and mucous membrane antibiotic, skin and mucous membrane antifungal, skin and mucous membrane antiviral, skin and mucous membrane miscellaneous anti-infective, skin and mucous membrane scabicide and pedulicide, skin and mucous membrane antineoplast, nitrofuran, or oxazolidinone.",
"47.The method of claim 42 wherein the mammal is a human."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Antibiotics are low-molecular weight antimicrobial agents that are produced as secondary metabolites by microorganisms that inhabit soil.",
"For instance, Penicillium and Cephalosporium produce beta-lactam antibiotics (e.g., penicillin, cephalosporin, and their relatives).",
"Actinomycetes (e.g., the Streptomyces species) produce tetracyclines, aminoglycosides (e.g., streptomycin and its analogs), macrolides (e.g., erythromycin and its analogs), chloramphenicol, ivermectin, rifamycins, and most other clinically-useful antibiotics that are not beta-lactams.",
"Bacillus species (e.g., B. polymyxa and Bacillus subtilis ) produce polypeptide antibiotics (e.g.",
"polymyxin and bacitracin), while B. cereus produces zwittermicin.",
"The modern era of antibiotic therapy began with Fleming's 1929 discovery of penicillin, and Domagk's 1935 discovery of synthetic sulfonamides.",
"Spurred by the need for antibacterial drugs during World War II, penicillin was isolated, purified and injected into experimental animals.",
"The substance was found to not only cure infections, but also to possess low toxicity.",
"This finding marked the beginning of the era of antibiotic use in human drug therapy and the intense search for similar antimicrobial agents of low toxicity that could be used to treat infectious diseases.",
"The rapid isolation of streptomycin, chloramphenicol and tetracycline followed, and these and several other antibiotics were in clinical usage by the 1950's.",
"Antibiotics are used therapeutically to treat bacterial infections.",
"Several types of antibiotics, classified according to their mechanism of action, are currently employed.",
"The known types of antibiotics include, e.g., cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, and inhibitors that bind to or affect the synthesis of DNA or RNA.",
"Cell wall synthesis inhibitors, such as beta lactam antibiotics, generally inhibit some step in the synthesis of bacterial peptidoglycan.",
"Penicillin is generally effective against non-resistant streptococcus, gonococcus and staphylococcus.",
"Amoxycillin and Ampicillin have broadened spectra against Gram-negative bacterias.",
"Cephalosporins are generally used as penicillin substitutes, against Gram-negative bacteria, and in surgical prophylaxis.",
"Monobactams are generally useful for the treatment of allergic individuals.",
"Cell membrane inhibitors disorganize the structure or inhibit the function of bacterial membranes.",
"Polymyxin, produced by Bacillus polymyxis, is a cell membrane inhibitor that is effective mainly against Gram-negative bacteria and is usually limited to topical usage.",
"Protein synthesis inhibitors include the tetracyclines, chloramphenicol, the macrolides (e.g.",
"erythromycin) and the aminoglycosides (e.g.",
"streptomycin ).",
"Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria.",
"Aminoglycosides bind to the bacterial RNA in manifestation of their activity.",
"Davis, B. D. Microbiol.",
"Rev.",
"1987, 51, 341-350.Mingeot-Leclercq, M.-P.; Glupczynski, Y.; Tulkens, P. M. Antimicrob.",
"Agents Chemother.",
"1999, 43, 727-737.Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.Shaw, K. J., Rather, P. N.; Hare, R. S.; Miller, G. H. Microbiol.",
"Rev.",
"1993, 57, 138-163.Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis.",
"Gentamicin is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Pseudomonas aeruginosa.",
"Kanamycin is active at low concentrations against many Gram-positive bacteria, including penicillin-resistant staphylococci.",
"The structures of two aminoglycoside antibiotics, paromomycin and gentamicin C 1a , bound to the template sequences of ribosomal RNA (rRNA), have been determined recently by NMR.",
"Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996, 274, 1367-1371; (a) Fourmy, D.; Recht, M. I.; Puglisi J. D. J. Mol.",
"Biol.",
"1998, 277, 347-362; (b) Fourmy, D.; Yoshizawa, S.; Puglisi, J. D. J. Mol.",
"Biol.",
"1998, 277, 333-345; (c) Recht, M. I.; Fourmy, D.; Blanchard, S. C.; Dahlquist, K. D.; Puglisi, J. D. J. Mol.",
"Biol.",
"1996, 262, 421-436; (d) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. EMBO J.",
"1998, 17, 6437-6448.These and other studies show that the neamine-class of aminoglycosides bind specifically to the A-site region on the 16S subunit of rRNA.",
"Hence, neamine serves as a minimal structural motif for such binding.",
"Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996, 274, 1367-1371; Kotra, L. P.; Haddad, J.; Mobashery, S. Antimicrob.",
"Agents Chemother.",
"2000 (in press)).",
"As such, aminoglycosides are a suitable class of compounds that could be effective antibiotics.",
"Neamine itself is a poor antibiotic and is not clinically useful.",
"However, clinically useful aminoglycosides include, e.g., gentamicin, amikacin and neomycin.",
"These compounds, however, face the possibility of clinical obsolescence because of the function of aminoglycoside-modifying enzymes, such as what already happened with kanamycins.",
"(Wright, G. D.; Berghuis, A. M.; Mobashery, S. Aminoglycoside antibiotics: Structures, functions and resistance; Rosen, B. P., Mobashery, S., Eds.",
"; Plenum Press: New York, 1998; pp.",
"27-69.The tetracyclines are protein synthesis inhibitors that consist of eight related antibiotics which are all natural products of Streptomyces, although some can now be produced semisynthetically.",
"Tetracycline, chlortetracycline and doxycycline are the best known.",
"The tetracyclines are broad-spectrum antibiotics with a wide range of activity against both Gram-positive and Gram-negative bacteria.",
"Tetracyclines have some important uses, such as in the treatment of Lyme disease.",
"Chloramphenicol is a protein synthesis inhibitor that has a broad spectrum of activity but it exerts a bacteriostatic effect.",
"It is effective against intracellular parasites such as the rickettsiae.",
"It is infrequently used in human medicine except in life-threatening situations (e.g.",
"typhoid fever).",
"Macrolide antibiotics, such as erythromycin, are protein synthesis inhibitors that are active against most Gram-positive bacteria.",
"Some antibiotics affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read.",
"For example, nalidixic acid is a synthetic quinoloid antibiotic which is active mainly against Gram-negative bacteria.",
"The main use of nalidixic acid is in treatment of lower urinary tract infections (UTI).",
"In addition, the rifamycins has greater bactericidal effect against the bacteria that causes tuberculosis than other anti-tuberculosis drugs and is also useful for treatment of tuberculosis meningitis and meningitis caused by Neisseria meningitidis.",
"Growth factor analogs are structurally similar to bacterial growth factors, but do not fulfill their metabolic functions in cells.",
"For example, sulfonamides have been extremely useful in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis.",
"The worldwide exploitation of antibiotics to treat infectious diseases has grown dramatically over the last forty years.",
"In 1954, two million pounds of antibiotics were produced in the United States.",
"Today, the figure exceeds 50 million pounds.",
"According to the Centers Disease Control (CDC), humans consume 235 million doses of antibiotics annually.",
"Widespread misuse or overuse of antibiotics has fostered the spread of antibiotic resistance and has contributed to the development of a serious public health problem.",
"Antibiotic resistance occurs when bacteria that cause infection are not killed by the antibiotics taken to stop the infection.",
"The bacteria survive and continue to multiply, causing more harm.",
"For example, the bacterium Staphylococcus aureus is a major cause of hospital acquired infections that, historically, responded satisfactorily to the antibiotic vancomycin.",
"Recently, however, many strains of S. aureus have been found to be resistant to vancomycin.",
"Moreover, the death rate for some communicable diseases such as tuberculosis have started to rise again, in part because of increases in bacterial resistance to antibiotics.",
"The development of new drugs is an essential component to strategies designed to reverse the problem of bacterial resistance, particularly in treating infectious diseases (e.g., bacterial infections).",
"Accordingly, there is a need to identify additional compounds to treat infectious diseases (e.g., bacterial infections).",
"The compounds can preferably be administered orally."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides compounds (e.g., aminoglycosides) that are useful to treat or prevent infectious diseases (e.g., bacterial infections).",
"The compounds are orally available and/or intravenously available.",
"The compounds kill or effectively inhibit the growth of one or more Gram positive bacteria and/or one or more Gram negative bacteria.",
"For example, the compounds of the present invention can effectively kill or effectively inhibit the growth of one or more of the following bacterium: Escherichia coli; Pseudomonas spp., Proteus spp.",
"; Bacteroides spp.",
"; Haemophilus influenzae; Klebsiella spp.",
"; Enterobacter spp.",
"; Neisseria gonorrhoeae; Acinetobacter; Citrobacter spp.",
"; Serratia marcescens; Branhamella (Moraxella) catarrhalis; Morganella morganii; Providencia stuartii; Salmonella spp.",
"; Shigella spp.",
"; Campilobacter spp.",
"; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Streptococcus pyogenes; Streptococcus (alpha-hemolytic); Streptococcus pneumoniae; and Enterococcus faecium.",
"The present invention provides a compound of formula (I): wherein: R 1 is thio, sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is O or S; R 1 is hydrogen, (C 1 -C 12 )alkyl, (C 6 -C 10 )aryl (C 1 -C 12 )alkyl, heteroaryl (C 1 -C 12 )alkyl, or (C 3 -C 8 )cycloalkyl (C 1 -C 12 )alkyl; wherein any alkyl, aryl, heteroaryl, or cycloalkyl is substituted with one or more amino or hydroxy; R 3 is a direct bond or C═X, wherein X is O or S; R 4 is (C 1 -C 12 )alkyl optionally substituted with one or more NR a R b , wherein R a and R b are each independently hydrogen, (C 1 -C 12 )alkyl, or (C 6 -C 10 )aryl (C 1 -C 12 )alkyl; wherein any alkyl or aryl of R 4 , R a or R b is optionally substituted with one or more amino, guanidinyl, or hydroxy; R 5 is hydroxy, halo, or hydrogen and R 6 is a direct bond; or R 5 is (C 1 -C 12 )alkyl optionally substituted with one or more NR c R d , wherein R c and R d are each independently hydrogen, (C 1 -C 12 )alkyl, or (C 6 -C 10 )aryl (C 1 -C 12 )alkyl, wherein any alkyl or aryl of R 5 , R c or R d is optionally substituted with one or more amino, guanidinyl, or hydroxy; and R 6 is oxy, thio, or C═X, wherein X is O or S; R 7 is hydrogen, halo, or hydroxy; R 8 is hydrogen or hydroxy; and R 9 is hydrogen or hydroxy; wherein any alkyl is optionally interrupted with one or more (e.g., 1, 2, 3, or 4) oxy, thio, sulfinyl, or sulfonyl; and wherein any alkyl, aryl, heteroaryl, or cycloalkyl is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) halo, cyano, nitro, trifluoromethyl, hydroxy, (C 1 -C 6 )alkoxy, mercapto, oxo, thioxo, or NR e R f , wherein R e and R f are each independently hydrogen, (C 1 -C 12 )alkyl, or (C 6 -C 10 )aryl (C 1 -C 12 )alkyl; or a pharmaceutically acceptable salt thereof.",
"The present invention also provides a pharmaceutical composition.",
"The pharmaceutical composition comprises a compound of formula (I) and a diluent or carrier.",
"The pharmaceutical composition can optionally comprise one or more additional antibiotic agents.",
"The present invention also provides a method of preventing or treating a bacterial infection in a mammal (e.g., human).",
"The method comprises administering to the mammal an effective amount of a compound of the present invention.",
"The method can optionally comprise administering to the mammal one or more additional antibiotic agents.",
"The present invention also provides a method of preventing or treating a bacterial infection in a mammal (e.g., human).",
"The method comprises administering to the mammal an effective amount of a pharmaceutical composition of the present invention.",
"The method can optionally comprise administering to the mammal one or more additional antibiotic agents.",
"The present invention also provides a compound of formula (I) for use in medical therapy.",
"The present invention also provides the use of a compound of formula (I) for the manufacture of a medicament for treating or preventing a bacterial infection in a mammal (e.g., human).",
"The present invention also provides intermediates and methods of making (e.g., synthetically preparing) compounds of formula (I)."
],
[
"BACKGROUND OF THE INVENTION Antibiotics are low-molecular weight antimicrobial agents that are produced as secondary metabolites by microorganisms that inhabit soil.",
"For instance, Penicillium and Cephalosporium produce beta-lactam antibiotics (e.g., penicillin, cephalosporin, and their relatives).",
"Actinomycetes (e.g., the Streptomyces species) produce tetracyclines, aminoglycosides (e.g., streptomycin and its analogs), macrolides (e.g., erythromycin and its analogs), chloramphenicol, ivermectin, rifamycins, and most other clinically-useful antibiotics that are not beta-lactams.",
"Bacillus species (e.g., B. polymyxa and Bacillus subtilis) produce polypeptide antibiotics (e.g.",
"polymyxin and bacitracin), while B. cereus produces zwittermicin.",
"The modern era of antibiotic therapy began with Fleming's 1929 discovery of penicillin, and Domagk's 1935 discovery of synthetic sulfonamides.",
"Spurred by the need for antibacterial drugs during World War II, penicillin was isolated, purified and injected into experimental animals.",
"The substance was found to not only cure infections, but also to possess low toxicity.",
"This finding marked the beginning of the era of antibiotic use in human drug therapy and the intense search for similar antimicrobial agents of low toxicity that could be used to treat infectious diseases.",
"The rapid isolation of streptomycin, chloramphenicol and tetracycline followed, and these and several other antibiotics were in clinical usage by the 1950's.",
"Antibiotics are used therapeutically to treat bacterial infections.",
"Several types of antibiotics, classified according to their mechanism of action, are currently employed.",
"The known types of antibiotics include, e.g., cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, and inhibitors that bind to or affect the synthesis of DNA or RNA.",
"Cell wall synthesis inhibitors, such as beta lactam antibiotics, generally inhibit some step in the synthesis of bacterial peptidoglycan.",
"Penicillin is generally effective against non-resistant streptococcus, gonococcus and staphylococcus.",
"Amoxycillin and Ampicillin have broadened spectra against Gram-negative bacterias.",
"Cephalosporins are generally used as penicillin substitutes, against Gram-negative bacteria, and in surgical prophylaxis.",
"Monobactams are generally useful for the treatment of allergic individuals.",
"Cell membrane inhibitors disorganize the structure or inhibit the function of bacterial membranes.",
"Polymyxin, produced by Bacillus polymyxis, is a cell membrane inhibitor that is effective mainly against Gram-negative bacteria and is usually limited to topical usage.",
"Protein synthesis inhibitors include the tetracyclines, chloramphenicol, the macrolides (e.g.",
"erythromycin) and the aminoglycosides (e.g.",
"streptomycin).",
"Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria.",
"Aminoglycosides bind to the bacterial RNA in manifestation of their activity.",
"Davis, B. D. Microbiol.",
"Rev.",
"1987, 51, 341-350.Mingeot-Leclercq, M.-P.; Glupczynski, Y.; Tulkens, P. M. Antimicrob.",
"Agents Chemother.",
"1999, 43, 727-737.Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.Shaw, K. J., Rather, P. N.; Hare, R. S.; Miller, G. H. Microbiol.",
"Rev.",
"1993, 57, 138-163.Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis.",
"Gentamicin is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Pseudomonas aeruginosa.",
"Kanamycin is active at low concentrations against many Gram-positive bacteria, including penicillin-resistant staphylococci.",
"The structures of two aminoglycoside antibiotics, paromomycin and gentamicin C1a, bound to the template sequences of ribosomal RNA (rRNA), have been determined recently by NMR.",
"Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996, 274, 1367-1371; (a) Fourmy, D.; Recht, M. I.; Puglisi J. D. J. Mol.",
"Biol.",
"1998, 277, 347-362; (b) Fourmy, D.; Yoshizawa, S.; Puglisi, J. D. J. Mol.",
"Biol.",
"1998, 277, 333-345; (c) Recht, M. I.; Fourmy, D.; Blanchard, S. C.; Dahlquist, K. D.; Puglisi, J. D. J. Mol.",
"Biol.",
"1996, 262, 421-436; (d) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. EMBO J.",
"1998, 17, 6437-6448.These and other studies show that the neamine-class of aminoglycosides bind specifically to the A-site region on the 16S subunit of rRNA.",
"Hence, neamine serves as a minimal structural motif for such binding.",
"Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996, 274, 1367-1371; Kotra, L. P.; Haddad, J.; Mobashery, S. Antimicrob.",
"Agents Chemother.",
"2000 (in press)).",
"As such, aminoglycosides are a suitable class of compounds that could be effective antibiotics.",
"Neamine itself is a poor antibiotic and is not clinically useful.",
"However, clinically useful aminoglycosides include, e.g., gentamicin, amikacin and neomycin.",
"These compounds, however, face the possibility of clinical obsolescence because of the function of aminoglycoside-modifying enzymes, such as what already happened with kanamycins.",
"(Wright, G. D.; Berghuis, A. M.; Mobashery, S. Aminoglycoside antibiotics: Structures, functions and resistance; Rosen, B. P., Mobashery, S., Eds.",
"; Plenum Press: New York, 1998; pp.",
"27-69.The tetracyclines are protein synthesis inhibitors that consist of eight related antibiotics which are all natural products of Streptomyces, although some can now be produced semisynthetically.",
"Tetracycline, chlortetracycline and doxycycline are the best known.",
"The tetracyclines are broad-spectrum antibiotics with a wide range of activity against both Gram-positive and Gram-negative bacteria.",
"Tetracyclines have some important uses, such as in the treatment of Lyme disease.",
"Chloramphenicol is a protein synthesis inhibitor that has a broad spectrum of activity but it exerts a bacteriostatic effect.",
"It is effective against intracellular parasites such as the rickettsiae.",
"It is infrequently used in human medicine except in life-threatening situations (e.g.",
"typhoid fever).",
"Macrolide antibiotics, such as erythromycin, are protein synthesis inhibitors that are active against most Gram-positive bacteria.",
"Some antibiotics affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read.",
"For example, nalidixic acid is a synthetic quinoloid antibiotic which is active mainly against Gram-negative bacteria.",
"The main use of nalidixic acid is in treatment of lower urinary tract infections (UTI).",
"In addition, the rifamycins has greater bactericidal effect against the bacteria that causes tuberculosis than other anti-tuberculosis drugs and is also useful for treatment of tuberculosis meningitis and meningitis caused by Neisseria meningitidis.",
"Growth factor analogs are structurally similar to bacterial growth factors, but do not fulfill their metabolic functions in cells.",
"For example, sulfonamides have been extremely useful in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis.",
"The worldwide exploitation of antibiotics to treat infectious diseases has grown dramatically over the last forty years.",
"In 1954, two million pounds of antibiotics were produced in the United States.",
"Today, the figure exceeds 50 million pounds.",
"According to the Centers Disease Control (CDC), humans consume 235 million doses of antibiotics annually.",
"Widespread misuse or overuse of antibiotics has fostered the spread of antibiotic resistance and has contributed to the development of a serious public health problem.",
"Antibiotic resistance occurs when bacteria that cause infection are not killed by the antibiotics taken to stop the infection.",
"The bacteria survive and continue to multiply, causing more harm.",
"For example, the bacterium Staphylococcus aureus is a major cause of hospital acquired infections that, historically, responded satisfactorily to the antibiotic vancomycin.",
"Recently, however, many strains of S. aureus have been found to be resistant to vancomycin.",
"Moreover, the death rate for some communicable diseases such as tuberculosis have started to rise again, in part because of increases in bacterial resistance to antibiotics.",
"The development of new drugs is an essential component to strategies designed to reverse the problem of bacterial resistance, particularly in treating infectious diseases (e.g., bacterial infections).",
"Accordingly, there is a need to identify additional compounds to treat infectious diseases (e.g., bacterial infections).",
"The compounds can preferably be administered orally.",
"SUMMARY OF THE INVENTION The present invention provides compounds (e.g., aminoglycosides) that are useful to treat or prevent infectious diseases (e.g., bacterial infections).",
"The compounds are orally available and/or intravenously available.",
"The compounds kill or effectively inhibit the growth of one or more Gram positive bacteria and/or one or more Gram negative bacteria.",
"For example, the compounds of the present invention can effectively kill or effectively inhibit the growth of one or more of the following bacterium: Escherichia coli; Pseudomonas spp., Proteus spp.",
"; Bacteroides spp.",
"; Haemophilus influenzae; Klebsiella spp.",
"; Enterobacter spp.",
"; Neisseria gonorrhoeae; Acinetobacter; Citrobacter spp.",
"; Serratia marcescens; Branhamella (Moraxella) catarrhalis; Morganella morganii; Providencia stuartii; Salmonella spp.",
"; Shigella spp.",
"; Campilobacter spp.",
"; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Streptococcus pyogenes; Streptococcus (alpha-hemolytic); Streptococcus pneumoniae; and Enterococcus faecium.",
"The present invention provides a compound of formula (I): wherein: R1 is thio, sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is O or S; R1 is hydrogen, (C1-C12)alkyl, (C6-C10)aryl (C1-C12)alkyl, heteroaryl (C1-C12)alkyl, or (C3-C8)cycloalkyl (C1-C12)alkyl; wherein any alkyl, aryl, heteroaryl, or cycloalkyl is substituted with one or more amino or hydroxy; R3 is a direct bond or C═X, wherein X is O or S; R4 is (C1-C12)alkyl optionally substituted with one or more NRaRb, wherein Ra and Rb are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; wherein any alkyl or aryl of R4, Ra or Rb is optionally substituted with one or more amino, guanidinyl, or hydroxy; R5 is hydroxy, halo, or hydrogen and R6 is a direct bond; or R5 is (C1-C12)alkyl optionally substituted with one or more NRcRd, wherein Rc and Rd are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl of R5, Rc or Rd is optionally substituted with one or more amino, guanidinyl, or hydroxy; and R6 is oxy, thio, or C═X, wherein X is O or S; R7 is hydrogen, halo, or hydroxy; R8 is hydrogen or hydroxy; and R9 is hydrogen or hydroxy; wherein any alkyl is optionally interrupted with one or more (e.g., 1, 2, 3, or 4) oxy, thio, sulfinyl, or sulfonyl; and wherein any alkyl, aryl, heteroaryl, or cycloalkyl is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) halo, cyano, nitro, trifluoromethyl, hydroxy, (C1-C6)alkoxy, mercapto, oxo, thioxo, or NReRf, wherein Re and Rf are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; or a pharmaceutically acceptable salt thereof.",
"The present invention also provides a pharmaceutical composition.",
"The pharmaceutical composition comprises a compound of formula (I) and a diluent or carrier.",
"The pharmaceutical composition can optionally comprise one or more additional antibiotic agents.",
"The present invention also provides a method of preventing or treating a bacterial infection in a mammal (e.g., human).",
"The method comprises administering to the mammal an effective amount of a compound of the present invention.",
"The method can optionally comprise administering to the mammal one or more additional antibiotic agents.",
"The present invention also provides a method of preventing or treating a bacterial infection in a mammal (e.g., human).",
"The method comprises administering to the mammal an effective amount of a pharmaceutical composition of the present invention.",
"The method can optionally comprise administering to the mammal one or more additional antibiotic agents.",
"The present invention also provides a compound of formula (I) for use in medical therapy.",
"The present invention also provides the use of a compound of formula (I) for the manufacture of a medicament for treating or preventing a bacterial infection in a mammal (e.g., human).",
"The present invention also provides intermediates and methods of making (e.g., synthetically preparing) compounds of formula (I).",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1 illustrates representative compounds of the present invention.",
"FIG.",
"2 illustrates representative compounds of the present invention.",
"FIG.",
"3 illustrates a synthesis for representative compounds of the present invention.",
"FIG.",
"4 illustrates a synthesis for representative compounds of the present invention.",
"FIG.",
"5 illustrates a synthesis of reagents useful for praparing representative compounds of the present invention.",
"FIG.",
"6 illustrates a synthesis for representative compounds of the present invention.",
"FIG.",
"7 illustrates several known aminoglycosides.",
"FIG.",
"8 illustrates the minimum inhibitory concentrations (MIC) of representative compounds of the present invention and several known antibiotic agents against various bacterial strains.",
"FIG.",
"9 illustrates kinetic paramaters for turnover of representative compounds of the present invention and several known antibiotic agents by various enzymes.",
"FIG.",
"10 illustrates a stereo view of the A-site region of the 16S ribosomal RNA template occupied by a representative compound of the present invention.",
"DETAILED DESCRIPTION OF THE INVENTION Applicant has discovered certain compounds (e.g., aminoglycosides) that are useful to treat or prevent infectious diseases (e.g., bacterial infections).",
"The compounds are orally available and/or intravenously available.",
"The following definitions are used, unless otherwise described: halo is fluoro, chloro, bromo, or iodo.",
"Alkyl, alkylene, etc.",
"denote both straight and branched groups; but reference to an individual group such as “propyl” embraces only the straight chain group, a branched chain isomer such as “isopropyl” being specifically referred to.",
"Aryl denotes a phenyl group or an ortho-fused bicyclic carbocyclic group having about nine to ten ring atoms in which at least one ring is aromatic.",
"Heteroaryl encompasses a group attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C1-C4)alkyl, phenyl or benzyl, as well as a group of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.",
"It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms.",
"Some compounds may exhibit polymorphism.",
"It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine antibacterial or antibiotic activity using the standard tests described herein, or using other similar tests which are well known in the art.",
"Specific and preferred values listed below for groups, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the groups and substituents.",
"Specifically, (C1-C8)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl, or octyl; (C1-C6)alkylene can be methylene, ethylene, propylene, butylene, pentylene, or hexylene; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; and aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).",
"A specific value for R1 is sulfonyl, a direct bond, or C═X, wherein X is O or S. A specific value for R2 is hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl is substituted with one or more amino and wherein any alkyl is optionally substituted with one or more hydroxy or mercapto.",
"Another specific value for R2 is hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl is substituted with one or more amino and wherein any alkyl is optionally substituted with one or more hydroxy or mercapto; wherein the (C1-C8)alkyl is terminally substituted with an amino or the aryl of (C6-C10)aryl (C1-C12)alkyl is substituted with an amino.",
"A specific value for R1R2 is hydrogen or a group of the formula: wherein n is 0 to about 6; R10 is sulfinyl, sulfonyl, a direct bond, or C═X wherein X is O or S; and R11 is hydroxy, mercapto, or NRgRh, wherein Rg and Rh are each independently hydrogen, (C1-C8)alkyl, or (C6-C10)aryl (C1-C8)alkyl.",
"A specific value for R3 is a direct bond.",
"Another specific value for R3 is C═X, wherein X is S or O.",
"A specific value for R4 is (C1-C12)alkyl substituted with one or more NRaRb, wherein Ra and Rb are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; wherein any alkyl is optionally substituted with one or more hydroxy or mercapto.",
"Another specific value for R4 is (C1-C12)alkyl substituted with one or more NRaRb, wherein Ra and Rb are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl; wherein any alkyl or aryl of R4, Ra or Rb is substituted with one or more amino or guanidinyl.",
"A specific value for R3R4 is a group of the formula: wherein Ri is hydrogen, Rj is wherein R12 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R13 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R14 is hydrogen, hydroxy, mercapto, or NRiRj, wherein Ri and Rj are each independently hydrogen, (C1-C8)alkyl, or (C6-C10)aryl(C1-C8)alkyl; and m is 0 to about 8.A specific value for Ri is (CH2)n—NH2, wherein n is 2 to about 8; or Ri is guanidinyl.",
"A specific value for Ri is hydrogen or a group of the formula: wherein R15 is C═X, wherein X is O or S; R16 is hydrogen, hydroxy or amino; and q is 2.A specific value for R5 is hydroxy, halo, or hydrogen and R6 is a direct bond.",
"Another specific value for R5 is (C1-C12)alkyl optionally substituted with one or more oxo or NRmRn, wherein Rm and Rn are each independently hydrogen, (C1-C12)alkyl, or (C6-C10)aryl (C1-C12)alkyl, wherein any alkyl or aryl of R5, Rm or Rn is optionally substituted with one or more amino, hydroxy, or guanidinyl, and R6 is oxy, thio, or C═X, wherein X is O or S. A specific value for R5R6 is hydrogen, hydroxy, flouro, or a group of the formula: wherein Ro is hydrogen, Rp is wherein R17 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R18 is sulfinyl, sulfonyl, a direct bond, or C═X, wherein X is S or O; R19 is hydrogen, hydroxy, mercapto, or NRqRs, wherein Rq and Rs are each independently hydrogen, (C1-C8)alkyl, or (C6-C10)aryl(C1-C8)alkyl; and p is 0 to about 6.A specific value for R5R6 is hydroxy.",
"A specific value for R7 is hydrogen, fluoro, or hydroxy.",
"Another specific value for R7 is hydrogen.",
"A specific value for R8 is hydroxy or hydrogen.",
"Another specific value for R8 is hydroxy.",
"A specific value for R9 is hydroxy or hydrogen.",
"A specific value for R9 is hydroxy.",
"A specific compound of formula (I) is Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"Another specific compound of formula (I) is or a pharmaceutically acceptable salt thereof.",
"The present invention also provides intermediates as well as methods of making (e.g., synthetically preparing) compounds of the present invention (e.g., compounds of formula (I)).",
"The compounds of the present invention can be prepared from procedures that are known to those of skill in the art or as shown herein below.",
"Specifically, the compounds of the present invention (e.g., compounds of formula (I)) can be prepared from convenient starting materials, employing procedures (e.g., reagents and reaction conditions) known to those of skill in the art.",
"For example, suitable reagents and reaction conditions are disclosed, e.g., in Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Carey and Sundberg (1983); Advanced Organic Chemistry Reactions, Mechanisms, and Structure, Second Edition, March (1977); Greene, T. W., Protecting Groups In Organic Synthesis, Third Edition, 1999, New York, John Wiley & sons, Inc.; and Comprehensive Organic Transformations, Second Edition, Larock (1999).",
"Additionally, specific exemplary procedures are shown in the examples herein below.",
"Referring to FIG.",
"3, neamine hydrochloride 1 was prepared from methanolysis of the commercially available neomycin sulfate, as reported in Dutcher, J.; Donin, M. J.",
"Am.",
"Chem.",
"Soc.",
"1952, 74, 3420-3422; Ford, J. H.; Bergy, M. E.; Brooks, A.",
"A.; Garrett, E. R.; Alberti, J.; Dryer, J. R.; Carter, H. E. J.",
"Am.",
"Chem.",
"Soc.",
"1955, 77, 5311-5314; Grapsas, I.; Cho, Y. I.; Mobahsery, S. J. Org.",
"Chem.",
"1994, 59, 1918-1922.Treatment of this compound with benzylchloroformate in the presence of sodium carbonate afforded the tetra-N-Cbz protected neamine derivative 10.Kumar, V.; Remers, W. A. J. Org.",
"Chem.",
"1978, 43, 3327-3337.This compound was subjected to 1,1-dimethoxycyclohexane to provide the mono-cyclohexylidene-protected compound 11, along with some 5,6:3′,4′-di-cyclohexylidene-protected derivative, which was fully converted to 11 in the presence of p-toluenesulfonic acid and methanol in DMF.",
"Tohma, S.; Yoneta, T.; Fukatsu, S. J. Antibiot.",
"1980, 33, 671-673.Protection of the hydroxyl groups at positions 3′ and 4′ was achieved by treatment of compound 11 with chloromethyl methyl ether in the presence of Hünig's base and tetrabutylammonium iodide to give 12 in good yield.",
"Cyclohexylidene deprotection of this compound with acetic acid afforded intermediate 13, which was treated with sodium hydride in DMF to furnish the cyclic carbamate 14 in high yield.",
"Protection of the hydroxyl group at position 5 of 14 with triethylsilyl chloride gave the TES-protected derivative 15 (FIG.",
"3).",
"Treatment of 15 with di-tert-butyldicarbonate in the presence of triethylamine and DMAP afforded compound 16 in high yield.",
"Attempt at the opening of the oxazolidinone ring under mild basic condition (0.5 N aqueous LiOH) gave an isomeric mixture of products having the TES group either at position 5 or 6 (due to TES migration from position 5 to 6 in the ring-opened product).",
"Several attempts to protect the C5 hydroxyl group with a variety of different types of protective groups, prior to N-Boc protection of the cyclic carbamate, failed to give any acceptable result.",
"For instance, attempt at MOM protection resulted in formation of a compound having the MOM groups both at the C5 oxygen and the N1 of the cyclic carbamate.",
"Therefore, the TES group of 16 was removed by TBAF to give 17, which was treated with chloromethyl methyl ether to afford the derivative 18 in good yield.",
"Referring to FIG.",
"3, treatment of compound 18 with 0.5 N aqueous lithium hydroxide afforded the advanced intermediate 19, having the C6 hydroxyl group free for further manipulation.",
"Compound 19 was allowed to undergo reaction with allyl bromide in the presence of lithium bis(trimethylsilyl)amide and tetrabutylammonium iodide to give the allyl protected derivative 20, according to the procedure of Park et al.",
"Park, W. K. C.; Auer, M.; Jaksche, H., Wong, C.-H. J.",
"Am.",
"Chem.",
"Soc.",
"1996, 118, 10150-10155.Referring to FIG.",
"4, ozonolysis of 20, followed by reductive amination of the aldehyde 21 with the corresponding mono-N-Cbz-protected diaminoalkanes, afforded the aminated products 22-24, according to similar reported methodology.",
"Park, W. K. C.; Auer, M.; Jaksche, H., Wong, C.-H. J.",
"Am.",
"Chem.",
"Soc.",
"1996, 118, 10150-10155.Efficient deprotection of the Boc and MOM groups with 1.3 N methanolic HCl resulted in the formation of derivatives 25-27, having the N1 group free for the preparation of the corresponding amides.",
"Treatment of these compounds with activated ester of (S)-4-amino-2-hydroxybutanoic acid (FIG.",
"4), furnished 29-31 in good yields.",
"Finally, removal of the Cbz groups of 29-31 and 26 by catalytic transfer hydrogenation (Felix, A. M.; Heimer, E. P.; Lambros, T. J.; Tzougraki, C.; Meienhofer, J. J. Org.",
"Chem.",
"1978, 43, 4194-4196) with 1,4-cyclohexadiene over Pd—C afforded the title compounds 3-6 in good yields (FIG.",
"4).",
"Compound 32 was prepared by treatment of 30 with 28 in pyridine, which after removal of the Cbz groups gave compound 7 (FIG.",
"6).",
"Compound 35 was prepared by treatment of 27 with 1.0 equivalent of the active ester of 4-aminophenylacetic acid (33).",
"Deprotection of this derivative under the same condition described earlier afforded compound 8 in good yield (FIG.",
"6).",
"Similarly, compound 36 was synthesized by treatment of 27 with 34, which after removal of the Cbz groups furnished 9 (FIG.",
"6).",
"One of the major difficulties in the synthesis was the preparation of the intermediate 19 from its precursor, cyclic carbamate 14 (FIG.",
"3).",
"When compound 14 was treated with di-tert-butydicarbonate in the presence of Et3N and DMAP, the product of the reaction was a compound with Boc groups both at C5 oxygen and N1 nitrogen.",
"Attempts to deprotect the O-Boc group under mild basic conditions (Na2CO3 or LiOH) resulted in the opening of the oxazilidinone ring.",
"Another difficulty encountered in the final step of the synthesis was deprotection of the Cbz groups, which proved to be difficult under typical conditions.",
"Hydrogenolysis of the Cbz protected compounds over Pd—C at atmospheric hydrogen pressure required long reaction times and afforded very low yield of the desired compounds.",
"This problem was solved by the application of the catalytic transfer hydrogenation, using 1,4-cyclohexadiene in the presence of activated palladium on carbon and acetic acid as solvent.",
"Felix, A. M.; Heimer, E. P.; Lambros, T. J.; Tzougraki, C.; Meienhofer, J. J. Org.",
"Chem.",
"1978, 43, 4194-4196.Processes for preparing compounds of formula (I), and intermediates thereof, are provided as further embodiments of the invention and are illustrated by the procedures in the examples herein below, wherein the meanings of the generic radicals are as given above unless otherwise qualified.",
"Intermediates useful for preparing compounds of formula (I) are also provided as further embodiments of the invention.",
"Design Strategy The three-dimensional NMR structure for paromomycin bound to the A-site rRNA template (Protein Data Bank Code: 1PBR) was the beginning for the design.",
"The paromamine portion (6′-hydroxyl analogue of neamine) of paromomycin, as a minimal motif for RNA binding, was retained in the A-site template and the remainder of the paromomycin structure was removed.",
"This complex was defined as the receptor template, and a DOCK (DOCK version 4.0, University of California at San Francisco.",
"Ewing, T. J.",
"A.; Kuntz, I. D. J. Comput.",
"Chem.",
"1997, 18, 1175-1189) search was performed using the Cambridge structural database (CSD) and the National Cancer Institute (NCI) 3-D database for molecules that would bind to this template.",
"The two databases provided a sampling of 273,000 compounds for the three-dimensional search protocol.",
"The list of selected compounds that emerged from the search was reduced according to steric and energetic criteria.",
"A subset of these structures that was predicted to bind near the positions N1 and O6 of paromamine was kept.",
"In addition to this set of compounds, attention was paid to the A-site rRNA-paromamine complex itself.",
"The core moiety of paromamine was modified by an aminohydroxybutyryl group at position N1.At position O6, various amine-containing aliphatic substitutions were made by looking at the distance between O6 and the phosphate backbone of A-site of rRNA, using molecular graphics.",
"The outcome of these protocols was compounds 3-9.The rationale for selecting aminohydroxybutyryl group at position N1 was that amikacin 2 possesses such a substitution in its structure, and this particular substitution was known to impart resistance to modification by a number of aminoglycoside-modifying enzymes.",
"Kotra, L. P.; Haddad, J.; Mobashery, S. Antimicrob.",
"Agents Chemother.",
"2000 (in press); Wright, G. D.; Berghuis, A. M.; Mobashery, S. Aminoglycoside antibiotics: Structures, functions and resistance; Rosen, B. P., Mobashery, S., Eds.",
"; Plenum Press: New York, 1998; pp.",
"27-69; Haddad, J.; Kotra, L. P.; Mobashery, S. Aminoglycoside Antibiotics: Structures and Mechanism of Action, In Glycochemistry: Principles, Synthesis, and Applications; Wang, G., Bertozzi, C., Eds.",
"; Marcel Dekker, Inc: NY, 2000 (in press).",
"Neamine, the 6′-amino analog of paromamine, was used in the syntheses because of its ready availability from fragmentation of neomycin.",
"The terminal amine-containing substitutions at position O6 on neamine were selected because of potential ion-pairing interactions between the amine and the phosphate backbone in the target rRNA region.",
"Computer Modeling FIG.",
"8 shows the energy-minimized structure of compound 4 bound in the A-site of rRNA template.",
"This compound occupies the pocket that is formed by A1492, similar to the case of other neamine class of compounds, and interacts with the phosphate backbone of the nucleic acid via ion-pairing and hydrogen-bonding interactions.",
"In addition, the aminohydroxybutyryl moiety forms two hydrogen bonds and one ion-pair interaction with the rRNA template.",
"The O6 side chain has one hydrogen bond with the nucleic acid base (G1405) and one ion-pairing interaction with the phosphate backbone.",
"This model suggests that the side chains at O6 may be longer than the propylamine due to the presence of more functional groups and space to accommodate such side chains in this area.",
"These homologous side chains will have similar interactions, such as ionic and hydrogen-bonding interactions as was seen in the case of compound 4, and indeed compound 5 showed comparable binding affinity to F-AS (vide infra).",
"Antibiotic Activities of Synthetic Aminoglycosides The antibiotic activities of representative compound of the present invention are shown in Table 1.For the purpose of comparison of activities and demonstration of the properties of the bacterial strains used in this study, susceptibility studies were also performed with several aminoglycosides (neamine, kanamycin A, gentamicin, tobramycin, and amikacin) and β-lactam [ampicillin (a penicillin), ceftazidime (an expanded-spectrum cephalosporin, and imipenem (a carbapenem)] antibiotics.",
"Neamine 1 (FIG.",
"7) is a suitable control, since the compounds of the present invention utilize the structure of Neamine 1 as a template.",
"Kanamycin A has become clinically obsolete, since it is an excellent substrate for the ubiquitous resistance enzymes aminoglycoside phosphotransferases (APH(3′)s), and it serves as another control.",
"Gentamicin, tobramycin and amikacin are widely used presently in clinic, either by themselves or in synergistic combinations with β-lactam antibiotics.",
"Escherichia coli JM83 is an antibiotic-sensitive laboratory strain.",
"E. coli JM83 (APH(3′)-I) contains high copies (500-700 copies per cell) of plasmid pUC19 with the cloned kanamycin resistance gene from transposon Tn903 that produces the type I aminoglycoside 3′-phosphotransferase.",
"APH(3′)-I is the most common aminoglycoside phosphotransferase in gram-negative bacteria.",
"E. coli JM83 (AAC6′/APH2″) contains a plasmid with the gene for the bifunctional aminoglycoside-modifying enzyme AAC(6′)/APH(2″), which is produced by many cocci.",
"The enzyme is able to phosphorylate at the 2″ position and/or acetylate at the 6′ position of various aminoglycoside antibiotics to manifest resistance for the organisms that express it.",
"Serratia marcescens ATCC 13880, Enterobacter cloacae ATCC 3047, Pseudomonas aeruginosa 66 and C43 are representative important gram-negative pathogens.",
"Aminoglycosides by themselves or in combination with other antibiotics are often used to combat these microorganisms, especially P. aeruginosa that is often very difficult to treat.",
"Staphylococcus aureus 3 and Enterococcus faecium 119 are representatives of the cocci.",
"Most of these microorganisms are intrinsically resistant to aminoglycosides (poor permeability), but in combination with β-lactam antibiotics are often used to treat infections caused by these microorganisms.",
"Compounds 4 and 5 show good activity against E. coli strain hyper-expressing either APH(3′)-I or AAC(6′)/APH(2″) aminoglycoside-modifying enzymes (Table 1).",
"These activities are much more superior than those of kanamycin A and neamine (250- to 1000-fold higher activities) against E. coli hyper-expressing APH(3′)-I, and merely 8-fold lower than activities of tobramycin and amikacin.",
"The results indicate that 4 and 5 are poorly turned over by APH(3′)-I (supported by enzymology with purified enzyme; vide infra).",
"The activity for the synthetic compounds against E. coli hyper-expressing AAC(6′)/APH(2″) is even more impressive.",
"These compounds either have equal activity of clinical aminoglycosides, such as amikacin, or are clearly superior (8- to 250-fold) more active (as compared with kanamycin A, gentamicin, or tobramycin).",
"Compounds 4 and 5 demonstrate excellent activities against enterobacteria, such as S. marcescens, E. cloacae and also P. aeruginosa that are sensitive or moderately resistant to aminoglycoside antibiotics, including strains also highly resistant to β-lactam antibiotics (ampicillin, ceftazidime, imipenem).",
"In these cases, activities of compounds 4 and 5 are typically either equal or superior to those of the most active aminoglycoside(s) used in this study.",
"Compounds 4 and 5 also show good activities against strains of S. aureus 3 and E. faecium 119 that is moderately resistant to aminoglycosides.",
"Compound 3 (Table 1) showed the spectrum of antimicrobial activity similar to those of compounds 4 and 5 except that its MICs values were approximately four-fold higher.",
"Compound 6 (Table 1) gave lower activity than compound 3.It is not active against E. coli strains producing either APH(3′)-I or AAC(6′)/APH(2″) enzymes, although it demonstrates activity against some of the tested organisms (E. coli JM83, S. marcescens ATCC 13880, E. cloacae ATCC 3047, S. aureus 3) that are sensitive or moderately resistant to aminoglycoside antibiotics in general.",
"Compound 7 shows moderate activity against E. coli strains producing either APH(3′)-I or AAC(6′)/APH(2″) enzymes (MICs 64 and 32 μg/mL, respectively) and also against strains of enterobacteria and P. aeruginosa.",
"Finally, compounds 8 and 9 do not show any appreciable activity against any of the microorganisms tested (MICs>1000 μg/mL), despite the fact that they do bind to the RNA under the specific assay conditions used, which may indicate that these compounds may have difficulty being transported into the cytoplasm.",
"Kinetics of Turnover with Aminoglycoside-Modifying Enzymes Compounds 4, 5, 8, and 9 were studied with two of the most important aminoglycoside-modifying enzymes, namely an aminoglycoside 3′-phosphotransferase (APH(3′)) (Siregar, J. J.; Lerner, S. A.; Mobashery, S. Antimicrob.",
"Agents Chemother.",
"1994, 38, 641) and the bifunctional (“BF”) aminoglycoside-modifying enzyme (AAC(6′)/APH(2″)).",
"Azucena, E.; Grapsas, I.; Mobashery, S. J.",
"Am.",
"Chem.",
"Soc.",
"1997, 119, 2317; Daigle, D M; Hughes, D W; Wright, G D Chem.",
"& Biol.",
"1999, 6, 99-110.The latter possesses both phosphotransferase (APH(2″)) and acetyltransferase (AAC(6′)) activities.",
"These compounds are exceedingly poor substrates for these resistance enzymes under in vitro conditions (Table 2).",
"So, in effect, they are not affected by these resistance enzymes.",
"The kinetic parameters for turnover of two known aminoglycosides are noted herein.",
"Neamine is an excellent substrate for APH(3′) (kcat/Km of 2.9×107 M−1s−1), and a mid-range substrate for the acetyltransferase activity of the bifunctional enzyme (kcat/Km of 4.3×105 M−1s−1).",
"Despite the fact that neamine does not have the 2″-hydroxyl, it would appear that it accepts phosphate in a reaction catalyzed by the bifunctional enzyme (kcat/Km of 189 M−1s−1), but the process is so inefficient that it is irrelevant for manifestation of resistance.",
"On the other hand, the semisynthetic amikacin was prepared for its poor interactions with many resistance enzymes.",
"King, J. W.; White, M. C.; Todd, J. R.; Conrad, S. A. Clin.",
"Infect.",
"Dis.",
"1992, 14, 908-915; Kucers, A.; Bennett, N. McK.",
"The use of antibiotics; Kucers, A., Bennett, N.",
"McK., eds.",
"; William Heinemann Medical Books: London, U.K. 1987; Schmitz, F. J.; Verhoef, J.; Fluit, A. C. Eur.",
"J. Clin.",
"Microbiol.",
"Infect.",
"Dis.",
"1999, 18, 414-421; Doern, G. V.; Jones, R. N.; Pfaller, M. A.; Kugler, K. C.; Beach, M. L. Diagn.",
"Microbiol.",
"Infect.",
"Dis.",
"1999, 34, 65-72.Amikacin is certainly turned over by these enzymes, but the kinetics of turnover are sufficiently inefficient that the antibiotic is viable against organisms that harbor these enzymes, with a few exceptions.",
"For example, amikacin was made initially to counter the effect of the ubiquitous APH(3′)s. As shown in Table 2, amikacin is turned over by APH(3′)-II with kcat/Km of 3.8×104 M−1s−1.Similarly, a kcat/Km of 8.6×103 M−1s−1 and 3.5×105 M−1s−1 are measured for the AAC(6′) and APH(2″) activities, respectively, of the bifunctional enzyme for amikacin.",
"Compounds 4, 5, 8, and 9 are turned over by these enzymes at or below 104 M−1s−1.At best, they are turned over by APH(3′)-II with kcat/Km of 104 M−1s−1.The Km for these substrates is substantially elevated above that for the substrate neamine (i.e., 1 μM), so enzymic catalysis would not reach saturation until a substantially higher concentration of the antibiotic is reached within the bacterial cytoplasm.",
"The same is true for both activities of the bifunctional enzyme with 4, 5, 8, and 9.The Km values for these activities were consistently above 100 μM, often substantially so, such that within the organism these concentrations cannot be reached for effective turnover of these antibiotics by the resistance enzymes.",
"Therefore, it is observed that these antibiotics are not affected by the resistance enzymes in any significant manner, consistent with the findings of the biological activity disclosed above.",
"Additional Antibiotic Agents The compound of the present invention can be co-administered with one or more additional antibiotic agents.",
"As used herein, an “antibiotic agent” is any compound having activity against either Gram-positive or Gram-negative organisms (i.e., inhibits the growth or destroys the development of either Gram-positive or Gram-negative organisms).",
"Stedman's Medical Dictionary, Illustrated, (25th Ed.",
"), Williams & Wilkins: Baltimore (1990) and Mosby's Medical, Nursing, & Allied Health Dictionary, (5th Ed.",
"), Mosby: St. Louis (1998).",
"Suitable antibiotic agents are disclosed, e.g., in Physician's Desk Reference (PDR), Medical Economics Company (Montvale, N.J.), (53rd Ed.",
"), 1999; Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, (11th Ed.",
"), Merck & Co., Inc. (Rahway, N.J.), 1989; and references cited therein.",
"Suitable antibiotics include, e.g., aminoglycosides, β-lactam antibiotics, cephalosporins, macrolides, miscellaneous antibiotics, penicillins, tetracyclines, antifungals, antimalarial agents, antituberculosis agents, antivirals, leprostatics, miscellaneous anti-infectives, quinolones, sulfonamides, urinary anti-infectives, nasal antibiotics, opthalmic antibiotics, opthalmic antivirals, opthalmic quinalones, opthalmic sulfonamides, skin and mucous membrane antibiotics, skin and mucous membrane antifungals, skin and mucous membrane antivirals, skin and mucous membrane miscellaneous anti-infectives, skin and mucous membrane scabicides and pedulicides, skin and mucous membrane antinepolasts, nitrofurans, and oxazolidinones.",
"Physician's Desk Reference (PDR), Medical Economics Company (Montvale, N.J.), (53rd Ed.",
"), 1999.The additional antibiotic agent can effectively kill or effectively inhibit the growth of bacteria (e.g., Gram positive bacteria and/or Gram negative bacteria).",
"For example, the additional antibiotic agent can effectively kill or effectively inhibit the growth of one or more of the following bacterium: Escherichia coli; Pseudomonas spp.",
"; Proteus spp.",
"; Bacteroides spp.",
"; Haemophilus influenzae; Klebsiella spp.",
"; Enterobacter spp.",
"; Neisseria gonorrhoeae; Acinetobacter; Citrobacter spp.",
"; Serratia marcescens; Branhamella (Moraxella) catarrhalis; Morganella morganii; Providencia stuartii; Salmonella spp.",
"; Shigella spp.",
"; Campilobacter spp.",
"; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Streptococcus pyogenes; Streptococcus (alpha-hemolytic); Streptococcus pneumoniae; and Enterococcus faecium.",
"It is appreciated that those skilled in the art understand that the additional antibiotic agent useful in the present invention is the biologically active compound present in any of the additional antibiotic agents disclosed above.",
"For example, the additional antibiotic can be a β-lactam and the β-lactam can be azactam (aztreonam), which is typically available as an injectable solution.",
"The additional antibiotic agent, however, is (z)-2-[[[(2-amino-4-thiazolyl) [[(2S,-3S)-2-methyl-4-oxo-1-sulfo-3-azetidinyl]carbamoyl]methylene]amino]oxy]-2-methyl propionic acid.",
"Physician's Desk Reference (PDR), Medical Economics Company (Montvale, N.J.), (53rd Ed.",
"), pp.",
"820-823, 1999.In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate.",
"Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ketoglutarate, and glycerophosphate.",
"Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.",
"Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.",
"Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.",
"The compounds of formula (I) can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.",
"Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.",
"They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.",
"For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.",
"Such compositions and preparations should contain at least 0.1% of active compound.",
"The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.",
"The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.",
"The tablets, troches, pills, capsules, and the lice may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.",
"When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol.",
"Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form.",
"For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.",
"A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.",
"Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.",
"In addition, the active compound may be incorporated into sustained-release preparations and devices.",
"The active compound may also be administered intravenously or intraperitoneally by infusion or injection.",
"Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.",
"Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils.",
"Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.",
"The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.",
"In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.",
"The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.",
"The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.",
"The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.",
"In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride.",
"Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.",
"Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.",
"In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.",
"For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids.",
"However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.",
"Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.",
"Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.",
"Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.",
"The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.",
"Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.",
"Examples of useful dermatological compositions which can be used to deliver the compounds of formula (I) to the skin are known to the art; for example, see Jacquet et al.",
"(U.S. Pat.",
"No.",
"4,608,392), Geria (U.S. Pat.",
"No.",
"4,992,478), Smith et al.",
"(U.S. Pat.",
"No.",
"4,559,157) and Wortzman (U.S. Pat.",
"No.",
"4,820,508).",
"Useful dosages of the compounds of formula (I) can be determined by comparing their in vitro activity, and in vivo activity in animal models.",
"Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat.",
"No.",
"4,938,949.Generally, the concentration of the compound(s) of formula (I) in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.",
"The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.",
"The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.",
"In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.",
"The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.",
"Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about 30 μM.",
"This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient.",
"Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).",
"The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.",
"The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.",
"The invention will now be illustrated in the following non-limiting examples.",
"EXAMPLES Experimental Section Proton (1H) and carbon (13C) nuclear magnetic resonance spectra were recorded on either a Varian 400 or a Varian unity-500 MHz spectrometer.",
"Chemical shifts are recorded in parts per million (δ) relative to tetramethylsilane (δ 0.00).",
"Infrared (IR) spectra were recorded on a Nicolet 680 DSP FTIR spectrometer.",
"Low-resolution mass spectra (MS) were recorded on a Kratos MS 80RFA spectrometer.",
"High-resolution mass spectra were performed by the Michigan State University Mass Spectrometry Facility.",
"Melting points were obtained on an Electrothermal melting point apparatus and are uncorrected.",
"Thin layer chromatography (TLC) was performed with Whatman precoated K6F silica gel 60A (0.25 mm thickness plates).",
"The plates were visualized by either ninhydrin spray or immersion in a p-anisaldehyde solution and warming on a hot plate.",
"All chromatography solvents were reagent grade.",
"Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl, and dichloromethane was distilled from calcium hydride.",
"1,1-Dimethoxycyclohexane was purchased from the TCI America Co. N-Cbz protected diaminoalkanes were either prepared (Atwell, G. J.; Denny, W. A. Synthesis 1984, 1032-1033) or purchased from the Fluka chemical company as hydrochloride salt.",
"The amine free-bases were prepared by treatment of the HCl salts with Amberlite IRA-400 (OH−) ion-exchange resin.",
"3-Amino and 4-amino phenylacetic acids were purchased from Fluka chemical company and converted to the N-Cbz protected derivatives prior to use.",
"Pyruvate kinase (PK), lactic dehydrogenase (LD), phospho(enol)pyruvate (PEP), ATP and NADH were purchased from the Sigma Chemical Co. All other reagents were purchased from the Aldrich Chemical Co. Spectrophotometric studies were performed on a Hewlett-Packard 8453 diode array instrument.",
"All calculations were performed by the MS Excel program.",
"Determination of Minimum Inhibitory Concentration (MICs) The MICs of all antibiotics, including the compounds of the present invention (i.e., the compounds of formula (I)), were determined by microdilution method broth procedure.",
"Sequential twofold dilutions of antibiotics were performed in 100 μL of Luria-Bertani (LB) broth in sterile 96-well microtiter plates.",
"Overnight cultures of bacteria were diluted 100 times in LB broth and subgrown for several hours and 10 μL of diluted cultures were transferred into the antibiotic containing microtiter plates to bring final inoculums to 105 CFU/mL (CFU stands for the colony forming units).",
"Cultures were incubated overnight at 35° C. and microtiter plates were checked from below with a reflective viewer.",
"MICs were defined as the lowest concentrations of the drug at which the microorganism did not demonstrate visible growth.",
"Kinetic Determinations with Resistance Enzymes Kinetic studies were performed for phosphotransferase activities of the bifunctional enzyme AAC(6′)/APH(2″) and APH(3′)-IIa, as well as the acetyltransferase activity of AAC(6′)/APH(2″) using the methods described by Azucena et al.",
"(Azucena, E.; Grapsas, I.; Mobashery, S. J.",
"Am.",
"Chem.",
"Soc.",
"1997, 119, 2317.)",
"The assay mixture consisted of 66 mM PIPES, pH 7.5, 11 mM magnesium acetate, 22 mM potassium acetate, 1.76 mM phosphoenol pyruvate, 0.1 mM NADH, 6.1 units of pyruvate kinase, 21 units of lactate dehydrogenase, 100 nM enzyme, the aminoglycoside substrate (at various concentrations), and 150 μM ATP in 500 μL total volume.",
"The components of the assay mixture were mixed in a cuvette in the absence of ATP and enzyme.",
"The solution was allowed to equilibrate at room temperature for 2 minutes.",
"The reaction was started by the addition of ATP and enzyme.",
"The progress of the reaction was monitored spectrophotometrically at 340 nm.",
"Lineweaver-Burk plots were obtained to determine the Km and kcat values.",
"For the acetyltransferase activity assay, the method of Haas and Dowding was employed.",
"(Haas, M.; Dowding, J. E. Methods Enzymol.",
"1972, 72, 248.)",
"The reaction mixture was composed of 58 mM citric acid, 124 mM tripotassium citrate, 18 mM magnesium acetate, 6 mM dithiothreitol, the aminoglycoside substrate (at various concentrations), and 120 μM acetylcoenzyme A (specific activity, 21 mCi/mmol) in a total volume of 30 μL.",
"The reaction was started by the addition of 5 μL of enzyme (final concentration of 100 nM) and was stopped at 1, 2, 3, and 4 min by the addition of 10% tricholoroacetic acid.",
"Kinetic constants were determined from Lineweaver-Burk plots.",
"Docking and Molecular Modeling The NMR structure of paromomycin bound to the rRNA A-site was used as the starting template.",
"Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996, 274, 1367-1371.Rings I and II (2-deoxystreptamine and 2′-deoxy-2′-aminoglucose, respectively) in the NMR structure of paromomycin were retained and the remainder of the structure was removed.",
"These two rings constitute the aminoglycoside paromamine (a structurally similar compound to neamine having a hydroxyl group in place of the amine at position 6′).",
"With this structure at hand, the Connolly surface of the complex (i.e., A-site RNA template bound by paromamine) was computed, which defines the “receptor site”.",
"Two ligand databases, the NCI-3D database and Cambridge Structural Database, collectively containing a total of 273,000 compounds, were used to dock the individual compounds into the “receptor site” using the program DOCK version 4.0.The electrostatic and steric counterparts on the receptor site were matched with the docked compounds.",
"This data set was reduced to 40 compounds based on their best fit into the “receptor site”.",
"Each compound in the set of 40 compounds was considered in the receptor site individually.",
"These 40 compounds fit in the space near the aminoglycoside-binding site, and were scored by the program favorably for their ability to bind to the depressions and niches of the surface of the rRNA structure.",
"Then, neamine analogues that would be covalently tethered to these entities individually were envisioned.",
"Many of these compounds were predicted to bind the surface such that they were amenable for attachment to neamine at position N1 and O6 (marked in the structure 1).",
"The tethers were designed such that they themselves would have potential favorable electrostatic interactions with the rRNA A-site.",
"The visualization and structure editing were performed using Sybyl molecular modeling program.",
"(Sybyl version 6.5, Tripos Inc., St. Louis, Mo.)",
"This complex was energy-minimized using Amber 5.0 package.",
"Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham III, T. E.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Ferguson, D. M.; Radmer, R. J.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollman, P. A. AMBER 5.1997, University of California, San Francisco, Calif.",
"The point charges on compound 4 were obtained from ESP charges calculated by MOPAC package (PM3 hamiltonian) and the parameters for the carbohydrate rings were according to Woods et al.",
"Woods, R. J.; Dwek, R. A.; Edge, C. J.; Fraser-Reid, B. J. Phys.",
"Chem.",
"1995, 99, 3832-3846.Sodium ions were added to the complex to neutralize the system using xleap routine as implemented in Amber 5.0, and the complete molecular assembly was solvated with TIP3 waters at least 10 Å from the surface of the assembly.",
"Energy minimization was carried out for 10,000 iterations with a non-bonded cutoff of 12 Å. Refering to FIGS.",
"3-6, exemplary compounds of the present invention can be prepared as follows: 1,3,2′,6′-Tetrakis(N-benzyloxycarbonyl)-5,6-O-cyclohexylideneneamine (11) A solution of 10 (6.37 g, 7.43 mmol) and p-toluenesulfonic acid mono hydrate (210 mg, 1.11 mmol) in anhydrous DMF (150 mL) was concentrated to about 120 mL in vacuo to remove the residual water.",
"To this solution was added 1,1-dimethoxycyclohexane (5.0 mL, 33.16 mmol) and the mixture was stirred overnight at room temperature under an atmosphere of argon.",
"TLC of the mixture after 24 h showed the presence of some starting compound.",
"Therefore, the mixture was concentrated to about 110 mL in vacuo to remove methanol and the other volatiles generated during the course of the reaction.",
"To this mixture was added another portion of 1,1-dimethoxycyclohexane (2.00 mL, 13.26 mmol) and the mixture was allowed to stir at room temperature for 12 h. TLC of the mixture showed the product as a major spot (Rf 0.37, 1:20 CHCl3/MeOH) and the di-protected derivative (Rf 0.77, 1:20 CHCl3/MeOH) as a minor component.",
"The reaction mixture was quenched by the addition of triethylamine (1.0 mL) and was concentrated to dryness in vacuo to give a residue, which was diluted with dichloromethane, extracted with water and then brine, dried (Na2SO4), and concentrated to give a solid as the crude product.",
"The solid was purified on a column (SiO2, 50:1 CHCl3/MeOH) to afford 11 (3.95 g, 56.6%) and the 5,6:3′,4′-di-O-cyclohexylidene derivative (2.49 g, 33%).",
"This compound was fully converted to 11 by the following procedure: To a solution of the di-protected derivative (2.49 g, 2.22 mmol) in anhydrous DMF (35 mL) was added a solution of p-toluenesulfonic acid (28 mg, 0.15 mmol) in methanol (1 mL) and the mixture was kept at room temperature overnight.",
"The reaction mixture was worked up and purified as described earlier to furnish another portion of 11 (2.10 g, 30%).",
"Total yield (6.05 g, 86.6%), mp 115-117° C.; FTIR (film): 3390, 3353, 2935, 1703, 1693, 1530, 1278, 1056 cm−1; 1H NMR (500 MHz, aceton-d6) δ 7.39-7.26 (m, 20H, Ph), 6.60 (d, J=7.0 Hz, 1H, NH), 6.49 (d, J=9.5 Hz, 1H, NH), 6.34 (s, 1H, NH), 6.17 (s, 1H, NH), 5.28 (s, 1H, H1′), 5.11 (d, J=12.5 Hz, 1H, CH2Ph), 5.10 (d, J=12.0 Hz, 2H, CH2Ph), 5.06 (m, 5H, CH2Ph), 3.93 (t, J=8.5, 10.0 Hz, 1H), 3.86 (m, 2H), 3.81 (m, 1H), 3.73 (m, 2H), 3.69 (t, J=10.0, 9.5 Hz, 1H), 3.52 (t, J=10.0, 9.0 Hz, 1H), 3.49 (m, 1H), 3.53 (m, 1H), 3.44 (m, 1H), 3.38 (m, 1H), 2.19 (m, 1H, H2eq), 1.56 (m, 9H), 1.29 (m, 2H); 13C NMR (125 MHz, aceton-d6) δ 157.57, 156.68, 156.21, 155.85, 137.59 (4C), 128.55, 128.19, 128.07, 127.90, 111.82, 97.46, 80.91, 78.36, 77.60, 72.28, 71.69, 71.21, 66.19, 66.19, 66.07, 65.89, 56.08, 141.65, 49.28, 42.13, 36.40, 36.22, 24.99, 23.75; MS (FAB, NBA) calcd for C50H58O14N4Na (M+Na+) 961, found 961; (M+H+) 939, found 939.1,3,2′,6′-Tetrakis(N-benzyloxycarbonyl)-5,6-O-cyclohexyl-3′,4′-di-O-methoxymethylneamine (12) To an ice-cold solution of 11 (1.74 g, 1.86 mmol), tetra-butylammonium iodide (2.73 g, 7.42 mmol), and N,N-di-isopropylethylamine (12.9 mL, 74.20 mmol) in anhydrous DMF (26 mL) was added chloromethyl methyl ether (4.22 mL, 55.65 mmol) and the mixture was stirred at 32° C. under an atmosphere of argon for 40 hrs.",
"The reaction mixture was stirred and quenched with a saturated solution of sodium bicarbonate (5 mL) for 30 min and was concentrated to a syrup in vacuo.",
"The resultant residue was diluted with dichloromethane and extracted with water and then brine.",
"The organic layer was dried over magnesium sulfate, and concentrated in vacuo to give a syrup, which was purified on a column (SiO2, 50:1 CHCl3/MeOH) to afford 12 (1.50 g, 79%) as a white solid.",
"mp 65-66° C.; FTIR (film): 3389, 3325, 2938, 1722, 1712, 1517, 1217, 1028 cm−1; 1H NMR (500 MHz, aceton-d6) δ 7.39-7.29 (m, 20H, Ph), 6.63 (s, 1H, NH), 6.54 (d, J=8.5 Hz, 1H, NH), 6.25 (s, 1H, NH), 6.11 (d, J=8.5 Hz, 1H, NH), 5.30 (s, 1H, H1′), 5.16 (d, J=12.0 Hz, 1H, CH2Ph), 5.12 (d, J=11.0 Hz, 1H, CH2Ph), 5.07 (m, 5H, CH2Ph), 5.01 (d, J=12.0 Hz, 1H, CH2Ph), 4.86 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.74 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.68 (d, J=6.0 Hz, 2H, OCH2OCH3), 3.93 (m, 2H), 3.81 (m, 2H), 3.75 (t, J=9.0, 11.4 Hz, 1H), 3.71 (t, J=9.5, 11.5 Hz, 1H), 3.66 (m, 1H), 3.53 (m, 1H), 3.48 (t, J=9.5, 9.5 Hz, 1H), 3.34 (s, 3H, OCH2OCH3), 3.24 (s, 3H, OCH2OCH3), 2.21 (m, 1H, H2eq), 1.55 (m, 9H), 1.33 (m, 2H); 13C NMR (125 MHz, aceton-d6) δ 162.29, 156.76, 156.25, 156.25, 155.89, 137.74, 137.61, 137.141, 137.141, 128.58, 128.12, 128.00, 111.93, 98.59, 98.26, 97.24, 80.77, 79.06, 78.42, 78.33, 77.68, 69.99, 69.29, 66.18, 65.99, 65.99, 56.03, 55.46, 55.01, 141.69, 49.37, 42.04, 36.50, 36.37, 36.21, 25.07, 23.77; MS (FAB, NBA) 1049 (M+Na+); HRMS (FAB, NBA) calcd for C54H67O16N4 (MH+) 1027.4550, found 1027.4556.1,3,2′,6′-Tetrakis(N-benzyloxycarbonyl)-3′,4′-di-O-methoxymethylneamine (13) To a solution of 12 (1.50 g, 1.46 mmol) in 1:1 dioxane-water (40 mL) was added glacial acetic acid (20 mL) and the mixture was stirred at 60-65° C. for 10 hrs.",
"The reaction mixture was concentrated to dryness in vacuo to give a residue, which was purified by column chromatography (SiO2, 50:1 CHCl3/MeOH) to give 13 (1.32 g, 95%) as a white solid.",
"mp 120-122° C. FTIR (film): 3387, 3328, 2947, 1721, 1709, 1692, 1517, 1218, 1028 cm−1.1H NMR (500 MHz, pyridine-d5) δ 8.34 (s, 1H, NH), 8.26 (d, J=6.4 Hz, 1H, NH), 7.98 (d, J=7.6 Hz, 1H, NH), 7.69 (s, 1H, NH), 7.34-7.14 (m, 20H, Ph), 5.97 (s, 1H, H1′), 5.46 (d, J=12.8 Hz, 1H, CH2Ph), 5.35 (d, J=12.4 Hz, 1H, CH2Ph), 5.27 (d, J=12.4 Hz, 1H, CH2Ph), 5.22 (d, J=12.8 Hz, 1H, CH2Ph), 5.20 (d, J=12.0 Hz, 1H, CH2Ph), 5.17 (d, J=12.4 Hz, 1H, CH2Ph), 5.14 (d, J=12.4 Hz, 1H, CH2Ph), 4.99 (d, J=5.6 Hz, 1H, OCH2OCH3), 4.93 (d, J=11.2 Hz, 1H, CH2Ph), 4.88 (d, J=6.0 Hz, 1H, OCH2OCH3), 4.78 (d, J=6.0 Hz, 2H, OCH2OCH3), 4.42 (m, 1H), 4.31 (m, 1H), 4.25 (t, J=8.8, 10.4 Hz, 1H), 4.09 (m, 1H), 3.99 (m, 2H), 3.93 (m, 1H), 3.77 (m, 3H), 3.68 (m, 1H), 3.33 (s, 3H, OCH2OCH3), 3.24 (s, 3H, OCH2OCH3), 2.42 (m, 1H, H2eq), 1.82 (m, 1H, H2ax); 13C NMR (125 MHz, pyridine-d5) δ 157.29, 157.29, 157.10, 156.80, 138.01, 137.89, 137.89, 137.89, 128.72, 128.69, 128.66, 128.30, 128.15, 128.06, 128.01, 100.02, 98.80, 98.38, 82.55, 79.36, 78.28, 78.03, 76.57, 70.87, 66.54, 66.28, 66.23, 66.11, 56.39, 56.37, 55.85, 52.59, 141.35, 42.17, 36.00; MS (FAB, NBA) 969 (M+Na+); HRMS (FAB, NBA) calcd for C48H58O16N4Na (M+Na+) 969.3745, found 969.3778.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N,6-O-carbonyl-3′,4′-di-O-methoxymethylneamine 14 To a solution of 13 (1.30 g, 1.37 mmol) in anhydrous DMF (20 mL) was added sodium hydride (220 mg, 5.50 mmol, 60% dispersion in mineral oil) and the mixture was stirred at room temperature under an atmosphere of argon for 3 h. The reaction mixture was quenched with an aqueous solution of acetic acid (5 mL, 1.0 N) and concentrated to dryness in vacuo to give a residue, which was purified on a column (SiO2, 50:1 CHCl3/MeOH) to furnish 14 (1.02 g, 89%) as a pure white solid.",
"mp 94-95° C.; FTIR (film): 3380, 3332, 2940, 1764, 1720, 1710, 1528, 1251, 1216, 1018 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 8.89 (s, 1H, NH), 8.59 (s, 1H, NH), 8.21 (d, J=9.5 Hz, 1H, NH), 8.18 (d, J=10.0 Hz, 1H, NH), 7.32-7.16 (m, 15H, Ph), 6.08 (s, 1H, H1′), 5.46 (d, J=12.5 Hz, 1H, CH2Ph), 5.33 (d, J=12.5 Hz, 1H, CH2Ph), 5.29 (d, J=13.0 Hz, 1H, CH2Ph), 5.21 (d, J=13.0 Hz, 1H, CH2Ph), 5.16 (d, J=12.0 Hz, 1H, CH2Ph), 5.01 (d, J=12.5 Hz, 1H, CH2Ph), 4.98 (d, J=8.5 Hz, 1H, OCH2OCH3), 4.87 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.76 (d, J=6.5 Hz, 2H, OCH2OCH3), 4.38 (m, 1H), 4.31 (dt, J=10.0, 3.0 Hz, 1H), 4.22 (t, J=9.5, 10.5 Hz, 1H), 4.17 (m, 2H), 4.07 (t, J=8.5 Hz, 1H), 3.98 (t, J=10.5 Hz, 1H), 3.94 (m, 1H), 3.77 (t, J=9.5 Hz, 1H), 3.69 (m, 1H), 3.43 (t, J=9.5 Hz, 1H), 3.34 (s, 3H), OCH2OCH3), 3.20 (s, 3H, OCH2OCH3), 2.31 (m, 1H, H-2), 1.73 (m, 1H, H2); 13C NMR (125 MHz, pyridine-d5) δ 161.14, 157.38, 157.20, 156.89, 137.88, 137.74, 137.74, 128.73, 128.73, 128.68, 128.34, 128.26, 128.11, 128.06, 99.93, 98.83, 98.35, 85.01, 83.28, 79.08, 78.23, 73.94, 70.98, 66.77, 66.38, 66.29, 56.42, 56.08, 55.81, 54.41, 52.54, 42.13, 33.61; MS (FAB, NBA) 861 (M+Na+); HRMS (FAB, NBA) calcd for C41H50O15N4Na (M+Na+) 861.3170, found 861.3170.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N,6-O-carbonyl-5-O-triethylsilyl-3′,4′-di-O-methoxymethylneamine (15) To a mixture of 14 (1.02 g, 1.22 mmol), triethylamine (0.2 mL), imidazole (372 mg, 5.70 mmol), and N,N-dimethylaminopyridine (45 mg, 0.36 mmol) in anhydrous DMF (10 mL) was added triethylsilyl chloride (0.61 mL, 3.65 mmol).",
"The mixture was stirred at room temperature under an atmosphere of argon for 5 h and then quenched with an aqueous solution of saturated sodium bicarbonate (2 mL).",
"The mixture was concentrated to a syrup in vacuo, diluted with dichloromethane, washed with water and then brine, dried (Na2CO3), and concentrated to give a residue, which was purified on a column (SiO2, 50:1 CHCl3/MeOH) to afford 15 (925 mg, 80%) as a white solid.",
"mp 79-80° C.; FTIR (film): 3332, 3319, 2953, 2877, 1764, 1722, 1710, 1529, 1215 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 8.63 (s, 1H, NH), 8.18 (d, J=10.5 Hz, 1H, NH), 8.12 (d, J=9.0 Hz, 1H, NH), 7.83 (s, 1H, NH), 7.32-7.20 (m, 15H, Ph), 6.00 (s, 1H, H1′), 5.48 (d, J=12.0 Hz, 1H, CH2Ph), 5.41 (d, J=12.0 Hz, 1H, CH2Ph), 5.29 (d, J=13.0 Hz, 1H, CH2Ph), 5.16 (m, 3H, CH2Ph), 4.98 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.88 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.77-4.74 (m, 2H, OCH2OCH3), 4.45 (m, 1H), 4.35 (td, J=11.0, 2.4 Hz, 1H), 4.21 (t, J=9.5 Hz, 1H), 4.16 (m, 1H), 4.12 (t, J=9.5 Hz, 1H), 4.02 (t, J=8.5 Hz, 1H), 3.85 (m, 1H), 3.77 (m, 2H), 3.69 (m, 1H), 3.37 (m, 1H), 3.32 (s, 3H, OCH2OCH3), 3.17(s, 3H, OCH2OCH3), 2.26 (m, 1H, H2eq), 1.72 (m, 1H, H2ax), 0.97 [t, J=7.5 Hz, 9H, Si(CH2CH3)3], 0.82 [m, 6H, Si(CH2CH3)3]; 13C NMR (125 MHz, pyridine-d5) δ 160.48, 157.44, 157.08, 156.79, 137.86, 137.73, 137.50, 128.83, 128.79, 128.76, 128.73, 128.35, 128.30, 128.13, 128.05, 98.82, 98.52, 98.43, 84.01, 81.08, 79.03, 78.35, 76.44, 70.53, 66.90, 66.85, 66.23, 56.43, 55.89, 55.48, 54.23, 141.98, 42.24, 33.36, 7.29, 5.28; MS (FAB, Gly) 953 (MH+); HRMS (FAB, Gly) calcd for C47H65O15N4Si (MH+) 953.4215, found 953.4191.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-1-N,6-O-carbonyl-5-O-triethylsilyl-3′,4′-di-O-methoxymethylneamine (16) To a solution of 15 (910 mg, 0.96 mmol), 4-(N,N-dimethylamino)pyridine (23 mg, 0.19 mmol), and triethylamine (0.17 mL, 1.22 mmol) in dry THF (10 mL) was added di-tert-butyldicarbonate (271 mg, 1.24 mmol).",
"The mixture was stirred at room temperature under an atmosphere of argon for 4 h. The solution was concentrated to dryness in vacuo to give a residue, which was purified on a column (SiO2, 100:1 CHCl3/MeOH) to give 16 (965 mg, 96%) as a white solid.",
"mp 76-77° C.; FTIR (film): 3342, 2953, 2878, 1812, 1726, 1515, 1327, 1157, 1021 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 7.88 (d, J=10.0 Hz, 1H, NH), 7.85 (d, J=8.5 Hz, 1H, NH), 7.46 (m, 1H, NH), 7.13-6.74 (m, 15H, Ph), 5.60 (s, 1H, H1′), 5.08 (d, J=12.5 Hz, 1H, CH2Ph), 5.04 (d, J=12.0 Hz, 2H, CH2Ph), 4.93 (d, J=12.0 Hz, 1H, CH2Ph), 4.82 (d, J=13.0 Hz, 1H, CH2Ph), 4.79 (d, J=13.5 Hz, 1H, CH2Ph), 4.71 (d, J=11.5 Hz, 1H, CH2Ph), 4.60 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.52 (d, J=6.0 Hz, 1H, OCH2OCH3), 4.40 (d, J=7.0 Hz, 1H, OCH2OCH3), 4.35 (d, J=7.0 Hz, 1H, OCH2OCH3), 4.10 (m, 1H), 3.98 (td, J=10.5, 3.5 Hz, 1H), 3.83 (t, J=9.5 Hz, 1H), 3.74-3.70 (m, 1H), 3.47 (m, 1H), 3.39 (t, J=9.5 Hz, 1H), 3.30 (m, 1H), 2.94 (s, 3H, OCH2OCH3), 2.78 (s, 3H, OCH2OCH3), 2.56 (m, 1H, H2eq), 1.41 (m, 1H, H2ax), 0.98 (m, 9H, OCOtBu), 0.58 [t, 9H, Si(CH2CH3)3], 0.42 [m, 6H, Si(CH2CH3)3]; 13C NMR (125 MHz, pyridine-d5) δ 157.06, 156.72, 156.44, 152.23, 150.00, 137.55, 137.27, 137.11, 128.47, 128.43, 128.39, 128.37, 127.99, 127.86, 127.76, 127.69, 127.58, 98.43, 98.18, 98.04, 83.03, 80.37, 80.20, 78.59, 78.03, 75.56, 70.03, 66.58, 66.49, 65.78, 56.04, 55.97, 55.49, 55.09, 141.48, 41.86, 32.48, 27.36, 6.84, 4.88; MS (FAB, NBA) 1075 (M+Na+); HRMS (FAB, NBA) calcd for C52H72O17N4SiNa (M+Na+) 1075.4560, found 1075.4566.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-1-N,6O-carbonyl-3′,4′-di-O-methoxymethylneamine (17) To an ice-cold solution of 16 (1.01 g, 0.96 mmol) in THF (10 mL) was added a 1.0 M solution of tetrabutylammonium fluoride in THF (0.96 mL, 0.96 mmol) and the mixture was allowed to stir at this temperature for 10 min.",
"The reaction mixture was quenched by the addition of ice water (2 mL), concentrated to a syrup in vacuo, diluted with water and extracted with dichloromethane (3×30 mL).",
"The organic layer was washed with brine, dried over sodium sulfate, and concentrated to give a residue, which was purified on a column (SiO2, 70:1 CHCl3/MeOH) to afford 17 (770 mg, 85%) as a white solid.",
"mp 82-83° C.; FTIR (film): 3350, 2952, 1812, 1724, 1521, 1254, 1018 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 8.73 (s, 1H, NH), 8.38 (d, J=8.0 Hz, 1H, NH), 8.24 (d, J=9.0 Hz, 1H, NH), 7.45-7.25 (m, 15H, Ph), 6.19 (s, 1H, H1′), 5.55 (d, J=12.0 Hz, 1H, CH2Ph), 5.44 (d, J=12.5 Hz, 1H, CH2Ph), 5.40 (d, J=12.5 Hz, 1H, CH2Ph), 5.29 (m, 2H, CH2Ph), 5.09 (m, 2H), 5.00 (m, 1H), 4.95 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.86 (m, 2H), 4.51 (m, 1H), 4.41 (m, 1H), 4.28 (m, 4H), 4.06 (t, J=10.5 Hz, 1H), 4.00 (m, 1H), 3.87 (t, J=9.0 Hz, 1H), 3.82 (m, 2H), 3.44 (s, 3H, OCH2OCH3), 3.29 (s, 3H, OCH2OCH3), 3.10 (m, 1H), 1.93 (m, 1H, H2eq), 1.48 (s, 10H); 13C NMR (125 MHz, pyridine-d5) δ 155.85, 155.65, 155.37, 151.71, 148.95, 136.39, 136.17, 136.17, 127.27, 127.22, 127.17, 126.76, 126.62, 126.56, 98.33, 97.30, 90.83, 81.79, 81.05, 80.15, 77.55, 76.72, 71.98, 69.32, 65.31, 64.90, 64.73, 55.07, 54.90, 54.49, 54.29, 50.91, 40.59, 31.57, 26.26; MS (FAB, NBA) 959 (M+Na+); HRMS (FAB, NBA) calcd for C46H58O17N4Na (M+Na+) 961.3694, found 961.3683.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-1-N,6-O-carbonyl-5,3′,4′-tri-O-methoxymethylneamine (18) To an ice-cold solution of 17 (661 mg, 0.70 mmol), tetra-butylammonium iodide (520 mg, 1.41 mmol), and N,N-di-isopropylethylamine (2.46 mL, 14.09 mmol) in anhydrous DMF (5.00 mL) was added chloromethyl methyl ether (0.85 mL, 10.57 mmol) dropwise.",
"The mixture was allowed to stir at 32° C. under an atmosphere of argon for 34 h. The reaction mixture was then quenched by the addition of a saturated solution of sodium bicarbonate (0.5 mL), diluted with water, and was extracted with dichloromethane (3×10 mL).",
"The organic layer was washed with brine, dried (Na2SO4), and concentrated in vacuo to give a syrup, which was purified on a column (SiO2, 80:1 CHCl3/MeOH) to furnish 18 (673 mg, 97%) as a white solid.",
"mp 82-83° C.; FTIR (film): 3338, 2944, 1812, 1724, 1523, 1154, 1020 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 8.38 (d, J=9.0 Hz, 1H, NH), 8.32 (d, J=9.5 Hz, 1H, NH), 8.08 (m, 1H, NH), 7.67-7.30 (m, 15H, Ph), 5.84 (d, J=3.0 Hz, 1H, H1′), 5.55 (d, J=12.5 Hz, 1H, CH2Ph), 5.39 (d, J=13.0 Hz, 1H, CH2Ph), 5.35 (m, 2H), 5.31 (d, J=13.0 Hz, 1H, CH2Ph), 5.27 (d, J=12.5 Hz, 1H, CH2Ph), 5.07 (d, J=6.0 Hz, 1H, OCH2OCH3), 5.03 (d, J=6.0 Hz, 1H, OCH2OCH3), 4.98 (s, 1H), 4.95 (d, J=7.0 Hz, 1H, OCH2OCH3), 4.84 (m, 2H), 4.49 (m, 1H), 4.40-4.36 (m, 1H), 4.25 (m, 2H), 4.17 (t, J=8.0 Hz, 1H), 4.02 (t, J=9.5 Hz, 1H), 3.98 (m, 2H), 3.87 (t, J=9.0 Hz, 1H), 3.80 (m, 2H), 3.46 (s, 3H, OCH2OCH3), 3.42 (s, 3H, OCH2OCH3), 3.29 (s, 3H, OCH2OCH3), 3.07 (m, 1H), 1.88 (m, 1H), 1.47 (s, 9H, OCOtBu); 13C NMR (125 MHz, pyridine-d5) δ 157.01, 156.67, 156.43, 152.40, 150.03, 137.54, 137.27, 137.21, 128.42, 128.37, 128.29, 128.17, 127.94, 127.89, 127.78, 127.71, 127.63, 98.99, 98.43, 97.99, 97.09, 83.08, 79.75, 79.69, 78.98, 78.33, 77.87, 70.31, 66.54, 66.30, 65.84, 56.15, 55.81, 55.43, 55.16, 141.78, 41.67, 32.44, 27.36; MS (FAB, NBA) 983 (MH+); HRMS (FAB, NBA) calcd for C48H63O18N4 (MH+) 983.4138, found 983.4150; HRMS (FAB, NBA) calcd for C48H62O18N4Na (M+Na+) 1005.3960, found 1005.3964.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-5,3′,4′-tri-O-methoxymethylneamine (19) To a solution of 18 (673 mg, 0.69 mmol) in dioxane (10.0 mL) was added a 0.5 N aqueous solution of lithium hydroxide (5.0 mL) and the mixture was stirred at room temperature for 30 min.",
"The reaction mixture was quenched with 0.5 N acetic acid (5.0 mL) and concentrated in vacuo to give a syrup, which was purified by chromatography (SiO2, 80:1 CHCl3/MeOH) to afford 19 (574 mg, 88%) as a white solid.",
"Rf 0.25 (29:1 CHCl3/MeOH); mp 147-148° C.; FTIR (film): 3343, 3331, 2945, 1719, 1699, 1528, 1254, 1017 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 8.50 (d, J=8.4 Hz, 1H, NH), 8.09 (d, J=8.0 Hz, 1H, NH), 7.74 (m, 1H, NH), 7.43-7.22 (m, 15H, Ph), 6.31 (s, 1H, OH), 5.84 (s, 1H, H1′), 5.58 (d, J=12.8 Hz, 1H, CH2Ph), 5.38 (m, 3H), 5.30 (d, J=12.0 Hz, 1H, CH2Ph), 5.24 (d, J=12.4 Hz, 1H, CH2Ph), 5.17 (d, J=7.6 Hz, 1H, OCH2OCH3), 5.09 (d, J=4.0 Hz, 1H, OCH2OCH3), 5.00 (m, 1H), 4.90 (d, J=6.4 Hz, 1H, OCH2OCH3), 4.86 (d, J=6.0 Hz, 1H, OCH2OCH3), 4.53 (m, 1H), 4.44-4.40 (m, 1H), 4.32 (t, J=9.2 Hz, 1H), 4.15 (m, 1H), 4.00 (m, 2H), 3.88 (m, 2H), 3.75 (m, 2H), 3.49 (t, J=9.2 Hz, 1H), 3.43 (s, 3H, OCH2OCH3), 3.42 (s, 3H, OCH2OCH3), 3.35 (s, 3H, OCH2OCH3), 2.47 (m, 1H, H2eq), 1.82 (m, 1H, H2ax), 1.48 (s, 9H, OCOtBu); 13C NMR (100 MHz, pyridine-d5) δ 157.35, 157.13, 156.76, 156.141, 137.77, 137.77, 137.77, 128.78, 128.75, 128.70, 128.61, 128.47, 128.27, 128.24, 128.12, 128.05, 99.23, 99.20, 98.80, 98.43, 87.22, 79.84, 78.86, 78.26, 78.16, 75.34, 70.61, 66.67, 66.60, 66.29, 56.36, 56.06, 55.88, 55.78, 52.07, 50.89, 42.05, 35.68, 28.43; MS (FAB, NBA) 957 (MH+); HRMS (FAB, NBA) calcd for C47H65O17N4 (MH+) 957.4344, found 957.4319; HRMS (FAB, NBA) calcd for C47H64O17N4Na (M+Na+) 979.4164, found 979.4140.6-O-Allyl-3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-5,3′,4′-tri-O-methoxymethylneamine (20) To a solution of 19 (890 mg, 0.930 mmol) and tetra-butylammonium iodide (420 mg, 1.136 mmol) in anhydrous DMSO (2 mL) was added lithium bis(trimethylsilyl)amide (1.16 mL, 1.0 M solution in THF).",
"To this mixture was added allyl bromide (99 μL, 1.125 mmol) dropwise and the mixture was allowed to stir at room temperature for 1 h. The mixture was quenched by the addition of AcOH (20% aqueous solution), diluted with water and extracted with ethyl acetate (3×30 mL).",
"The organic layer was back extracted with brine, dried (Na2SO4) and concentrated in vacuo to give a yellow solid as crude product.",
"The solid was purified on a column (SiO2, 100:1:0.1 CHCl3/MeOH/NH4OH) to furnish 20 (643 mg, 69%) as a pure white solid.",
"Rf 0.42 (29:1 CHCl3/MeOH); mp 163-165° C.; FTIR (film): 3334, 2928, 1721, 1708, 1703, 1692, 1529, 1253, 1147, 1042, 1017 cm31 1; 1H NMR (500 MHz, pyridine-d5) δ 8.28 (d, J=8.0 Hz, 1H, NH), 8.14 (d, J=7.5 Hz, 1H, NH), 7.79 (d, J=7.5 Hz, 1H, NH), 7.65 (m, 1H, NH), 7.44-7.27 (m, 15H, Ph), 6.08 (m, 1H), 5.84 (s, 1H, H1′), 5.56 (d, J=12.0 Hz, 1H, CH2Ph), 5.41 (d, J=12.0 Hz, 1H, CH2Ph), 5.34 (m, 3H), 5.25 (d, J=12.0 Hz, 1H, CH2Ph), 5.08 (m, 3H), 4.99 (m, 3H), 4.99 (d, J=6.5 Hz, 1H, OCH2OCH3), 4.85 (d, J=5.5 Hz, 1H, OCH2OCH3), 4.53 (m, 1H), 4.40 (m, 2H), 4.32 (t, J=10.5 Hz, 1H), 4.02 (m, 2H), 3.89 (m, 2H), 3.78 (m, 1H), 3.50 (m, 1H), 3.48 (m, 1H), 3.48 (s, 3H, OCH2OCH3), 3.38 (s, 3H, OCH2OCH3), 3.38 (s, 3H, OCH2OCH3), 2.23 (m, 1H, H2eq), 1.95 (m, 1H, H2ax), 1.52 (s, 9H, OCOtBu); 13C NMR (125 MHz, pyridine-d5) δ 157.03, 156.77, 156.36, 155.67, 137.49, 137.49, 137.49, 135.58, 128.39, 128.35, 128.09, 127.86, 127.76, 127.69, 115.81, 98.81, 98.80, 98.44, 98.09, 83.53, 82.69, 79.48, 78.45, 77.97, 77.84, 73.26, 70.33, 66.29, 66.21, 65.94, 56.21, 55.99, 55.53, 55.44, 50.60, 50.38, 41.77, 35.08, 28.12; MS (FAB, NBA) 997 (MH+); HRMS (FAB, NBA) calcd for C50H69O17N4 (MH+) 997.4658, found 997.4699; HRMS (FAB, NBA) calcd for C50H68O17N4Na (M+Na+) 1019.4480, found 1019.4529.3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-5,3′,4′-tri-O-methoxymethyl-6-O-(2-oxoethyl)neamine (21) Ozone was bubbled into a solution of 20 (400 mg, 0.401 mmol) in dichloromethane (10 mL) at −78° C. until a light blue color persisted (˜10 min).",
"The reaction mixture was flushed with O2 and quenched by the addition of triphenyl phosphine (160 mg, 0.600 mmol), followed by warming up to room temperature.",
"The solution was stirred for 1 h prior to the removal of the solvent in vacuo.",
"The crude product can be used for the next step without further purification.",
"The analytical sample was prepared by purification of the solid by column chromatography (SiO2, 100:1:0.1 CHCl3/MeOH/NH4OH) to give 21 (327 mg, 81%) as a white solid.",
"Rf 0.53 (19:1 CHCl3/MeOH); mp 93-95° C.; FTIR (film): 3329, 2932, 1716, 1702, 1537, 1455, 1252, 1156, 1027 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 9.96 (s, 1H), 8.41-8.34 (m, 2H, NH), 8.05 (d, J=8 Hz, 1H, NH), 7.78 (s, 1H, NH), 7.57-7.32 (m, 15H, Ph), 6.16 (s, 1H), 5.86 (d, J=12.0 Hz, 1H, H1′), 5.52-5.21 (m, 6H), 5.14-4.97 (m, 4H), 4.9 (d, J=6.4 Hz, 1H), 4.86 (t, J=4.8 Hz, 1H), 4.52 (s, 1H), 4.41-4.29 (m, 2H), 4.18 (s, 2H), 4.12-4.00 (m, 2H), 3.87 (s, 1H), 3.79-3.71 (m, 1H), 3.60 (s, 1H), 3.51-3.33 (m, 10 H), 2.89 (s, 1H, H2eq), 1.87 (q, J=12.4 Hz, 1H, H2ax), 1.48 (s, 3H), 1.44 (s, 6H); 13C NMR (100 MHz, pyridine-d5) δ 199.0 (weak), 157.8, 157.5157.1, 154.6, 138.2, 138.1, 129.2, 129.1, 128.9, 128.6, 128.5, 128.4, 123.6, 99.7 (C1′), 99.2, 98.8, 82.7, 82.2, 81.7, 80.8, 80.6, 80.4, 80.2, 79.2, 78.6, 76.5, 75.7, 73.2, 72.7, 72.0, 71.0, 67.1, 67.0, 66.7, 56.8, 56.7, 56.5, 56.3, 56.1, 54.1, 51.8, 51.7, 51.1, 42.5, 35.7, 28.8, 28.5; MS (FAB, NBA) 999 (MH+); HRMS (FAB, NBA) calcd for C49H67N4O18 (MH+) 999.4451, found 999.4498.General Procedure for Reductive Amination of Aldehyde 21 and Synthesis of Compounds 22-24 A solution of the corresponding mono-Cbz-alkylenediamine (5 equivalent) in methanol (1 mL) was acidified with a 1 M methanolic solution of acetic acid (˜4 mL) to pH ˜6 (pH paper).",
"This solution was added to a solution of the crude aldehyde (˜400 mg, 0.400 mmol) in methanol (3 mL) at room temperature.",
"The mixture was subsequently treated with NaCNBH3 (100 mg, 1.6 mmol) and stirred at ambient temperature for 2 h. The reaction mixture was diluted with ethylacetate, extracted with 1 N NaOH, water and then with brine.",
"The organic layer was dried (Na2SO4) and concentrated to dryness in vacuo to give a solid, which was purified on a column (SiO2, 19:1 CHCl3/MeOH) to afford the corresponding aminated product as a white solid.",
"The reported yields are for the combined two steps of ozonolysis and reductive amination.",
"6-O-[2-N-[2-(benzyloxycarbonyl)ethylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-5,3′,4′-tri-O-methoxymethylneamine (22) Yield (258 mg, 55%); Rf 0.14 (19:1 CHCl3/MeOH); mp 126-28° C.; FTIR (film): 3332, 3064, 2939, 2898, 2825, 1716, 1532, 1455, 1252, 1150, 1044, 1025 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 8.35 (d, J=8.8 Hz, 1H, NH), 8.18 (s, 1H, NH), 8.06 (d, J=7.2 Hz, 1H, NH), 7.84 (s, 1H, NH), 7.56-7.31 (m, 20H), 5.79 (s, 1H), 5.53 (d, J=12.0 Hz, 1H), 5.38 (d, J=4.8 Hz, 2H), 5.32 (s, 3H), 5.27 (d, J=7.6 Hz, 2H), 5.07 (d, J=4.8 Hz, 1H), 5.04 (s, 2H), 4.99 (d, J=5.6 Hz, 1H), 4.90 (d, J=5.6 Hz, 1H), 4.83 (d, J=4.8 Hz, 1H), 4.48 (s,1H), 4.40 (t, J=9.6 Hz, 2H), 3.75 (s, 2H), 3.58 (s, 2H), 3.48 (s, 3H), 3.41 (s, 4H), 3.36 (s, 4H), 2.90-2.86 (m, 4H), 2.42 (br s, 1H, H2eq), 1.80 (q, J=12.0 Hz, 1H, H2ax), 1.52 (s, 9H); 13C NMR (100 MHz, pyridine-d5) δ 157.3, 157.2, 157.1, 156.7, 156.3, 138.0, 137.8 (2C), 137.7, 128.8, 128.7, 128.5, 128.2, 128.1, 128.0, 99.2 (CH2OCH3), 99.1 (C1′), 98.8 (CH2OCH3), 98.4 (CH2OCH3), 84.1, 83.5, 79.8, 78.8, 78.4, 72.2 (CH2), 70.7, 66.7 (CH2Ph), 66.6 (CH2Ph), 66.3 (CH2Ph), 66.2 (CH2Ph), 56.6 (CH2OCH3), 56.3 (CH2OCH3), 55.9 (CH2OCH3), 55.8, 51.2, 50.8, 50.3 (CH2), 49.6 (CH2), 42.2 (CH2), 41.3 (CH2), 35.4 (CH2), 28.5 (CH3); MS (FAB, NBA) 1177 (MH+); HRMS (FAB, NBA) calcd for C59H81N6O19 (MH+) 1177.5560, found 1177.5515.6-O-[2-N-[3-(benzyloxycarbonyl)propylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-5,3′,4′-tri-O-methoxymethylneamine (23) Yield (318 mg, 67%); Rf 0.15 (19:1 CHCl3/MeOH); mp 71-73° C.; FTIR (film): 3329, 2932, 1716, 1531, 1455, 1253, 1149, 1026 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 8.44 (d, J=8.8 Hz, 1H, NH), 8.24-8.16 (m, 4H, NH), 7.70 (s, 1H, NH), 7.49-7.28 (m, 20H), 5.81 (s, 1H), 5.56 (d, J=12.0 Hz, 1H), 5.43-5.24 (m, 7H), 5.08 (d, J=5.6 Hz, 1H), 5.05 (s, 2H), 5.00 (d, J=5.6 Hz, 1H), 4.90 (d, J=5.6 Hz, 1H), 4.84 (d, J=5.6 Hz, 1H), 4.51 (s,1H), 4.42 (t, J=10.0 Hz, 1H), 4.31 (t, J=9.2 Hz, 1H), 4.09-3.78 (m, 9H), 3.54 (ABq, J=14.2, 5.6 Hz, 2H), 3.48 (s, 3H), 3.41 (s, 3H), 3.38 (s, 1H), 3.35 (s, 3H), 2.95-2.90 (m, 3H), 2.39 (br s, 1H, H2eq), 2.01 (br s, 2H), 1.85 (q, J=12.4 Hz, 1H, H2ax), 1.52 (s, 9H), 1.36-1.26 (m, 1H); 13C NMR (100 MHz, pyridine-d5) δ 157.4, 157.3, 157.2, 156.7, 156.3, 138.1, 137.8 (2C), 128.8, 128.7, 128.5, 128.3, 128.2, 128.1, 128.0, 99.4 (CH2OCH3), 99.1 (C1′), 98.8 (CH2OCH3), 98.4 (CH2OCH3), 84.1, 79.6, 78.8, 78.3, 70.7 (CH2), 66.7 (CH2Ph), 66.6 (CH2Ph), 66.3 (CH2Ph), 66.2 (CH2Ph), 56.7 (CH2OCH3), 56.4 (CH2OCH3), 55.9 (CH2OCH3), 55.8, 51.1, 50.7, 50.0 (CH2), 46.9 (CH2), 42.1 (CH2), 39.2 (CH2), 35.5 (CH2), 29.9 (CH2), 29.6 (CH2), 28.5 (CH3); MS (FAB, NBA) 1191 (MH+); HRMS (FAB, NBA) calcd for C60H83N6O19 (MH+) 1191.5710, found 1191.5660.6-O-[2-N-[4-(benzyloxycarbonyl)butylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)-1-N-tert-butoxylcarbonyl-5,3′,4′-tri-O-methoxymethylneamine (24) Yield (311 mg, 63%); Rf 0.17 (19:1 CHCl3/MeOH); mp 66-68° C.; FTIR (film): 3329, 3033, 2940, 2902, 2825, 1708, 1529, 1455, 1254, 1148, 1028 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 8.36 (d, J=9.0 Hz, 1H, NH), 8.18-8.10 (m, 4H, NH), 7.56-7.30 (m, 20H), 5.76 (s, 1H), 5.54 (d, J=12.0 Hz, 1H), 5.42-5.23 (m, 7H), 5.06 (d, J=5.5 Hz, 1H), 5.00 (d, J=4.5 Hz, 1H), 4.96-4.94 (m, 2H), 4.86 (d, J=6.5 Hz, 1H), 4.83 (d, J=6.5 Hz, 1H), 4.49 (d, J=9.0 Hz, 1H), 4.38 (td, J=10.5, 3.0 Hz, 1H), 4.26 (t, J=10.0 Hz, 1H), 4.16-3.94 (m, 5H), 3.86 (t, J=9.5 Hz, 1H), 3.81-3.72 (m, 2H), 3.50-3.23 (m, 14H), 3.10 (s, 2H), 2.29 (d, J=10.5 Hz, 1H, H2eq), 1.92-1.84 (m, 3H), 1.76 (quint, J=7.0 Hz, 2H), 1.51 (s, 9H), 1.50-1.46 (m, 1H); 13C NMR (125 MHz, pyridine-d5) δ 157.4, 157.3, 157.1, 156.7, 156.4, 138.0, 137.7 (2C), 128.8, 128.7, 128.6, 128.3, 128.2, 128.1, 99.6 (CH2OCH3), 99.0 (C1′), 98.8 (CH2OCH3), 98.4 (CH2OCH3), 84.4, 84.2, 79.4, 78.8, 78.3, 70.6 (CH2), 66.7 (CH2Ph), 66.6 (CH2Ph), 66.3 (CH2Ph), 66.1 (CH2Ph), 56.5 (CH2OCH3), 56.3 (CH2OCH3), 55.9 (CH2OCH3), 55.7, 51.0, 50.6, 48.8 (CH2), 48.0 (CH2), 42.0 (CH2), 40.6 (CH2), 35.4 (CH2), 28.4 (CH3), 29.8 (CH2), 27.9 (CH2), 25.1(CH2); MS (FAB, NBA) 1205 (MH+); HRMS (FAB, NBA) calcd for C61H85N6O19 (MH+) 1205.5870, found 1205.5821.6-O-[2-N-[2-(benzyloxycarbonyl)ethylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (25) To a solution of 22 (145 mg, 0.123 mmol) in chloroform (2 mL) was added a solution of 2 N hydrochloric acid in methanol (4 mL) and the mixture was kept at room temperature for 12 h. The reaction mixture was quenched with triethylamine and concentrated to dryness in vacuo to give a solid as the crude product.",
"The solid was washed with ether, suspended in water, filtered and purified on a column (SiO2, 15:2:0.1 CHCl3/MeOH/NH4OH) to give 25 (85 mg, 73%) as a pure white solid.",
"Rf 0.17 (15:2:0.2 CHCl3/MeOH/NH4OH); mp 193-95° C.; FTIR (KBr): 3401, 3332, 3063, 2935, 1697, 1536, 1454, 1261, 1139, 1052, 1015, 735, 697 cm−1; 1H NMR (500 MHz, DMSO-d6) δ 7.35-7.28 (m, 20H), 7.20-7.18 (m, 2H, NH), 6.94 (s, 2H, NH), 5.09 (s, 1H), 5.06-4.99 (m, 8H), 4.89 (d, J=12.0 Hz, 1H), 3.73 (d, J=4.5 Hz, 1H), 3.68-3.62 (m, 2H), 3.47-3.42 (m, 4H), 3.37 (t, J=9.0 Hz, 1H), 3.34 (t, J=9.5 Hz, 1H), 3.16-3.10 (m, 2H), 3.05 (q, J=6.0 Hz, 2H), 2.69 (t, J=8.5 Hz, 1H), 2.62-2.49 (m, 4H), 1.71 (d, J=10.5 Hz, 1H, H2eq), 1.22-1.17 (m, 1H, H2ax); 13C NMR (125 MHz, DMSO-d6) δ 157.2, 156.9, 156.8, 156.5, 138.1, 137.9, 137.7, 129.0 (2C), 128.9, 128.5, 128.4, 128.3 (2C), 128.1, 127.9, 99.4 (C1′), 87.9, 83.3, 76.2, 72.1, 71.8, 71.5 (CH2), 71.4, 66.1 (CH2Ph), 66.0 (CH2Ph), 65.9 (CH2Ph), 65.8 (CH2Ph), 56.9, 51.2, 51.1, 49.3 (CH2), 49.0 (CH2), 42.7 (CH2), 40.7 (CH2), 37.2 (CH2); MS (FAB, Gly) 945 (MH+); HRMS (FAB, Gly) calcd for C48H61N6O14 (MH+) 945.4246, found 945.4248.6-O-[2-N-[3-(benzyloxycarbonyl)propylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (26) A solution of 23 (260 mg, 0.218 mmol) in chloroform (3 mL) was treated with a solution of 2 N hydrochloric acid in methanol (6 mL), as described for 25.Yield (182 mg, 87%).",
"Rf 0.18 (15:2:0.2 CHCl3/MeOH/NH4OH); mp 253-55° C.; FTIR (KBr): 3422, 3344, 3062, 3032, 2927, 2881, 1693, 1536, 1287, 1262, 1048 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 7.35-7.30 (m, 20H), 6.93 (s, 2H, NH), 6.69 (s, 2H, NH), 5.09 (s, 1H), 5.06-5.00 (m, 8H), 4.88 (d, J=11.6 Hz, 1H), 4.08 (s, 1H), 3.85 (s, 1H), 3.61 (s, 1H), 3.53 (t, J=9.2 Hz, 2H), 3.44-3.37 (m, 4H), 3.31 (t, J=9.6 Hz, 1H), 3.12-3.02 (m, 6H), 2.85 (d, J=4.8 Hz, 2H), 1.96 (s, 1H, H2eq), 1.80 (t, J=6.4 Hz, 2H), 1.51 (q, J=11.6, 1H, H2ax); 13C NMR (100 MHz, DMSO-d6) δ 157.2, 156.8 (2C), 156.4, 138.0, 137.9, 137.8, 137.6, 129.0 (2C), 128.9, 128.4, 128.3 (2C), 128.2, 128.0, 99.3 (C1′), 82.2, 76.3, 72.1, 71.7, 71.2, 67.9 (CH2), 66.2 (CH2Ph), 66.0 (2C, CH2Ph), 65.9 (CH2Ph), 56.8, 50.4, 49.3, 47.7 (CH2), 45.5 (CH2), 42.6 (CH2), 38.4 (CH2), 32.9 (CH2), 27.0 (CH2); MS (FAB, Gly) 959 (MH+); HRMS (FAB, Gly) calcd for C49H63N6O14 (MH+) 959.4402, found 959.4403.6-O-[2-N-[4-(benzyloxycarbonyl)butylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (27) A solution of 24 (318 mg, 0.264 mmol) in chloroform (4 mL) was treated with a solution of 2 N hydrochloric acid in methanol (8 mL), as described for 25.Yield (191 mg, 74%).",
"Rf 0.18 (15:2:0.2 CHCl3/MeOH/NH4OH); mp 225-27° C.; FTIR (KBr): 3400, 3341, 3027, 2930, 1695, 1535, 1456, 1259, 1140, 1266, 1044 cm−1; 1H NMR (500 MHz, DMSO-d6) δ 7.36-7.28 (m, 20H), 6.99 (s, 2H, NH), 6.92 (d, J=6.5 Hz, 2H, NH), 5.12 (s, 1H), 5.07-4.98 (m, 8H), 4.89 (d, J=12.5 Hz, 1H), 3.79 (s, 2H), 3.65 (s, 1H), 3.48-3.37 (m, 4H), 3.36 (q, J=9.0 Hz, 1H), 3.16-3.13 (m, 2H), 2.98 (s, 2H), 2.92 (t, J=9.0 Hz, 1H), 2.77 (t, J=7.0 Hz, 1H), 2.70 (s, 2H), 2.53 (s, 2H), 1.81 (d, J=8.5 Hz, 1H, H2eq), 1.40-1.34 (m, 5H); 13C NMR (125 MHz, DMSO-d6) δ 157.2, 156.9, 156.8, 156.5, 138.0 (2C), 137.9, 137.7, 129.0, 128.5, 128.4, 128.3, 128.0, 99.3 (C1′), 85.3, 82.7, 76.2, 72.1, 71.8, 71.4, 69.7 (CH2), 66.1 (CH2Ph), 66.0 (CH2Ph), 65.8 (2C, CH2Ph), 56.8, 50.9, 50.6, 48.5 (CH2), 48.2 (CH2), 42.7 (CH2), 40.7 (CH2), 36.1 (CH2), 27.7 (CH2), 25.9 (CH2); MS (FAB NBA) 973 (MH+); HRMS (FAB, NBA) calcd for C50H65N6O14 (MH+) 973.4559, found 973.4515.N-[(S)-4-(Benzyloxycarbonylamino)-2-hydroxybutanoyloxy]succinimide (28) A mixture of the N-Cbz protected (S)-4-amino-2-hydroxybutanoic acid (76 mg, 0.300 mmol, prepared from treatment of 1.0 eq.",
"of the acid with 1.2 eq.",
"of benzyl chloroformate in the presence of 2.1 equiv.",
"of sodium carbonate at 0-5° C. overnight, [α]D25=−61°), N-hydroxysuccinimide (38 mg, 0.318 mmol), and DCC (63 mg, 0.300 mmol) in anhydrous THF (3.5 mL) was stirred at room temperature under an atmosphere of argon for 2 h. This solution was directly used for the syntheses of 29-31.General Procedure for Synthesis of 29-31 A solution of the starting material (100 mg, ˜0.104 mmol) was made in 3:1 dioxane-water (4 mL).",
"To this solution was added a saturated solution of NaHCO3 (100 μL) and a solution of 28 (2.5 mL 0.086 M in anhydrous THF, 0.215 mmol) and the mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated to dryness in vacuo.",
"The resultant residue was suspended in water, filtered and washed with water to give a white solid as a crude product.",
"The solid was dissolved in 3:1 CHCl2/MeOH, pre-absorbed on silica gel (˜1 g), dried and purified on a column (SiO2, 15:2:0.1 CHCl3/MeOH/NH4OH) to afford the product as a pure white solid.",
"1-N-[(S)-4-(Benzyloxycarbonylamino)-2-hydroxybutanoyl]-6-O-[2-N-(2-(benzyloxycarbonyl)ethylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (29) Yield (63 mg, 50%); Rf 0.36 (15:3:0.2 CHCl3/MeOH/NH4OH); mp 223-25° C.; FTIR (KBr): 3414, 3326, 3064, 3033, 2942, 1686, 1531, 1454, 1263, 1140, 1053, 1015, 736, 969 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 8.62 (s, 1H, NH), 8.28 (d, J=8.0 Hz, 1H, NH), 8.21 (s, 1H, NH), 8.14 (s, 1H, NH), 7.91 (s, 1H, NH), 7.81 (s, 1H, NH), 7.56-7.28 (m, 25H), 6.02 (s, 1H), 5.53 (d, J=12.0 Hz, 1H), 5.36-5.19 (m, 10H), 4.71 (s, 1H), 4.64 (s, 1H), 4.58-4.46 (m, 2H), 4.33 (s, 1H), 4.14-4.02 (m, 5H), 3.95-3.80 (m, 3H), 3.70 (t, J=8.8 Hz, 1H), 3.65-3.52 (m, 3H), 2.80-2.72 (m, 3H), 2.58 (s,1H), 2.40-2.30 (m, 2H), 2.10-2.04 (m, 1H), 1.27 (br s, 1H); 13C NMR (100 MHz, pyridine-d5) δ 174.7, 157.8 (2C), 157.4, 157.3, 156.8, 138.0, 137.9, 137.8, 128.7, 128.6, 128.4, 128.2, 128.1, 128.0, 127.9, 100.6 (C1′), 84.5, 83.1, 77.0, 73.2, 72.9, 72.5, 70.8 (CH2), 70.4, 66.5 (CH2Ph), 66.3 (2C, CH2Ph), 66.1 (2C, CH2Ph), 57.8, 51.3, 49.4 (CH2), 49.3, 49.1 (CH2), 42.8 (CH2), 40.7 (CH2), 38.3 (CH2), 35.8 (CH2), 35.2 (CH2); MS (FAB, NBA) 1180 (MH+); HRMS (FAB, NBA) calcd for C60H74N7O18 (MH+) 1180.5090, found 1180.5084.1-N-[(S)-4-(Benzyloxycarbonylamino)-2-hydroxybutanoyl]-6-O-[2-N-[3-(benzyloxycarbonyl)propylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (30) Yield (58 mg, 46%); Rf 0.36 (15:3:0.2 CHCl3/MeOH/NH4OH); mp 201-203° C.; FTIR (KBr): 3328, 2929, 1695, 1532, 1261, 1027, cm−1; 1H NMR (400 MHz, pyridine-d5) δ 8.62 (d, J=7.2 Hz, 1H, NH), 8.33 (d, J=8.0 Hz, 1H, NH), 8.23 (s, 1H, NH), 8.16 (d, J=6.4 Hz, 1H, NH), 7.98 (s, 1H, NH), 7.83 (s, 1H, NH), 7.56-7.26 (m, 25H), 6.04 (s, 1H), 5.53 (d, J=12.8 Hz, 1H), 5.34-5.21 (m, 10H), 4.71 (t, J=2.4 Hz, 1H), 4.64 (s, 1H), 4.55 (q, J=8.8 Hz, 1H), 4.50 (q, J=9.2 Hz, 1H) 4.34 (s, 1H), 4.18-4.05 (m, 5H), 3.96-3.88 (m, 3H), 3.80 (t, J=5.6 Hz, 1H), 3.72 (t, J=9.6 Hz, 1H), 3.42 (q, J=5.6 Hz, 2H), 2.71 (s,1H), 2.62 (s,3H), 2.40-2.31 (m, 2H), 2.06 (q, J=11.6 Hz, 1H), 1.84 (s, 2H), 1.33-1.27 (m, 1H); 13C NMR (100 MHz, pyridine-d5) δ 174.8, 157.8 (2C), 157.3, 156.8 (2C), 138.1, 138.0, 137.9, 128.7, 128.6, 128.4, 128.2, 128.1, 128.0, 127.8, 100.5 (C1′), 84.4, 82.9, 77.0, 73.1, 72.9, 72.5, 70.7 (CH2), 70.4, 67.1 (CH2Ph), 66.5 (CH2Ph), 66.3 (CH2Ph), 66.1 (2C, CH2Ph), 57.7, 51.3, 49.6 (CH2), 49.3, 46.8 (CH2), 42.8 (CH2), 39.2 (CH2), 38.3 (CH2), 35.8 (CH2), 35.2 (CH2), 29.9 (CH2); MS (FAB, NBA) 1194 (MH+); HRMS (FAB, NBA) calcd for C61H76N7O18 (MH+) 1194.5250, found 1194.5246.1-N-[(S)-4-(Benzyloxycarbonylamino)-2-hydroxybutanoyl]-6-O-[2-N-[4-(benzyloxycarbonyl)butylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (31) Yield (67 mg, 54%); Rf 0.37 (15:3:0.2 CHCl3/MeOH/NH4OH); mp 207-209° C.; FTIR (KBr): 3318, 3064, 2933, 1712, 1686, 1535, 1454, 1267, 1140, 1055, 1016 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 8.61 (d, J=8.0 Hz, 1H, NH), 8.31 (d, J=8.0 Hz, 1H, NH), 8.17 (t, J=5.5 Hz, 1H, NH), 8.10 (d, J=7.5 Hz, 1H, NH), 7.92 (t, J=5.5 Hz, 1H, NH), 7.77 (s, 1H, NH), 7.55-7.27 (m, 25H), 6.02 (s, 1H), 5.53 (d, J=13.0 Hz, 1H), 5.38-5.19 (m, 10H), 4.70 (q, J=4.0 Hz, 1H), 4.63 (s, 1H), 4.53 (q, J=9.0 Hz, 1H), 4.50 (qd, J=9.0, 3.0 Hz, 1H) 4.34 (d, J=10.0 Hz, 1H, NH), 4.15-3.97 (m, 6H), 3.91-3.87 (m, 2H), 3.80 (q, J=6.0 Hz, 1H), 3.72 (t, J=9.5 Hz, 1H), 3.36 (s, 2H), 2.74-2.53 (m,5H), 2.38-2.32 (m, 2H), 2.06 (q, J=12.0 Hz, 1H), 1.60 (br s, 4H); 13C NMR (125 MHz, pyridine-d5) δ 174.7, 157.8 (2C), 157.4, 157.2, 156.8, 138.2, 138.0, 137.9, 128.7, 128.6, 128.4, 128.1, 128.0, 127.9, 100.5 (C1′), 84.5, 83.1, 77.0, 73.0, 72.9, 72.5, 70.5 (CH2), 70.4, 66.5 (CH2Ph), 66.3 (2C, CH2Ph), 66.1 (2C, CH2Ph), 57.7, 51.3, 49.5 (CH2), 49.3, 48.9 (CH2), 42.8 (CH2), 41.1 (CH2), 38.3 (CH2), 35.8 (CH2), 35.2 (CH2), 28.0 (CH2), 26.6 (CH2); MS (FAB, NBA) 1208 (MH+); HRMS (FAB, NBA) calcd for C62H78N7O18 (MH+) 1208.5400, found 1208.5392.General Procedure for Deprotection of Compounds 29-31 and 26 To a solution of the Cbz protected compound in glacial acetic acid (˜0.05 mmol/ml) was added palladium (Pd) on activated carbon (10%, 3-fold excess by weight; e.g., 90 mg for 30 mg of the starting compound).",
"To this mixture was added 1,4-cyclohexadiene (10 mole equivalent per each protecting group in the compound) and the mixture was allowed to stir at ambient temperature under an atmosphere of argon for 4 h. The reaction mixture was filtered (Celite), the filter-cake was thoroughly washed with water and the filtrate was concentrated to dryness in vacuo.",
"The crude product was purified on a short column (Amberlite CG-50 ion-exchange resin, NH4+ form, 3×1 cm) using 0.2-1.0 M aqueous ammonia as eluent.",
"Fractions containing the product were pooled and concentrated to dryness in vacuo and the resultant residue was dissolved in 0.1 N aqueous hydrochloric acid (1-2 mL) and lyophilized to give the product as hydrochloride salt.",
"Analytical samples for NMR spectroscopy were prepared by dissolving the solids in D2O and lyophilizing them again (this process was repeated twice).",
"The reported yields are based on the hydrochloride salts.",
"1-N-[(S)-4-Amino-2-hydroxybutanoyl]-6-O-[2-N-(2-ethylamino)ethylamino]neamine (3) Yield (10 mg, 51%) (based on 32 mg of 29); Rf 0.06 (9:4:4 MeOH/H2O/NH4OH, cf.",
"neamine 1: Rf 0.52); FTIR (KBr): 3394 (br), 2994 (br), 1604, 1497, 1395, 1123, 1058, 1029, 579 cm−1; 1H NMR (500 MHz, D2O) δ 5.77 (d, J=4.0 Hz, 1H, H1′), 4.15 (dd, J=10.0, 3.5 Hz, 1H), 3.94-3.79 (m, 6H), 3.70 (t, J=9.0 Hz, 1H), 3.41-3.35 (m, 2H), 3.34 (ABq, J=9.5, 6.0 Hz, 2H), 3.31-3.25 (m, 5H), 3.23-3.20 (m, 1H), 3.14 (ABq, J=13.5, 7.0 Hz, 2H), 3.02 (t, J=7.5 Hz, 2H), 2.08 (dt, J=12.5, 4.0 Hz, 1H, H2eq), 2.03 (m, J=3.5 Hz, 1H), 1.76 (qd, J=9.5, 2.5 Hz, 1H), 1.73 (q, J=12.5 Hz, 1H, H2ax); 13C NMR (125 MHz, D2O) δ 176.6, 96.1 (C1′), 82.8, 78.0, 75.3, 70.8, 69.6, 69.3, 68.3, 67.3 (CH2), 53.7, 48.9, 48.3 (CH2), 48.1, 44.3 (CH2), 40.2 (C6′), 37.0 (CH2), 35.5 (CH2), 31.1 (CH2), 30.2 (C2); MS (FAB, NBA) 509 (M+); HRMS (FAB, NBA) calcd for C20H44N7O8 (MH+) 510.3250, Found 510.323% 1-N-[(S)-4-Amino-2-hydroxybutanoyl]-6-O-[2-N-(3-propylamino)ethylamino]neamine (4) Yield (11 mg, 52%) (based on 34 mg of 30); Rf 0.06 (9:4:4 MeOH/H2O/NH4OH); FTIR (KBr): 3378 (br), 2976 (br), 1647, 1605, 1497, 1398, 1122, 1057, 1031, 575 cm−1; 1H NMR (500 MHz, D2O) δ 5.80 (d, J=2.5 Hz, 1H, H1′), 4.17 (dd, J=9.5, 1.5 Hz, 1H), 3.97-3.80 (m, 6H), 3.71 (t, J=9.5 Hz, 1H), 3.43-3.34 (m, 5H), 3.20-3.15 (m, 2H), 3.10 (d, J=7.0 Hz, 1H), 3.07-3.04 (m, 4H), 2.98 (t, J=7.5 Hz, 2H), 2.11 (dt, J=13.5, 4.5 Hz, 1H, H2eq), 2.07-2.03 (m, 1H), 1.98 (quint, J=7.5 Hz, 2H), 1.83-1.75 (m, 1H), 1.74 (q, J=13.0 Hz, 1H, H2ax); 13C NMR (125 MHz, D2O) δ 175.6, 96.2 (C1′), 82.9, 78.1, 75.3, 70.9, 69.7, 69.4, 68.4, 67.4 (CH2), 53.7, 49.0, 48.1, 47.9 (CH2), 44.8 (CH2), 40.3 (C6′), 37.1 (CH2), 36.7 (CH2), 31.1 (CH2), 30.2 (C2), 23.8 (CH2); MS (FAB, NBA) 523 (M+); HRMS (FAB, NBA) calcd for C21H46N7O8 (MH+) 524.3407, Found 524.3404.1-N-[(S)-4-Amino-2-hydroxybutanoyl]-6-O-[2-N-(4-butylamino)ethylamino]neamine (5) Yield (27 mg, 68%) (based on 63 mg of 31); Rf 0.06 (9:4:4 MeOH/H2O/NH4OH); FTIR (KBr): 3410 (br), 2976 (br), 1652, 1647, 1616, 1506, 1124, 1056, 1029, 576 cm−1; 1H NMR (500 MHz, D2O) δ 5.80 (s, 1H, H1′), 4.17 (dd, J=9.5, 3.0 Hz, 1H), 3.93-3.82 (m, 6H), 3.71 (t, J=9.5 Hz, 1H), 3.42-3.34 (m, 5H), 3.16 (ABq, J=14.0, 6.5 Hz, 2H), 3.08-3.03 (m, 3H), 2.99 (t, J=6.5 Hz, 2H), 2.90 (t, J=6.5 Hz, 2H), 2.09 (dt, J=13.0, 5.0 Hz, 1H, H2eq), 2.05-2.01 (m, J=3.5 Hz, 1H), 1.82-1.70 (m, 2H), 1.64 (s, 4H); 13C NMR (125 MHz, D2O) δ 175.6, 96.1 (C1′), 82.9, 78.1, 75.3, 70.1, 69.8, 69.4, 68.5, 67.4 (CH2), 53.8, 49.1, 48.2, 47.8 (CH2), 47.2 (CH2), 40.4 (C6′), 39.1 (CH2), 37.2 (CH2), 31.2 (CH2), 30.3 (C2), 24.2 (CH2), 22.9 (CH2); MS (FAB, NBA) 537 (M+); HRMS (FAB, NBA) calcd for C22H48N7O8 (MH+) 538.3563, found 538.3566.6-O-[2-N-(3-Propylamino)ethylamino]neamine (6) Yield (6 mg, 44%) (based on 20 mg of 26); Rf 0.08 (9:4:4 MeOH/H2O/NH4OH); FTIR (KBr): 3394 (br), 2982 (br), 1601, 1497, 1457, 1410, 1124, 1058, 1026, 540 cm−1; 1H NMR (500 MHz, D2O) δ 5.76 (d, J=3.5 Hz, 1H, H1′), 4.10 (dd, J=11.0, 6.0 Hz, 1H), 3.90-3.82 (m, 4H), 3.76 (t, J=9.0 Hz, 1H), 3.46 (t, J=9.5 Hz, 1H), 3.42-3.29 (m, 5H), 3.21 (s, 2H), 3.14 (dd, J=13.5, 6.5 Hz, 1H), 3.04 (t, J=8.0 Hz, 2H), 2.94 (t, J=8.0 Hz, 2H), 2.35 (dt, J=12.0, 4.5 Hz, 1H, H2eq), 1.96 (quint, J=8.0 Hz, 2H), 1.78 (q, J=12.5 Hz, 1H, H2ax); 13C NMR (125 MHz, D2O) δ 96.1 (C1′), 80.8, 77.4, 75.6, 70.7, 69.4, 68.2, 67.9 (CH2), 53.6, 48.8, 48.4, 47.9 (CH2), 44.9 (CH2), 40.2 (C6′), 36.7 (CH2), 28.3 (C2), 23.8 (CH2); MS (FAB, NBA) 422 (M+); HRMS (FAB, NBA) calcd for C17H39N6O6 (MH+)423.2930, found 423.2941.1-N,6-O-(2-N)-Bis[(S)-4-(benzyloxycarbonylamino)-2-hydroxybutanoyl]-6-O-[2-N-[3-(benzyloxycarbonyl)propylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (32) To a solution of 30 (60 mg, 0.050 mmol) in anhydrous pyridine (200 μL) was added dropwise a solution of 28 in anhydrous THF (0.5 mL 0.450 M, 0.225 mmol) and the mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with water and extracted with methylene chloride (3×10 mL).",
"The organic layer was back extracted with brine, dried (Na2SO4) and concentrated to dryness in vacuo.",
"The resultant solid was purified on a column (SiO2, 14:1:0.1 CHCl3/MeOH/NH4OH) to afford recovered starting compound (22 mg, 37%) and 32 (19 mg, 26%); Rf 0.67 (15:3:0.2 CHCl3/MeOH/NH4OH); mp 228-30° C.; FTIR (KBr): 3328, 2930, 1699, 1653, 1533, 1437, 1260, 1136, 1028, 737, 696 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 8.30 (d, J=8.0 Hz, 1H, NH), 8.22-8.18 (m, 3H, NH), 7.87-7.79 (m, 3H, NH), 7.56-7.27 (m, 30H), 6.19 (d, J=9.2 Hz, 1H), 6.05 (s, 1H), 5.54 (d, J=12.0 Hz, 1H), 5.34-5.17 (m, 12H), 5.12-488 (m, 2H), 4.65 (s, 2H), 4.53 (s, 3H), 4.22-3.98 (m, 7H) 3.92-3.76 (m, 3H), 3.60-3.38 (m, 4H), 3.24-3.14 (m, 1H), 2.94 (d, J=15.2 Hz, 1H), 2.74 (td, J=10.4, 4.0 Hz, 1H), 2.64-2.56 (m, 1H), 2.42 (d, J=11.2 Hz, 1H), 2.33 (d, J=11.2 Hz, 1H), 19.2 (q, J=11.6 Hz, 1H), 1.80-1.71 (m, J=6.4 Hz, 2H), 1.28 (brs, 1H); 13C NMR (100 MHz, pyridine-d5) δ 172.1, 159.3, 157.8, 157.7, 157.3, 156.8 (2C), 138.0, 137.9, 128.7, 128.6, 128.4, 128.2, 128.1, 128.0, 127.9, 100.8 (C1′), 85.9, 83.2, 78.9, 74.3 (CH2), 72.8 (2C), 72.6, 70.4, 66.5 (CH2Ph), 66.3 (2C, CH2Ph), 66.2 (3C, CH2Ph), 57.8, 51.5, 48.1, 47.6 (CH2), 43.9 (CH2), 42.8 (CH2), 38.8 (CH2), 38.3 (CH2), 38.1 (CH2), 35.9 (CH2), 35.2 (CH2), 29.9 (CH2), 28.6 (CH2); MS (FAB, NBA) 1451 (M+Na+); HRMS (FAB, NBA) calcd for C73H88N8O22Na (M+Na+) 1451.5910, found 1451.5950.1-N,6-O-(2-N)-Bis[(S)-4-amino-2-hydroxybutanoyl]-6-O-[2-N-(3-propylamino)ethylamino]neamine (7) To a solution of 32 (18 mg, 0.0125 mmol) in glacial acetic acid was added palladium (Pd) on activated carbon (60 mg, 10%) and the mixture was treated with 1,4-cyclohexadiene (60 μL, 0.646 mmol) at room temperature under an atmosphere of argon for 10 h. Purification of the mixture (as described for 3-5) gave the title compound (5.5 mg, 52%, a mixture of two rotainers by NMR) as a hydrochloride salt.",
"Rf 0.12 (9:4:4 MeOH/H2O/NH4OH); FTIR (KBr): 3409 (br), 2976 (br), 1648, 1492, 1120, 1060, 1027, 576 cm−1; 1H NMR (500 MHz, D2O) δ 5.76 (d, J=4.5 Hz, 1H, H1′), 4.14-4.09 (m, 1H), 3.86-3.77 (m, 5H), 3.73-3.58 (m, 3H), 3.38-3.29 (m, 8H), 3.26-3.16 (m, 2H), 3.13 (dd, J=12.0, 6.5 Hz, 1H), 3.01 (t, J=7.0 Hz, 3H), 2.94 (t, J=8.0 Hz, 1H), 2.81 (t, J=7.5 Hz, 1H), 2.08-2.04 (m, 1H), 2.02-1.96 (m, 1H), 1.87-1.62 (m, 4H); 13C NMR (125 MHz, D2O) δ 175.6, 175.4, 96.2 (C1′), 83.0 (w), 82.8, 82.5 (w), 82.2, 78.2 (w), 78.1, 75.5, 75.3, 70.8, 69.9 (CH2), 69.6, 69.3, 68.3, 67.4 (CH2), 53.6, 48.1 (w), 47.7 (w), 45.8 (w, CH2), 45.1 (CH2), 42.7 (CH2), 40.2 (C6′), 39.6 (w, CH2), 37.0 (CH2), 36.9 (w, CH2), 36.8 (CH2), 35.8 (w, CH2), 34.8 (CH2), 31.2 (CH2), 31.1 (w, CH2), 30.3 (C2), 30.2 (w, C2), 25.8 (w, CH2), 25.6 (CH2), 25.0 (w, CH2), 24.6 (CH2); MS (FAB, NBA) 624 (M+); HRMS (FAB, NBA) calcd for C25H53N8O10 (MH+) 625.3884, found 625.3892.The designated weak (w) signals in the 13C NMR spectrum belong to the minor rotamer.",
"N-[4-(Benzyloxycarbonylamino)phenylethanoyloxy]succinimide (33) To an ice-cold solution of 4-aminophenylacetic acid (500 mg, 3.307 mmol) in 1:1 dioxane-water (10 mL) was added a solution of 2 N NaOH (˜2.2 equivalent, pH 9-10).",
"This mixture was treated with benzylchloroformate (0.5 mL, 3.681 mmol) and the solution was allowed to stir in an ice-bath for 2 h. The reaction mixture was acidified with an aqueous solution of 1 N HCl (pH ˜4), extracted with ethyl acetate, dried (Na2SO4), and concentrated in vacuo to give a solid as crude product.",
"Purification of the solid (SiO2, 10:1 CHCl3/MeOH) gave N-Cbz protected phenylacetic acid (856 mg, 91%) as a pure white solid.",
"To make the succinimide active ester, a mixture of N-Cbz protected acid (120 mg, 0.421 mmol), N-hydroxysuccinimide (53 mg 97%, 0.445 mmol), and dicyclohexylcarbodiimide (88 mg, 0.421 mmol) in dry THF (4 mL) was stirred at room temperature under an atmosphere of argon for 2 h. This solution was directly used for preparation of compound 35.N-[3-(Benzyloxycarbonylamino)phenylethanoyloxy]succinimide (34) This active ester was made in a similar way as described for 33 and was directly used for preparation of compound 36.1-N-[4-(benzyloxycarbonylamino)phenylethanoyl]-6-O-[2-N-[4-(benzyloxycarbonyl)butylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (35) To an ice-cold solution of 27 (100 mg, 0.103 mmol) in 2:1 THF-water (3 mL) was added a saturated solution of NaHCO3 (150 μL).",
"To this mixture was added dropwise a solution of the active ester 33 (1.0 mL 0.105 M solution in THE, 0.105 mmol) over 30 min and the reaction mixture was stirred for 1 h at room temperature.",
"Concentration of the mixture gave a solid, which was suspended in water, filtered and washed with water and ether to provide a solid as crude product.",
"The solid was dissolved in MeOH—CH2Cl2, pre-absorbed on silica gel (˜1 g), dried and purified on a column (SiO2, 25:1:0.1 to 15:3:0.2 CHCl3/MeOH/NH4OH) to give 35 (115 mg, 74%) as a pure white solid.",
"Rf 0.38 (15:3:0.2 CHCl3/MeOH/NH4OH); mp 232-34° C.; FTIR (KBr): 3315, 3063, 3034, 2944, 1698, 1610, 1533, 1454, 1415, 1228, 1132, 1050, 740, 696 cm−1; 1H NMR (400 MHz, pyridine-d5) δ 10.83 (s, 1H, ArNHCbz), 8.98 (d, J=6.8 Hz, 1H, NH), 8.29 (d, J=8.4 Hz, 1H, NH), 8.05-8.02 (m, 2H, NH), 7.87 (d, J=8.0 Hz, 2H), 7.83 (s, 1H, NH), 7.56-7.25 (m, 27H), 6.04 (s, 1H, H1′), 5.54 (d, J=12.0 Hz, 1H), 5.35-5.34 (m, 7H), 5.29-5.19 (m, 3H), 4.63 (d, J=8.8 Hz, 1H), 4.52 (t, J=8.0 Hz, 2H), 4.28-4.21 (m, 1H), 4.13-4.07 (m, 4H), 4.07-4.00 (m, 2H), 3.89-3.78 (m, 3H), 3.68 (t, J=10.0 Hz, 1H), 3.36 (d, J=4.0 Hz 2H), 2.80 (br s, 2H), 2.75-2.65 (m, 2H), 2.44 (d, J=12.4 Hz, 1H, H2eq), 1.91 (q, J=12.0 Hz, 1H, H2ax), 1.74 (br s, 2H), 1.65 (d, J=6.4 Hz, 2H); 13C NMR (100 MHz, pyridine-d5) δ 171.2, 157.8, 157.2, 156.8, 154.4, 138.8, 138.1, 138.0, 137.9, 137.5, 131.2, 130.2, 130.1, 128.8, 128.7 (2C), 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 119.1, 100.4 (C1′), 84.7, 82.8, 77.1, 76.3, 73.0, 72.9, 72.5, 69.7 (CH2), 66.5 (CH2Ph), 66.4 (CH2Ph), 66.3 (CH2Ph), 66.1 (CH2Ph), 57.7, 51.2, 49.5, 49.2 (CH2), 48.7 (CH2), 43.2 (CH2), 42.8 (CH2), 40.9 (CH2), 35.2 (CH2), 27.9 (CH2), 25.9 (CH2); MS (FAB, NBA) 1239 (M+); HRMS (FAB, NBA) calcd for C66H78N7O17 (MH+) 1240.5453, found 1240.5474.1-N-[4-(benzyloxycarbonylamino)phenylethanoyl]-6-O-[2-N-[4-(benzyloxycarbonyl)butylamino]ethylamino]-3,2′,6′-Tris(N-benzyloxycarbonyl)neamine (36) To an ice-cold solution of 27 (150 mg, 0.154 mmol) in 2:1 THF-water (4.5 mL) was added a saturated solution of NaHCO3 (200 μL) and the mixture was treated with a solution of the active ester 34 (1.5 mL 0.105 M solution in THF, 0.158 mmol) as described for 35.Yield 175 mg, 75%.",
"Rf 0.38 (15:3:0.2 CHCl3/MeOH/NH4OH); mp 223-25° C.; FTIR (KBr): 3318, 3063, 3033, 2941, 1696, 1646, 1539, 1454, 1230, 1132, 1055, 1012, 736, 696 cm−1; 1H NMR (500 MHz, pyridine-d5) δ 10.93 (s, 1H, ArNHCbz), 8.94 (d, J=8.0 Hz, 1H, NH), 8.32 (d, J=8.5 Hz, 1H, NH), 8.05 (s, 2H), 7.97 (t, J=6.0 Hz, 1H, NH), 7.88 (d, J=8.0 Hz, 1H, NH), 7.80 (s, 1H, NH), 7.56-7.25 (m, 27H), 6.00 (s, 1H, H1′), 5.54 (d, J=12.5 Hz, 1H), 5.38-5.33 (m, 6H), 5.30-5.26 (m, 2H), 5.24-5.19 (m, 2H), 4.63 (d, J=8.5 Hz, 1H), 4.56-4.47 (m, 2H), 4.30 (br s, 1H), 4.14-4.05 (m, 3H), 4.00 (t, J=8.5 Hz, 2H), 3.93 (br s, 1H), 3.87 (t, J=9.0 Hz, 1H), 3.58 (t, J=9.0 Hz, 1H), 3.36 (t, J=6.0 Hz, 2H), 2.71 (br s, 1H), 2.65 (br s, 1H), 2.54-2.47 (m, 2H), 2.41 (d, J=12.5 Hz, 1H, H2eq), 1.93 (q, J=12.5 Hz, 1H, H2ax), 1.59 (q, J=7.0 Hz, 2H),1.56-1.52 (m, 2H), 1.34-1.28 (m, 1H); 13C NMR (125 MHz, pyridine-d5) δ 170.7, 157.8, 157.2, 156.8, 154.4, 140.4, 138.2, 138.0, 137.8, 137.7, 137.4, 135.8, 129.3, 128.8, 128.7 (2C), 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 124.0, 123.8, 119.8, 117.3, 100.5 (C1′), 84.8, 83.0, 77.0, 73.1, 72.9, 72.5, 70.9 (CH2), 66.5 (CH2Ph), 66.4 (CH2Ph), 66.3 (CH2Ph), 66.1 (CH2Ph), 57.7, 51.2, 49.9 (CH2), 49.6, 49.2 (CH2), 44.0 (CH2), 42.8 (CH2), 41.2 (CH2), 35.3 (CH2), 28.1 (CH2), 27.0 (CH2); MS (FAB, NBA) 1239 (M+); HRMS (FAB, NBA) calcd for C66H78N7O17 (MH+) 1240.5453, found 1240.5448.1-N-[4-Aminophenylethanoyl]-6-O-[2-N-(4-butylamino)ethylamino]neamine (8) To a solution of 35 (28 mg, 0.022 mmol) in glacial acetic acid (400 μL) was added palladium (Pd) on activated carbon (120 mg, 10%).",
"This mixture was treated with 1,4-cyclohexadiene (240 μL, 2.60 mmol) at room temperature under an atmosphere of argon for 4 h. The mixture was diluted with water, filtered (Celite), and the filter cake was thoroughly washed with water.",
"Concentration of the filtrate gave a residue, which was purified on a column (Amberlite CG-50 NH4+ form, 3×1 cm, 0.0-0.8 M aqueous ammonia) to furnish the product as a free base.",
"Treatment of this free base with excess 0.1 N aqueous HCl (1-2 mL) followed by lyophilization of the solution afforded 8 (10 mg, 56%) as a hydrochloride salt.",
"Rf 0.22 (9:4:4 MeOH/H2O/NH4OH); FTIR (KBr): 3419 (br), 2966 (br), 1651, 1599, 1500, 1451, 1118, 1054, 1028, 687, 571 cm−1; 1H NMR (500 MHz, D2O) δ 7.32-7.18 (m, 4H), 5.74 (d, J=3.5 Hz, 1H, H1′), 3.89-3.79 (m, 3H), 3.78-3.73 (m, 1H), 3.62 (t, J=9.5 Hz, 1H), 3.59-3.55 (m, 1H), 3.52 (s, 2H), 3.35-3.28 (m, 3H), 3.25 (t, J=10 Hz, 1H), 3.13 (q, J=6.5 Hz, 1H), 2.95 (dq, J=13.0, 3.0 Hz, 1H), 2.85 (t, J=7.0 Hz, 4H), 2.71-2.66 (m, 1H), 2.07 (dt, J=13.0 4.5 Hz, 1H, H2eq), 1.64 (q, J=13.0 Hz, 1H, H2ax), 1.56 (t, J=3.5 Hz, 2H), 1.42 (s, 2H); 13C NMR (125 MHz, D2O) δ 173.9, 136.0, 131.0, 129.3, 128.2, 123.5, 115.0, 96.1 (C1′), 83.2, 78.0, 75.8, 75.2, 70.8, 69.3, 68.3, 67.4 (CH2), 53.6, 48.9, 48.2, 47.5 (CH2), 46.9 (CH2), 42.1 (CH2), 40.2 (C6′), 38.9 (CH2), 30.2 (C2), 24.0 (CH2), 22.8 (CH2); MS (FAB, NBA) 569 (M+); HRMS (FAB, NBA) calcd for C26H48N7O7 (MH+) 570.3591.1-N-[3-Aminophenylethanoyl]-6-O-[2-N-(4-butylamino)ethylamino]neamine (9) This compound was prepared from 36 (53 mg, 0.043 mmol) in a similar manner as described for 8 to give the title compound (21 mg, 62%) as a hydrochloride salt.",
"Rf 0.22 (9:4:4 MeOH/H2O/NH4OH); FTIR (KBr): 3419 (br), 2960 (br), 1657, 1631, 1599, 1484, 1451, 1119, 1055, 1029, 687, 524, 453 cm−1; 1H NMR (400 MHz, D2O) δ 7.48 (t, J=8.0 Hz, 1H), 7.40 (d, J=8.0 Hz, 1H), 7.33 (d, J=10.0 Hz, 2H), 5.86 (d, J=4.0 Hz, 1H, H1′), 3.99-3.92 (m, 4H), 3.84 (t, J=8.0 Hz, 1H), 3.75 (t, J=9.2 Hz, 1H), 3.64 (s, 3H), 3.47-3.34 (m, 5H), 3.23 (dd, J=12.8, 9.2 Hz, 1H), 3.04-2.94 (m, 3H), 2.75-2.70 (m, 1H), 2.19 (dt, J=12.8, 4.8 Hz, 1H, H2eq), 1.77 (q, J=12.8 Hz, 1H, H2ax), 1.66 (s, 4H); 13C NMR (100 MHz, D2O) δ 173.8, 137.3, 130.8, 130.1, 124.1, 122.3, 96.1 (C1′), 83.2, 77.9, 75.3, 70.9, 69.4, 68.4, 67.5 (CH2), 53.8, 49.0, 48.3, 47.5 (CH2), 47.0 (CH2), 42.4 (CH2), 40.4 (C6′), 39.1 (CH2), 30.3 (C2), 24.2 (CH2), 22.9 (CH2); MS (FAB, NBA) 569 (M+); HRMS (FAB, NBA) calcd for C26H48N7O7 (MH+) 570.3611.The following illustrate representative pharmaceutical dosage forms, containing a compound of the present invention (‘Compound X’), for therapeutic, and/or prophylactic use in humans.",
"(i) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 300.0 (ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0 (iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg/mL) mg/mL ‘Compound X’ (free acid form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0 N Sodium hydroxide solution q.s.",
"(pH adjustment to 7.0-7.5) Water for injection q.s.",
"ad 1 mL (v) Injection 2 (10 mg/mL) mg/mL ‘Compound X’ (free acid form) 10.0 Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.0 01 N Sodium hydroxide solution q.s.",
"(pH adjustment to 7.0-7.5) Water for injection q.s.",
"ad 1 mL (vi) Aerosol mg/can ‘Compound X’ 20.0 Oleic acid 10.0 Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0 Dichlorotetrafluoroethane 5,000.0 (vii) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 300.0 (viii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0 (ix) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0 (x) Injection 1 mg/mL ‘Compound X’ (free acid form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0 N Sodium hydroxide solution q.s.",
"(pH adjustment to 7.0-7.5) Water for injection q.s.",
"ad 1 mL (xi) Injection 2 mg/mL ‘Compound X’ 10.0 Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.0 01 N Sodium hydroxide solution q.s.",
"(pH adjustment to 7.0-7.5) Water for injection q.s.",
"ad 1 mL (xii) Aerosol mg/can ‘Compound X’ 20.0 Oleic acid 10.0 Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0 Dichlorotetrafluoroethane 5,000.0 The above formulations may be obtained by conventional procedures well known in the pharmaceutical art.",
"Although specific quantities of “Compound X” are shown in the above illustrative examples, it is to be understood that the compounds can be present in any ratio provided the final formulation possesses the desired biological properties.",
"All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference.",
"The invention has been described with reference to various specific and preferred embodiments and techniques.",
"However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention."
]
] | Patent_10415732 |
[
[
"Method for the detection of viable microorganisms",
"Method for the detection and enumeration of viable microorganisms.",
"A liquid that comprises one or more markers incorporated in a liquid sol-gel precursor, is provided.",
"A transparent slide is coated with a thin uniform layer of the liquid sol-gel precursor composition.",
"The microorganisms are separated from liquid sample to be analyzed by passing the sample through a filter, and then bringing the filter into close contact with the sol-gel coated slide.",
"The filter is co-incubated with the sol-gel coated slide for a period of time and at a temperature suitable to promote uptake of the markers by the microorganisms.",
"The gel-coated slide irradiated with an external energy source, so as to generate detectable signals emitted from the markers uptaken by the microorganisms.",
"Image of the detectable signals emitted from the microorganisms are acquired, and analyzed using a computer system, in order to provide the identification and enumeration of the microorganisms."
],
[
"1-14.",
"(canceled) 15.A method for detecting and enumerating viable microorganisms in a sample comprising: providing a smooth transparent porous material containing a marker therein; separating the microorganisms from a sample to be analyzed and contacting the microorganisms with the smooth transparent porous material; incubating the contacted microorganisms and smooth transparent porous material for a time sufficient to promote uptake of the marker by the microorganisms; irradiating the marker containing microorganisms with an energy source to generate detectable signals emitted by the marker, and detecting the signals emitted and analyzing the signals to identify and enumerate the microorganisms.",
"16.A method according to claim 15 wherein the microorganisms are bacteria.",
"17.A method according to claim 15 wherein the energy source is selected from the group consisting of visible light, fluorescent light, UV light, infrared light, an electro-magnetic field, sonar, ultrasonic waves, radio waves, and short wave radiation.",
"18.A method according to claim 15, further comprising isolating the microorganisms from a sample selected from the group consisting of water, milk, food, saliva, urine, throat swab tests, wounds, sputum, stomach content, and feces.",
"19.The method according to claim 15, wherein the marker is selected from the group consisting of 3-carboxyumbelliferyl β-D-galactopyranoside, 4-chloromethyl-6,8-difluoroumbelliferyl β-D-galactopyranoside, 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di β3-D-galactopyranoside, 6,8-difluoroumbelliferyl β-D-glucuronide, 5-(pentafluorobenzoylamino) fluorescein di-β-D-glucuronide, and β-trifluoromethylumbelliferyl β-D-glucuronide.",
"20.The method according to claim 15 further comprising attaching antibiotic substances to the marker.",
"21.The method according to claim 20, wherein the antibiotic substance is selected from the group consisting of Chloramphenicol, Erythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethorprin, Rifanapicin and Penicillin.",
"22.The method according to claim 15, wherein the markers are provided in a form selected from the group consisting of liposomes; films; multilayers; braided; lamellar, helical, tubular, and fiber shapes; solvated rods; solvated coils; and combination thereof.",
"23.The method according to claim 15, further comprising providing pyridine or K2SO4 in the smooth transparent porous material.",
"24.The method according to claim 15, further comprising detecting microorganisms present at less than 103 ml−1.25.The method according to claim 15, wherein separating the microorganisms from a sample comprises filtering the sample to capture microorganisms on a filter media, and placing the filter media in contact with the smooth transparent porous material containing the marker.",
"26.The method of claim 25 further comprising co-incubating the filter media with the smooth transparent porous material.",
"27.The method of claim 26 wherein the co-incubating is conducted at 0.5 to 6 hours at a temperature of between 35 and 44° C. 28.A system for identifying and enumerating viable microorganisms in a sample comprising; a smooth transparent porous material containing at least one marker in the pores therein; separation means for separating the microorganisms from a sample to be analyzed; means for incubating the microorganisms with the marker-containing smooth transparent porous material such that the microorganisms ingest the markers; means for irradiating the marker containing microorganisms for generating detectable signals emitted by the marker; means for detecting the signals; and means for analyzing the detected signals to identify and enumerate the microorganisms.",
"29.The system of claim 28, wherein the irradiating means comprise an energy source selected from the group consisting of visible light, flourescent light, UV light, infrared light, an electrochemical field, sonar, ultrasonic waves, radio waves and short wave radiation.",
"30.The system of claim 28, wherein the detecting means comprise a frame grabber, a CCD array camera, a microscope, and an autofocus system.",
"31.The system of claim 28, further comprising a mechanical x-y table.",
"32.The system of claim 30, wherein the frame grabber has a rate of 30 frames per second."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>A major goal of microorganism detection research is to develop inexpensive, fast, reliable, and sensitive detectors.",
"Standard laboratory procedures are currently available for the detection of microorganisms.",
"The vast majority of procedures are based on the use of agar media on which specific microorganisms grow over a period of time.",
"The normal incubation period is between 24 to 48 hours.",
"After the microorganisms multiply their presence can be identified and quantified.",
"The main drawback of the existing tests is the time required for obtaining their results.",
"Bacterial contamination in water sources results in the shutdown of water sources and systems and requires the use of more expensive water supply alternatives.",
"Fast detection of microorganisms is needed to allow for shorter shutdown periods.",
"In the medical sector, bacterial identification and antibiotic sensitivity tests are required in any medical situation in which antibiotics are to be administrated.",
"The time required for obtaining test results is between 72 to 96 hours.",
"Reducing this time period will produce better results and faster patient recovery.",
"In the food and beverage industry, raw materials and manufactured goods are routinely inspected for bacterial contamination.",
"The required incubation period for test results does not allow for immediate process treatment and causes delays both in the manufacturing and supplying of goods.",
"Reducing the taking time period can result in savings in infrastructure and labor.",
"In the last few years several methods were developed in order to identify and enumerate bacteria in 1.5 to 11.0 hours.",
"Methods that are relatively simple require a large amount of bacteria (above 10 4 in 1 ml sample), while methods that can detect small numbers of bacteria are expensive and cannot be used as large scale systems.",
"U.S. Pat.",
"No.",
"5,811,251 discloses a system for counting the number of viable microorganisms based on a CCD system.",
"However this system cannot differentiate between different types of bacteria and provides only a total number of bacteria.",
"U.S. Pat.",
"Nos.",
"5,972,641 and 5,518,894 disclose a rapid coliform detection systems using a statistic methods for determining the number of bacteria.",
"Said methods require up to 11 hours for obtaining the results in low number of bacteria.",
"Other patents disclose a method for detection of microorganisms using fluorescence and laser light source (U.S. Pat.",
"Nos.",
"5,891,394, 5,858,697, 5,763,203, 5,751,839 and U.S. Pat.",
"No.",
"5,663,057).",
"The disadvantages of said methods is the use of an expensive laser light source and the detection of microorganisms directly from the filter which is not smooth and causes problems during analysis.",
"In addition, these systems are not portable and are relatively expensive.",
"Immunoassay methods are also used for detecting certain types of microorganisms (Lee et al., App.",
"Environ.",
"Microbiol., Vol.",
"56, pp.1541-1546).",
"In these methods, specific antibodies labeled with a fluorescent or radioactive dye are used to detect the microorganism.",
"However, immunoassay methods are limited in that they require the production of antibodies against each microorganism of interest, which is time-consuming and expensive.",
"“Sol-gel” is the term used to indicate inorganic glass manufactured at room temperatures based on metal oxides.",
"A certain process involving ceramic materials in which the sol (solution) is transformed to a gel phase through hydrolysis, condensation and polymerization.",
"The common starting materials for the sol-gel preparation are ormosils or metal oxides.",
"In recent years sol-gel has been applied to organosilanes to create “glass at room temperature”.",
"Sol-gel type materials comprise pores ranging from tens of angstroms to tens of nanometers, and exhibit a large area to mass ratios e.g., hundreds of square meters per gram.",
"Sol-gel materials are transparent even at UV wavelengths, and are simple to prepare in different shapes, such as powders, monolithic blocks, thin sheets, fibers etc.",
"The use of sol-gel-based materials to entrap various organic molecules in a matrix media was described in the art.",
"(Avnir et al., Supramolecular architecture in two and three dimensions Bein T.",
"(ed.)",
"American Chemical Society Symposium Series XXX, 1992).",
"Using said technology, organic molecules are entrapped at room temperatures within the sol-gel matrix without impairing the structure of the relatively sensitive organic molecule.",
"In addition, the entrapped molecule retains almost all of the original physical and chemical characteristics, and is available to outside reactants as a result of the massive pore system inside the sol-gel.",
"U.S. Pat.",
"No.",
"6,022,748 discloses a method for the direct detection of analytes using color changes in response to selective binding of analytes to a surface.",
"Said detection occurs in immobilized biopolymeric material encapsulated into metal oxide glass using the sol-gel method.",
"The disadvantages of this method are that only large amounts of bacteria can be detected or enumerated, since only high counts are able to cause a visible color change in the sol-gel.",
"Furthermore, said method cannot differentiate between viable and non-viable microorganisms, since it is based on the binding of the microorganisms to the sol-gel surface, independent whether said microorganisms are viable or not.",
"Armon et al.",
"(J. of Biotechnology 51, 279-285) disclose a method for detecting large quantities of E. coli bacteria by spreading them on sol-gel doped with specific compounds, said compounds being uptaken into the bacteria, which consequently causes the bacteria to glow at specific wavelengths.",
"However, this method does not provide a method suitable to count bacteria present in a given sample.",
"In addition, detection of low number of microorganisms is not possible using said method.",
"The art has so far failed to provide a fast method for the enumeration of microorganisms, which is sensitive enough to provide a reliable count at low microorganism concentrations.",
"It is a purpose of this invention to provide a fast and sensitive method for the detection of viable microorganisms.",
"It is another object of the invention to provide a method and compositions useful in providing an enumeration of microorganisms found in low-count samples.",
"Other objects and advantages of the invention will become apparent as the description proceeds."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>It has now been surprisingly found, and this is an object of the present invention, that viable microorganisms at a concentration lower than 10 3 ml −1 may be detected and enumerated in a 2-hours period, using organic or inorganic substances that are entrapped in porous sol-gel glass (hereinafter referred to as “markers”, for the sake of brevity).",
"The present invention relates to methods and compositions for the detection and enumeration of viable microorganisms using organic or inorganic substances that are incorporated in porous sol-gel glass (markers).",
"The microorganisms metabolize said markers, and thereby emit detectable radiation, electromagnetism or fluorescence.",
"Thus, a fast visualization of viable microorganisms is obtained on the sol-gel glass.",
"The sol-gel surface is extremely smooth and transparent and allows for an almost uniform focal point for high resolution microscopic scanning.",
"According to a preferred embodiment of the invention, the following steps are carried out: a) Providing a liquid composition comprising one or more marker(s) incorporated in a liquid sol-gel precursor; b) Coating a transparent slide, e.g., a glass slide, with a thin uniform layer of said liquid sol-gel precursor composition; c) Separating the microorganisms from a liquid sample to be analyzed by passing said sample through a filter, and then bringing said filter into close contact with the sol-gel-coated slide; d) Co-incubating said filter with said sol-gel-coated slide for a period of time and at a temperature suitable to promote uptake of the marker(s) by the microorganisms, e.g.",
"at 35-44° C. for 0.5 to 6 hours; e) Irradiating said sol-gel-coated slide with an external energy source such as to generate detectable signals emitted from the marker(s) metabolized by the microorganisms; and f) Acquiring images of said detectable signals emitted from the microorganisms, and analyzing said images using a computer system, thereby to provide the identification and enumeration of said microorganisms.",
"The present invention further relates to a method for preparation of a liquid sol-gel mixture containing organic or inorganic substances (markers).",
"Said markers are metabolized by the microorganisms that are to be identified.",
"Coliform bacteria that can be detected according to the present invention are actually a broad group of bacteria that include E. coli, Enterobacter spp., Klebsiella spp.",
"and Citrobacter spp.",
"Coliform bacteria are identified by detecting the activity of an enzyme, β-Galactosidase (E.C.",
"3.2.1.23), using fluorogenic or chromogenic substances 3-carboxyumbelliferyl β-D-galactopyranoside (CUG) or 4-chloromethyl-6,8-difluoroumbelliferyl β-D-galactopyranoside (CMDi FUG).",
"According to the present invention, E. coli are identified by detecting the activity of an E. coli -specific-enzyme, β-Glucuronidase (GUS or EC 3.2.1.31), using the following fluorogenic or chromogenic substances: 4-methylumbelliferyl β-D-galactopyranoside (MUG), fluorescein di β-D-galactopyranoside (FDG), 6,8-difluoro-4-methylumbelliferyl β-D-glucuronide, lithium salt (DiFMUGGlcU), 2-dodecylresorufin, Elf-97, fluorescein di-β-glucoronide (FDGlcU), 5-(pentafluorobenzoylamino) fluorescein di-β-D-glucoronide (PFB-FDGlcU) and β-trifluoromethylumbelliferyl β-D-glucoronide.",
"According to another preferred embodiment of the invention, an antibiotic material is incorporated into the sol-gel mixture for identifying bacterial antibiotic resistance.",
"It may be appreciated that the emission of fluorescence from bacteria in spite of the presence of a specific antibiotic, indicates that said bacteria is antibiotic-resistant.",
"Partial resistance may be indicated when the presence of the specific antibiotic leads to a partial reduction in the number of fluorescent bacteria.",
"The following antibiotics are added to the sol-gel mixture: Chloramphenicol, Erythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethoprin, Rifanapicin and Penicillin.",
"According to the present invention, it is possible to provide the markers as liposomes; films; multilayers; braided, lamellar, helical, tubular, and fiber-like shapes; solvated rods; solvated coils; and combinations thereof.",
"According to the present invention, it is also possible to detect injured or stressed microorganisms by incorporating pyruvate or K 2 SO 4 within the sol-gel.",
"It is known in the art that pyruvate or K 2 SO 4 can be used to resuscitate or improve chlorinated injured coliform bacteria (“enumeration and differentiation of chlorine-stressed total coliform bacteria” Robert A Duncanson—Ph.D.",
"dissertation—University of Rhode Island 1993).",
"According to the present invention it is possible to add polylysine or similar substances at a concentration of between 10 to 100 parts per million to the sol-gel solution to allow for enhancing bacteria absorption.",
"All the above and other characteristics and advantages of the invention will be further understood from the following illustrative and non-limitative examples of preferred embodiments thereof."
],
[
"FIELD OF THE INVENTION The present invention is concerned with a method and compositions for the fast detection and enumeration of low concentration of viable microorganisms using organic or inorganic substances that are entrapped in porous sol-gel glass.",
"BACKGROUND OF THE INVENTION A major goal of microorganism detection research is to develop inexpensive, fast, reliable, and sensitive detectors.",
"Standard laboratory procedures are currently available for the detection of microorganisms.",
"The vast majority of procedures are based on the use of agar media on which specific microorganisms grow over a period of time.",
"The normal incubation period is between 24 to 48 hours.",
"After the microorganisms multiply their presence can be identified and quantified.",
"The main drawback of the existing tests is the time required for obtaining their results.",
"Bacterial contamination in water sources results in the shutdown of water sources and systems and requires the use of more expensive water supply alternatives.",
"Fast detection of microorganisms is needed to allow for shorter shutdown periods.",
"In the medical sector, bacterial identification and antibiotic sensitivity tests are required in any medical situation in which antibiotics are to be administrated.",
"The time required for obtaining test results is between 72 to 96 hours.",
"Reducing this time period will produce better results and faster patient recovery.",
"In the food and beverage industry, raw materials and manufactured goods are routinely inspected for bacterial contamination.",
"The required incubation period for test results does not allow for immediate process treatment and causes delays both in the manufacturing and supplying of goods.",
"Reducing the taking time period can result in savings in infrastructure and labor.",
"In the last few years several methods were developed in order to identify and enumerate bacteria in 1.5 to 11.0 hours.",
"Methods that are relatively simple require a large amount of bacteria (above 104 in 1 ml sample), while methods that can detect small numbers of bacteria are expensive and cannot be used as large scale systems.",
"U.S. Pat.",
"No.",
"5,811,251 discloses a system for counting the number of viable microorganisms based on a CCD system.",
"However this system cannot differentiate between different types of bacteria and provides only a total number of bacteria.",
"U.S. Pat.",
"Nos.",
"5,972,641 and 5,518,894 disclose a rapid coliform detection systems using a statistic methods for determining the number of bacteria.",
"Said methods require up to 11 hours for obtaining the results in low number of bacteria.",
"Other patents disclose a method for detection of microorganisms using fluorescence and laser light source (U.S. Pat.",
"Nos.",
"5,891,394, 5,858,697, 5,763,203, 5,751,839 and U.S. Pat.",
"No.",
"5,663,057).",
"The disadvantages of said methods is the use of an expensive laser light source and the detection of microorganisms directly from the filter which is not smooth and causes problems during analysis.",
"In addition, these systems are not portable and are relatively expensive.",
"Immunoassay methods are also used for detecting certain types of microorganisms (Lee et al., App.",
"Environ.",
"Microbiol., Vol.",
"56, pp.1541-1546).",
"In these methods, specific antibodies labeled with a fluorescent or radioactive dye are used to detect the microorganism.",
"However, immunoassay methods are limited in that they require the production of antibodies against each microorganism of interest, which is time-consuming and expensive.",
"“Sol-gel” is the term used to indicate inorganic glass manufactured at room temperatures based on metal oxides.",
"A certain process involving ceramic materials in which the sol (solution) is transformed to a gel phase through hydrolysis, condensation and polymerization.",
"The common starting materials for the sol-gel preparation are ormosils or metal oxides.",
"In recent years sol-gel has been applied to organosilanes to create “glass at room temperature”.",
"Sol-gel type materials comprise pores ranging from tens of angstroms to tens of nanometers, and exhibit a large area to mass ratios e.g., hundreds of square meters per gram.",
"Sol-gel materials are transparent even at UV wavelengths, and are simple to prepare in different shapes, such as powders, monolithic blocks, thin sheets, fibers etc.",
"The use of sol-gel-based materials to entrap various organic molecules in a matrix media was described in the art.",
"(Avnir et al., Supramolecular architecture in two and three dimensions Bein T.",
"(ed.)",
"American Chemical Society Symposium Series XXX, 1992).",
"Using said technology, organic molecules are entrapped at room temperatures within the sol-gel matrix without impairing the structure of the relatively sensitive organic molecule.",
"In addition, the entrapped molecule retains almost all of the original physical and chemical characteristics, and is available to outside reactants as a result of the massive pore system inside the sol-gel.",
"U.S. Pat.",
"No.",
"6,022,748 discloses a method for the direct detection of analytes using color changes in response to selective binding of analytes to a surface.",
"Said detection occurs in immobilized biopolymeric material encapsulated into metal oxide glass using the sol-gel method.",
"The disadvantages of this method are that only large amounts of bacteria can be detected or enumerated, since only high counts are able to cause a visible color change in the sol-gel.",
"Furthermore, said method cannot differentiate between viable and non-viable microorganisms, since it is based on the binding of the microorganisms to the sol-gel surface, independent whether said microorganisms are viable or not.",
"Armon et al.",
"(J. of Biotechnology 51, 279-285) disclose a method for detecting large quantities of E. coli bacteria by spreading them on sol-gel doped with specific compounds, said compounds being uptaken into the bacteria, which consequently causes the bacteria to glow at specific wavelengths.",
"However, this method does not provide a method suitable to count bacteria present in a given sample.",
"In addition, detection of low number of microorganisms is not possible using said method.",
"The art has so far failed to provide a fast method for the enumeration of microorganisms, which is sensitive enough to provide a reliable count at low microorganism concentrations.",
"It is a purpose of this invention to provide a fast and sensitive method for the detection of viable microorganisms.",
"It is another object of the invention to provide a method and compositions useful in providing an enumeration of microorganisms found in low-count samples.",
"Other objects and advantages of the invention will become apparent as the description proceeds.",
"SUMMARY OF THE INVENTION It has now been surprisingly found, and this is an object of the present invention, that viable microorganisms at a concentration lower than 103 ml−1 may be detected and enumerated in a 2-hours period, using organic or inorganic substances that are entrapped in porous sol-gel glass (hereinafter referred to as “markers”, for the sake of brevity).",
"The present invention relates to methods and compositions for the detection and enumeration of viable microorganisms using organic or inorganic substances that are incorporated in porous sol-gel glass (markers).",
"The microorganisms metabolize said markers, and thereby emit detectable radiation, electromagnetism or fluorescence.",
"Thus, a fast visualization of viable microorganisms is obtained on the sol-gel glass.",
"The sol-gel surface is extremely smooth and transparent and allows for an almost uniform focal point for high resolution microscopic scanning.",
"According to a preferred embodiment of the invention, the following steps are carried out: a) Providing a liquid composition comprising one or more marker(s) incorporated in a liquid sol-gel precursor; b) Coating a transparent slide, e.g., a glass slide, with a thin uniform layer of said liquid sol-gel precursor composition; c) Separating the microorganisms from a liquid sample to be analyzed by passing said sample through a filter, and then bringing said filter into close contact with the sol-gel-coated slide; d) Co-incubating said filter with said sol-gel-coated slide for a period of time and at a temperature suitable to promote uptake of the marker(s) by the microorganisms, e.g.",
"at 35-44° C. for 0.5 to 6 hours; e) Irradiating said sol-gel-coated slide with an external energy source such as to generate detectable signals emitted from the marker(s) metabolized by the microorganisms; and f) Acquiring images of said detectable signals emitted from the microorganisms, and analyzing said images using a computer system, thereby to provide the identification and enumeration of said microorganisms.",
"The present invention further relates to a method for preparation of a liquid sol-gel mixture containing organic or inorganic substances (markers).",
"Said markers are metabolized by the microorganisms that are to be identified.",
"Coliform bacteria that can be detected according to the present invention are actually a broad group of bacteria that include E. coli, Enterobacter spp., Klebsiella spp.",
"and Citrobacter spp.",
"Coliform bacteria are identified by detecting the activity of an enzyme, β-Galactosidase (E.C.",
"3.2.1.23), using fluorogenic or chromogenic substances 3-carboxyumbelliferyl β-D-galactopyranoside (CUG) or 4-chloromethyl-6,8-difluoroumbelliferyl β-D-galactopyranoside (CMDi FUG).",
"According to the present invention, E. coli are identified by detecting the activity of an E. coli-specific-enzyme, β-Glucuronidase (GUS or EC 3.2.1.31), using the following fluorogenic or chromogenic substances: 4-methylumbelliferyl β-D-galactopyranoside (MUG), fluorescein di β-D-galactopyranoside (FDG), 6,8-difluoro-4-methylumbelliferyl β-D-glucuronide, lithium salt (DiFMUGGlcU), 2-dodecylresorufin, Elf-97, fluorescein di-β-glucoronide (FDGlcU), 5-(pentafluorobenzoylamino) fluorescein di-β-D-glucoronide (PFB-FDGlcU) and β-trifluoromethylumbelliferyl β-D-glucoronide.",
"According to another preferred embodiment of the invention, an antibiotic material is incorporated into the sol-gel mixture for identifying bacterial antibiotic resistance.",
"It may be appreciated that the emission of fluorescence from bacteria in spite of the presence of a specific antibiotic, indicates that said bacteria is antibiotic-resistant.",
"Partial resistance may be indicated when the presence of the specific antibiotic leads to a partial reduction in the number of fluorescent bacteria.",
"The following antibiotics are added to the sol-gel mixture: Chloramphenicol, Erythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethoprin, Rifanapicin and Penicillin.",
"According to the present invention, it is possible to provide the markers as liposomes; films; multilayers; braided, lamellar, helical, tubular, and fiber-like shapes; solvated rods; solvated coils; and combinations thereof.",
"According to the present invention, it is also possible to detect injured or stressed microorganisms by incorporating pyruvate or K2SO4 within the sol-gel.",
"It is known in the art that pyruvate or K2SO4 can be used to resuscitate or improve chlorinated injured coliform bacteria (“enumeration and differentiation of chlorine-stressed total coliform bacteria” Robert A Duncanson—Ph.D.",
"dissertation—University of Rhode Island 1993).",
"According to the present invention it is possible to add polylysine or similar substances at a concentration of between 10 to 100 parts per million to the sol-gel solution to allow for enhancing bacteria absorption.",
"All the above and other characteristics and advantages of the invention will be further understood from the following illustrative and non-limitative examples of preferred embodiments thereof.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1 illustrates a system for identifying and enumerating microorganisms, according to a preferred embodiment of the invention; FIG.",
"2 is a fluorescence picture of E. coli bacteria on a sol-gel surface at ×400 magnification; and FIG.",
"3 is a flow-chart of a process according to a preferred embodiment of the invention.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS According to a preferred embodiment, the system for enumeration of bacteria comprises the following components shown in FIG.",
"1: standard frame grabber coupled with a computer, processing and controlling software, and motor and light control apparatus- collectively indicated at (1), standard CCD array camera with frame rate of 30 fps and frame dimensions of 640×480 (2), an epi-fluorescence or fluorescence microscope with a 400 and/or 1000 magnification and with an autofocus system (3), a light source (4), a beam splitter (5), a mechanical x-y table (6) on which a filter (7) is placed.",
"In another preferred embodiment of the invention, instead of utilizing a standard CCD system, a CCD Line Approach is used comprising a CCD line camera, Line dimension: 512 pixels, Line rate: 70 kHz equivalent to 135 frames of 512×512 pixel2 per second, Custom-design signal synchronization hardware, Custom-design digital/analog signal processing hardware For the purpose of clarity, and as an aid in the understanding of the invention, as disclosed and claimed herein, the following abbreviations are defined below: CFU—colony forming unit.",
"MUG—4-methylumbelliferyl β-D-galactopyranoside FDG—fluorescein di-β-D-galactopyranoside CUG—3-carboxyumbelliferyl β-D-galctopyranoside EXAMPLE 1 Preparation of Sol-gel-coated Glass Slides Standard microscope slides were washed in soap water by gentle shaking for 30 minutes, and were then initially rinsed with tap water, followed by an additional rinse with tri-distilled water.",
"The slides were then dried over night in an incubator at (36° C.).",
"The cleansed microscopic slides were then stored in soft paper for further use.",
"A typical starting solution for the thin sol-gel film preparation was as follows: 0.9 ml pure pyridine+0.2 ml CUG solution (100 mM)+0.1 ml H2O were stirred for 1 minute (with a small magnet).",
"If the substrate did not completely dissolve only the upper phase of the mixture was used.",
"0.8 ml of the above mentioned solution were mixed with 0.4 ml of methyltrimethoxy silane (MTMS, Fluka).",
"The whole solution was well mixed by magnetic stirrer for additional 2 minutes.",
"For reaction initiation, 30 μl of HCl 0.1 M were added to the above solution, followed by 5 minutes magnetic stirring.",
"The slides were then coated with 40 μl of the resulting solution by using a fine pipette.",
"The solution was spread equally along the glass by means of a spin coating.",
"The coated glass was dried in darkness for a period of 24 hours under dry conditions and at room temperatures.",
"After the initial drying period, the sol-gel-coated slides were wrapped with aluminium foil and stored in a cool place.",
"EXAMPLE 2 Example 1 was repeated, using a 0.1 ml solution of 4′-6-diamino-2-phenyl indole at a 0.016 mg/ml concentration.",
"The results obtained was similar to that of Example 1.EXAMPLE 3 E. coli Identification and Enumeration An E. coli bacteria culture was grown in a nutrient broth for 24 hours at 36° C. A seeded solution of bacteria with an estimated concentration of 108 bacteria/ml was prepared in sterile distilled water.",
"A series of different solutions was prepared (350 ml of each solution) with an anticipated bacteria concentration as follows: 109 bacteria in 100 ml, 108 bacteria in 100 ml, 107 bacteria in 100 ml, 106 bacteria in 100 ml 105 bacteria in 100 ml.",
"All bacteria concentrations were verified by performing parallel membrane filtration (MF) test in water samples.",
"This was done by diluting the solution with sterile distilled water in sterile plastic bottles so that a dilution of up to 1:108 was accurately achieved.",
"In order to count the bacteria in a given sample the water sample was first filtered through a Millipore filter (0.47 μm, 13 mm).",
"The Millipore filter was then placed upside down on the sol-gel surface.",
"In order to create full contact with the filter and the sol-gel, a minute quantity of sterile water (10 μl) was placed over the filter surface and pressure (up to 0.5 kg/cm2) was applied.",
"The bacteria were then incubated together with the sol-gel for a period of 2.0 hours at 36° C. in a humid container (consisting of a large petri dish with a moist Wattman pad, in order to prevent the filter from drying and breaking away from the sol-gel).",
"The Millipore filter was then discarded and the slide was dried during 10-15 minutes at 44.5° C. For the purpose of enumerating the bacteria, the sol-gel slide was viewed under a microscope system (Zeiss Axiolab with an epiflourescence illumination system and a 50 W mercury lamp) with a magnification of ×400 that utilizes a LP420 filter.",
"In order to eliminate natural fluorescent algae which may create false positive results, specific light wavelengths were used (e.g., a LP420 filter).",
"Pictures of the fluorescent spots were taken at 5 various random locations on the sol-gel-coated slide.",
"FIG.",
"2 is a picture taken at 400 magnification of E. coli bacteria on a sol-gel surface.",
"The number of fluorescence spots were then counted by using image processing software (Image-Pro Plus, Media Cybernetics).",
"The number of counts obtained following the analysis is indicated in Table I.",
"TABLE I Initial Number of Estimated Average Coliform Fluorescent Number of Number of Count Spots Bacteria Bacteria Test (CFU/ Picture Counted (CFU/ (CFU/ Number 1 ml) Number (Units) 1 ml) 1 ml) 1 106 1 550 980,000 1,014,000 2 500 890,000 3 600 1,070,000 4 575 1,020,000 5 625 1,110,000 2 105 1 57 83,000 96,800 2 56 99,000 3 50 89,000 4 58 103,000 5 62 110,000 3 104 1 5 8,900 9,600 2 6 10,700 3 8 14,200 4 5 8,900 5 3 5,300 Since the area of vision of the microscope was known, the number of fluorescent spots counted in each slide gives an exact estimate of the initial amount of bacteria that was in the original solutions.",
"EXAMPLE 4 Description of Algorithm to Enumerate the Number of Fluorescence Spots Using Image-Pro Plus The algorithm described here is an example used for detecting and enumerating bacteria on sol-gel images.",
"The purpose of this algorithm is to detect bright spots with the expected size: Low-pass filtering was used for high frequency noise reduction.",
"Image segmentation was used leading to a binary image of white blobs of bacteria on black background.",
"The segmentation in this implementation was done using a simple threshold application, with a constant pre-defined threshold.",
"The analysis comprises the following steps: A) Binary image labeling, resulting in a list of blob objects, candidates for being indicated as bacteria.",
"B) Geometry features calculation of the blob list elements (area, contrast, etc.).",
"C) Elimination of candidates of which size and contrast are outside the expected range.",
"D) Enumeration of the candidates left after the geometrical filtering.",
"p An example script of the above algorithm is shown in FIG.",
"3, which illustrates a process comprising the following stages: Stage 1: Low-pass filtering for high frequency noise reduction.",
"Stage 2: Image segmentation that results in a binary image of white blobs of bacteria on black background.",
"The segmentation in this implementation is done using a simple threshold application, with a constant pre-defined threshold.",
"If necessary, an automatic and/or dynamic threshold calculation can be used.",
"Stage 3: Binary image labeling, resulting in a list of blob objects, candidates for being indicated as bacteria.",
"Stage 4: Geometry features calculation of the blob list elements (area, contrast, etc.).",
"Stage 5: Elimination of candidates the size and contrast of which are outside the expected range.",
"Stage 6: Enumeration of the candidates left after the geometrical filtering.",
"While specific embodiments of the invention have been described for the purpose of illustration, it will be understood that the invention may be carried out in practice by skilled persons with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims."
]
] | Patent_10415844 |
[
[
"Extension arm for a sheet-processing printing machine",
"A sheet delivery mechanism for a sheet-fed printing machine which is adapted to reduce changes in the position of sheet material in a stack during a stack changeover operation and which facilitates the formation of a stack with properly aligned edges.",
"The sheet-delivery mechanism includes at least one length adjustable edge guide 16 supported between a stack support plate 14 and a cross element 15 which can be detachably connected to the frame of the sheet delivery mechanism."
],
[
"1-10.",
"(canceled) 11.A sheet delivery mechanism for a sheet-fed printing machine comprising an endless conveyor for transporting printed sheet material to a delivery stack formed by stacking said sheet material, a separating device for separating the delivery stack into an auxiliary stack and a main stack, a stack lifting device including a vertically movable stack support plate for supporting the delivery stack, at least one edge guiding device connected to said support plate and extending in parallel vertical relation adjacent to an edge of the main stack formed by stacking the sheet material, and said edge guide device having a vertical length that varies depending upon the vertical position of said support plate.",
"12.The sheet delivery mechanism of claim 11 including at least two said edge guiding devices along a common edge of said main stack.",
"13.The sheet delivery mechanism of claim 11 in which said edge guiding device is connected in vertically extended fashion between said support plate and a frame of said edge guiding device.",
"14.The sheet delivery mechanism of claim 11 in which said edge guiding device is connected at one end to a crossarm detachably mounted to said frame.",
"15.The sheet delivery mechanism of claim 11 in which said edge guiding device includes a flexible material vertically positioned adjacent an edge of said main stack and a winding real for winding said flexible material upon itself during raising movement of said support plate.",
"16.The sheet delivery mechanism of claim 15 in which said flexible material is a band.",
"17.The sheet deliver system of claim 11 in which said edge guiding device is arranged in parallel adjacent relation to a front edge of the main stack formed by stacking the sheet material.",
"18.The sheet delivery mechanism of claim 11 in which said edge guiding device is arranged in parallel adjacent relation to a lateral edge of a main stack formed by the sheet material.",
"19.The sheet delivery mechanism of claim 11 in which said edge guiding device is arranged in parallel adjacent relation to a rear edge of the main stack formed by stacking the sheet material .",
"20.The sheet delivery mechanism of claim 11 in which said sheet guiding device changes in length as an incident to vertical movement of the support plate.",
"21.The sheet delivery mechanism of claim 11 in which the vertical length of said edge guiding device is changeable in synchronism with vertical movement of the stack support plate.",
"22.The sheet delivery mechanism of claim 11 in which said edge guiding device is arranged a predetermined short distance from the edge of the main stack.",
"23.The sheet delivery mechanism of claim 11 in which said edge guiding device includes a tensioned band material that is wound upon itself to shorten its length upon raising movement of said support plate.",
"24.The sheet delivery mechanism of claim 11 in which said edge guiding device includes a winding device mounted on the stack for winding the edge guiding device to change its length.",
"25.The sheet delivery mechanism of claim 24 in which said winding device is removably mounted on said stack support plate.",
"26.The sheet delivery mechanism of claim 11 in which said main stack of sheet material is supported by said support plate in inclined relation to the horizontal toward an adjacent edge guiding device."
],
[
"The invention pertains to a sheet delivery mechanism for a sheet-fed printing machine with a device for changing over from one stack to another according to the preamble of claim 1.PRIOR ART A sheet delivery mechanism of this type is known from DE 195 16 071 A1.This sheet delivery mechanism contains a device for changing over from one stack to another in a sheet-fed printing machine with continuous sheet feeding.",
"In this case, a separating element is used which is composed of a shaft arranged outside the stacking region and one or more wedges, wherein said separating element produces the triangular space required for inserting the stack rake.",
"The separating element lies on the top sheet of the main stack in the region of the front edge.",
"The additional sheets received are simultaneously placed on the auxiliary stack now being formed above the separating element.",
"The sheets accumulated above the separating element are released when the separating element is turned about the shaft and follows the main stack being lowered, wherein the separating element is subsequently pivoted out of the stacking region.",
"This solution can only be used with stack rakes because the wedges must contact the upper edge of the main stack through the intermediate spaces of the stack rake.",
"A sheet delivery mechanism with a device for changing over from one stack to another in a sheet-fed printing machine with continuous sheet feeding is also known from DE 198 19 491 C1.This device is arranged upstream of a delivery stack (first stacking system) and functions as a switch, wherein said device selectively deposits the sheets on the delivery stack (first stacking system) or on a second stacking system arranged upstream of the first stacking system.",
"During nonstop stack changing mode, for example, the switch is activated in such a way that the sheets can be deposited on the second stacking system.",
"In the meantime, the main stack of the first stacking system, for example, can be lowered and transported away from the sheet delivery mechanism, wherein a new stack board or stack rake is previously inserted, and wherein the sheets that were transported to the second stacking system are now fed to the delivery stack (first stacking system).",
"The stack of the second stacking system can be exchanged in the meantime.",
"When changing a stack, it is disadvantageous that sheet material stacked properly aligned may change its position in the stack when the stack is changed.",
"The lowering movement and/or the air enclosed between the sheets, particularly, in the upper region of the stack (reduction in static friction) may cause the sheets to change their position in the stack.",
"OBJECTIVE OF THE INVENTION The invention is based on the objective of developing a sheet delivery mechanism of the initially described type which eliminates the aforementioned disadvantages and, in particular, noticeably reduces changes in position in the sheet material when changing from stack to stack, such that the formation of a stack with properly aligned edges can be achieved.",
"According to the invention, this objective is attained with the characteristics of claim 1.Additional refinements are disclosed in the dependent claims.",
"A first advantage of the invention is that the sheet delivery mechanism contains an edge guiding device of variable length that is assigned to and located against at least one edge of the sheet material being stacked, for example, the front edge, wherein said edge guiding device is arranged on a vertically movable stack support plate and on a crossarm that preferably is mounted separably on the frame.",
"This edge guiding device guides the sheet material into its predetermined stacking position and, in particular, guides the sheet material when the main stack is lowered.",
"This prevents the material being printed from changing its position in the stack, for example, by shifting, and the proper alignment of the stack edges achieved during the sheet delivery is preserved when (the stack of) sheet material is lowered.",
"It is also advantageous if the length of the edge guiding device is variable during the vertical upward and downward movement of the stack.",
"Independently of the stack size, this ensures a continuous guidance in the region of one edge, for example, the front edge, of the stacked sheet material when a stack is changed, particularly during the lowering movement.",
"It is also advantageous if the edge guiding device is arranged a short distance from the edge of the sheet material forming the stack.",
"This makes it possible to prevent friction between one edge of the sheet material and the edge guiding device during the lowering (relative movement) of the stack.",
"This friction could negatively influence the desired position of the sheet material in the stack.",
"It is equally advantageous if the edge guiding device is preferably arranged on a crossarm, and if this crossarm is separably connected to the frame of the sheet delivery mechanism.",
"The edge guiding device preferably is separably connected to the crossarm.",
"This makes it possible also to realize the stacking operation without an edge guiding device, wherein required free space is not blocked during the stack change.",
"According to another embodiment, it is advantageous if the separable crossarm can be detached from the frame of the sheet delivery mechanism, preferably in a manual fashion, and separably fixed, for example, on the stack support plate together with the edge guiding device (parking position).",
"The sheet delivery mechanism according to the invention can be universally used, for example, for the non-stop stack change mode or for the packaging mode (separated partial stacks containing a certain quantity of sheet material).",
"The invention is not limited to the use of only one edge guiding device.",
"On the contrary, it would also be possible to arrange several edge guiding devices on the given edge(s) of the stack.",
"EXAMPLES The invention is described in greater detail below with reference to an embodiment example.",
"The figures schematically show: FIG.",
"1, a sheet delivery mechanism of a sheet-fed printing machine; FIG.",
"2, a front view of a sheet delivery mechanism with two edge guiding devices, and FIG.",
"3, a side view of the sheet delivery mechanism according to FIG.",
"2.A sheet delivery mechanism 1 for the non-stop stack change mode essentially consists of a revolving conveyor system 4 with gripper systems 3 arranged thereon, wherein said gripper systems feed the sheet material 2 arriving from an upstream printing station or converting station to a delivery stack 5.During this process, the sheet material 2 is guided along stationary sheet guiding elements 8 and sheet guiding elements 10 that can be adjusted depending on the format and the print image.",
"The sheet guiding elements 8, 10 preferably can be selectively controlled by means of blasting air and suction air.",
"A pneumatic system 6 that assists in depositing the arriving sheet material 2 on the delivery stack 5 is arranged above the delivery stack 5.Conventionally, the sheet material 2 arriving in the transport direction 9 is decelerated by a sheet decelerating device 7 and deposited onto the delivery stack 5 such that its front edges contact limit stops 11.Pivoted guides that, for example, consist of angle rails and serve for accommodating an auxiliary stack support 12, e.g., a board or a rake, are arranged parallel to the lateral frames of the sheet delivery mechanism 1.The auxiliary stack support 12 can be horizontally inserted in the inserting direction 13 and-retracted opposite to the inserting direction 13.In order to remove sample sheets or to form an auxiliary stack in the delivery stack 5, the delivery mechanism is provided with known front holding elements for holding up the sheets which extend over the width of the sheets and can be selectively acted upon by blown air or suction air.",
"The front sheet holding elements are arranged such that they protrude into the region of the delivery stack 5 at an acute angle referred to the horizontal line.",
"The front sheet holding elements can be directly moved from a waiting position in the front edge region outside the delivery stack 5 into the stacking region underneath the arriving gripper systems 5 and lowered together with the arriving sheet material 2 in order to form an auxiliary stack.",
"A crossarm 15 that is preferably mounted separably on the frame of the sheet delivery mechanism on both sides and extends at least over the format width is situated underneath the guides for the auxiliary stack support 12 in the region of the front edge of the delivery stack 5, particularly in the region of the main stack.",
"The delivery stack 5 also contains a stack support plate 14 for receiving a pallet 19 with sheet material 2 stacked on top of one another.",
"The stack support plate 14 is preferably provided with limit stops 18, wherein the pallet 19 is aligned relative to these limits stops.",
"At least one, preferably two edge guiding devices 16 of variable length are arranged adjacent to one another (aligned) on the stack support plate 14 and on the crossarm 15.Each edge guiding device 16 is assigned to and located against the front edge 21 of the main stack being formed by the sheet material 2 as part of the delivery stack 5, wherein the respective edge guiding devices are arranged parallel to and separated from the front edge of the stack by a predetermined distance 20.The length of each edge guiding device 16 can be varied when the stack support plate 14 is raised or lowered by means of a stack lifting device in order to follow the vertical movement of the stack support plate 14.The change in length of each edge guiding device 16 preferably can be realized synchronously with the vertical movement of the stack support plate 14.In order to prevent changes in position of the stacked sheet material 2, the edge guiding device 16 is arranged a slight distance 20 from the front edge 21 of the main stack.",
"The distance 20 is chosen such that any friction between the front edge 21 of the sheet material 2 and the edge guiding devices 16 which could impair the position of the sheet material 2 is prevented when the main stack is lowered.",
"According to one embodiment, the edge guiding device 16 consists of at least one band that can be wound and preferably is under tension.",
"In an alternative embodiment, the edge guiding device 16 contains at least one cable or a corresponding belt that can be wound and is under tension.",
"Each edge guiding device 16 preferably contains a winding device 17 that is arranged on the stack support plate 14.Alternatively, the winding device 17 may also be arranged on the crossarm 15.The winding device 17 serves for winding and unwinding the band material.",
"In one preferred embodiment, the crossarm 15 can be separated from the frame of the sheet delivery mechanism and deposited on the stack support plate 14 so that it is secured in its position.",
"The band material stored on the winding device 17 is rigidly or separably connected to the crossarm 15 in this embodiment.",
"When depositing the crossarm 15 on the stack support plate 14, the winding device 17 winds the band material, preferably with the aid of a spring.",
"This also applies to the embodiment in which the edge guiding device 16 or the band material can be separated from a crossarm 15 that remains rigidly connected to the frame.",
"The invention is not limited to only one edge guiding device 16 that is assigned to the front edge 21 of the delivery stack 5.On the contrary, a single or multiple arrangement of (aligned) edge guiding devices 16 can be provided parallel and adjacent to at least one lateral edge and/or the rear edge of the main stack being formed by stacking sheet material 2 on top of one another.",
"When assigning an edge guiding device 16 to the lateral edges and/or the rear edge of the delivery stack 5, the processing of different sheet formats should be taken into consideration.",
"For this purpose, the crossarms 15 and the edge guiding devices 16 are preferably arranged on the frame of the sheet delivery mechanism or the stack support plate 14 or on the pallet 19 such that they can be adapted to the respective formats.",
"In principle, the winding device 17 for the edge guiding device 16 may be selectively arranged on the stack support plate 14 (and also on the pallet 19 in the region of the lateral and rear edges) or on the crossarm 15.In another embodiment, the stacked sheets 2 of the main stack can be pivoted in the transport direction 9 from the essentially horizontal stacking plane, such that their front edges are inclined toward an adjacent vertical edge guiding device 16.This pivoting motion of the main stack may be realized, e.g., by pivoting the stack support plate 14 out of the horizontal plane by means of an actuating device that acts upon the stack lifting device at least while the stack is lowered.",
"For example, the stack support plate 14 can be raised in the vertical direction by a defined amount with tension means of the stack lifting device which only act upon the rear edge of the delivery stack 5.This causes the stack support plate 14 (with the delivery stack 5) to become inclined in the direction of the edge guiding device 16 assigned to the front edge of the delivery stack 5.In another embodiment, at least one wedge can be placed onto the stack support plate 14 in the region of the rear edge of the delivery stack 5 (before the stack is formed).",
"This wedge engages underneath the pallet 19 and thus inclines the pallet 19 in the transport direction 9.This causes the front edge of the delivery stack 5 to become slightly inclined toward the edge guiding device 16.In another embodiment, a tilting device is arranged on the stack support plate 14 in the region of the rear edge of the delivery stack 5.This tilting device acts upon the pallet 19 in the region of the rear edge by means of an actuating device, for example, a pneumatic or hydraulic actuating device, and causes the delivery stack 5 to become inclined toward the edge guiding device 16.The inclination of the delivery stack 5 must be realized in such a way that the guidance along the respective edges takes place in a largely contactless fashion and that a possible excursion of the edge guiding device 16 from the vertical line due to a contact with the sheet material 2 is prevented."
]
] | Patent_10415871 |
[
[
"Arrangement in ventilatory treatment of the lungs",
"The invention relates to a valve arrangement in the ventilatory treatment of the lungs of a living creature (2) under a state of anaesthesia, comprising valve equipment (4) devised to carry an insufflation gas and/or an insufflation gas mixture from a lung ventilation unit (3) into the lungs and airways (2a) of the living creature (2) and allow expiration of gas and/or gas mixture held in the airways.",
"The said valve equipment (4) and a combination of sensor units (61, 61A, 62) are arranged in close proximity to one another.",
"They are inter-connected by an end section for a bundle of tubing (30) from the lung ventilation unit (3), and the said sensor units (61, 61a, 62) are located closer to the said living creature (2) than the said valve equipment (4)."
],
[
"1.A valve arrangement in the ventilatory treatment of the lungs of a living creature under a state of anaesthesia, comprising valve equipment devised to carry an insufflation gas and/or an insufflation gas mixture from a lung ventilation unit into the lungs and airways of the living creature and allow expiration of gas and/or gas mixture held in the airways, a sensor unit in gas-measuring equipment, the sensor unit being devised to sense the velocity-related pressure gradient in measuring the velocity of the insufflation gas and the duration of the insufflation phase, a sensor unit devised to sense the velocity-related pressure gradient in measuring the velocity of expired gas and the duration of the expiration phase, a sensor unit for measuring the pressure of the gas and its variation in pressure over time and/or a sensor unit for diverting part of the gas mixture for measuring the presence of and/or percentage of one or more gas components in the gas mixture, characterized in that valve equipment has a valve housing consisting of at least two parts, and these parts can be conjoined or separated by means of an end-related rotary movement.",
"2.An arrangement according to claim 1, characterized in that the said rotary movement is arranged around an axis of rotation at the end of a section of tubing intended for e.g.",
"insufflation gas.",
"3.An arrangement according to claim 1, characterized in that the end of at least one section of tubing has the shape of part of a sphere.",
"4.An arrangement according to claim 1, characterized in that a ring-shaped gasket is arranged between the parts in one of the parts and has the external shape of part of a sphere.",
"5.An arrangement according to claim 3, characterized in that part of the sphere formed by the ring-shaped gasket is truncated by two planes located on either side of the centre of the sphere, forming small circles with the same or different diameters.",
"6.An arrangement according to claim 1, characterized in that one of the said two parts has a system of channels for connection to a means for coupling a plurality of tubes, preferably gathered in a bundle of tubing.",
"7.An arrangement according to claim 1, characterized in that one of the parts has a lid devised for sealed contact with a membrane in a valve located between the parts.",
"8.An arrangement according to claim 1, characterized in that one of the parts has a number of channel-closing first coupling means.",
"9.An arrangement according to claim 8, characterized in that the said first coupling means is devised to enclose the second part's second coupling means.",
"10.An arrangement according to claim 4, characterized in that the elasticity of the said ring-shaped gasket is such that the first and second coupling means can made to interact axially during relative movement of the first and second part.",
"11.An arrangement according to claim 1, characterized in that one of the said parts is provided with a locking arrangement for firm locking to the second part when these parts are conjoined."
],
[
"<SOH> FIELD OF INVENTION <EOH>The present invention relates to an arrangement for ventilatory treatment of the lungs of a living creature and has been devised primarily for use during a state of anaesthesia caused by intravenous infusion of a liquid anaesthesia-inducing drug.",
"The arrangement in accordance with the present invention comprises valve equipment devised to carry an insufflation gas and/or an insufflation gas mixture from a lung ventilator unit to the lungs and airways of the living creature and allow controlled expiration of the gas and/or gas mixture held in the airways.",
"The arrangement and equipment can additionally comprise one or more sensors belonging to gas measuring equipment.",
"One of these sensors can then be devised to sense a pressure gradient related to gas velocity in measuring the velocity of insufflation gas and the duration of the insufflation phase.",
"Another sensor unit, or the same one as above, can be devised to sense a pressure gradient related to gas velocity in measuring the velocity of expired gas and the duration of the expiratory phase.",
"Yet another sensor can be devised to measure the pressure and/or time-related variations in the pressure of insufflation gas and/or expired gas.",
"Yet another sensor can be devised to divert part of the gas mixture for analysis for measurement of the presence of an/or percentage of one or more gas components in the gas mixture.",
"The invention relates more specifically to a refinement of the arrangement taught and disclosed in the Swedish patent application 99 02051-3, filed on 3 Jun.",
"1999 and entitled “Arrangemang vid en lungventilatorisk behandling” (Eng.",
"=Arrangement in ventilatory treatment of the lungs.)"
],
[
"<SOH> SUMMARY OF THE PRESENT INVENTION <EOH>Technical Problems When the technical deliberations that a person skilled in this particular art must make in order to provide a solution to one or more technical problems she/he encounters are taken into consideration, it will be seen that it is initially necessary to realise the measures and/or sequence or measures that must be undertaken to this end and to realise which means is/are required.",
"On this basis, the technical problems listed below should be relevant to development of the present invention.",
"When the prior art, as described above, is considered, it will be seen that a technical problem resides, in the ventilatory treatment of the lungs of a living creature under a state of anaesthesia created by intravenous infusion of a liquid anaesthesia-inducing drug, in disclosing a valve arrangement consisting of control-lable valve equipment, combined with a plurality of sensor units which can be located very close to the creature.",
"It will also be seen that a technical problem resides in the provision of conditions, with simple means, enabling the arrangement and the valve equipment to offer less dead space in relation to known equipment for this application.",
"It will also be seen that a technical problem resides in realising the significance of and the advantages for a patient under a state of deep anaesthesia, induced by an intravenous infusion of a liquid drug, of being able to offer utilisation of only one insufflation tube, devised with one cross-section for being able to carry an insufflation gas or gas mixture from the lung ventilator to the patient and, additionally, a plurality of tubes with a much smaller cross-section for determining in the lung ventilator different criteria for the gas or gas mixture and for controlling a valve in the valve equipment from the lung ventilator via a lung ventilator valve.",
"It will also be seen that a technical problem resides in realising the importance of and the advantages afforded by development of a valve arrangement made from a few simple parts, the parts being devised for easy cleaning, rinsing and/or replacement.",
"It will also be seen that a technical problem resides in realising the importance of and advantages afforded by having the valve equipment comprise a valve housing, enclosing sensor units, which can be made in two parts that attach to one another with a rotary movement.",
"These parts can be conjoined or separated by means of this rotary movement.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by arranging the said rotary movement around an end-oriented valve housing's axis of rotation.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by devising the area around the end-oriented axis of rotation with a passage for gas and an elastic ring-shaped gasket.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by having the said elastic gasket absorb the relative, axial movement between the said two parts forming the valve housing in order to provide a tight seal between the respective connector means for each part for sensor unit channel tubes sections when the parts are conjoined.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by using an elastic ring-shaped gasket between the parts.",
"The gasket can be mounted on one of the parts and have the shape of part of a sphere.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by truncating the part of the sphere with two planes, located on either side of a sphere's centre, forming small circles with the same or different diameters.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by having one of the said two parts contain a system of channels for interconnection to a coupling means for a plurality of tubes, preferably arranged in a single tubing bundle.",
"It will also be seen that a technical problem resides in realising the importance of and advantages afforded by orienting the system of channels so as to connect openings in the coupling means with the openings in one, the first, of the said two parts.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by equipping one of the parts with a lid, devised to seal against a peripheral area of a valve membrane.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by devising one of the parts with a number of channel system first coupling means, arranged in a row, in a two-part coupling arrangement, preferably with an intermediate gasket.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by devising the said first coupling means on one part to enclose a second coupling means on the second part in the same coupling arrangement.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by using a said ring-shaped elastic gasket with an elasticity allowing the first and second coupling means, in a coupling arrangement, to interconnect during cam-guided relative movement between the first section and the second section.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by providing one of the said two parts with a locking means, such as one or two clamps, so the first part locks firmly to the second part.",
"Solution With the intention of solving one or more of the aforesaid technical problems, the present invention takes as its starting point a valve arrangement in ventilatory treatment of the lungs of a living creature under a state of anaesthesia.",
"It comprises valve equipment devised for passing an insufflation gas and/or an insufflation gas mixture from a lung ventilation unit into the creature's lungs and airways and allowing expiration of gas and/or gas mixture held in the airways, and one or more sensor units in gas measuring equipment, one of the sensor units being devised for sensing the velocity-related pressure gradient in measurement of the velocity of the insufflation gas and the duration of the insufflation phase.",
"One, another or the same sensor unit is devised for sensing the velocity related pressure gradient in measuring the velocity of expired gas and the duration of the expiratory phase.",
"A second sensor is devised for measuring gas pressure and/or variations in pressure over time.",
"One sensor can be devised to divert part of the gas mixture for gas analysis in measuring the presence of and/or percentage of one or more gas components in the gas mixture.",
"With the intention of solving one or more of the aforesaid technical problems, the present invention proposes that the said valve equipment comprise a valve housing, which can consist of two parts.",
"These parts can be conjoined or separated by means of an end-related rotary movement.",
"As proposed embodiments that lie within the scope of the inventive concept, it is also proposed that the said rotary movement be arranged around an axis of rotation at the end of a section of tubing for e.g.",
"insufflation gas.",
"It is further proposed that the end of the section of tubing be in the shape of part of a sphere.",
"A ring-shaped gasket is arranged on one of the parts and has an external shape corresponding to part of a sphere.",
"It is further proposed that the part of the sphere be truncated by two planes on either side of a sphere's centre, forming small circles with the same or different diameters.",
"It is further proposed that one of the said two parts comprise a system of channels for interaction with a coupling means for a plurality of tubes, preferably arranged in a single bundle of tubing.",
"It is further proposed than one part be equipped with a lid, devised for sealed interaction with a membrane in a valve.",
"One part has a number of channel-closing and channel-coupling first coupling means, said first coupling means being devised to enclose a coupling means on the second part, thereby jointly forming a coupling arrangement.",
"The invention also teaches the use of a ring-shaped elastic gasket with an elasticity allowing the first and second coupling means to interconnect during cam-guided relative, axial movement between the first part and the second part.",
"The invention also teaches that at least one of the said parts is provided with an arrangement, such as clamps, for locking the two parts together and achieving firm locking to the second part.",
"Advantages Those advantages primarily obtained for an arrangement, in accordance with the present invention, for ventilatory treatment of the lungs reside in the provision of features making possible effective co-ordination of valve equipment and an array of sensor units, so they can be placed near the living creature, preferably with the aid of a connector part.",
"They also make possible the utilisation of only one tube for ventilatory gas between a lung ventilator unit and the living creature or patient being treated by intravenous administration of a liquid anaesthesia-inducing drug, thereby allowing expired gas from the creature to pass via the valve equipment into atmosphere in the immediate vicinity of the creature without contaminating of the lung ventilator with expired gas.",
"Specially shaped coupling means and a coupling arrangement that eliminate the risk of erroneous coupling are also provided.",
"The interacting parts are also devised for easy cleaning, and a valve membrane for the parts is easily removed.",
"The primary characteristic features of a valve arrangement in accordance with the present invention are set forth in the characterising clause of the following claim 1 ."
],
[
"FIELD OF INVENTION The present invention relates to an arrangement for ventilatory treatment of the lungs of a living creature and has been devised primarily for use during a state of anaesthesia caused by intravenous infusion of a liquid anaesthesia-inducing drug.",
"The arrangement in accordance with the present invention comprises valve equipment devised to carry an insufflation gas and/or an insufflation gas mixture from a lung ventilator unit to the lungs and airways of the living creature and allow controlled expiration of the gas and/or gas mixture held in the airways.",
"The arrangement and equipment can additionally comprise one or more sensors belonging to gas measuring equipment.",
"One of these sensors can then be devised to sense a pressure gradient related to gas velocity in measuring the velocity of insufflation gas and the duration of the insufflation phase.",
"Another sensor unit, or the same one as above, can be devised to sense a pressure gradient related to gas velocity in measuring the velocity of expired gas and the duration of the expiratory phase.",
"Yet another sensor can be devised to measure the pressure and/or time-related variations in the pressure of insufflation gas and/or expired gas.",
"Yet another sensor can be devised to divert part of the gas mixture for analysis for measurement of the presence of an/or percentage of one or more gas components in the gas mixture.",
"The invention relates more specifically to a refinement of the arrangement taught and disclosed in the Swedish patent application 99 02051-3, filed on 3 Jun.",
"1999 and entitled “Arrangemang vid en lungventilatorisk behandling” (Eng.",
"=Arrangement in ventilatory treatment of the lungs.)",
"DESCRIPTION OF THE PRIOR ART Arrangements of this kind are previously known from a number of different embodiments and are normally connected to a lung ventilator and/or unit for ventilatory treatment of the lungs.",
"One previously known lung ventilator, used for ventilating the lungs and for patients under a state of anaesthesia, has a first tube, running between the lung ventilator and a patient, devised for insufflation of a gas or gas mixture for the patient and a second tube connected to the lung ventilator's insufflation valve.",
"Lung ventilators of this kind for the stated use also have a second tube, running between the lung ventilator and a patient, with the same cross-section as the first tube.",
"The second tube carries expired gas or a gas mixture from the patient to the lung ventilator and to an expiratory valve, connected to the tube, on the lung ventilator.",
"The ends of tubes, away from the lung ventilator, are joined and connected to a patient connector part for the purpose of forming a sealed, controlled path of flow for both insufflation gas and expired gas.",
"Connector parts of this kind are available in different versions, such as e.g.",
"a tracheal tube and laryngeal mask.",
"In lung ventilators of this kind and for the aforementioned use, an insufflation valve must be open during an initial part of the insufflation phase, whereas an expiratory valve must be closed throughout the insufflation phase.",
"The expiratory valve must be open throughout the expiratory phase, whereas the insufflation valve must be closed.",
"An ‘inspiratory pause’, during which the insulation valve and the expiratory valve are both closed, can also occur.",
"In this manner, the valves are both actuated in controlling the insufflation phase and the expiratory phase and are built into the lung ventilator's basic unit.",
"Therefore, two different tubes, a first carrying insufflation gas and a second carrying expired gas, leave the lung ventilator's basic unit.",
"When the significant feature of the present invention is taken into consideration, it can be noted that the use of one or more gas-measuring units and one or more sensor units connected to same are previously known in anaesthesial applications.",
"Examples of such sensor units were previously mentioned under “Field of invention”.",
"When the measures associated with the present invention are also taken into consideration, it can be noted that in the field for utilisation of units for ventilating the lungs of a living creature which is not under a state of anaesthesia, the introduction of a control valve in the direction of ventilator gas flow and a measuring device, with a requisite sensor in the form of a device for evaluating gas flow and gas pressure, in the vicinity of the creature or patient is known from e.g.",
"the patent publication U.S. Pat.",
"No.",
"3,961,627.The valve's drive mechanism has e.g.",
"a servomotor, which receives control signals from a valve control unit.",
"SUMMARY OF THE PRESENT INVENTION Technical Problems When the technical deliberations that a person skilled in this particular art must make in order to provide a solution to one or more technical problems she/he encounters are taken into consideration, it will be seen that it is initially necessary to realise the measures and/or sequence or measures that must be undertaken to this end and to realise which means is/are required.",
"On this basis, the technical problems listed below should be relevant to development of the present invention.",
"When the prior art, as described above, is considered, it will be seen that a technical problem resides, in the ventilatory treatment of the lungs of a living creature under a state of anaesthesia created by intravenous infusion of a liquid anaesthesia-inducing drug, in disclosing a valve arrangement consisting of control-lable valve equipment, combined with a plurality of sensor units which can be located very close to the creature.",
"It will also be seen that a technical problem resides in the provision of conditions, with simple means, enabling the arrangement and the valve equipment to offer less dead space in relation to known equipment for this application.",
"It will also be seen that a technical problem resides in realising the significance of and the advantages for a patient under a state of deep anaesthesia, induced by an intravenous infusion of a liquid drug, of being able to offer utilisation of only one insufflation tube, devised with one cross-section for being able to carry an insufflation gas or gas mixture from the lung ventilator to the patient and, additionally, a plurality of tubes with a much smaller cross-section for determining in the lung ventilator different criteria for the gas or gas mixture and for controlling a valve in the valve equipment from the lung ventilator via a lung ventilator valve.",
"It will also be seen that a technical problem resides in realising the importance of and the advantages afforded by development of a valve arrangement made from a few simple parts, the parts being devised for easy cleaning, rinsing and/or replacement.",
"It will also be seen that a technical problem resides in realising the importance of and advantages afforded by having the valve equipment comprise a valve housing, enclosing sensor units, which can be made in two parts that attach to one another with a rotary movement.",
"These parts can be conjoined or separated by means of this rotary movement.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by arranging the said rotary movement around an end-oriented valve housing's axis of rotation.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by devising the area around the end-oriented axis of rotation with a passage for gas and an elastic ring-shaped gasket.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by having the said elastic gasket absorb the relative, axial movement between the said two parts forming the valve housing in order to provide a tight seal between the respective connector means for each part for sensor unit channel tubes sections when the parts are conjoined.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by using an elastic ring-shaped gasket between the parts.",
"The gasket can be mounted on one of the parts and have the shape of part of a sphere.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by truncating the part of the sphere with two planes, located on either side of a sphere's centre, forming small circles with the same or different diameters.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by having one of the said two parts contain a system of channels for interconnection to a coupling means for a plurality of tubes, preferably arranged in a single tubing bundle.",
"It will also be seen that a technical problem resides in realising the importance of and advantages afforded by orienting the system of channels so as to connect openings in the coupling means with the openings in one, the first, of the said two parts.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by equipping one of the parts with a lid, devised to seal against a peripheral area of a valve membrane.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by devising one of the parts with a number of channel system first coupling means, arranged in a row, in a two-part coupling arrangement, preferably with an intermediate gasket.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by devising the said first coupling means on one part to enclose a second coupling means on the second part in the same coupling arrangement.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by using a said ring-shaped elastic gasket with an elasticity allowing the first and second coupling means, in a coupling arrangement, to interconnect during cam-guided relative movement between the first section and the second section.",
"It will be seen that a technical problem resides in realising the importance of and advantages afforded by providing one of the said two parts with a locking means, such as one or two clamps, so the first part locks firmly to the second part.",
"Solution With the intention of solving one or more of the aforesaid technical problems, the present invention takes as its starting point a valve arrangement in ventilatory treatment of the lungs of a living creature under a state of anaesthesia.",
"It comprises valve equipment devised for passing an insufflation gas and/or an insufflation gas mixture from a lung ventilation unit into the creature's lungs and airways and allowing expiration of gas and/or gas mixture held in the airways, and one or more sensor units in gas measuring equipment, one of the sensor units being devised for sensing the velocity-related pressure gradient in measurement of the velocity of the insufflation gas and the duration of the insufflation phase.",
"One, another or the same sensor unit is devised for sensing the velocity related pressure gradient in measuring the velocity of expired gas and the duration of the expiratory phase.",
"A second sensor is devised for measuring gas pressure and/or variations in pressure over time.",
"One sensor can be devised to divert part of the gas mixture for gas analysis in measuring the presence of and/or percentage of one or more gas components in the gas mixture.",
"With the intention of solving one or more of the aforesaid technical problems, the present invention proposes that the said valve equipment comprise a valve housing, which can consist of two parts.",
"These parts can be conjoined or separated by means of an end-related rotary movement.",
"As proposed embodiments that lie within the scope of the inventive concept, it is also proposed that the said rotary movement be arranged around an axis of rotation at the end of a section of tubing for e.g.",
"insufflation gas.",
"It is further proposed that the end of the section of tubing be in the shape of part of a sphere.",
"A ring-shaped gasket is arranged on one of the parts and has an external shape corresponding to part of a sphere.",
"It is further proposed that the part of the sphere be truncated by two planes on either side of a sphere's centre, forming small circles with the same or different diameters.",
"It is further proposed that one of the said two parts comprise a system of channels for interaction with a coupling means for a plurality of tubes, preferably arranged in a single bundle of tubing.",
"It is further proposed than one part be equipped with a lid, devised for sealed interaction with a membrane in a valve.",
"One part has a number of channel-closing and channel-coupling first coupling means, said first coupling means being devised to enclose a coupling means on the second part, thereby jointly forming a coupling arrangement.",
"The invention also teaches the use of a ring-shaped elastic gasket with an elasticity allowing the first and second coupling means to interconnect during cam-guided relative, axial movement between the first part and the second part.",
"The invention also teaches that at least one of the said parts is provided with an arrangement, such as clamps, for locking the two parts together and achieving firm locking to the second part.",
"Advantages Those advantages primarily obtained for an arrangement, in accordance with the present invention, for ventilatory treatment of the lungs reside in the provision of features making possible effective co-ordination of valve equipment and an array of sensor units, so they can be placed near the living creature, preferably with the aid of a connector part.",
"They also make possible the utilisation of only one tube for ventilatory gas between a lung ventilator unit and the living creature or patient being treated by intravenous administration of a liquid anaesthesia-inducing drug, thereby allowing expired gas from the creature to pass via the valve equipment into atmosphere in the immediate vicinity of the creature without contaminating of the lung ventilator with expired gas.",
"Specially shaped coupling means and a coupling arrangement that eliminate the risk of erroneous coupling are also provided.",
"The interacting parts are also devised for easy cleaning, and a valve membrane for the parts is easily removed.",
"The primary characteristic features of a valve arrangement in accordance with the present invention are set forth in the characterising clause of the following claim 1.BRIEF DESCRIPTION OF THE DRAWINGS The present proposed embodiment of a valve arrangement devised for use in the ventilatory treatment of the lungs of a living creature will now be described in greater detail with reference to the accompanying drawings, in which FIG.",
"1 is a side view of a living creature onto whose mouth and nasal area a connecting part, in the form of facemask with mounted valve equipment and a combination of sensor arrays, has been applied in accordance with the present invention's features.",
"FIG.",
"2 is a perspective view of an equipment coupling means for connecting a bundle of tubing from a lung ventilator unit.",
"The cross-section of the bundle of tubing between the valve equipment and the lung ventilatory unit can be regarded as evident.",
"FIG.",
"3 is a perspective cross-section side view of valve equipment, in accordance with the present invention with two rotating interacting parts fully conjoined.",
"FIG.",
"4 is a perspective cross-section side view of valve equipment, in accordance with FIG.",
"3 with the two rotating, interacting parts separated.",
"FIG.",
"5 shows on a slight enlarged scale, compared to FIG.",
"4, the first part's end-oriented interaction with the second part's corresponding end section.",
"FIG.",
"6 shows a first and a second coupling means in a coupling arrangement in a position before being conjoined according to FIG.",
"3.FIG.",
"7 shows a sphere truncated by two planes in order to illustrate an elastic, ring-shaped gasket with a shape significant to the invention, and FIG.",
"8 is an exploded view of the valve equipment.",
"DESCRIPTION OF EMBODIMENTS AT PRESENT PROPOSED Thus, FIG.",
"1 depicts the principles of the use of a valve arrangement 1 with valve equipment 4 constructed in accordance with the present invention.",
"The arrangement 1 is devised for use in ventilatory treatment of the lungs of a living creature 2.The ventilatory treatment of the lungs is to take place during anaesthesia induced by intravenous administration of a liquid anaesthesia-inducing drug into the patient's 2 blood.",
"In these circumstances, utilisation of a lung ventilation unit 3 is required as well as checks on various therapeutic and diagnostic criteria.",
"In accordance with the invention, only one tube or a bundle of tubing 30 leaves the lung ventilation unit 3.One tube, i.e.",
"an insufflation tube 3a, illustrated in the embodiment example as two tubes connected in parallel to the arrangement 1, has a relatively large cross-section.",
"The unit 3 with requisite means for evaluating therapeutic and/or diagnostic criteria could be devised as is taught and disclosed in Swedish patent application 9901688-3.The contents of the said patent application shall accordingly be regarded as a part of the contents of the present application.",
"The arrangement 1 according to the present invention comprises valve equipment 4.This equipment is devised for carrying an insufflation gas and/or an insufflation gas mixture from a lung ventilation unit 3 to the lungs and airways 2a of the living creature 2 and allowing expiration of gas and/or a gas mixture held under positive pressure in the airways.",
"The insufflation gas can consist of pure air, air mixed with oxygen to a greater or lesser degree or just oxygen and/or additives thereto.",
"This insufflation gas passes a short section of tubing 3b, the arrangement 1, valve equipment 4 and a short section of tubing 3c, in addition to the tube 3a, enters the airways 2a through a connection part 5 and descends into the lungs.",
"During this sequence, a valve 40 in the valve equipment 4 remains closed by pressurisation from the unit 3 via a tube 14′, a coupling means 100 and a channel 14.When insufflation gas held in the airways 2a is expired, the expired gas is allowed to pass the said connection part 5, the section of tubing 3c and, with the aid of the valve 40 in the valve arrangement 4, through an outlet 4a into atmosphere.",
"Expired gas follows this route because an insufflation valve 3e in the lung ventilation unit 3 is closed, thereby preventing expired gas from passing through the section of tubing 3b and the tube 3a into the unit 3.Pressure on the valve 40 is released during this sequence or phase.",
"The valve equipment 4 can also be controlled in such a way that the valve equipment serves, during an insulation phase, as a pressure relief device by having the unit 3, via a valve 3f, pressurise a tube 14′ with a pre-set maximum pressure.",
"In this manner, a pressure of e.g.",
"60-120 cm H2O could activate the pressure relief device.",
"The valve equipment 4 is also devised to create a lower excess pressure, i.e.",
"a PEEP pressure, of e.g.",
"0-30 cm H2O, by means of reduced pressure on the tube 14′ during the expiration phase.",
"The arrangement 1 and its valve equipment 4, designated 50 in FIG.",
"3, also has gas-measuring equipment in a unit 3 sensor unit or sensor unit array 6 comprising a plurality of sensor units in a section of tubing 52a in the second part 52.One or more gas-measuring units can advantageously be built into the lung ventilation unit 3, as shown in block 6a.",
"Here, the array of sensor units 6 with associated gas-measuring units in block 6a consists of a plurality of known, co-ordinated and integrated sensor units.",
"The embodiment according to FIG.",
"3 illustrates the possibility of devising a measurement chamber 60 in the second part's section of tubing 52a in which a plurality of sensors are combined to form the said array of sensor units 6.The array of sensor units 6 and the measurement chamber 60 incorporate and co-ordinate a plurality of sensor units in combination.",
"The following is a description of an example of such a combination.",
"One sensor 61 can accordingly be devised for measuring the velocity of insufflation gas and the duration of the insufflation phase by sensing the gas velocity-related pressure gradient.",
"This has been illustrated as openings 61, 61a, facing one another, in connection with channels 10, 11 in the first part 51 through the coupling means 71, 72 in a coupling arrangement 70, the channels being connected to the tubes 10′, 11′ via the coupling means 100.Here, it is assumed that the flow of gas in one direction (3b-3c) causes an increase in pressure in channel 10 and a drop in pressure in channel 11, and a momentary pressure gradient provides a measure of the momentary gas velocity.",
"Another sensor unit or sensor unit 61, 61a above can be devised for measuring the velocity of the expired gas and the duration of the expiration phase by sensing the gas velocity-related pressure gradient or a momentary pressure value and/or variation in pressure over time.",
"An additional sensor 62 can be devised for measuring gas pressure and/or the variation in pressure over time via a channel 12 and a tube 12′.",
"Gas sampling and/or pressure measurement via the channel 12 and tube 12′ may be employed here.",
"The sensor unit 62 shall be primarily devised for measuring the presence of and/or percentage of one or more gas components in the gas mixture by diverting part of the gas mixture through the channel 12 and tube 12′ for analysis.",
"The sensors 61, 61a and 62 are therefore arrayed in a measurement chamber 60 for measuring velocity and pressure, sampling gas etc.",
"Measurement values received in the form of values, sensed via the channels 10, 11 and 12 and connected tubes 10′, 11′ and 12′, and part of the gas mixture are sent, via the channel 12 and tube 12′, to the unit 3 which contains requisite means and equipment for evaluating therapeutic and/or diagnostic criteria.",
"The invention is based upon the concept that the said valve equipment 50 (4) consists of two parts, i.e.",
"a first upper part 51 and a second lower part 52.The said second part 52 contains a combination of sensor unit arrays 6 in the said measurement chamber 60, the arrays being arranged in close proximity to one another.",
"The tube 3a is connected by the coupling means 100 to a channel 15 by utilising two tubes 15a′ and 15b′ in the bundle of tubes, as shown in FIGS.",
"2 and 3.The said channel 15 carries a flow of air through an elastic gasket 55 and the section of tubing 52a in the second part 52.The bundle of tubing and the tubing therein are e.g.",
"connected to an end section 3a′ of a tube 3a carrying insufflation gas from the lung ventilation unit 3, and the said array of sensor units 6 is located closer to the living creature 2 than the said valve equipment 4.FIG.",
"3 shows that the said valve equipment 4 and the said array of sensor units 6 are integrated into a single unit assigned the reference designation 50.FIG.",
"1 shows that a bundle of tubes 30 is arranged between the equipment 4 and the unit 3.This bundle of tubes 30 can consist of a plurality of individual tubes, attached to one another or separated, dominated by the insufflation gas tube 3a with a large cross-section.",
"It is primarily proposed that all the tubes be integrated (or bundled) in a way that is evident from a study of FIG.",
"2 and the utilised coupling means 100.This will show that the tubes 15a′ and 15b′ constitute the insufflation tube (3a), that a tube 14′ is a tube for controlling the expiratory valve 40, tubes 10′ and 11′ respectively are intended for measuring flow and a tube 12′ is intended for sampling and measuring a gas, such as carbon dioxide FIG.",
"2 shows the coupling means 100 of the first part 51, and the cited tubes are obviously intended for connection to corresponding coupling means (not shown).",
"Therefore, five separate tube cross-sections are required for the proposed application and with the sensor units 61, 62 and 63 combined in the measurement chamber 60.These cross-sections are devised for connection to channels 10, 11, 12, 14 and 15 respectively in the equipment's 50 first part 51.The channels 10 and 11 are connected to the measurement chamber 60 in order to measure the velocity and duration of insufflation gas and the velocity of expired gas and duration of the expiration phase, both channels being arranged for measurement of opposite directions of flow.",
"The channel 14 is devised for generating varying positive pressure, ranging between two or three values, generated by the lung ventilation unit 3.These positive pressures cause the valve 40 to open at various pre-selected positive pressures on the basis of the positive pressure in a section of tubing 52a.",
"The valve equipment 50, in accordance with the invention, consists of two parts, a first part 51 and a second part 52.These parts 51, 52 can be conjoined, as shown in FIG.",
"3, and separated, as shown in FIG.",
"4, by means of a rotary movement.",
"The rotary movement is arranged around an axis of rotation 53, and this axis of rotation 53 is arranged at the end of a section of tubing 52a for e.g.",
"insufflation gas.",
"FIG.",
"5 shows that the interior of the end-oriented section of tubing, assigned the reference designation 54a, has the internal shape of part of a sphere, and this partial sphere defines an interior area 54b.",
"A ring-shaped gasket 55 is arranged on one of the parts 51 between the parts 51 and 52 and has an external shape 55a like part of a sphere.",
"The partially spherical part 54a and the partially spherical part 55a are truncated by two planes located on or on either side of the centre of a sphere, forming small circles with the same or different diameters.",
"It also shows that one of the parts 51 has a ring-shaped lid 58 devised for sealed closure against a circular peripheral area 40d of a membrane 40a in the valve 40.The membrane 40a is shaped like a cup with a flat lower surface 40c, a peripheral area 40e of which is devised to form a tight seal with a narrow, ring-shaped seat 40b as soon as pressure above the lower surface 40c exceeds the pressure under the lower surface 40c.",
"The first part 51 also has a number of first channel-closing means 71, and the second part 52 has a number of second coupling means 72 in a coupling arrangement 70 in which the said first coupling means 71 is devised to enclose the second part's 52 second coupling means 72.The second part 52 has been equipped with a cam profile 70c with a knee 70d located over a straight centre line 52a′, perpendicular to a section of tubing 52a over a distance 70e, and the first part 51 is equipped with a guide flange 70f.",
"When the first part 51 and the second part 52 are fully conjoined, the guide flange 70f follows the cam profile 70c, passes the said knee 70d and joins the straight distance 70e, causing the tube parts for the channel segments to connect in parallel to the second part's 52 opposite tube parts.",
"The elastic ring gasket 55 absorbs this relative movement.",
"A flat gasket 72a shown in FIG.",
"6 may be used for sealing two coupling means 71, 72 in the coupling arrangement 70.The elasticity and, in particular, the thickness of the said ring-shaped gasket 55 in the tube area 52a′ of the tube 52a are such that the first and second coupling means 71, 72 are able to interconnect axially during the relative movement of the first part 51 and second part 52 back and forth.",
"The fig.",
"also discloses that one of the said parts, i.e.",
"the first part 51, is provided with a locking mechanism, such as one or more clamps 56, for firm connection to the second part 52.FIG.",
"7 shows that a plane P1 through a centre “0” truncates the area 54b formed by part of a sphere, thereby forming a large circle “C1”, and a plane P2 forms a small circle “C2”.",
"The gasket 55 forming part of a sphere with its area 55a is formed by a plane P3 in the form of a small circle C3, a small circle C2 and a small circle C3 lying on either side of the plane P1 and the large circle C1.However, it should be noted that the plane P3 with the small circle C3 must be located near the large circle C1 through the plane P1.FIG.",
"8 is an exploded view of the valve equipment 4, in accordance with the invention, and the equipment's five parts (the gasket 72a excluded).",
"Corresponding parts have been assigned the same reference designations as in the previous figures.",
"FIG.",
"8 shows that one of the parts 51 consists of two complementary conjoined parts 51d and 51e.",
"It should be noted that a valve 400 for spontaneous inspiration has been shown in FIG.",
"4 with a membrane 401 which seals off a number of holes 402, and the membrane 401 is attached to the tube at a seat 403.This valve 400 can be used when required.",
"It will be understood that the invention is not restricted to the aforesaid exemplifying embodiment thereof and that modifications are possible within the scope of the inventive concept as defined in the following claims."
]
] | Patent_10415942 |
[
[
"Diamido alkoxides as polymerisation initiators",
"The present invention relates to a complex having the formula (I) wherein M is selected from Sn(II), Sn(IV), Al(III) and Mg(II); each of R1 and R2 is independently hydrocarbyl; R3 is H or hydrocarbyl; and X is a linker group.",
"A further aspect of invention relates to the use of said complex as a polymerisation initiator, particularly in the polymerisation of lactides."
],
[
"1.A complex having the formula I wherein M is selected from Sn(II), Sn(IV), Al(III) and Mg(II); each of R1 and R2 is independently hydrocarbyl; R3 is H or hydrocarbyl; and X is a linker group: 2.A complex according to claim 1 wherein linker group X comprises a C1-4 carbon backbone, or a heteroatom-containing C1-4 carbon backbone.",
"3.A complex according to claim 1 or claim 2 wherein each of R1 and R2 is independently selected from alkyl, cycloalkyl, haloalkyl, aryl, haloaryl or heteroaryl; R3 is H, alkyl, cycloalkyl, haloalkyl, aryl or haloaryl.",
"4.A complex according to any preceding claim wherein X comprises a vinylene or an arylene group.",
"5.A complex according to any preceding claim, having the formula II, wherein each of R4, R5 and R6 is independently selected from hydrogen, alky, cycloalkyl, haloalkyl, aryl, haloaryl and heteroaryl, and R1-3 and M are as defined in claim 1.6.A complex according to claim 5 wherein each of R4, R5 and R6 is independently selected from H, methyl, t-butyl, phenyl and trifluoromethyl.",
"7.A complex according to any preceding claim wherein M is Sn(II).",
"8.A complex according to any one of claims 1 to 7 having the formula III wherein Ar is o-diisopropylphenyl.",
"9.A complex according to any one of claims 1 to 6 wherein M is Mg(II).",
"10.A complex according to any one of claim 9 having the formula IV wherein Ar is o-diisopropylphenyl.",
"11.A complex according to any one of claims 1 to 8 having the formula V wherein R1-3 and M are as defined in claim 1, and wherein each of R7 and R8 is independently selected from H, alkyl, cycloalkyl, haloalkyl, aryl, haloaryl and heteroaryl.",
"12.A complex according to claim 11 wherein R7 is H and R8 is methyl.",
"13.A complex according to claim 11 or claim 12 having the formula VI wherein Ar is o-diisopropylphenyl.",
"14.A complex comprising a dimer of a complex according to any preceding claim.",
"15.A dimer according to claim of formula VII 16.A dimer according to claim 15 of formula VIII wherein Ar is o-diisopropylphenyl.",
"17.Use of a complex according to any one of claims 1 to 16 as a polymerisation initiator.",
"18.Use according to claim 17 in the polymerisation of lactide.",
"19.Use according to claim 18 wherein the lactide is rac-lactide.",
"20.A process for the polymerisation of lactide, said process comprising contacting an initiating amount of a complex according to any one of claims 1 to 16 with a lactide monomer in the presence of a suitable solvent.",
"21.A process according to claim 20 wherein the ratio of lactide monomer to the complex is between 100:1 and 20000:1.22.An article prepared by a process according to 20 or claim 21.23.An article according to claim 22 wherein said article is a medical article.",
"24.A composition comprising lactide monomer and a complex according to any one of claims 1 to 16.25.A composition comprising poly(lactide) and a complex according to any one of claims 1 to 16.26.A composition comprising poly(lactide) and a complex according to any one of claims 1 to 16, wherein said complex is deactivated, for example, by the addition of methanol.",
"27.A process for preparing a complex of formula I, said process comprising the steps of: (i) reacting a compound of formula IX, wherein each of Z′ and Z″ is independently H (where —NR1 and/or —NR2 is an amino group) or absent (where —NR1 and/or —NR2 is an imino group), with nBuLi to form a compound of formula X; (ii) reacting said compound of formula X with MCln to form a compound of formula XI, wherein M is selected from Sn(II), Mg(II), Al(Ill) and Sn(IV) and n is 2, 2, 3 or 4 respectively; (ii) reacting said compound of formula XI with LiOR3 to form a complex of formula I; wherein X and R1-3 are as defined in claim 1.28.A process for preparing a complex of formula XIII, said process comprising the steps of: (i) reacting a compound of formula IX, wherein each of Z′ and Z″ is independently H (where —NR1 and/or —NR2 is an amino group) or absent (where —NR1 and/or —NR2 is an imino group), with MgMe2 to form a compound of formula XII; (ii) reacting said compound of formula XII with R3OH to form a compound of formula XIII; wherein X and R1-3 are as defined in claim 1.29.A process for preparing a complex of formula XIII, said process comprising the steps of: (i) reacting a compound of formula IX, wherein each of Z′ and Z″ is independently H (where —NR1 and/or —NR2 is an amino group) or absent (where —NR1 and/or —NR2 is an imino group), with (a) nBuLi and (b) R3MgCl to form a compound of formula XIV; (ii) reacting said compound of formula XIV with O2 to form a complex of formula XIII; wherein X and R1-3 are as defined in claim 1."
],
[
"The present invention relates to a series of discrete, well-defined coordination complexes.",
"More specifically, the invention relates to complexes that are usefull as polymerisation initiators, and in particular, in lactide polymerisation.",
"Over recent years, there has been an increasing interest in the design of polymers for in vivo applications such as sutures, artificial tissue networks and drug delivery agents.",
"For these applications it is desirable for the polymer to be non-toxic, biocompatible and resorbable.",
"One of the most promising classes of polymer in this field are the poly(lactide)s [J. C. Middleton, A. J. Tipton, Biomaterials, 2000, 21, 2335].",
"Initiators for lactide polymerisation are typically based on alkoxide or alkanoate complexes of metals such as Al, Mg, Sn, Zn and the rare earths.",
"Tin(II) catalysts such as Sn(ethylhexanoate)2 are generally preferred in the commercial production of poly(lactide)s [E. E. Schmitt and R. A. Rohistina, U.S. Pat.",
"No.",
"3,297,033, 1967 (Chem.",
"Abstr.",
"1967, 66, P38656u); E. E. Schmitt and R. A. Rohistina, U.S. Pat.",
"No.",
"3,463,158, 1969 (Chem.",
"Abstr.",
"1969, 71, P92382t); H. R. Kricheldorf, I. Kreiser-Saunders and C. Boettcher, Polymer, 1995, 36, 1253, and references therein] for medical or pharmaceutical applications due to the low toxicity of Sn(II) compared to other metal ions.",
"Indeed, Sn(ethylhexanoate)2 is a permitted food additive in many countries.",
"To date, the ring opening polymerisation of cyclic ethers and esters are usually carried out with the aid of so-called “coordinate catalysts” which are ill-defined with regard to their exact mechanism and structure.",
"In the polymerisation of lactide, the exact structure and detailed mode of reaction of the above-mentioned tin carboxylates, M(O2CR)2, are not fully understood.",
"Indeed, some evidence suggests that the active species in the polymerisation of lactides using tin octanoates may actually be a hydrolysis product, present only as a minor component.",
"A number of systems that initiate the living ring-opening polymerisation of lactide have been disclosed in the art.",
"By way of example, systems based on aluminium [P. Dubois, C. Jacobs, R. Jérome, P. Teyssié, Macromolecules 1991, 24, 2266; P. A. Cameron, D. Jhurzy, V. C. Gibson, A. J. P. White, D. J. Williams, S. Williams, Macromol.",
"Rapid.",
"Commun.",
"1999, 20, 616; N. Spassky, M. Wisniewski, C. Pluta, A. LeBorgne, Macromol.",
"Chem.",
"Phys.",
"1996, 197, 2627], and more recently, magnesium [M. H. Chisholm, N. W. Eilerts, J. Chem.",
"Soc., Chem.",
"Commun., 1996, 853] and zinc [M. Cheng, A.",
"B. Attygalle, E. B. Lobkovsky, G. W. Coates, J.",
"Am.",
"Chem.",
"Soc.",
"1999, 121, 11583], have all been described.",
"Another important advance in modern polymer synthesis is the control achievable over the tacticity of polymers derived from prochiral monomers or monomers bearing stereogenic centres.",
"This is most dramatically exemplified by the family of propylenes obtained using ansa-metallocene polymerisation catalysts, leading to materials with new and markedly different properties.",
"By comparison, relatively little research has been directed towards poly(lactide)s derived from the polymerisation of rac- and meso-lactides, which are the cyclic diesters of lactic acid.",
"These are gaining increasing attention due to their medicinal, pharmaceutical and agricultural applications as well as uses as environmentally benign packaging materials.",
"Unsurprisingly, the physical properties of the poly(lactide)s are also strongly dependent upon their tacticity.",
"For example, isotactic poly(R,R-lactide) is a semi-crystalline thermoplastic of high mechanical strength and toughness (Tg ˜57° C., Tm ˜174° C.), while syndiotactic poly(R,S-lactide) has a lower softening temperature (Tg ˜45° C., Tm ˜152° C.) and has found applications in controlled drug release.",
"Atactic poly(lactide), usually obtained upon polymerisation of rac-(R,R/S,S)-lactide is an amorphous polymer with a glass transition temperature near room temperature.",
"In recent years there have been a number of studies on the stereoselective polymerisation of lactide monomers using single-site catalysts.",
"Spassky and co-workers have described the selective polymerisation of one of the enantiomers of rac-lactide (essentially a kinetic resolution) to isotactic poly(lactide) using a resolved tetradentate Schiff base complex of aluminium [Spassky et al, ibid].",
"Baker and Smith have shown that a racemic mixture of a similar complex can be used to polymerise both enantiomers of rac-lactide to the isotactic stereocomplex which has the added advantage of possessing a melting point approximately 50° C. higher than the homochiral polymers [C. P. Radano, G. L. Baker, M. R. Smith III, J.",
"Am.",
"Chem.",
"Soc.",
"2000, 122, 1552].",
"Moreover, studies on meso-lactide have revealed that syndiotactic poly(lactide) can also be obtained [Cheng et al, ibid].",
"The aluminium catalyst systems, however, tend to give low rates of propagation, and more highly active catalysts would be beneficial for the commercial production of such materials.",
"A significant development was the recent introduction of a zinc catalyst bearing β-diketininate ligands which acts as a highly efficient catalyst for the polymerisation of rac-lactide to heterotactic poly(lactide) via a chain end control mechanism [Cheng et al, ibid].",
"The present invention seeks to provide alternative, well-defined, single-site metal complexes that are suitable for use as polymerisation initiators.",
"More specifically, the invention seeks to provide well-defined complexes that are suitable for initiating the polymerisation of lactide.",
"In a broad aspect, the present invention provides a series of well-defined metal complexes.",
"More specifically, the invention provides a complex having the formula I wherein M is selected from Sn(II), Sn(IV), Al(III) and Mg(II); each of R1 and R2 is independently hydrocarbyl; R3 is H or hydrocarbyl; and X is a linker group.",
"Preferably, each of R1, R2 and R3 is independently hydrocarbyl.",
"As used herein, the term “hydrocarbyl” refers to a group comprising at least C and H that may optionally comprise one or more other suitable substituents.",
"Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, or a cyclic group.",
"In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group.",
"If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other.",
"For example, at least two of the carbons may be linked via a suitable element or group.",
"Thus, the hydrocarbyl group may contain heteroatoms.",
"Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon.",
"In a preferred aspect of the invention, the linker group X comprises a C1-4 carbon backbone, or a heteroatom-containing C1-4 carbon backbone.",
"Preferably, the linker group X is a C2 or C3 carbon backbone.",
"In a further preferred aspect of the invention, each of R1 and R2 is independently selected from alkyl, cycloalkyl, haloalkyl, aryl, haloaryl or heteroaryl; and R3 is hydrogen, alkyl, cycloalkyl, haloalkyl, aryl or haloaryl.",
"As used herein, the term “alkyl” refers to a saturated carbon-containing chain which may be straight or branched, and substituted (mono- or poly-) or unsubstituted.",
"Suitable substituents include those which do not have any significant adverse effect on the activity of the complex as a polymerisation initiator.",
"Preferably, the alkyl group is a C1-20 alkyl group, more preferably a C1-10 alkyl group.",
"Accordingly, the term “haloalkyl” refers to an alkyl group substituted by at least one halogen, for example, chlorine, bromine, fluorine or iodine.",
"As used herein, the term “aryl” refers to an aromatic, substituted (mono- or poly-) or unsubstituted.",
"Again, suitable substituents include those which do not have any significant adverse effect on the activity of the complex as a polymerisation initiator.",
"Accordingly, the term “haloaryl” refers to an aryl group substituted by a halogen, for example, chlorine, bromine, fluorine or iodine.",
"As used herein, the term “cycloalkyl” refers to a cyclic alkyl group.",
"As used herein, the term “heteroaryl” refers to an aromatic heterocycle comprising one or more heteroatoms.",
"Preferred heteroaryl groups include pyrrole, pyrmidine, pyrazine, pyridine, quinoline and furan.",
"In a preferred aspect, the linker group X is conjugated.",
"In one preferred aspect of the invention, the linker group X comprises a vinylene or an arylene group.",
"In a preferred aspect, the complex of the invention has the formula II, wherein each of R4, R5 and R6 is independently selected from hydrogen, alkyl, cycloalkyl, haloalkyl, aryl, haloaryl and heteroaryl, and R1-3 and M are as defined hereinbefore.",
"In a more preferred aspect of the invention, each of R4, R5 and R6 is independently selected from H, methyl, t-butyl, phenyl and trifluoromethyl.",
"Even more preferably, RW is H and R4 and R6 are alkyl, more preferably methyl.",
"The skilled person will appreciate that where the metal is Al(III), the complex will have an overall charge of 1+, and where the metal is Sn (IV), the ligand will be dianionic and the complex will have an overall charge of 1+.",
"Accordingly, when the metal is Sn(II) or Mg(II), the complex is neutral.",
"In one particularly preferred aspect, M is Sn(II).",
"In another particularly preferred aspect, M is Mg(II).",
"In one particularly preferred embodiment, the complex of the invention has the formula III wherein Ar is o-diisopropylphenyl.",
"In contrast to the ill-defined tin “coordinate catalysts” of the prior art, the present invention provides a tin complex with a well-defined structure that is suitable for use as a polymerisation initiator.",
"The complex of formula III is prepared according to the scheme shown in FIG.",
"1.Treatment of SnCl2 with [HC{C(Me)NAr}2]Li in diethylether followed by crystallisation from pentane affords [HC{C(e)NAr}2]SnCl, IIIa as a yellow crystalline solid.",
"This is converted to III by treatment with LiOPri followed by re-crystallisation from pentane.",
"Crystals of III suitable for an X-ray structure determination were grown from pentane; the structure is shown in FIG.",
"2.Further details of the X-ray structure may be found in the accompanying examples.",
"The complex of formula III has non-crystallographic CS symmetry with the three-coordinate tin atom adopting a tripodal geometry with inter-bond angles in the range 83.6(2)-94.1(2)°, the most acute being associated with the bite of the chelating N,N′ ligand.",
"The Sn—N and SnO distances are unexceptional, and there is the expected pattern of delocalisation within the β-diketiminate ligand.",
"The six-membered chelate ring has a boat conformation with C(2) and Sn lying 0.12 and 0.87 Å “above” the N(1), C(1), C(3), N(3) plane; the isopropoxide oxygen atom lies 0.74 Å “below” this plane.",
"As a consequence of the folded chelate ring conformation, and retention of near trigonal planar geometries at the two nitrogen centres, the C(12) and C(27) isopropyl groups are drawn together, and those associated with C(15) and C(24) are folded away exposing the non-coordinated “face” containing the stereochemically active lone pair on the tin atom; the shortest intermolecular approach to the tin centre is 3.84 Å from C(13)—H.",
"A further aspect of the present invention relates to the use of the above-described complexes as polymerisation initiators.",
"As used herein, the term “polymerisation initiator” refers to an agent used to start the polymerisation of a monomer.",
"In a preferred aspect, the invention relates to the use of the complexes described herein as initiators in the polymerisation of lactide.",
"The term “lactide” encompasses rac-lactide, meso-lactide and the resolved L and D forms of lactide.",
"In particular, the complexes of the invention may be used as initiators in the “living polymerisation” of lactide.",
"As used herein, the term “living polymerisation” refers to a polymerisation in which there is no termination step, i.e.",
"the initiator complex is associated with the end of the propagating polymer chain and is not released.",
"Even more preferably, the lactide is in the form of rac-lactide.",
"As used herein, the term “rac-lactide” refers to a racemic mixture of L (S,S) and D (R,R) lactide.",
"By way of example, the polymerisation of rac-lactide was initially investigated using complex III in CH2Cl2 at ambient temperature.",
"Under these conditions it was found that 100 equivalents of monomer required 96 hours for complete conversion (>99% by 1H NMR).",
"The resultant polymer has a molecular weight close to that calculated from the monomer:initiator ratio (observed Mn=17,100; calculated Mn=14,400) and exhibits a narrow polydispersity (Mw/Mn=1.11), characteristics of a living process.",
"The polymerisation was then repeated at 60° C. in toluene affording 85% conversion after 4 hours, again resulting in a narrow molecular weight distribution product (Mw/Mn=1.05).",
"The activity of the tin catalyst is lower than that observed for the related zinc system [Cheng et al, ibid] which may be in part due to the lower electrophilicity of the tin centre, and partly a consequence of the stereochemically active lone pair which may disfavour monomer binding.",
"The living characteristics of the polymerisation are confirmed by the linear increase in Mn with conversion giving in each case a low polydispersity product (FIG.",
"3).",
"In order to confirm that the initiator is indeed the iso-propoxide complex III, a 1H NMR study was carried out in which increasing amounts of lactide were added to III in CDCl3.The iso-propoxide methine septet resonance at the end of the propagating chain is shifted slightly to higher frequency (0.005 ppm) relative to the unconsumed initiator (δ4.01).",
"New resonances for the β-diketiminate ligand substituents are also observed for the propagating species.",
"As the number of monomer equivalents is increased, the intensities of the signals attributable to the propagating species increase relative to those of unconsumed III.",
"Due to the overlapping nature of the resonances, accurate integration has not been possible, but the addition of 5 equivalents of monomer leads to an approximately 1:1 mixture of initiator: propagating species.",
"This indicates a favourable kp/ki ratio (the rate constant of propagation to rate constant of initiation) which is desirable for minimising the polydispersity.",
"The 1H NMR spectrum of the poly(lactide) derived from m (FIG.",
"4) differs from the spectrum predicted from a Bernouillian analysis of totally random poly(rac-lactide), with the rmr and mrm tetrads much more intense than expected.",
"These observations are consistent with a heterotactic-biased product since the rmr microstructure can only arise from two consecutive D-L or L-D interchanges; each rmr tetrad is accompanied by two mrm tetrads in agreement with the NMR integration.",
"This represents the first example of tacticity bias arising from the polymerisation of rac-lactide using a tin catalyst.",
"As used herein, the term “heterotactic” refers to a polymer in which the adjacent monomer units alternate in the form [-L-D-L-D-L-D .",
".",
".",
"].",
"A true heterotactic polymer is one in which the monomer units alternate perfectly in this fashion.",
"A polymer exhibiting a “heterotactic bias” therefore refers to a polymer in which there is a bias towards heterotacticity, compared to an “atactic” polymer in which there is no regular arrangement of monomer units along the chain.",
"In another particularly preferred embodiment, the complex of the invention has the formula IV wherein Ar is o-diisopropylphenyl.",
"In one preferred embodiment, the complex of the invention has the formula V wherein R1-3 and M are as defined hereinabove, and wherein each of R7 and R8 is independently selected from H, alkyl, cycloalkyl, haloalkyl, aryl, haloaryl and heteroaryl.",
"Preferably, each of R7 and R8 is independently selected from H and alkyl, more preferably, H and methyl.",
"In one particularly preferred embodiment, R7 is H and R8 is methyl.",
"Even more preferably, the complex has the formula VI wherein Ar is o-diisopropylphenyl.",
"In a further preferred aspect, the complex of the invention comprises a dimer of a complex as described herein.",
"Preferably, the dimer has the formula VII In a particularly preferred embodiment of the invention, the complex has the formula VIII The complex of formula VIII is prepared according to either of the two procedures outlined in FIG.",
"5.Reaction of dimethyl magnesium with 2-(2,6-di-iso-propylphenylamino)-4-(2,6-di-iso-propylphenylimino)-2-pentene in toluene affords the bridged methyl dimer, VIIIa.",
"Alcoholysis proceeds smoothly upon addition of 2 equivalents of iPrOH in toluene, to give the bridged alkoxide species, VIII.",
"Alternatively, VIII may be prepared by stirring a toluene solution of [HC(CMeNAr)2]MgiPr, VIIIb, under a dioxygen atmosphere.",
"The crystal structure of complex VIII is shown in FIG.",
"6.Both magnesium centres exhibit a distorted tetrahedral geometry (N—Mg—N=92°; O—Mg—O=81°).",
"Recent studies by the applicant have investigated the synthesis of a range of magnesium alkyl complexes bearing the β-diketiminate ligand [V. C. Gibson, J.",
"A. Segal, A. J. P. White, D. J. Williams, J.",
"Am.",
"Chem.",
"Soc.",
"2000, 122, 7120].",
"The present invention discloses that the alkoxide derivatives of these magnesium complexes, as described herein are, in fact, highly effective catalysts for the living polymerisation of lactide.",
"Yet another aspect of the invention relates to a process for the polymerisation of lactide, said process comprising contacting an initiating amount of a complex according to the invention with lactide monomer in the presence of a suitable solvent.",
"As used herein, the term “initiating amount” refers to a sufficient amount of an initiator to commence the chemical reaction of polymerisation.",
"In a preferred aspect, the ratio of lactide monomer to the initiator complex of the invention is between 100:1 and 20000:1.By way of example, suitable solvents include dichloromethane and toluene.",
"Other solvents suitable for the purposes of the present invention will be familiar to those skilled in the relevant art.",
"A further aspect of the invention relates to an article produced by the process described hereinbefore.",
"Preferably, the article is a medical article.",
"Typical articles may include sutures, drug delivery devices and medical implants, for example, orthopedic fixation devices.",
"Yet another aspect of the invention provides a process for preparing a complex of formula I, said process comprising the steps of: (i) reacting a compound of formula IX, wherein each of Z′ and Z″ is independently H (where —NR1 and/or —NR2 is an amino group) or absent (where —NR1 and/or —NR2 is an imino group), with nBuLi to form a compound of formula X; (ii) reacting said compound of formula X with MCln to form a compound of formula XI, wherein M is selected from Sn(II), Mg(II), Al(III) and Sn(IV) and n is 2, 2, 3 or 4 respectively; (ii) reacting said compound of formula XI with LiOR3 to form a complex of formula I; wherein X and R1-3 are as defined hereinbefore.",
"Another aspect of the invention relates to a process for preparing a complex of formula XIII, said process comprising the steps of: (i) reacting a compound of formula IX, wherein each of Z′ and Z″ is independently H (where —NR1 and/or —NR2 is an amino group) or absent (where —NR1 and/or —NR2 is an imino group), with MgMe2 to form a compound of formula XII; (ii) reacting said compound of formula XII with R3OH to form a compound of formula XIII; wherein X and R1-3 are as defined hereinbefore.",
"A further aspect of the invention provides a process for preparing a complex of formula XIII, said process comprising the steps of: (i) reacting a compound of formula IX, wherein each of Z′ and Z″ is independently H (where —NR1 and/or —NR2 is an amino group) or absent (where —NR1 and/or —NR2 is an imino group), with (a) aBuLi and (b) R3MgCl to form a compound of formula XII; (ii) reacting said compound of formula XII with O2 to form a complex of formula XIII; wherein X and R1-3 are as defined hereinbefore.",
"The present invention will now be described by way of example and with reference to the following figures wherein: FIG.",
"1 shows the reaction scheme for the preparation of the Sn(II) complex III.",
"FIG.",
"2 shows the molecular structure of complex III.",
"In more detail, the selected bond lengths (Å) and angles (°) in the molecular structure of III are as follows; Sn—O 2.000(5), Sn—N(1) 2.206(4), Sn—N(3) 2.208(4), C(1)-N(1) 1.323(6), C(1)-C(2) 1.404(7), C(2)-C(3) 1.387(7), C(3)-N(3) 1.331(6), O—Sn—N(1) 94.1(2), O—Sn—N(3) 92.5(2), N(1)-Sn—N(3) 83.6(2).",
"FIG.",
"3 shows Mn of the resulting polymer against the percentage conversion.",
"In particular, FIG.",
"3 confirms the living charactersitics of polymerisation, showing a linear increase in Mn with conversion, giving a low polydispersity product.",
"FIG.",
"4 shows the 1H NMR spectrum of the poly(lactide) derived from III.",
"FIG.",
"5 shows the reaction scheme for the preparation of the magnesium (II) complex VIII.",
"FIG.",
"6 shows the crystal structure of the magnesium (II) complex VIII.",
"EXAMPLES Synthesis of [HC{C(Me)NAr}2]SnCl, Complex IIIa To a 75 cm3 Et2O solution of 5.00 g ArN═C(Me)CH═C(Me)NHAr (1.19 mmol) at 0° C. was added 7.5 cm3 n-BuLi (1.6M in hexanes; 1.20 mmol).",
"After stirring at room temperature for 16 hours the mixture was added to a 40 cm3 Et2O suspension of SnCl2 (2.26 g; 1.19 mmol) and the reaction was then stirred for 18 hours.",
"Removal of volatiles in vacuo afforded a yellow-orange solid which was recrystallised from cold pentane as pale yellow crystals (3.70 g; 0.72 mmol; 60%).",
"Spectroscopic Data Complex IIIa: 1H NMR (250 MHz, C6D6, 25° C.) δ 1.06 (d, 6H, 3JHH=6.8 Hz, CHMeMe), 1.18 (d, 6H, 3JHH=6.9 Hz, CHMeMe), 1.22 (d, 6H, 3JHH=6.8 Hz, CHMe′Me′), 1.45 (d, 6H,3JHH=6.6 Hz, CHMe′Me′), 1.61 (s, 6H, HC{C(Me)NAr}2), 3.12 (sept, 2H, 3JHH=6.8 Hz, CHMe2), 3.95 (sept, 2H, 3JHH=6.8 Hz, CHMe2), 5.06 (s, 1H, HC{C(Me)NAr}2), 7.15 (m, 6H, Haryl).",
"MS: m/z 572 M]+.",
"Anal.",
"Calc.",
"(found) for C29H41ClN2Sn: C, 60.91 (60.77); H, 7.22 (7.32); N, 4.90 (5.07).",
"Synthesis of [HC{C(Me)NAr}2]Sn(OiPr) Complex III Lithium iso-propoxide (0.4365 g, 7.27 mmol) in 70 cm3 toluene was added to a rapidly stirred solution of [HC{C(Me)NAr}2]SnCl (2.0811 g, 3.64 mmol) in 50 cm3 toluene at 0° C. After 18 hours stirring at ambient temperature volatiles were removed to afford a bright yellow solid which was recrystallised from heptane at −30° C. (1.8857 g, 3.16 mmol, 87%).",
"Spectroscopic Data Complex III: 1H NMR (250 MHz, C6D6, 25° C.) δ 0.90 (d, 6H, 3JHH =6.8 Hz, OCHMe2), 1.14 (d, 3H, 3JHH=6.8 Hz, CHMeMe), 1.24 (d, 3H, 3JHH=6.9 Hz, CHMeMe), 1.27 (d, 3H, CHMe′Me′), 1.54 (d, 3H, CHMe′Me′), 1.59 (s, 6H, HC{C(Me)NAr}2), 3.25 (sept, 2H, 3JHH=6.8 Hz, CHMe2), 3.86 (sept, 2H, 3JHH=6.8 Hz, CHMe2), 4.15 (sept, 1H, 3JHH=6.0 Hz, OCHMe2), 4.73 (s, 1H, HC{C(Me)NAr}2), 7.16 (m, 6H, Haryl).",
"Anal.",
"Calc.",
"(found) for C32H48N2OSn: C, 64.55 (64.28); H, 8.13 (8.08); N, 4.70 (4.90).",
"Crystallographic Data for Complex III C32H48N2OSn, M=595.4, monoclinic, space group P21/n (no.",
"14), a=13.205(2), b=16.680(2), c=15.527(2) Å, β=107.42(1)°, V=3263.1(6) Å3, Z=4, Dc=1.212 g cm−3, μ(Mo—Kα)=8.07 cm−1, T=293 K, yellow blocks; 5742 independent measured reflections, F2 refinement, R1=0.049, wR2=0.112, 4038 independent observed reflections [|Fo|>4σ(|Fo|), 2θ<50°], 326 parameters.",
"Synthesis of [{HC(CMeNAr)2}Mg(OiPr)]2 Complex VIII Route A 0.15 cm3 propan-2-ol (1.96×10−3 mol) was added dropwise to a solution of [{HC(CMeNAr)2}MgMe]2 (1.001 g, 1.067×10−3 mol in 50 cm3 toluene).",
"The reaction was then stirred for 60 minutes at room temperature, during which time a white precipitate formed which was recovered by filtration (0.410 g, 0.400×10−3 mol, 37.5%).",
"Crystals suitable for x-ray diffraction studies were grown from benzene.",
"Route B A schlenk tube containing 0.604 g {HC(CMeNAr)2}Mg(iPr) (1.215×10−3 mol) dissolved in 15 cm3 toluene under a dinitrogen atmosphere was evacuated and an atmosphere of dioxygen was then introduced.",
"The evacuation/refill cycle was repeated a total of three times.",
"Almost immediately the solution turns cloudy, with more precipitate forming during the time-scale of the reaction.",
"After 30 minutes stirring a room temperature the solution was filtered to afford a white solid.",
"Spectroscopic Data Complex VIII: 1H NMR δ (CDCl3, 250 MHz): 0.15 (d, 6H, 3JHH=6.84 Hz, OCHMe2), 0.94 (d, 3H, 3JHH=6.58 Hz, CHMeMe), 1.02 (d, 3H, 3JHH=6.58 Hz, CHMeMe), 1.03 (d, 3H, 3JHH=6.58 Hz, CHMe′Me′), 1.26 (d, 3H, 3JHH=6.58 Hz, CHMe′Me′), 1.42 (s, 6H, Meα), 2.89 (sept, 3JHH=6.84 Hz, CHMeMe), 3.28 (sept, 3JHH=6.84 Hz, CHMe′Me′), 3.63 (sept, 3JHH=6.84 Hz, OCHMe2), 4.77 (s, 1H, Hβ), 7.11 (m, 6H, Haryl).",
"Anal.",
"Calc.",
"for C7OH102N4Mg2O2: C 77.83, H 9.52, N 5.19%.",
"Found C 77.18, H 9.27, N 5.78%.",
"Crystallographic data for Complex VIII The crystallographic data for Complex VIII is attached in Annex 1.Synthesis of (NN)Sn(OiPr), Complex VI (NN)Sn(OiPr) was prepared by the synthetic scheme shown below.",
"Synthesis of 3-(N-(2,6-di-iso-propylphenlamino)-2-(N-(2,6-di-iso-propylphenyl)imino)propane, (NN)H 86.5 g of ArNCHCHNAr (0.230 mol) was dissolved in 300 cm3 toluene.",
"To this solution a 2.0 M toluene solution of Me3Al (120 cm3, 0.240 mol) was added dropwise over 30 minutes at room temperature, furnishing a deep red colouration.",
"The reaction was stirred at 80° C. for 16 hours, then chilled in a solid CO2/acetone bath at −78° C. The product, [{ArN═C(Me)CH2NAr}AlMe2], was then slowly hydrolysed by the addition of 200 cm3 degassed, distilled water.",
"The resultant suspension was filtered and the organic layer decanted under nitrogen.",
"The aqueous layer was extracted with 3×50 cm3 portions of diethyl ether.",
"The ether extracts were combined with the toluene solution and dried over anhydrous MgSO4.This mixture was then filtered and volatiles removed in vacuo to give a viscous yellow oil, which solidifies under prolonged vacuum.",
"Spectroscopic Data (NN)H: 1H NMR (C6D6, 250 MHz, 298K): 7.20-7.11 (m, 6H) Hmeta and Hpara, 5.24 (t, 3JHH=4.6 Hz, 1H)NH, 3.70 (d, 3JHH =4.7 Hz, 2H)NCH2, 3.59 (sept, 2H) and 2.85 (sept, 2H) CHMe2, 1.29 (d, 12H), 1.19 (d, 6H), 1.12 (d, 6H), all CHMe2, 1.24 (s, 3H) N═C(Me) Synthesis of (NN)Li Handling difficulties associated with the oily nature of (NN)H may be overcome by converting it into its lithium salt, a pale yellow powder, which can be readily recrystallised from hot hydrocarbon solvents (e.g.",
"heptane, toluene).",
"45.46 g (NN)H (0.118 mol) was dissolved in 200 cm3 Et2O and chilled in a solid CO2/acetone bath at −78° C. To this stirring solution was added 50 cm3 nBuLi (2.5M in hexanes, 0.125 mol) dropwise over 20 minutes.",
"After stirring at room temperature for 3 hours the reaction mixture was filtered and the precipitate washed with 2×50 cm3 pentane.Yield: 42.92 g 93%.",
"Spectroscopic Data (NN)Li: 1H NMR (d8-THF, 250 MHz, 298K): 7.16-6.91 (m, 3H), 6.72 (m, 2H), 6.27 (m, 1H) Hmeta and Hpara, 4.57 (s, 2H) NCH2, 3.49 (sept, 2H) and 3.04 (sept, 2H) CBMe2, 1.65 (s, 3H) N═C(Me),1.17 (d, 6H), 1.15 (d, 6H), 1.14 (d, 12H), all CHMe2.Synthesis of (NN)SnCl A 50 cm3 Et2O solution of (NN)Li (1.4680 g, 3.68×10−3mol) was added to a 30 cm3 Et2O suspension of 0.6983 g SnCl2 (3.68×10−3 mol) at −78° C. The mixture was allowed to reach room temperature and then stirred for 2 hours to afford a pale yellow suspension.",
"The reaction was filtered and concentrated in vacuo to give a pale yellow solid.",
"Recrystallisation from Et2O gave a pale yellow crystalline material.Yield: 1.192 g, 59%.",
"Spectroscopic Data (NN)SnCl: 1H NMR (C6D6, 250 MHz, 298K): δ 7.22-7.00 (m, 6H) Hmeta and Hpara, 4.65 (s, br, 2H) NCH2, 3.89 (br, 2H) and 3.19 (sept, 2H) CHMe2, 1.35 (d, 6H), 1.34 (d, 6H), 1.30 (d, 6H), 1.10 (d, 6H) all CHMe2, 1.21 (s, 3H) N═C(Me) Synthesis of (NN)Sn(OiPr).",
"Complex VI 1.1427 g (NN)SnCl and 0.2765 g Li(OiPr) (2.094×10−3mol and 4.187×10−3 mol respectively; 2.00 equivalents) were weighed into a schlenk tube and chilled in a solid CO2/acetone bath at −78° C. 20cm3 of Et2O was added and the stirring mixture was allowed to warm to room temperature, affording an orange-brown suspension.",
"This was stirred for a further 30minutes, filtered and concentrated under reduced pressure to a sticky orange solid.",
"The solid was purified by stirring in 5 cm3 pentane at −78° C. for 1hour.",
"Yield: 0.417 g, 35%.",
"Typical Polymerisation Procedure Using [{HC(CMeNAr)2}Mg(OiPr)]2 as an Initiator (Complex VIII) 200 equivalents of rac-lactide (0.121 g, 0.840×103 mol, previously recrystallised from ethyl acetate) were dissolved in 2 cm3 CH2Cl2, and this solution was chilled to −30° C. It was then added to a 2 cm3 CH2Cl2 solution of [{HC(CMeNAr)}2Mg(OiPr)]2 (0.0043 g, 0.419×10−6 mol; [M]/[I]=100) also at −30° C. The reaction mixture was then stirred for the allotted time period, then opened to the atmosphere to destroy the initiator.",
"The reaction mixture was then reduced in vacuo to afford a sticky white oil, which was analysed by 1H NMR to determine conversion.",
"The oil was purified by dissolving in 2cm3 CH2Cl2 and then reprecipitating from excess cold MeOH.",
"Using (NN)Sn(OiPr) as an Initiator (Complex VI) 0.0057 g (NN)Sn(OiPr) (1.00×10-5 mol) and 0.2885 g rac-lactide (2.00×10−3; 200 equivalents) were weighed into an ampoule and 4 cm3 toluene was added.",
"The ampoule was then placed in an oil-bath at 60° C. and stirred for 60 minutes.",
"The polymerisation was terminated by opening the ampoule to air.",
"Volatiles were removed under reduced pressure to yield a cream-coloured foamy solid.",
"1H NMR spectroscopy confirms that the conversion of lactide to poly(lactide) is 40% (i.e.",
"80 equivalents of monomer consumed in 60 minutes).",
"GPC (CHCl3): Mn=4.980, Mw=6110, pdi=1.23.Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.",
"Although the invention has been described in connection with specific preferred embodiments, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.",
"TABLE 1 Crystal data and structure refinement for 1.Identification code VG0059 Empirical formula C64H96N1O2Mg2 phH Formula weight 1080.18 Temperature 203 (2) k Diffractometer Used Siemens P4/RA Wavelength 1.54178 A Crystal system Monoclinic Space group P21/n Unit cell dimensions a = 13.5668 (8) Å alpha = 90° b = 16.1541 (8) Å beta = 98.339 (4)° c = 14.8933 (7) Å gamma = 90° Volume, Z 3229.5 (3) Å3, 2 Density (calculated) 1.111 Mg/m3 Absorption Coefficient 0.674 mm−1 F (000) 1180 Crystal colour/morphology Clear prisms Crystal size 0.40 × 0.33 × 0.27 mm θ range for data collection 1.06 to 59.99° Limiting indices −15 ≦ h < 0, 0 ≦ k < 18 −16 ≦ 1 ≦ 16 Scan type ω-scans Reflections collected 5027 Independent reflections 4800 (Rint 0.1057) Observed reflections [ F > 4σ (F) ] 4066 Absorption correction None Structure solution method Direct Refinement method Full matrix least-squares on F2 Data/restraints/parameters 4632/0/353 Goodness of-fit on F2 1.023 Final R indicates [ F > 4σ (F) ] R1 = 0.0498, wR2 = 0.1298 R indices (all data) R1 = 0.0596, wR2 = 0.1401 Extinction coefficient 0.00024 (3) Largest diff peak and hole 0.330 and −0.369 eÅ3 Mean and maximum shift/error 0.000 and 0.000 TABLE 3 Bond lengths [A] and angles [°] for 1.Mg—O 1.9803 (14) Mg.0#1 1.9863 (14) Mg—N(1) 2.114 (2) Mg—N(3) 2.126 (2) Mg—Mg#1 3.0138 (12) O—C(30) 1.425 (2) O—Mg#1 1.9863 (14) C(1)—N(1) 1.339 (3) C(1)—C(2) 1.102 (3) C(1)—C(4) 1.516 (3) N(1)—C(6) 1.416 (3) C(2)—C(3) 1.404 (3) C(3)—N(3) 1.331 (3) C(3)—C(5) 1.523 (3) N(3)—C(18) 1.451 (2) C(6)—C(7) 1.407 (3) C(6)—C(11) 1.116 (3) C(7)—C(8) 1.381 (3) C(7)—C(12) 1.520 (4) C(8)—C(9) 1.387 (5) C(9)—C(10) 1.375 (5) C(10)—C(11) 1.393 (3) C(11)—C(15) 1.507 (4) C(12)—C(14) 1.529 (4) C(12)—C(13) 1.529 (3) C(15)—C(17) 1.523 (3) C(15)—C(16) 1.530 (1) C(18)—C(19) 1.403 (3) C(18)—C(23) 1.405 (3) C(19)—C(20) 1.394 (3) C(19)—C(24) 1.517 (3) C(20)—C(21) 1.373 (3) C(21)—C(22) 1.372 (4) C(22)—C(23) 1.401 (3) C(23)—C(27) 1.519 (3) C(24)—C(26) 1.525 (4) C(24)—C(25) 1.530 (3) C(27)—C(29) 1.521 (4) C(27)—C(28) 1.527 (3) C(30)—C(31) 1.498 (3) C(30)—C(32) 1.511 (3) O—MgO#1 81.11 (6) OMg—N(1) 125.90 (7) O#1-MgN(1) 117.94 (6) O—MgN(3) 123.28 (6) O#1 Mg—N(3) 120.75 (7) N(1)—MgN(3) 91.40 (6) O—Mg Mg#1 40.63 (4) O#1-Mg-#1 40.48 (4) N(1)—Mg—Mg#1 133.95 (5) N(3)—Mg—Mg#1 134.23 (6) C(30)—O—Mg 133.38 (12) C(30) OMg#1 127.08 (12) Mg—O—Mg#1 98.89 (6) N(1)—C(1)—C(2) 124.4 (2) N(1)—C(1)—C(4) 120.9 (2) C(2)—C(1)—C(4) 114.7 (2) C(1)—N(1)—C(6) 115.0 (2) C(1) N(1)—Mg 119.04 (13) C(6)—N(1)—Mg 125.87 (12) C(1)—C(2)—C(3) 130.1 (2) N(3)—C(3)—C(2) 125.2 (2) N(3) C(3)—C(5) 120.9 (2) C(2)—C(3)—C(5) 113.9 (2) C(3)—N(3)—C(18) 114.7 (2) C(3)—N(3)—Mg 119.05 (13) C(18)—N(3)—Mg 126.14 (12) C(7)—C(6)—C(11) 120.8 (2) C(7)—C(6)—N(1) 118.4 (2) C(11)—C(6)—N(1) 120.7 (2) C(8)—C(7)—C(6) 118.9 (2) C(8)—C(7)C(12) 119.5 (2) C(6)—C(7)—C(12) 121.5 (2) C(7)—C(8)—C(9) 121.0 (3) C(10)—C(9)—C(8) 119.7 (2) C(9)—C(10)—C(11) 122.1 (3) C(10)—C(11)—C(6) 117.5 (2) C(10)—C(11)—C(15) 119.6 (2) C(6)—C(11)—C(15) 122.9 (2) C(7)—C(12)—C(14) 111.5 (2) C(7)—C(12)—C(13) 113.7 (2) C(14)—C(12)—C(13) 108.7 (2) C(11)—C(15)—C(17) 110.6 (2) C(11)—C(15)—C(16) 113.2 (2) C(17)—C(15)—C(16) 109.1 (2) C(19)—C(18)—C(23) 120.6 (2) C(19)—C(18)—N(3) 121.0 (2) C(23)—C(18)—N(3) 118.4 (2) C(20)—C(19)—C(18) 118.4 (2) C(20)—C(19)—C(21) 119.0 (2) C(18)—C(19)—C(24) 122.6 (2) C(21)—C(20)—C(19) 121.6 (2) C(22)—C(21)—C(20) 119.8 (2) C(21)—C(22)—C(23) 121.2 (2) C(22)—C(23)—C(18) 118.3 (2) C(22)—C(23)—C(27) 119.7 (2) C(18)—C(23)—C(27) 121.9 (2) C(19)—C(21)—C(26) 110.1 (2) C(19)—C(24)—C(25) 113.2 (2) C(26)—C(24)—C(25) 109.6 (2) C(23)—C(27)—C(29) 111.3 (2) C(23)—C(27)—C(28) 113.6 (2) C(29)—C(27)—C(28) 110.7 (2) OC(30)—C(31) 111.5 (2) OC(30)—C(32) 111.2 (2) C(31)—C(30) C(32) 110.5 (2)"
]
] | Patent_10416284 |
[
[
"System, process and software arrangement for recognizing handwritten characters",
"A process and system are provided for determining a most likely combination of characters.",
"In particular, character data which includes information indicative of at least one handwritten character is obtained.",
"The character data includes at least one set of segmentation points for the handwritten character.",
"Then, a score can be provided for each particular character of a set of previously stored characters based on a comparison between the character data and the previously stored particular character.",
"In addition, it is possible to compare visual aspects of the handwritten character to visual aspects of each of the previously stored characters for determining likely characters.",
"Also, a Fisher Matching procedure can be used on the character data to ascertain the likely characters.",
"A plurality of handwritten characters can include a first character that is connected to a second character of the handwritten characters via a ligature.",
"A further score of the ligature can be determined based on a starting point of the ligature and an ending point of the ligature.",
"Furthermore, the first character can be connected to the second character via a transition.",
"A particular score of the transition can be ascertained based on a difference in length of the first character and the second character.",
"This particular score can be combined with a score corresponding to the particular handwritten characters."
],
[
"1.A process for determining a most likely combination of characters, comprising the steps of: a) obtaining character data which includes information indicative of at least one handwritten character, the character data including at least one set of segmentation points for the at least one handwritten character; and b) providing a score for each particular character of a set of previously stored characters based on a comparison between the character data and the respective stored particular character.",
"2.The process according to claim 1, further comprising the step of: c) prior to steps (a) and (b), generating the character data after a user completes entering handwritten characters on an input device.",
"3.The process according to claim 2, wherein the character data corresponds to a handwritten word.",
"4.The process according to claim 1, wherein the score is provided for each of the previously stored characters by analyzing a predetermined number of the segmentation points of the character data.",
"5.The process according to claim 2, wherein the character data is generated when the user disconnects a writing instrument from the input device.",
"6.The process according to claim 1, wherein the character data includes cursive data corresponding to at least one handwritten cursive character and script data corresponding to at least one handwritten script character.",
"7.The process according to claim 1, wherein the score for each of the previously stored characters is recorded at predetermined time intervals.",
"8.The process according to claim 1, wherein the score for a current previously stored character is based on previous scores of a word path associated with a current previously stored character.",
"9.The process according to claim 8, wherein the score is the average of total scores for the previously stored characters corresponding to the word path.",
"10.The process according to claim 8, further comprising the step of: d) removing the word path from being considered as the most likely combination of characters, the word path corresponding to a characters sequence.",
"11.The process according to claim 10, wherein step (d) includes the substep of determining whether the character sequence is to be filtered out based on the score associated with the word path of the character sequence.",
"12.The process according to claim 11, wherein the determining substep is performed based on a total number of the character sequences.",
"13.The process according to claim 1, further comprising the step of: e) normalizing the score, and recording the normalized score in a storage arrangement.",
"14.The process according to claim 13, wherein step (e) is performed for each of the previously stored characters.",
"15.The process according to claim 1, wherein the character data corresponds to a sequence of handwritten characters, and further comprising the step of: f) determining if the sequence exists in a dictionary.",
"16.The process according to claim 15, wherein step (f) is performed for each of sequences of a plurality of handwritten characters at predetermined time intervals.",
"17.The process according to claim 15, wherein the dictionary has a tree-type structure, and wherein step (f) is performed by evaluating whether every character of the sequence is provided at a particular node of the tree-type structure of the dictionary.",
"18.The process according to claim 15, further comprising the step of: g) if the sequence is not in the dictionary, removing the sequence from being considered as the most likely combination of characters.",
"19.The process according to claim 15, further comprising the step of: h) if the sequence is not in the dictionary, arranging the score for the sequence to be at least one of lowered or unchanged.",
"20.The process according to claim 1, wherein the at least one handwritten character includes a plurality of handwritten characters, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a ligature, and further comprising the step of: i) determining a further score of the ligature based on a starting point of the ligature and an ending point of the ligature.",
"21.The process according to claim 20, wherein the further score is combined with the score corresponding to the previously stored characters to form a combined score.",
"22.The process according to claim 1, further comprising the steps of: j) assigning a vector corresponding to each of the handwritten characters; and k) comparing each of the vectors to recorded vectors which correspond to the previously stored characters.",
"23.The process according to claim 22, wherein step (k) is performed by applying a Fisher technique to the vectors corresponding to the handwritten characters.",
"24.The process according to claim 23, further comprising the step of: l) comparing the vectors corresponding to the handwritten characters to a cluster of vectors corresponding to the previously stored characters.",
"25.The process according to claim 24, wherein step (l) is performed by measuring a Euclidean distance from the vector corresponding to the handwritten characters to each of the clusters.",
"26.The process according to claim 25, wherein the Euclidean distance is determined from a center of each of the clusters.",
"27.The process according to claim 1, wherein the at least one handwritten character includes a plurality of handwritten characters, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a transition, and further comprising the steps of: m) determining a further score of the transition based on a difference in length of the first character and the second character; and n) combining the further score with the score corresponding to the particular characters to form a combined score.",
"28.The process according to claim 27, wherein step (m) is determined based on a height difference ratio (MH), a top difference ratio (MT) and a bottom difference ratio (MB) of the first character and the second character.",
"29.The process according to claim 28, wherein each of the previously stored handwritten characters are assigned to at least one of three classes of characters based on vertical extension dimensions thereof.",
"30.The process according to claim 29, wherein a first class of the three classes includes first characters which extend beyond a predetermined top position, wherein a second class of the three class includes second characters which extend below a predetermined bottom position, and wherein a third class of the three classes includes third characters which do not extend above the predetermined top position or below the predetermined bottom position.",
"31.The process according to claim 28, wherein the further score (VBScore) is determined according to the following: VBScore(<c1, c2>)=kH·MH(<c1, c2>)+kT·MT(<c1, c2>)+kB·MB(<c1, c2>), where c1 is the first character, c2 is the second character, kH is a height position coefficient, kT is a top position coefficient, and kB is a bottom position coefficient.",
"32.The process according to claim 31, wherein the height, top and bottom position coefficients are estimated.",
"33.The process according to claim 31, wherein the height, top and bottom position coefficients are the same for each pair of the three classes.",
"34.The process according to claim 31, further comprising the steps of: o) generating a histogram for each pair of the three classes; and p) estimating at least one of the height, top and bottom position coefficients based on the histograms.",
"35.The process according to claim 1, further comprising the step of: q) prior to steps (a) and (b), segmenting the at least one handwritten character to produce a plurality of segmentation points for the character data.",
"36.The process according to claim 35, further comprising the step of: r) adding a further segmentation point between two neighboring segmentation points of the plurality of segmentation points if a Euclidean distance between the two neighboring segmentation points if the Euclidean distance is greater than a predetermined threshold.",
"37.The process according to claim 35, further comprising the steps of: s) establishing a segment between two neighboring segmentation points of each pair of the plurality of segmentation points; and t) assigning a predetermined code to each of the segments using a previously stored list of codes.",
"38.The process according to claim 37, wherein the predetermined code is assigned to each respective segment based on a similarity of a curvature of the respective segment and a list of previously stored extensions.",
"39.The process according to claim 38, wherein each respective segment is normalized to be comparable with the previously stored extensions.",
"40.A process for ascertaining one or more particular characters from character data associated with a handwriting, comprising the steps of: a) obtaining the character data which includes information indicative of at least one handwritten character; and b) comparing visual aspects of the at least one handwritten character to visual aspects of each of previously stored characters to ascertain the one or more particular characters.",
"41.The process according to claim 40, wherein the at least one handwritten character includes a plurality of handwritten characters, and wherein step (b) includes comparing at least one of a respective angle and a position of two neighboring characters of the handwritten characters.",
"42.The process according to claim 40, further comprising the step of: c) prior to steps (a) and (b), segmenting the at least one handwritten character to produce a plurality of segmentation points for the character data.",
"43.The process according to claim 42, further comprising the step of: d) providing a score for each of previously stored characters based on a comparison between the character data and the respective previously stored particular character.",
"44.The process according to claim 42, further comprising the steps of: e) establishing a segment between two neighboring segmentation points of each pair of the plurality of segmentation points; and f) assigning a predetermined code to each of the segments using a previously stored list of codes.",
"44.The process according to claim 44, wherein the predetermined code is assigned to each respective segment based on a similarity of a curvature of the respective segment and a list of previously stored extensions.",
"45.The process according to claim 43, wherein the score is provided for each of the previously stored characters by analyzing the a predetermined number of the segmentation points of the character data.",
"46.The process according to claim 45 wherein the character data is generated when the user disconnects a writing instruments from the input device.",
"47.The process according to claim 43, wherein the score for each previously stored character is recorded at a predetermined time intervals.",
"48.The process according to claim 43, wherein the score for a current previously stored character is based on previous scores of a word path associated with a current previously stored character.",
"49.The process according to claim 48, wherein the score is an average of total scores for the previously stored characters corresponding to the word path.",
"50.The process according to claim 40, wherein the character data includes cursive data corresponding to at least one handwritten cursive character and script data corresponding to at least one handwritten script character.",
"51.The process according to claim 40, further comprising the steps of: g) assigning a vector corresponding to each of the handwritten characters; and h) comparing each of the vectors to recorded vectors which correspond to the previously stored characters.",
"52.The process according to claim 51, further comprising the step of: i) comparing the vectors corresponding to the handwritten characters to a cluster of vectors corresponding to the previously stored characters.",
"53.The process according to claim 52, wherein step (i) is performed by measuring a Euclidean distance from the vector corresponding to the handwritten characters to each of the clusters.",
"54.The process according to claim 53, wherein the Euclidean distance is determined from a center of each of the clusters.",
"55.The process according to claim 43, wherein the at least one handwritten character includes a plurality of handwritten characters, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a transition, and further comprising the steps of: j) determining a further score of the transition based on a difference in length of the first character and the second character; and k) combining the further score with the score corresponding to the previously stored characters to form a combined score.",
"56.The process according to claim 55, wherein step (k) is determined based on a height difference ratio (MH), a top difference ratio (MT) and a bottom difference ratio (MB) of the first character and the second character.",
"57.The process according to claim 56, wherein each of the previously stored handwritten characters are assigned to at least one of three classes of characters based on vertical extension dimensions thereof.",
"58.The process according to claim 57, wherein a first class of the three classes includes first characters which extend beyond a predetermined top position, wherein a second class of the three class includes second characters which extend below a predetermined bottom position, and wherein a third class of the three classes includes third characters which do not extend above the predetermined top position or below the predetermined bottom position.",
"59.The process according to claim 56, wherein the further score (VBScore) is determined according to the following: VBScore(<c1, c2>)=kH·MH(<c1, c2>)+kT·MT(<c1, c2>)+kB·MB(<c1, c2>), where c1 is the first character, c2 is the second character, kH is a height position coefficient, kT is a top position coefficient, and kB is a bottom position coefficient.",
"60.The process according to claim 61, wherein the height, top and bottom position coefficients are estimated.",
"61.The process according to claim 61, wherein the height, top and bottom position coefficients are the same for each pair of the three classes.",
"62.The process according to claim 61, further comprising the steps of: l) generating a histogram for each pair of the three classes; and m) estimating at least one of the height, top and bottom position coefficients based on the histograms.",
"63.The process according to claim 40, further comprising the step of: n) prior to steps (a) and (b), segmenting the at least one handwritten character to produce a plurality of segmentation points for the character data.",
"64.The process according to claim 63, further comprising the step of: o) adding a further segmentation point between two neighboring segmentation points of the plurality of segmentation points if a Euclidean distance between the two neighboring segmentation points is greater than a predetermined threshold.",
"65.The process according to claim 63, further comprising the steps of: p) establishing a segment between two neighboring segmentation points of each pair of the plurality of segmentation points; and q) assigning a predetermined code to each of the segments using a previously stored list of codes.",
"66.The process according to claim 65, wherein the predetermined code is assigned to each respective segment based on a similarity of a curvature of the respective segment and a list of previously stored extensions.",
"67.The process according to claim 66, wherein each respective segment is normalized to be comparable with the previously stored extensions.",
"68.The process according to claim 40, further comprising the step of: r) performing a training procedure to obtain at least two variations of the same character.",
"69.The process according to claim 68, wherein step (r) is performed using a Fisher technique.",
"70.A process for ascertaining one or more particular characters from character data associated with a handwriting, comprising the steps of: a) obtaining the character data which includes information indicative of at least one handwritten character; and b) applying a Fisher Matching procedure on the character data to as certain the one or more particular characters.",
"71.A process for ascertaining at least one particular character from character data associated with a handwriting, comprising the steps of: a) obtaining the character data associated with handwritten characters, a first character of the handwritten characters being connected to a second character of the handwritten characters via a ligature; and b) determining a further score of the ligature based on a start point of the ligature and an end point of the ligature.",
"72.The process according to claim 71, further comprising the steps of: c) assigning a vector corresponding to each of the handwritten characters; and d) comparing each of the vectors to recorded vectors which correspond to previously stored characters.",
"73.The process according to claim 72, wherein step (d) is performed by applying a Fisher technique to the vectors corresponding to the handwritten characters.",
"74.The process according to claim 73, further comprising the step of: e) comparing the vectors corresponding to the handwritten characters to a cluster of vectors corresponding to the previously stored characters.",
"75.The process according to claim 74, wherein step (e) is performed by measuring a Euclidean distance from the vector corresponding to the handwritten characters to each of the clusters.",
"76.The process according to claim 75, wherein the Euclidean distance is determined from a center of each of the clusters.",
"77.A process for ascertaining at least one particular handwritten character from character data associated with handwritten characters, comprising the steps of: a) obtaining the character data, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a transition; b) determining a particular score of the transition based on a difference in length of the first character and the second character; and c) combining the particular score with a score corresponding to the handwritten characters to form a combined score.",
"78.The process according to claim 77, wherein step (b) is determined based on a height difference ratio (MH), a top difference ratio (MT) and a bottom difference ratio (MB) of the first character and the second character.",
"79.The process according to claim 78, further comprising the step of: d) assigning each of previously stored handwritten characters to at least one of three classes of characters based on vertical extension dimensions thereof.",
"80.The process according to claim 79, wherein a first class of the three classes includes first characters which extend beyond a predetermined top position, wherein a second class of the three class includes second characters which extend below a predetermined bottom position, and wherein a third class of the three classes includes third characters which do not extend above the predetermined top position or below the predetermined bottom position.",
"81.The process according to claim 78, wherein the particular score (VBScore) is determined according to the following: VBScore(<c1, c2>)=kH·MH(<c1, c2>)+kT·MT(<c1, c2>)+kB·MB(<c1, c2>), where c1 is the first character, c2 is the second character, kH is a height position coefficient, kT is a top position coefficient, and kB is a bottom position coefficient.",
"82.The process according to claim 81, wherein the height, top and bottom position coefficients are estimated.",
"83.The process according to claim 81, wherein the height, top and bottom position coefficients are the same for each pair of the three classes.",
"84.The process according to claim 81, further comprising the steps of: e) generating a histogram for each pair of the three classes; and f) estimating at least one of the height, top and bottom position coefficients based on the histograms.",
"85.A software system which, when executed on a processing device, configures the processing device to determine a most likely combination of characters, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following: a) obtain character data which includes information indicative of at least one handwritten character, the character data including at least one set of segmentation points for the at least one handwritten character, and b) provide a score for each particular character of a set of previously stored characters based on a comparison between the character data and the respective previously stored particular character.",
"86.The software system according to claim 85, wherein, when executed on the processing device, the processing subsystem is capable of generating the character data after a user completes entering handwritten characters on an input device.",
"87.The software system according to claim 86, wherein the character data corresponds to a handwritten word.",
"88.The software system according to claim 86, wherein the score is provided for each of the previously stored characters by analyzing a predetermined number of the segmentation points of the character data.",
"89.The software system according to claim 86, wherein the character data is generated when the user disconnects a writing instrument from the input device.",
"90.The software system according to claim 85, wherein the character data includes cursive data corresponding to at least one handwritten cursive character and script data corresponding to at least one handwritten script character.",
"91.The software system according to claim 85, wherein the score for each of the previously stored characters is recorded at predetermined time intervals.",
"92.The software system according to claim 85, wherein the score for a current previously stored character is based on previous scores of a word path associated with a current previously stored character.",
"93.The software system according to claim 92, wherein the score is the average of total scores for the previously stored characters corresponding to the word path.",
"94.The software system according to claim 92, wherein, when in operation, the processing subsystem is configured to remove the word path from being considered as the most likely combination of characters, the word path corresponding to a characters sequence.",
"95.The software system according to claim 94, wherein, when in operation, the processing subsystem is configured to determines whether the character sequence is to be filtered out based on the score associated with the word path of the character sequence.",
"96.The software system according to claim 95, wherein, when in operation, the processing subsystem is configured to make the determination regarding the character sequences based on a total number of the character sequences.",
"97.The software system according to claim 85, wherein, when in operation, the processing subsystem is configured to normalize the score, and record the normalized score in a storage arrangement.",
"98.The software system according to claim 85, wherein the character data corresponds to a sequence of handwritten characters, and wherein, when in operation, the processing subsystem is configured to determine if the sequence exists in a dictionary.",
"99.The software system according to claim 98, wherein the determination regarding the existence of the sequence is performed for each of sequences of a plurality of handwritten characters at predetermined time intervals.",
"100.The software system according to claim 98, wherein the dictionary has a tree-type structure, and wherein the determination regarding the existence of the sequence is performed by evaluating whether every character of the sequence is provided at a particular node of the tree-type structure of the dictionary.",
"101.The software system according to claim 98, wherein, when in operation, the processing subsystem is configured to remove the sequence from being considered as the most likely combination of characters if the sequence is not in the dictionary.",
"102.The software system according to claim 98, wherein, when in operation, the processing subsystem is configured to arrange the score for the sequence to be at least one of lowered or unchanged if the sequence is not in the dictionary.",
"103.The software system according to claim 85, wherein the at least one handwritten character includes a plurality of handwritten characters, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a ligature, and wherein, when in operation, the processing subsystem is configured to determine a further score of the ligature based on a starting point of the ligature and an ending point of the ligature.",
"104.The software system according to claim 102, wherein the further score is combined with the score corresponding to the previously stored characters to form a combined score.",
"105.The software system according to claim 85, wherein, when in operation, the processing subsystem is configured to: c) assign a vector corresponding to each of handwritten characters, and d) compare each of the vectors to recorded vectors which correspond to the previously stored characters.",
"106.The software system according to claim 105, wherein the comparison is performed by applying a Fisher technique to the vectors corresponding to the handwritten characters.",
"107.The software system according to claim 106, wherein, when in operation, the processing subsystem is configured to compare the vectors corresponding to the handwritten characters to a cluster of vectors corresponding to the previously stored characters.",
"108.The software system according to claim 107, wherein the comparison of the vectors is performed by measuring a Euclidean distance from the vector corresponding to the handwritten characters to each of the clusters.",
"109.The software system according to claim 108, wherein the Euclidean distance is determined from a center of each of the clusters.",
"110.The software system according to claim 85, wherein the at least one handwritten character includes a plurality of handwritten characters, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a transition, and wherein, when in operation, the processing subsystem is configured to: e) determine a further score of the transition based on a difference in length of the first character and the second character, and f) combine the further score with the score corresponding to the particular characters to form a combined score.",
"111.The software system according to claim 110, wherein the further score is determined based on a height difference ratio (MH), a top difference ratio (MT) and a bottom difference ratio (MB) of the first character and the second character.",
"112.The software system according to claim 111, wherein each of the previously stored handwritten characters are assigned to at least one of three classes of characters based on vertical extension dimensions thereof.",
"113.The software system according to claim 112, wherein a first class of the three classes includes first characters which extend beyond a predetermined top position, wherein a second class of the three class includes second characters which extend below a predetermined bottom position, and wherein a third class of the three classes includes third characters which do not extend above the predetermined top position or below the predetermined bottom position.",
"114.The software system according to claim 111, wherein the further score (VBScore) is determined according to the following: VBScore(<c1, c2>)=kH·MH(<c1, c2>)+kT·MT(<c1, c2>)+kB·MB(<c1, c2>), where c1 is the first character, c2 is the second character, kH is a height position coefficient, kT is a top position coefficient, and kB is a bottom position coefficient.",
"115.The software system according to claim 114, wherein the height, top and bottom position coefficients are estimated.",
"116.The software system according to claim 114, wherein the height, top and bottom position coefficients are the same for each pair of the three classes.",
"117.The software system according to claim 114, wherein, when in operation, the processing subsystem is configured to: g) generate a histogram for each pair of the three classes, and h) estimate at least one of the height, top and bottom position coefficients based on the histograms.",
"118.The software system according to claim 85, wherein, when in operation, the processing subsystem is configured to segment the at least one particular character to produce a plurality of segmentation points for the character data.",
"119.The software system according to claim 118, wherein, when in operation, the processing subsystem is configured to add a further segmentation point between two neighboring segmentation points of the plurality of segmentation points if a Euclidean distance between the two neighboring segmentation points is greater than a predetermined threshold.",
"120.The software system according to claim 11 8, wherein, when in operation, the processing subsystem is configured to: i) establish a segment between two neighboring segmentation points of each pair of the plurality of segmentation points and j) assign a predetermined code to each of the segments using a previously stored list of codes.",
"121.The software system according to claim 120, wherein the predetermined code is assigned to each respective segment based on a similarity of a curvature of the respective segment and a list of previously stored extensions.",
"122.The software system according to claim 121, wherein each respective segment is normalized to be comparable with the previously stored extensions.",
"123.A software system which, when executed on a processing device, configures the processing device to ascertain one or more particular characters from character data associated with a handwriting, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) obtaining the character data which includes information indicative of at least one handwritten character, and b) comparing visual aspects of the at least one handwritten character to visual aspects of each of previously stored characters to ascertain the one or more particular characters.",
"124.A software system which, when executed on a processing device, configures the processing device to ascertain one or more particular characters from character data associated with a handwriting, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) obtaining the character data which includes information indicative of at least one handwritten character, and b) applying a Fisher Matching procedure on the character data to ascertain the one or more particular characters.",
"125.A software system which, when executed on a processing device, configures the processing device to ascertain at least one particular character from character data associated with handwritten characters, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) obtaining the character data associated with handwritten characters, a first character of the handwritten characters being connected to a second character of the handwritten characters via a ligature, and b) determining a further score of the ligature based on a starting point of the ligature and an ending point of the ligature.",
"126.A software system which, when executed on a processing device, configures the processing device to ascertain at least one particular handwritten character from character data associated with handwritten characters, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) obtaining the character data, wherein a first character of the handwritten characters is connected to a second character of the handwritten characters via a transition, b) determining a particular score of the transition based on a difference in length of the first character and the second character, and c) combining the particular score with a score corresponding to the particular handwritten characters to form a combined score."
],
[
"<SOH> BACKGROUND INFORMATION <EOH>Currently, technologies such as microcomputers, word processors, fax machines and electronic mails utilize electronic handwriting recording and recognition techniques to enable a useful and versatile communication between such devices.",
"In particular, conventional computer-based handwriting analysis methods include a recognition and interpretation of characters, as well as a verification of the handwritten data.",
"The known electronic handwriting recognition procedures generally transcribe a message, represented in a spatial form of graphical marks, into a computer text, e.g., into a sequence of 8-bit ASCII characters.",
"These handwriting interpretation techniques generally determine the most likely meaning of a particular handwritten text, e.g., a mailing address written on an envelope.",
"The handwriting verification techniques are used to determine whether the handwritten text belongs to a particular individual, and can be used in, e.g., the forensic investigations.",
"The handwriting recognition techniques can be considered as being in two separate categories—on-line recognition and off-line recognition.",
"The on-line handwriting recognition techniques are generally used with a transducer/input device is connected to a computer which is available to the user.",
"One such arrangement is shown in FIG.",
"1 , which illustrates an on-line handwriting recognition system 5 that can also be used with conventional techniques and techniques according to the present invention.",
"The transducer/input device converts the user's writing motion into a sequence of signals, and sends this signal information to a computer 50 .",
"The computer 50 generally includes a handwriting recognition system.",
"An exemplary transducer can be a tablet digitizer 10 .",
"This tablet digitizer 10 generally includes a plastic or electronic pen 15 and a pressure or electrostatic-sensitive writing surface 20 on which the user provides the handwritten information using the pen 15 .",
"By sampling or tracking the movement of a tip of the pen 15 on the writing surface 20 , the tablet digitizer 10 is able to detect certain information when the pen 15 is in contact with the writing surface 20 e.g., the x and y coordinates of a sampled point on the writing surface 20 , providing information indicative of whether the pen 15 touches the writing surface 20 (“pen-down state” or has been removed therefrom—“pen-up state”, etc.).",
"The information is transmitted to the connected computer 50 for recognition processing by the handwriting recognition system.",
"A “stroke” in the data in the “on-line” recognition system can be defined as a sequence of sampled points from the pen-down state to the pen-up state of the pen 15 .",
"Thus, the completed writing of a word would likely consist of a sequence of one or more strokes.",
"The tablet digitizer 10 then captures the temporal (dynamic) data of the word when it samples the points on the contours that the user is forming.",
"The off-line handwriting recognition techniques are generally related to the field of Optical Character Recognition (“OCR”).",
"In contrast to the on-line handwriting recognition techniques, these off-line techniques are not interactive.",
"In the exemplary OCR system, a machine-printed material is scanned into a computer file in two-dimensional image representation using a scanner.",
"Then, the off-line handwriting recognition technique of this conventional OCR system attempts to recognize the scanned handwritten data.",
"One of the benefits of the on-line handwriting recognition techniques which set them apart from the off-line handwriting OCR or other image recognition techniques is their ability to utilize the temporal and dynamic input sequence information which is provided directly by the user in real-time.",
"This dynamic information obtained by the on-line handwriting recognition techniques provides a vivid separation of the foreground from the background, and thus can bypass the pre-processing procedures that are required to be performed by the off-line handwriting recognition techniques.",
"Also, the obtained on-line dynamic information is generally more compact than the off-line information because of the different dimensionalities in representation.",
"The difference in the data size also leads to the difference in the processing time.",
"Another advantage of the on-line handwriting recognition techniques is their use of the sequence information of the data received thereby which allows the character boundary segmentation easier to be performed.",
"After the preprocessing stage, most handwriting recognition systems and methods attempt to separate their received data into intervals/segments (which correspond to hypothetical characters), and apply an evaluation process to such intervals/segments.",
"The recognition performance of such system and process is substantially dependent on the quality and robustness of the character segmentation.",
"Due to the cues available from the temporal ordering of its input data, the on-line handwriting recognizer may generate the segmentations in a reliable and efficient manner.",
"For example, when the two neighboring characters overlap in the respective occupying regions, it is significantly more difficult for an off-line recognition system and method to segment such characters correctly.",
"This is because any simple geometric separation would likely contain a portion of at least one of the characters.",
"Using the on-line handwriting recognition system, it would be easier to handle the above-described scenario.",
"As known to those having ordinary skill in the art, the handwriting recognition systems (whether on-line or off-line) are designed to support three different styles, i.e., a printed style, a cursive style and a mixed style.",
"Recognizing the printed style of handwriting is, most likely, simpler than recognizing other handwriting styles.",
"This is because each character of such style has clearer boundaries with its neighboring characters.",
"For example, the characters in the printed style are usually separated by the “pen-up” signal in the on-line handwriting recognition system.",
"In recognizing the cursive handwritten script, however, most of the component characters are connected to their neighbors by a sub-stroke (i.e., a “ligature”) which is not a part of any character or letter, but only a connecting pattern between two characters/letters.",
"In this situation, it is more difficult to hypothesize about the character segmentation since there is less information regarding the likely segmentation boundaries of each character.",
"Handwritings having a printed style can be regarded as a subset of the cursive mode recognition, and the mixed mode can be obtained as a by-product of obtaining both printed and cursive modes support.",
"Therefore, one having ordinary skill in the art would understand that it is the hardest task to recognize characters in the cursive mode.",
"Conventional handwriting recognition systems and methods can be writer-independent or writer-dependent.",
"For example, writer-independent systems can handle the idiosyncrasies of user's writing styles, and writer-dependent systems are trained to recognize a single user's writing style.",
"It is possible to have the same character (or a class of character) written in different ways, e.g., so that they are in different subclasses or allographs.",
"Therefore, each character class usually consists of one or more subclasses.",
"Correctly identifying a good set of allographs is a challenging task which requires a recording of a huge number of samples, which usually cannot be done by the conventional systems and methods.",
"Also, a larger number of subclasses/allographs would require additional time for processing for such conventional systems which would not be preferable, especially when using an on-line character recognition system or method."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of a process and system according to the present invention overcome the above-described deficiencies of the conventional systems and methods.",
"In particular, a process and system are provided which determine the most likely combination of characters.",
"In particular, character data which includes information indicative of at least one handwritten character is obtained, which is preferably on-line data or real-time data.",
"The character data includes at least one set of segmentation points for the handwritten character.",
"Then, a score can be determined for each particular character of a set of previously stored characters based on a comparison between the character data and the respective particular character.",
"In addition, it is possible to generate the character data after a user completes entering handwritten characters on an input device.",
"The character data may correspond to a handwritten word.",
"Also, the score can be provided for each of the previously stored characters by analyzing a predetermined number of the segmentation points of the character data.",
"The character data may be generated when the user disconnects a writing instrument from the input device.",
"The character data can also include cursive data corresponding to at least one handwritten cursive character, and script data corresponding to at least one handwritten script character.",
"According to another embodiment of the present invention, the score for each of the previously stored characters can be recorded at predetermined time intervals.",
"The score for a current previously stored character may be based on previous scores of a word path associated with the current previously stored character.",
"This score can preferably be the average of the total scores for the previously stored characters corresponding to the word path which includes the score for the current previously-stored character.",
"The word path can be removed from being considered to be the most likely combination of characters.",
"Also, the word path preferably corresponds to a characters sequence of the certain characters.",
"A determination can also be made as to whether the character sequence is to be filtered out based on the score associated with the word path of the character sequence.",
"This determination is preferably made based on a total number of the character sequences.",
"In yet another embodiment of the present invention, the score can be normalized, and the normalized score may be stored in a storage arrangement.",
"The character data may also correspond to a sequence of handwritten characters, and it is possible to determine whether the sequence exists in a dictionary.",
"This determination regarding the existence of the sequence may be performed for each of the sequences of a plurality of handwritten characters at predetermined time intervals.",
"Also, the dictionary may have a tree-type structure, and the determination regarding the existence of the sequence can be performed by evaluating whether every character of the sequence is provided at a particular node of this tree-type structure of the dictionary.",
"The sequence can be removed from consideration as being the most likely combination of characters if the sequence is not in the dictionary.",
"In addition, the score for the sequence can be lowered and/or unchanged if the sequence is not in the dictionary.",
"In still another embodiment of the present invention, a first character of the handwritten characters is connected to a second character of the handwritten characters via a ligature, and a further score of the ligature is determined based on a starting and ending points of the ligature.",
"This further score may be combined with the score corresponding to the previously stored characters so as to form a combined score.",
"According to another embodiment of the present invention, a vector corresponding to each of the handwritten characters can be assigned, and a comparison of each of the vectors can be made to vectors corresponding to the previously-stored characters.",
"This comparison can be performed by applying a Fisher technique to the vectors corresponding to the handwritten characters.",
"The vectors corresponding to the handwritten characters can preferably be compared to a cluster of vectors corresponding to the previously stored characters.",
"This comparison of the vectors may be performed by measuring a Euclidean distance from the vector corresponding to the handwritten characters to each of the clusters.",
"The Euclidean distance can be measured from a center of each of the clusters.",
"According to still another embodiment of the present invention, a first character of the handwritten characters may be connected to a second character of the handwritten characters via a transition.",
"A further score of the transition can also be determined based on a difference in length of the first character and the second character, and the further score combined with the score corresponding to the particular characters to form a combined score.",
"This further score is preferably determined based on a height difference ratio (M H ), a top difference ratio (M T ) and a bottom difference ratio (M B ) of the first and the second characters.",
"Each of the previously stored handwritten characters may be assigned to at least one of three classes of characters based on vertical extension dimensions thereof.",
"A first class of the three classes may include first characters which extend beyond a predetermined top position, a second class of the three class includes second characters which extend below a predetermined bottom position, and a third class of the three classes includes third characters which do not extend above the predetermined top position or below the predetermined bottom position.",
"The above-referenced further score (VBScore) can be determined according to the following equation: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"VB Score(< c 1 , c 2 >)= k H ·M H (< c 1 , c 2 >)+ k T ·M T (< c 1 , c 2 >)+ k B ·M B (< c 1 , c 2 >), in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"where c 1 is the first character, c 2 is the second character, k H · is a height position coefficient, k T is a top position coefficient and k B is a bottom position coefficient.",
"The height, top and bottom position coefficients can preferably be estimated, and may be the same for each pair of the three classes.",
"A histogram can be generated for each pair of the three classes, and the height, top and/or bottom position coefficients may be estimated based on such histograms.",
"According to yet another embodiment of the present invention, the particular character can be segmented to produce a plurality of segmentation points for the character data.",
"It is possible to insert a further segmentation point on the respective segment between two neighboring segmentation points if the Euclidean distance between two such neighboring segmentation points is this distance greater than a predetermined threshold.",
"Also, the segment is preferably established between two neighboring segmentation points of each pair of the plurality of segmentation points, and a predetermined code is assigned to each of the segments using a previously stored list of codes.",
"This predetermined code can be assigned to each respective segment based on a similarity of a curvature of the respective segment and a list of previously stored extensions.",
"Furthermore, each respective segment is normalized to be comparable with the previously stored extensions."
],
[
"FIELD OF THE INVENTION The present invention relates to a system, process and software arrangement for recognizing handwritten characters, and more particularly to recognizing the handwritten characters on-line using, e.g., character segmentation techniques.",
"BACKGROUND INFORMATION Currently, technologies such as microcomputers, word processors, fax machines and electronic mails utilize electronic handwriting recording and recognition techniques to enable a useful and versatile communication between such devices.",
"In particular, conventional computer-based handwriting analysis methods include a recognition and interpretation of characters, as well as a verification of the handwritten data.",
"The known electronic handwriting recognition procedures generally transcribe a message, represented in a spatial form of graphical marks, into a computer text, e.g., into a sequence of 8-bit ASCII characters.",
"These handwriting interpretation techniques generally determine the most likely meaning of a particular handwritten text, e.g., a mailing address written on an envelope.",
"The handwriting verification techniques are used to determine whether the handwritten text belongs to a particular individual, and can be used in, e.g., the forensic investigations.",
"The handwriting recognition techniques can be considered as being in two separate categories—on-line recognition and off-line recognition.",
"The on-line handwriting recognition techniques are generally used with a transducer/input device is connected to a computer which is available to the user.",
"One such arrangement is shown in FIG.",
"1, which illustrates an on-line handwriting recognition system 5 that can also be used with conventional techniques and techniques according to the present invention.",
"The transducer/input device converts the user's writing motion into a sequence of signals, and sends this signal information to a computer 50.The computer 50 generally includes a handwriting recognition system.",
"An exemplary transducer can be a tablet digitizer 10.This tablet digitizer 10 generally includes a plastic or electronic pen 15 and a pressure or electrostatic-sensitive writing surface 20 on which the user provides the handwritten information using the pen 15.By sampling or tracking the movement of a tip of the pen 15 on the writing surface 20, the tablet digitizer 10 is able to detect certain information when the pen 15 is in contact with the writing surface 20 e.g., the x and y coordinates of a sampled point on the writing surface 20, providing information indicative of whether the pen 15 touches the writing surface 20 (“pen-down state” or has been removed therefrom—“pen-up state”, etc.).",
"The information is transmitted to the connected computer 50 for recognition processing by the handwriting recognition system.",
"A “stroke” in the data in the “on-line” recognition system can be defined as a sequence of sampled points from the pen-down state to the pen-up state of the pen 15.Thus, the completed writing of a word would likely consist of a sequence of one or more strokes.",
"The tablet digitizer 10 then captures the temporal (dynamic) data of the word when it samples the points on the contours that the user is forming.",
"The off-line handwriting recognition techniques are generally related to the field of Optical Character Recognition (“OCR”).",
"In contrast to the on-line handwriting recognition techniques, these off-line techniques are not interactive.",
"In the exemplary OCR system, a machine-printed material is scanned into a computer file in two-dimensional image representation using a scanner.",
"Then, the off-line handwriting recognition technique of this conventional OCR system attempts to recognize the scanned handwritten data.",
"One of the benefits of the on-line handwriting recognition techniques which set them apart from the off-line handwriting OCR or other image recognition techniques is their ability to utilize the temporal and dynamic input sequence information which is provided directly by the user in real-time.",
"This dynamic information obtained by the on-line handwriting recognition techniques provides a vivid separation of the foreground from the background, and thus can bypass the pre-processing procedures that are required to be performed by the off-line handwriting recognition techniques.",
"Also, the obtained on-line dynamic information is generally more compact than the off-line information because of the different dimensionalities in representation.",
"The difference in the data size also leads to the difference in the processing time.",
"Another advantage of the on-line handwriting recognition techniques is their use of the sequence information of the data received thereby which allows the character boundary segmentation easier to be performed.",
"After the preprocessing stage, most handwriting recognition systems and methods attempt to separate their received data into intervals/segments (which correspond to hypothetical characters), and apply an evaluation process to such intervals/segments.",
"The recognition performance of such system and process is substantially dependent on the quality and robustness of the character segmentation.",
"Due to the cues available from the temporal ordering of its input data, the on-line handwriting recognizer may generate the segmentations in a reliable and efficient manner.",
"For example, when the two neighboring characters overlap in the respective occupying regions, it is significantly more difficult for an off-line recognition system and method to segment such characters correctly.",
"This is because any simple geometric separation would likely contain a portion of at least one of the characters.",
"Using the on-line handwriting recognition system, it would be easier to handle the above-described scenario.",
"As known to those having ordinary skill in the art, the handwriting recognition systems (whether on-line or off-line) are designed to support three different styles, i.e., a printed style, a cursive style and a mixed style.",
"Recognizing the printed style of handwriting is, most likely, simpler than recognizing other handwriting styles.",
"This is because each character of such style has clearer boundaries with its neighboring characters.",
"For example, the characters in the printed style are usually separated by the “pen-up” signal in the on-line handwriting recognition system.",
"In recognizing the cursive handwritten script, however, most of the component characters are connected to their neighbors by a sub-stroke (i.e., a “ligature”) which is not a part of any character or letter, but only a connecting pattern between two characters/letters.",
"In this situation, it is more difficult to hypothesize about the character segmentation since there is less information regarding the likely segmentation boundaries of each character.",
"Handwritings having a printed style can be regarded as a subset of the cursive mode recognition, and the mixed mode can be obtained as a by-product of obtaining both printed and cursive modes support.",
"Therefore, one having ordinary skill in the art would understand that it is the hardest task to recognize characters in the cursive mode.",
"Conventional handwriting recognition systems and methods can be writer-independent or writer-dependent.",
"For example, writer-independent systems can handle the idiosyncrasies of user's writing styles, and writer-dependent systems are trained to recognize a single user's writing style.",
"It is possible to have the same character (or a class of character) written in different ways, e.g., so that they are in different subclasses or allographs.",
"Therefore, each character class usually consists of one or more subclasses.",
"Correctly identifying a good set of allographs is a challenging task which requires a recording of a huge number of samples, which usually cannot be done by the conventional systems and methods.",
"Also, a larger number of subclasses/allographs would require additional time for processing for such conventional systems which would not be preferable, especially when using an on-line character recognition system or method.",
"SUMMARY OF THE INVENTION Various embodiments of a process and system according to the present invention overcome the above-described deficiencies of the conventional systems and methods.",
"In particular, a process and system are provided which determine the most likely combination of characters.",
"In particular, character data which includes information indicative of at least one handwritten character is obtained, which is preferably on-line data or real-time data.",
"The character data includes at least one set of segmentation points for the handwritten character.",
"Then, a score can be determined for each particular character of a set of previously stored characters based on a comparison between the character data and the respective particular character.",
"In addition, it is possible to generate the character data after a user completes entering handwritten characters on an input device.",
"The character data may correspond to a handwritten word.",
"Also, the score can be provided for each of the previously stored characters by analyzing a predetermined number of the segmentation points of the character data.",
"The character data may be generated when the user disconnects a writing instrument from the input device.",
"The character data can also include cursive data corresponding to at least one handwritten cursive character, and script data corresponding to at least one handwritten script character.",
"According to another embodiment of the present invention, the score for each of the previously stored characters can be recorded at predetermined time intervals.",
"The score for a current previously stored character may be based on previous scores of a word path associated with the current previously stored character.",
"This score can preferably be the average of the total scores for the previously stored characters corresponding to the word path which includes the score for the current previously-stored character.",
"The word path can be removed from being considered to be the most likely combination of characters.",
"Also, the word path preferably corresponds to a characters sequence of the certain characters.",
"A determination can also be made as to whether the character sequence is to be filtered out based on the score associated with the word path of the character sequence.",
"This determination is preferably made based on a total number of the character sequences.",
"In yet another embodiment of the present invention, the score can be normalized, and the normalized score may be stored in a storage arrangement.",
"The character data may also correspond to a sequence of handwritten characters, and it is possible to determine whether the sequence exists in a dictionary.",
"This determination regarding the existence of the sequence may be performed for each of the sequences of a plurality of handwritten characters at predetermined time intervals.",
"Also, the dictionary may have a tree-type structure, and the determination regarding the existence of the sequence can be performed by evaluating whether every character of the sequence is provided at a particular node of this tree-type structure of the dictionary.",
"The sequence can be removed from consideration as being the most likely combination of characters if the sequence is not in the dictionary.",
"In addition, the score for the sequence can be lowered and/or unchanged if the sequence is not in the dictionary.",
"In still another embodiment of the present invention, a first character of the handwritten characters is connected to a second character of the handwritten characters via a ligature, and a further score of the ligature is determined based on a starting and ending points of the ligature.",
"This further score may be combined with the score corresponding to the previously stored characters so as to form a combined score.",
"According to another embodiment of the present invention, a vector corresponding to each of the handwritten characters can be assigned, and a comparison of each of the vectors can be made to vectors corresponding to the previously-stored characters.",
"This comparison can be performed by applying a Fisher technique to the vectors corresponding to the handwritten characters.",
"The vectors corresponding to the handwritten characters can preferably be compared to a cluster of vectors corresponding to the previously stored characters.",
"This comparison of the vectors may be performed by measuring a Euclidean distance from the vector corresponding to the handwritten characters to each of the clusters.",
"The Euclidean distance can be measured from a center of each of the clusters.",
"According to still another embodiment of the present invention, a first character of the handwritten characters may be connected to a second character of the handwritten characters via a transition.",
"A further score of the transition can also be determined based on a difference in length of the first character and the second character, and the further score combined with the score corresponding to the particular characters to form a combined score.",
"This further score is preferably determined based on a height difference ratio (MH), a top difference ratio (MT) and a bottom difference ratio (MB) of the first and the second characters.",
"Each of the previously stored handwritten characters may be assigned to at least one of three classes of characters based on vertical extension dimensions thereof.",
"A first class of the three classes may include first characters which extend beyond a predetermined top position, a second class of the three class includes second characters which extend below a predetermined bottom position, and a third class of the three classes includes third characters which do not extend above the predetermined top position or below the predetermined bottom position.",
"The above-referenced further score (VBScore) can be determined according to the following equation: VBScore(<c1, c2>)=kH·MH(<c1, c2>)+kT·MT(<c1, c2>)+kB·MB(<c1, c2>), where c1 is the first character, c2 is the second character, kH· is a height position coefficient, kT is a top position coefficient and kB is a bottom position coefficient.",
"The height, top and bottom position coefficients can preferably be estimated, and may be the same for each pair of the three classes.",
"A histogram can be generated for each pair of the three classes, and the height, top and/or bottom position coefficients may be estimated based on such histograms.",
"According to yet another embodiment of the present invention, the particular character can be segmented to produce a plurality of segmentation points for the character data.",
"It is possible to insert a further segmentation point on the respective segment between two neighboring segmentation points if the Euclidean distance between two such neighboring segmentation points is this distance greater than a predetermined threshold.",
"Also, the segment is preferably established between two neighboring segmentation points of each pair of the plurality of segmentation points, and a predetermined code is assigned to each of the segments using a previously stored list of codes.",
"This predetermined code can be assigned to each respective segment based on a similarity of a curvature of the respective segment and a list of previously stored extensions.",
"Furthermore, each respective segment is normalized to be comparable with the previously stored extensions.",
"BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the present invention and its advantages, reference is now made to the following description, taken in conjunction with the accompanying drawings, in which: FIG.",
"1 is an exemplary embodiment of an on-line handwriting recognition system which can be used with conventional techniques and advantageous techniques according to the present invention; FIG.",
"2 is a high-level block diagram of an exemplary process for recognizing handwritten characters according to the present invention; FIG.",
"3 is a flow diagram of another exemplary embodiment of the process for recognizing handwritten characters according to the present invention; FIG.",
"4A is an illustration of the original distribution of interval segmentation points in an exemplary handwriting; FIG.",
"4B is an illustration of a resultant distribution of the interval points of FIG.",
"4A after being subjected to a local filtering technique of the present invention.",
"FIG.",
"5A is an exemplary illustration of a cursively-written word “eye” with only high-curvature points provided therein, according to a particular embodiment of the present invention; FIG.",
"5B is an exemplary illustration of the cursively-written word “eye” with the high-curvature points and augmentation points provided therein, according to the present invention; FIG.",
"6 is an illustration of an exemplary embodiment of directional convex templates according to the present invention which may represent direction and convexity of categories of feature-links; FIG.",
"7 is a flow diagram of an exemplary embodiment of the process according to the present invention, in which indexes representing the directional template of FIG.",
"6 are assigned to particular feature-links; FIG.",
"8 is an exemplary illustration of a word “day” showing its respective feature links and corresponding codes as scored using the templates of FIG.",
"6; FIG.",
"9 is a flow diagram of an exemplary embodiment of a Fisher Projection Training technique as used with the handwriting data of the present invention; FIG.",
"10 is a high-level flow diagram of an exemplary technique for training handwritten character data vectors according to an exemplary embodiment of the present invention; FIG.",
"11 is a high-level flow diagram of an exemplary technique for recognizing handwritten characters according to an exemplary embodiment of the present invention; FIG.",
"12 is a flow diagram of an exemplary embodiment of a feature-fusion character recognition system according to the present invention; FIG.",
"13 is a flow diagram of an exemplary embodiment of a Fisher fusion technique according to the present invention; FIG.",
"14 is an illustration of a hypothetical word input whose middle part is poorly shaped; FIG.",
"15A is a segmentation graph of a word input having segmentation points provided thereon; FIG.",
"15B is an illustration of an exemplary transformation of an edge from the segmentation graph of FIG.",
"15A to an edge which is expanded into multiple interpreted edges; FIG.",
"16 is a lattice structure of an exemplary embodiment of a Hypotheses Propagation Network (“HPN”) according to the present invention, with an example of an edge provided therein; FIG.",
"17 is an illustration of look-back windows of the HPN of FIG.",
"16, ending at a particular current processing time; FIG.",
"18 is a sample partial recognition path for recognizing handwritten words using the HPN according to an exemplary embodiment of the present invention; FIG.",
"19 is an illustration of an exemplary instance of the HPN of FIGS.",
"16 and 18 in which a hypotheses list is generated for each particular time and input; FIG.",
"20A is an illustration of an incorrect segmentation of the letter “g” into the word “oj”; FIG.",
"20B is an illustration of an incorrect segmentation of the word “Jie”; FIG.",
"20C is an illustration of a correct segmentation of the word “pie” using an embodiment of the present invention which recognizes ligatures; FIG.",
"21 is an illustration of different segmentations of the handwritten characters which can be subjected to visual bigram modeling techniques according to the present invention; FIG.",
"22 is a flow diagram for a process to compute a height difference ratio according to an exemplary embodiment of the present invention; FIG.",
"23 is a flow diagram for a process to compute a histogram score of a model according to an exemplary embodiment of the present invention; and FIG.",
"22 is a flow diagram for a process to compute a distribution discrepancy of two bigram classes according to an exemplary embodiment of the present invention.",
"DETAILED DESCRIPTION I. Overview of Exemplary Embodiments of System and Process FIG.",
"2 illustrates an exemplary embodiment of a technique for recognizing handwritings according to the present invention, which can be used by the system of FIG.",
"1.In this exemplary embodiment, input data can be received by the handwriting recognition system 50 (step 110).",
"This input data preferably includes coordinate information (i.e., x, y) and time information for the handwritten characters produced by the user using the pen 15 on the pad 20.The input data is normalized in step 120.The normalization procedure may include noise suppression and regularization of the input data's variability (e.g., size, translation, rotation, etc.).",
"Then, in step 130, the target features are computed by a feature extraction technique, which plugs these features into an internal representation or previously-stored handwritten characters of the system 50.The plugged features are forwarded to a segmentation module in step 140 to generate a hypothetical character.",
"In addition, a component character recognition module preferably evaluates a particular segmentation to assign, e.g., class-labeled scores to hypotheses derived by the system 50 (step 150).",
"Thereafter, the resultant data is forwarded to a handwriting recognition engine.",
"In particular, this handwriting recognition engine integrates various information received from other modules and engines of the system 50 to generate and propagate word level hypotheses (step 160).",
"Thereafter, the hypotheses filtering technique can be used in step 170 to improve the accuracy and accelerate the recognition performance by, e.g., controlling the search which is being performed by the handwriting recognition system 50.Preferably, the handwriting recognition system 50 can generate a large number of the hypotheses, so that the filtering technique may apply pre-arranged models to eliminate or rearrange the “worthiness” of the hypotheses.",
"It should be understood that the segmentation module according to an exemplary embodiment of the present invention can be a “stand-alone” module or may be merged with the handwriting recognition system 50.It is also possible for the character recognizer module of the handwriting character system 50 to communicate with a feature extraction module each time the character recognizer module is evoked by the system 50.In the exemplary embodiment of the present invention, the sampling rate of the user's writing used by the tablet digitizer 10 can be, e.g., at least 100 Hz (i.e., one hundred points per second).",
"However, it should be understood that using other sampling rates is conceivable, and clearly within the scope of the present invention.",
"In addition, the tablet digitizer 10 provides “pen-down” and “pen-up” signals indicating when the user contacts the pen 15 to the tablet surface 10, and when the user removes it therefrom, respectively.",
"This information can be used to determine the start and the end of the stroke and may provide other information (e.g., the data regarding a sequence of strokes).",
"According to another embodiment of the present invention, it is also conceivable to obtain other data, such as the pressure on the tablet surface 20 and the tilt of the pen 15, as well as the speed of the pen movement.",
"The point coordinates provided by the tablet digitizer 10 can be integer or decimal values in reference to the origin of the tablet surface 20's coordinate system.",
"FIG.",
"3 shows a flow diagram of another exemplary embodiment of the process for the handwriting recognition according to the present invention, which can also be used with the handwriting recognition system 50 of FIG.",
"1.It should be noted that the steps of FIG.",
"3 illustrate additional details to (and alternative techniques for) the process described above with reference to FIG.",
"2.As provided above, the sequence of (x, y) coordinate pairs is obtained from the tablet digitizer 10 in step 210, which forwards this information to the handwriting recognition system 50.This occurs, in real-time, as the user writes on the tablet surface 20 with the input device (e.g., the pen 15) to generate the input data, which is sampled by the tablet digitizer 10 (step 215).",
"Steps 210 and 215 may be substantially similar to step 110 of FIG.",
"2.The points represented by the coordinates are provided from the contour formed by the user's writing on the tablet surface 20.In most circumstances, the digital handwritten “ink” entered on the tablet surface 20, when magnified, has jagged lines.",
"Noises can originate from the digitizing process, hardware error, or erratic hand motion and pen-down indication which limit the accuracy of the tablet digitizer 10.The same handwritten characters or words entered by different users can vary greatly in size, shape and distortion.",
"Even the same user may write in substantially different ways depending on situations or over time.",
"Thus, in order to suppress various noises and reduce the variability in the raw handwritten data for easier and standardized processing, the handwriting recognition system according to the present invention preferably receives a list of point coordinates (or sequences of coordinate pairs) from the tablet digitizer 10.Then, the handwriting recognition system subjects the received coordinates to a normalization technique, such as Gaussian Smoothing process (step 220).",
"Thereafter, in step 225 (which is substantially similar to step 130 of FIG.",
"2), the handwriting recognition system of the present invention can determine curvature information of each point on the stroke contour or segment of the input data using the normalized or smoothed coordinates.",
"Thereafter, the generation of segmentation points is initiated in step 230, which is similar to the step 140 of FIG.",
"2.The segmentation points are candidate points that indicate if a character stroke has began or ended within a handwritten word.",
"In particular, these segmentation points are candidates for segmenting or breaking a word into different characters.",
"In one exemplary embodiment of the present invention, the segmentation points which have a curvature value larger than a predetermined or selected threshold value may be selected into the set of candidates for the segmentation points.",
"In step 235, a Hypotheses Propagation Network (“HPN”) of the exemplary embodiment of the system according to the present invention receives the handwritten input data and the corresponding segmentation points, and uses this information to generate sets of hypothesis for each of the received segmentation points to obtain the resultant list of hypotheses of words.",
"The HPN uses techniques which may be substantially similar to those of step 170 of FIG.",
"2.Also, the HPN preferably utilizes certain character training and recognition techniques to effectively generate accurate handwriting word recognition results.",
"In particular, the system and process of the present invention can use Fisher Normalization and Vector Generation techniques on the handwritten characters, thereby training the system and process of the present invention with different variations of the same handwritten characters (step 240).",
"Thereafter, in step 245, the Fisher character matching technique is executed on the handwritten data to obtain the best matches for such data based on the Fisher-trained character data.",
"In addition, it is possible to utilize a lexicon or a dictionary of permissible words to filter the combination of characters which are most unlikely to be able to form a word.",
"This can be accomplished by matching the set of possible combination of the handwritten words to a tree-type structure of the dictionary (step 250).",
"Next, in step 255, the system and process according to the present invention preferably uses a hypothesis generator to determine the characteristics and the curves of two segmentation points in a given handwritten word (or character), which can be used to compute a Feature Link Code (“FLC”).",
"The FLC can be a non-negative integer which is based on the characteristics of predetermined curvature characteristics.",
"The type of an interval between two segmentation points is then computed in terms of the FLCs.",
"Further, in step 260, a ligature modeling procedure is executed.",
"In particular, a ligature can be defined as a part of a stroke, which does not belong to any characters.",
"Instead, it can be identified as a connector between the characters in a cursive-type writing.",
"By appropriately modeling away the ligatures in the input handwritten data, additional regularity in the character recognition by the HPN can be established.",
"Finally, in step 265 (which can correspond to step 170 of FIG.",
"2), the list of the candidate words generated by the HPN can be sorted according to their respective hypothesis scores.",
"Then, the HPN combines the “best” entries into a single sorted list that is the list of candidate words hypothesized by the system of the present invention.",
"Generally, the top ranking word in the list would be the final output of this exemplary embodiment of the system and process according to the present invention.",
"Provided below are further details of the steps and techniques of the system and process of the present invention that were referred to be above.",
"II.",
"Details of Normalization Techniques A. Gaussian Smoothing As known to those having ordinary skill in the art, a smoothing procedure is the technique used to suppress a quantization noise of point sampling, which also averages an input point with its neighboring points based on a predetermined weighting scheme.",
"In the exemplary embodiment of the system and process of the present invention, the smoothing procedure can be utilized to, e.g., obtain more fine-grained real number coordinates (instead of the integer numbers in the raw data) so that the point curvatures can be reliably determined.",
"It is preferable to compute the point curvatures accurately and reliably.",
"This task can be accomplished using Gaussian distributions controlled by the size of the window (ρ) and the spread (σ) of the distribution (step 120 of FIG.",
"2 and step 220 of FIG.",
"3).",
"For a contour C=<p1, p2, .",
".",
".",
", pn>, the Gaussian smoothing of the contour C transforms it to a transformed contour C′=<p1, p2, .",
".",
".",
", pn> where p i = ( x i , y i ) , p i ′ = ( x i ′ , y i ′ ) x i ′ = ∑ j = - ρ j = ρ x i + j · G ( j ) ∑ j = - ρ j = ρ G ( j ) , y i ′ = ∑ j = - ρ j = ρ y i + j · G ( j ) ∑ j = - ρ j = ρ G ( j ) and the Gaussian mask G(k) can be defined as G ( k ) = exp ( - k 2 2 · σ 2 ) , for k = - ρ to k = ρ , .",
"B.",
"Global and Local Filtering Filtering technique can eliminate duplicate handwritten data points, and normalize the irregularity in a data point density that may be caused by the relative speed of the user handwriting.",
"For example, in the handwriting recognition systems, when the handwriting speed is slower in a particular interval, it would likely contain more points in such interval.",
"It follows that when the writing speed is faster, the interval would likely possess sparser distribution of the points.",
"A conventional technique for executing such filtering procedure is called “equidistant re-sampling” procedure, which forces a minimum Euclidean distance between two data points.",
"The results of this prior art procedure likely provides approximately equidistant data points.",
"In the fast handwriting interval, there may be a smaller number of data points, and an interpolation technique may be used to fill the gaps between these points.",
"Conventionally, this filtering procedure can be performed at a global level as part of the data acquisition process.",
"In the exemplary embodiment of the system and process according to the present invention, two level filtering (i.e., global and local level filtering) procedures can be employed.",
"The global level filtering procedure operates in substantially the same manner as the conventional filtering techniques.",
"Moreover, the local level filtering uses a handwriting character hypothesis interval as the input data.",
"This character hypothesis interval is a sequence of points contained in one or more consecutive sub-strokes which is used because a character may span across several strokes, with the starting and ending portions possibly covering partial strokes.",
"As shown in FIG.",
"6A, the character segmentation procedure utilized by the exemplary embodiment of the system and process according to the present invention generates a set of proposed segmentation points 300 based on the curvatures.",
"In order to determine the appropriate interval between the two segmentation points for attempting to determine the hypothetical character, the local level filtering procedure is performed on the target interval.",
"The re-sampling size of the local level filtering is maintained as a constant, and the spacing between two re-sampled segmentation points is preferably dependent on the arc/curvature length of the particular interval.",
"Thus, the local level filtering generates, as an output, a fixed size sequence of points.",
"This is done regardless of how many points the input interval has, nor does it depend on the length of the interval (in terms of arc/curvature length).",
"FIG.",
"4B shows an exemplary output 305 of the local level filtering procedure according to the present invention, which was based on the input of the segmentation point 300 illustrated in FIG.",
"4A.",
"Because the size of the interval can vary significantly (from the minimum to the maximum sizes), it is possible for the input interval to contain less points than those in the re-sampled size.",
"In such case, an equidistant interpolation is performed to make up for the missing points by adding additional points between the starting and ending points of the arc curvature.",
"C. Translation and Scale Normalization It should be understood that the coordinate representation of the raw input data is generally not translation-invariant.",
"To achieve the invariance of the coordinate values, it is preferable to recompute the point positions with reference to a new standard origin.",
"The determination as to what origin is assigned as the standard is facilitated by ascertaining whether the system and process of the present invention can hypothesize the definite boundaries of the characters.",
"At that point, it is possible to use a fixed boundary point as the origin.",
"Depending on the segmentation methodology being used, however, this information may not be available.",
"According to the exemplary embodiment of the present invention, it is possible to compute the graphical bounding box of a writing interval hypothesized by the recognition module as being a possible character, because the interval has predetermined starting and ending points.",
"The lower left corner of the interval (of the particular handwriting character or word) can be selected as a new origin, and the coordinates therefore are recomputed with reference thereto.",
"In particular, with the interval of coordinate pairs being I=<(x1, y1), (x2, y2), .",
".",
".",
", (xn, yn), >, the new origin can be O=(x′, y′), where x′=min{xi} and y′=min{yi}, i=1 .",
".",
".",
"n. Then, the translation invariant version of the interval becomes I′=<(x1−x′, y1−y′), (x2−x′, y2−y′), .",
".",
".",
", (xn−x′, yn−y′), >.",
"It is preferable to reduce the size variability of the target objects, which can be applied for each character or for the entire word.",
"The size normalization in the system and process of the present invention is preferably applied at the character level, which utilizes the local level filtering procedure described above.",
"The output S of the local level filtering procedure applied to the character can preferably be a fixed length sequence of re-sampled points.",
"After the translation normalization technique is applied to the output S, this output S is converted as follows: S′=<(x1, y1), (x2, y2), .",
".",
".",
", (xn, yn)>.",
"Thereafter, the converted output S′ can be transformed into an Expanded Coordinate Vector (“ECV”) by expanding the point coordinate pairs into a vector form, as follows: ECV(Ŝ′)=V=<x1, y1, x2, y2, .",
".",
".",
", xn, yn>.",
"The Extended Coordinate Vector V can then be applied to the vector magnitude normalization to become a normalized vector magnitude representation U such that |U|=1.In particular, the normalized vector magnitude representation U=V/|V|, where U can also be referred to as a translation and scale invariant representation of the input character interval.",
"The vector generation shall be discussed in further detail below.",
"III.",
"Feature Extraction and Representation After the handwritten data is normalized in step 130 of FIG.",
"2, it is preferable to obtain or extract prominent information from the normalized data, and represent it in a standard form to be used by the exemplary embodiment of the system and process of the present invention for recognizing handwritten data (step 140 of FIG.",
"2).",
"In particular, the exemplary embodiment of the system and process of the present invention determines local geometric features of the handwritten data (e.g., the point curvatures and the point tangent angles of such data).",
"Initially, the tangent angle at each point of the handwriting is determined, which is approximated as the direction angle from the current point to the next point.",
"As an example, for two consecutive points pi=(xi, yi) and pi+1=(xi+1, yi+1), the tangent angle θi of pi is θ i = acos ( x i - x i + 1 dist ( p i , p i + 1 ) ) where dist(pi, pi+1) is the Euclidean distance between points pi, pi+1.The curvature ki at point pi is approximated as the amount of the direction angle change around point pi.",
"Thus, the curvature ki is computed as the absolute amount of the angle change from θi−1 to θi, multiplied by a plus or minus sign.",
"The plus would be utilized if the angle change is clockwise, and the minus would be utilized if the angle change is counter-clockwise.",
"Along with the curvature ki of point pi (i.e., xi, yi), it is also possible to determine the arc-length of the sub-stroke starting from a point p0, which is the first point of the stroke, and ending at point pi.",
"This can be computed incrementally by adding the Euclidean distance between points pi−1 and pi to the sub-stroke up to pi−1.IV.",
"Character Segmentation The exemplary embodiment of the system and method of the present invention then segment the handwritten data into segments, e.g., corresponding to individual characters (step 140 of FIG.",
"2 and step 230 of FIG.",
"3).",
"A.",
"Segmentation Point Generation The generation of the candidate segmentation points according to the exemplary embodiment of the present invention can be based on the detection of high curvature points.",
"This is because the high curvature points (e.g., corner-points or turning-points) of a particular segment are the locations where the information regarding the dynamics and geometry of the particular handwritten data is most condensed, thus providing better information for the character boundaries.",
"An exemplary illustration of the word “eye” 320 having the set of such high curvature points is illustrated in FIG.",
"5A.",
"However, this set of high curvature points may be incomplete at least because certain real segmentation points can lie in the middle of a smooth interval whose points have only low curvatures.",
"Hence, an augmentation technique may be utilized according to the present invention to generate a complete candidate set of the curvature points.",
"This can be effectuated by adding an intermediary point in a long interval (determined by a predetermined threshold) between two high curvature points.",
"For example, based on the word “eye” and the high curvature points illustrated in FIG.",
"5A, it is possible to insert the intermediary point (a transparent point) between the high curvature points which span for a length that is longer that such threshold, as illustrated in the further segmented word “eye” 325 shown in FIG.",
"5B.",
"B. Feature-Link Coding The segmentation points generated, as described above, segment the handwritten data input into a sequence of intervals (or “feature links”), each of which can be determined by the two consecutive segmentation points.",
"In particular, with a stroke C=<p1, p2, .",
".",
".",
", pn>, the segmentation procedure according to the exemplary embodiment of the present invention computes a sequence of the respective segmentation points S=<pk1, pk2, .",
".",
".",
", pkm>, where pk1<pk2< .",
".",
".",
"<pkm.",
"Thereafter, the stroke C is subdivided into the intervals Ii=<pki, pki+1, pki+1, .",
".",
".",
", pki+1>, for i=1 to m-1.The interval determined in this manner preferably contains no corners or cusps.",
"Otherwise, the high curvature points would be detected at such corner or cusps, and the interval would have been further subdivided.",
"The feature links can be considered as sub-stroke or sub-character level primitives which can be associated with the line or the direction that the intervals form.",
"According to the present invention, it may be preferable to obtain a graph providing base line and reference information for all possible lines or curves formed by the component points of such intervals which can be used to compare the segments or strokes thereto.",
"Thus, as shown in FIG.",
"6, it is possible to define certain templates, e.g., 24-directional convex templates 330, each representing the direction and the convexity of the corresponding categories of feature links that defines a respective feature link code (“FLC”).",
"The FLC can be a non-negative integer, and, according to one exemplary embodiment, can include 24 of such FLCs ranging from type-0 to type-23, as shown in the FIG.",
"6.Thus, during a template matching process of the present invention, the strokes of the handwritten data input can be compared with each template, and the label of the closest matching template can be established.",
"For example, with a sub-stroke C=<p1, p2, .",
".",
".",
", pn>, as with feature-link, the “cross-length” of C can be defined as the Euclidean distance between the first point and the last point of the sub-stroke C, such that a cross_length(C)=dist(p1, pn).",
"The templates have a standard scale in terms of the cross-length, and are stored in a storage arrangement of the system.",
"The feature-link templates are also normalized to have the same number of data points using the local level filtering procedures described above.",
"With the feature link templates established as described above, it is possible to assign an index to a particular feature link that represents a particular feature link template which closely matches the normalized input.",
"This task can be performed by the template matching process described briefly above, whose high level steps are provided as follows (and shown as a flow diagram in FIG.",
"7): normalize an input feature link to form a normalized input (I) so as to have the standard cross length and the standard number N of data points by local level filtering (step 350); determine a subset T of the templates that can be the candidates to match the normalized input I (step 355); set a first template T1 of subset T to be the current template Ti (step 360); determine the amount of a rotation angle θ(Ti, I) needed to align the current template Ti with the normalized input I in terms of the starting and ending points (step 365); in the aligned state, determine an area A(Ti, I) between the current template Ti and the normalized input I (step 370); determine a matching metric score E(Ti, I) by combining the rotation angle θ(Ti, I) and the area A(Ti, I) (step 375); if all templates T have been reviewed (step 380), assign the next template to be the current template Ti, and go back to step 365 (step 385); and otherwise, output an index k such that index k=arg mini{E(Ti, I)} (step 390).",
"In step 355, each template can first be translated to have the same starting position as that of the normalized input I, and the distance between the ending points of the template and the normalized input I can be determined.",
"This distance provides an indication regarding the offset of the template from the normalized input I.",
"For example, the longer the distance, the more discrepancy can exist between the directions of the template and the normalized input I.",
"Therefore, it is not preferable for the template matching process according to the present invention to consider all its feature-link templates.",
"Indeed, only a few of these templates which have the end-point distances smaller than a predetermined threshold are preferably selected as candidates for further processing.",
"Also, in step 370, the area A(Ti, I) between the current template Ti and the normalized input I can be an approximation of the gap between them.",
"In one embodiment of the present invention, the current template Ti=<T1, T2, .",
".",
".",
", TN> and the normalized input I=x1, x2, .",
".",
".",
", xN>.",
"Then, the current template Ti and the normalized input I can be further normalized to have the same representation length N. Then, the area A(Ti, I) can be defined as: A ( T i , I ) = ∑ j = 1 N dist ( t j , x j ) where dist(tj, xj) is preferably the Euclidean distance between the start point and the end point of the particular segment.",
"This metric provides a reasonable approximation of the gap between the current template Ti and the normalized input I because the feature links are smooth intervals without having sharp or complex curves.",
"In step 375, the rotation angle θ(Ti, I) and the area A(Ti, I) can be combined to determine the matching metric score, which can be determined as follows: E(Ti, I)=A(T1, I)3×exp(c·θ(Ti, I)) where c is a normalizing constant.",
"The lower the value of the matching metric score E(Ti, I), the better the current template Ti matches the normalized input I.",
"This is because the rotation angle θ(Ti, I) is more significant in determining a match since it measures the level of the directional alignment, and functionally grows more rapidly.",
"Therefore, as the value of the rotation angle θ(Ti, I) grows, the matching metric score E(Ti, I) would likely to be dominated by such rotation angle θ(Ti, I).",
"If the rotation angles θ(Ti, I) are within a close range, then the matching metric score E(Ti, I) can be determined by the magnitude of, e.g., the area A(Ti, I).",
"FIG.",
"8 shows an exemplary word “day” 340, with the segmentation points identified thereon, and the feature links and their respective FLCs being determined by the template matching procedure described above.",
"V. Component Character Recognition In the exemplary embodiment of the system and process according to the present invention, particular character recognition procedure are implemented (step 150 of FIG.",
"2) are provided so as to decipher the handwritten text input on the tablet surface 20 of FIG.",
"1.A.",
"Introduction of Fisher Analysis Fisher's linear discriminant analysis was successfully used for improving the performance of a face recognition task under an extensive variation of lighting conditions and facial expressions.",
"It was used to reduce the large number of dimensions typically involved in face images.",
"B.",
"Construction of Fisher Projection Matrix For example, for a number C of classes V1, V2, V3, .",
".",
".",
", VC, each class Vi may a population of Ni vectors Vi=v′1, v′2, .",
".",
".",
", v′ni.",
"Each data vector can have n dimensions.",
"The Fisher analysis generally considers two types of scatter matrices: one for between-class distribution and the other for within-class distribution.",
"The between-class scatter ΦB can be defined as Φ B = ∑ i = 1 C N i ( μ i - μ ) ( μ i - μ ) T where μi is the centroid of the class Vi, and μ is the global centroid.",
"The within-class scatter ΦW can be defined as Φ W ∑ i = 1 C ∑ j = 1 N i ( v j i - μ i ) ( v j i - μ i ) T .",
"Therefore, the Fisher technique can use the class label information to describe two comparative kinds of distributions.",
"Given a projection matrix W (of size n by m) and its linear transformation p=WTv, the between-class scatter in the projection space is as follows: Ψ B = ∑ i = 1 C N i ( μ i ′ - μ ′ ) ( μ i ′ - μ ′ ) T = ∑ i = 1 C N i ( W T μ i - W T μ ) ( W T μ i - W T μ ) T = ∑ i = 1 C N i ( W T μ i - W T μ ) ( μ i T W - μ T W ) = ∑ i = 1 C W T N i ( μ i - μ ) ( μ i T - μ T ) W = W T ( ∑ i = 1 C N i ( μ i - μ ) ( μ i - μ ) T ) W = W T Φ B W , where μ′i and μ′ are the class centroid and the global centroid in the projection space, respectively.",
"Similarly, the within-class scatter ΨW=WTΨWW, is likely in the projection space.",
"It is preferable to select a projection matrix W′ so as to maximize the between-class scatter, while minimizing the within-class scatter in the projection space, thus widened gaps between the class boundaries which can lead to a better class separability.",
"The Fisher Projection Matrix (“FMP”) can be provided in the following form: W ′ = arg max w { Ψ B Ψ W } = arg max w { Ψ B Ψ W } The construction of the FPM W′ can be implemented by solving the generalized eigen value equation Φww=λΦww and computing the largest eigen values λ1, λ2, .",
".",
".",
", λm of the FPM, as well as their corresponding eigen vectors w1, w2, .",
".",
".",
", wm.",
"Thereafter, the respective eigen vectors wi's form the orthonormal columns of the target projection matrix, that is W′=[w1, w2, .",
".",
".",
", wm].",
"The wj's are orthonormal columns that maximizes the quantity of the above ratio, and can be formulated as generalized eigen vectors.",
"FIG.",
"9 shows a detailed illustration of the Fisher matrix generation procedure according to the exemplary embodiment of the present invention.",
"In particular, character handwritten samples are collected in step 450.In step 455, each sample character is read-in, and the Gaussian smoothing technique is applied thereto.",
"Then, the Fisher Vector can be generated (step 460) as described in detail above.",
"Using this vector, the between-class scatter matrix iterating over the read-in samples can be computed in step 465.In addition, using the generated Fisher character vectors, the within-class scatter matrix iterating over the read-in samples can be computed in step 470.The matrixes computed in steps 465 and 470 can be forwarded to be utilized in the solution of the generalized eigen value problem in step 475.Thereafter, in step 480, the Fisher Projection Matrix (FPM) can be determined based on the results provided in step 475.Finally, the mean class model vectors described above are computed in the projection space using the FPM in step 485.C.",
"Fisher Training and Character Recognition The determination of the Fisher projection matrix using a set of character data vectors can be equated to the training procedure for recognizing the characters using a metric in the Fisher projection space.",
"For example, it is possible to utilize a fixed length ECV (i.e., Expanded Coordinate Vector) described above having particular character samples for this procedure.",
"Accordingly, given a sample S, ECVN(S)=<x1, y1, x2, y2, .",
".",
".",
", xm, ym> for a constant N. The expanded coordinate values xi's and yi's of ECVN(S) can preferably be re-sampling results of the local level filtering procedure after applying the data normalization procedures (as described above) to the original data.",
"The training data are complied by computing ECVN(Si) for each character sample, which is then subjected to the Fisher analysis to construct the projection matrix.",
"In addition, the training process determines the model centroid mi for each class i.",
"The class vectors are normalized to be unit vectors for the character matching, which can be used at a later point.",
"A vector V, F(V) can be defined as the vector in the projection space mapped by the projection matrix that has been trained by the Fisher analysis.",
"Given an input ECV(y), its Fisher matching score (FMScore) for a class c can be defined as: FMScore ( y , c ) = 2 - dist ( F ( m c ) , F ( y ) ) 2 where dist( ) is the Euclidean distance.",
"Thus, FMScore( ) can have a range from 0 to 1 because the model centroid and the input data are previously normalized to unit vectors.",
"For example, the FMScore of 1 corresponds to the perfect match, and the FMscore of 0 would indicate a complete mismatch.",
"FIG.",
"10 shows a summary of the exemplary Fisher training steps according to an exemplary embodiment of the present invention.",
"In particular: the training data can be computed and converted into a standard representation (step 510); the model centroid is computed and recorded in the standard representation (step 515); and the Fisher projection matrix is computed (e.g., see FIG.",
"9 and the corresponding description provided above) and this matrix is stored (step 515).",
"FIG.",
"11 shows a high level diagram of the Fisher character recognition technique according to an exemplary embodiment of the present invention.",
"In particular: an input y is obtained, and then converted into the standard representation y (step 550); for each class c, the score fc=FMScore(y, c) is computed and the pair <c, fc> is generated (step 555); the pairs <c, fc> are sorted based on a decreasing order into a list L based on the score fc (step 560); and Return the sorted list L (step 565).",
"In the embodiment when the handwriting character recognition system and process of the present invention can operate as a stand-alone system and process, and when performing a letter-by-letter handwriting recognition, only the pair <c, fc> or the index c can be returned such that the score fc is the maximum, instead of performing steps 560 and 565.However, when the handwriting character recognition system and process of the present invention operates as a component of at least a word-level recognition system and process, a more detailed information for generating and managing the string hypotheses shall be used.",
"Instead, an output of a set of candidate characters with the corresponding confidence values which are worth considering can be more desirable for the recognition system and process according to the present invention.",
"D. Feature-Fusion Character Recognition In one exemplary embodiment of the present invention shown in FIG.",
"12, ECV and TFV described above can be used as the two different base representations.",
"For example, ECV-FCR 620 can be the Fisher character recognition system/module based on ECV representation, and TFV-FCR 610 can be the Fisher character recognizer based on TFV representation.",
"These systems/modules can receive handwritten input data 600, and generate outputs which may be forwarded to a Fisher Fusion Module (“FFM”) 630 by integrating the outputs of the ECV-FCR and TFV-FCR at the FFM 620, thus outputting the result which is the final candidate list.",
"For example, when the handwritten input data 600 has been segmented, the ECV and the TFV vectors are extracted and then provided to the corresponding base recognition modules.",
"In each of these base recognition modules, the Fisher matching technique can be implemented on the candidates so that the candidates that have the scores being within the top 50% will be filtered, and output in a sorted candidate list.",
"Then, these lists can be identified as LECV and LTFV, respectively, for ECV and TFV representations.",
"The lists LECV and LTFV may include different elements and different lengths.",
"In order to allow the ECV-FCR recognition system/module to perform in a more reliable level, the list LECV can be used as the primary information source, and the list LTFV may be compared with the list LECV so as to proceed with the fusion process.",
"For each element pair <c, fc> in the list LECV, where c is a class label and the score fc is its Fisher matching score, the pair <c, gc> is determined in the list LTFV.",
"If such pair <c, gc> does not exist in the list LTFV, that means that the TFV-FCR recognition system/module missed the class c as a candidate, and thus the score fc is penalized by reducing it by 50%.",
"Otherwise, it is possible to reevaluate the score fc as follows.",
"TopTFV can be the top score of the list LTFV, so as to compute the ratio of gc with topTFV.",
"In particular, the score fc can be updated to be an updated score fc′ as follows: f c ′ = f c · g c top TFV .",
"The updated pair <c, fc′> can be sorted into the final list F. Then, the final list F may be reduced to include items whose scores are within the top 40% of the top score of the final list F. FIG.",
"13 shows the exemplary steps implemented by the Fisher Fusion Module according to the present invention which are provided as follows: Input the expanded coordinate list LECV (step 630) and the tangential feature vector list LTFV (step 635); Assign a first score pair <c0, fc0> in the list LECV as a current score pair <c, fc′> (step 640); Locate the current pair <c, fc′> in the list LTFV (step 645); Determine the Fisher Matching score fc′ (step 650); Sort the current score pair <c, fc′> the list F (step 655); If all score pairs have not been reviewed (step 660), the next score pair in the list LECV is assigned as the current score pair <c, fc′> (step 665); and Otherwise, remove the score pairs in the sorted list F whose score is below a predetermined threshold (e.g., 60% from the top score)−step 670.VI.",
"Handwritten Word Recognition A.",
"Word Recognition using a Graph Search Technique A word level hypothesis can consist of a string of character hypotheses and a confidence score of such list.",
"According to the exemplary embodiment of the present invention, the character hypothesis can include the interpreted character class label, the evaluation score computed by the character recognizing module, and other relevant information (e.g., starting and ending points of the character or curvature, the bounding box thereof, etc.).",
"In particular, the likelihood score is determined as the average of the scores of the component character hypotheses.",
"Also, the scores at each tentative recognized character are added, and the result of such summation is divided by the number of the characters which contributed to the score.",
"Accordingly, the word hypothesis score according to the exemplary embodiment of the present invention can be a measure of how well the handwritten characters are shaped in relation with the Fisher character models stored in a storage arrangement (e.g., a hard disk, CD-ROM, tape drive, etc.)",
"of the system of the present invention.",
"FIG.",
"14 shows a hypothetical word input 700 whose middle portions is poorly shaped, which is provided to illustrate the above-described score determination technique.",
"In particular, the string C1C2C3 can provide the correct interpretation of the input.",
"The character recognition score for C1 is 0.9, for C2 it is 0.1 and for C3 it is again 0.9.An incorrect interpretation is provided by the string T1T2.The character recognition score for T1 is 0.6, and for T2 it is 0.5.The conventional systems and process generally multiply the character recognition scores in the respective strings to determine the “best” string.",
"In the present exemple, such traditional systems and methods would obtain the score as 0.9×0.1×0.9=0.081 for the string C1C2C3, while the score for the string T1T2 would be 0.6×0.5=0.3.Accordingly, the score for the string T1T2 would be larger than that of the string C1C2C3.Therefore, the string C1C2C3 would likely be improperly rejected, and the incorrect interpretation string T1T2 determined to be the “best” string.",
"In contrast, the determination procedure of the present invention provides the character recognition score as (09+0.1+0.9)/3=0.63 for the correct hypothesis string C1C2C3, which is higher than the score (0.6+0.5)/2=0.55 assigned to the incorrect hypothesis string T1T2.Using the segmentation points and the rules to form the character segments according to the present invention, the structure of the curves generated by the handwriting recognition procedure can be formulated as a graph 710, as shown in FIG.",
"15A.",
"For example, the segmentation points 702, along with the starting and ending points of the handwritten input data, can be regarded as the set of vertices.",
"The starting point may correspond to a vertex s, and the ending point to a vertex t. A directed edge s→t between two nodes s and t can be regarded as representative of the segment starting from the segmentation point represented by the vertex s, to the segmentation point represented by the vertex t. The techniques of the present invention described above determine which edges are present in the graph 710.It should be noted that the graph 710 is a directed acyclic graph (“DAG”) because the edges of the input data are directed forward, and do not return with respect to time.",
"Thereafter, the segmentation graph can be expanded into an “interpretation graph,” as shown in FIG.",
"15B.",
"In this interpretation graph, each unlabeled directed edge in the segmentation graph can be interpreted by invoicing the character recognition procedure according to the present invention with the corresponding segment 720 as the input.",
"Then, this segment is replaced with a set of edges 730 having substantially the same source and destination vertices as shown in FIG.",
"15A, being labeled with the character class index (and possibly the evaluation score).",
"In this manner, the handwritten words can be recognized by locating the optimal path from the starting vertex s to the ending vertex t in the interpretation graph of FIG.",
"15B.",
"The character recognition score of the path is preferably a function of the scores of the edges contained in the path, which is performed by the summation and averaging procedure (i.e., of the scores) described above.",
"According to the present invention, it may be preferably to utilize a modified topological-sort algorithm for the performing the search of the handwritten character data, in which a list of multiple predecessors can be established at each propagation point, instead of a single point.",
"B.",
"Hypotheses Propagation Network The handwriting recognition engine/module of the system and process according to the present invention can be referred to as a “Hypotheses Propagation Network” (“HPN”) which is shown as block 235 of FIG.",
"3.As shown in FIG.",
"16, the HPN may have a two-dimensional lattice structure 750, which applies the above-referenced interpretation graph and the search procedure thereon as shall be described in further detail below.",
"One dimension or axis of the HPN is the dimension of time in terms of the segmentation points—the first segmentation point starting at time 1, the second segmentation point starting at time 2, etc.",
"The other dimension or axis provides the indices of the character classes.",
"The intersections of the MPN's lattice structure corresponds to a node N(t, m), where t is the time and m is the index of a character class.",
"Given two nodes N(t′, m′) and N(t, m) where t′<t, the edge N(t′, m′)→N(t, m) corresponds to the segment from time t′ to time t in the input that can be interpreted as the class m, having m′ as its predecessor.",
"The class m is the index of a character class (e.g., m=1, .",
".",
".",
", 23 for the alphabet, m=1, .",
".",
".",
", 46 if upper and lower case characters are considered as separate classes, and m=1, .",
".",
".",
", 92 if cursive and type characters are being considered separately, etc.).",
"Each node may have its character hypothesis score determined by, e.g: a Fisher matching technique for the interval between the predetermined time w and time t, and a hypothesis score for all the letters up to time w (H[q, w] with the number of characters up to time w, divided by (1+ number of characters up to time w), and/or a visual bigram hypothesis that determines whether the visual parameters are consistent with a bigram (q,m) at time stages (w,t), the details of which shall be described in further details below.",
"In effect, the node N may represent a string of characters, and has the score of the path leading to such node N associated therewith.",
"That score is preferably the average of all the Fisher matching scores of the characters contained in the path that the node N represents, with the procedure according to the exemplary embodiment of the present to calculate the scores in such manner has been described above.",
"In particular, in order to compute the path score, the node N includes two fields.",
"The first field is the total accumulated character scores W in the path.",
"The second field is the number of characters x in the path of the node N. Thus, the path score can be calculated by dividing the value of the first field (i.e., the total score) by the value of the second field (i.e., the total number of characters in the path of the node N)—W/x.",
"FIG.",
"18 shows the exemplary HPN lattice structure 770 in which the two-dimensional matrix or hypothesis H[m, t] represents all the partial recognition paths of the HPN ending at time t with the last character m, with each of the nodes having its own number of characters and path scores.",
"C. HPN Search Using the HPN, the interpreted edges are constructed dynamically by the segment generation rule described above.",
"As shown in FIG.",
"17, at each processing time t, a portion 760 of the HPN's lattice structure looks back in time and ranges over the look-back windows of sizes from 1 to a predetermined size w which can be considered a maximum size.",
"According to a preferred embodiment of the present invention, the predetermined size w can be equal to 6, however other sizes are conceivable, and are within the scope of the present invention.",
"In this manner, the i-th look-back window Wi starts the look back at time t-i and ends at t. Thus, the look back windows can analyze up to the predetermined number w to recognize a possible character.",
"In particular, for each Wi, the HPN transmits the feature vector (extracted from the corresponding data segment) to the component character recognizer, which in turn returns the list of candidates m1, m2, .",
".",
".",
"mk.",
"For each of the candidate mj, the HPN iterates over the nodes N(t-i, m′), and determines whether or not to place the edge/segment N(t-i, m′)→N(t, mj) in the graph.",
"It is also possible to score the additional edge/segment when it is placed in the graph.",
"The decision of whether to place the edge/segment into the graph and/or the scoring thereof can be based on the information coming from various hypothesis-filtering models.",
"One example of such model can be is the use of the lexicon or dictionary.",
"In particular, if the string corresponding to a hypothesis that has been propagated to the node N(t-i, m′) which forms a legal prefix of the lexicon if mj, which is concatenated to thereto, then the new prefix is identified to be legal in the dictionary and the edge/segment is permissible and/or the score can be higher therefore.",
"In contrast to the conventional programming search techniques, the HPN of the exemplary embodiment of the present invention preferably uses multiple predecessors.",
"For example, at each node N(t, m), the HPN of the present invention preferably stores a list of the word level hypotheses H(t, m), each of whose elements being a hypothesis which ends at time t (with the character of class m as the last character of its string).",
"For the edge/segment N(t1, m′)→N(t2, m), the HPN can iterate on each element of H(t1, m′), compute a new hypothesis with the score of the edge/segment and the class label m, and insert the score and the hypothetical character into the list H(t2, m).",
"FIG.",
"19 shows an exemplary score diagram 780 generated in this manner, with the path scores being inserted therein.",
"After the last time T, the lists H(T, *) can be merged into a single sorted list H. This list H can be the sequence of candidate words recognized by the system, ordered according to the confidence or score values.",
"D. Trimming Hypothesis List By allowing multiple hypotheses to end on the same propagation node (without any limit on the length of the lists H(t, m)) can lead to intractable computation.",
"This is because the hypothesis and propagating process may likely encounter exponentially growing number of predecessors.",
"The hypotheses filtering procedures and models described in further detail below can be used to trim the HPN search by possibly blocking the propagation of the hypotheses that are determined to be inconsistent with other information provided for the hypotheses.",
"With respect to the HPN, all H(t, m) can be restricted to have, at most, a predetermined number of items.",
"At the other level, the maximum number of hypotheses at each time t is can also be restricted to a predetermined number U.",
"This can be achieved by maintaining, at most, U number of hypotheses at time t (in terms of their hypothesis scores) that are distributed over H(t, *)'s.",
"The predetermined numbers C and U are part of the parameters controlling the accuracy and the execution speed of the system and process according to the present invention.",
"VII.",
"Hypotheses Filtering Models/Techniques By relying only on the scores assigned by the component character recognition system and process of the present invention, a large number of hypotheses may be generated, many of which may be unusable.",
"For example, certain English characters can be inherently ambiguous: “o” vs. “0,” “1” vs. “I,” “1” (one) vs. “1” (lowercase “L”),.etc.",
"In many situations, the ambiguities of such characters can preferably be resolved by taking a particular context into consideration.",
"With a contextual perspective, however, many of the generated hypotheses may not necessarily make sense, and may be eliminated from further propagation and consideration.",
"The economy of the hypothesis propagation, consideration and processing time obtained using such filtering procedures substantially contributes to the performance of the system and process according to the present invention.",
"The preferred embodiments of such advantageous filtering models/procedures are described in further detail below.",
"A.",
"Dynamic Lexicon The most common models/techniques of context information utilize a lexicon, e.g., a dictionary of permissible words.",
"The lexicon filtering procedures can be applied either after the generation of word candidates or during the propagation process.",
"In still another conventional approach, the lexicon can be dynamically reduced at an early-recognition stage using a fast recognizer-type method as described in G. Seni et al., “Large Vocabulary Recognition of On-Line Handwritten Cursive Words,” IEEE Trans.",
"Pattern Analysis and Machine Intelligence, Vol.",
"18, No.",
"7, July 1996.The purpose of using such early-recognition procedure is to determine a small set of plausible candidate words (instead of single most-likely word), thereby reducing the size of the lexicon.",
"Thereafter, it would be possible to focus primarily on the reduced lexicon.",
"A statistical n-gram modeling of character sequences can also be used for the system and process according to the present invention, and may be preferable if it is necessary to recognize certain words which are not in the lexicon.",
"In a preferred embodiment of the present invention, the dictionary can be organized into a tree-type data structure, and dynamically accessed when the HPN attempts to propagate a word hypothesis.",
"For example, a non-leaf node of such lexicon tree may correspond to a legal prefix of the system, such as a proper prefix of a full word.",
"The full words are generally represented by the leaves of this tree data structure.",
"Each word level hypothesis may include a pointer to the node of the tree, which corresponds to the prefix string representing the particular word hypothesis.",
"Thus, when the HPN according to the exemplary embodiment of the present invention processes a hypothesis h that ends at a particular HPN node for propagation (with an edge/segment interpreted as a character class x), the HPN looks up the tree node of the hypothesis h. If this HPN node has the hypothesis x as a successor, the hypotheses h can be extended to another hypotheses h′ which includes x as the last character.",
"In this manner, the system and process according to the present invention can dynamically prevent a hypothesis from being extended to a non-permissible string, and all word level hypotheses generated and propagated can be limited to the “legal” prefixes of the lexicon.",
"B. Ligature Modeling The way the character written in cursive script connects with its surrounding characters (i.e., via the ligatures) provides the variability of such character.",
"This concept is similar to the “co-articulation” in speech recognition, in which a phoneme has greater variation in the pattern around the border with the neighboring phonemes.",
"Ligatures, are not necessary, but generally present in the continuous cursive writing used by many users because they allow a faster writing to be implemented.",
"The ligatures can be modeled since there are certain regularities in their formation, and they can be used to measure how well the hypothesis may be formed.",
"By appropriately modeling or filtering away these dummy “bridges” or ligatures, it is possible to obtain more regularity in the shapes of the characters.",
"The difficulty of modeling ligatures may arise, however, at least because they may be context sensitive.",
"Also, taking the full contexts into consideration may lead to a proliferation of the models.",
"In the English language, there may be few alphabetic constraints on the formation of the ligatures that could be exploited for more concise modeling.",
"C. Use of Feature-Link Code for Model Ligatures The feature-link code (“FLC”) and the 24 convexity-directional feature-link templates are shown in FIG.",
"6 (along with the indices assigned to the individual templates) and described above in great detail.",
"Also, FIG.",
"8 and the accompanying text provides the details for a sample handwriting of the word “day,” its feature-link intervals and the FLCs.",
"From this exemplary handwritten data, it can be seen that the FLC is a compact representation which can be used to describe a smooth contour interval.",
"According to the exemplary embodiment of the system and process of the present invention, the feature-link interval can be the smallest unit along which the entire input may be broken down.",
"As described above, the character segment is generally formed as a consecutive sequence of the feature-link intervals.",
"Thus, a ligature (if present in the handwritten data) can also be a sequence of the feature-link intervals between two character segments.",
"According to the present invention, it is preferable to use the feature-link intervals to obtain a hypotheses regarding the ligatures.",
"The ligature segment, however, does not need to contain many feature-link intervals in normal handwriting.",
"This is because, unlike the characters, the ligature segment serves merely as a connector between the characters, and its shape does not usually have a complex structure, being smooth interval with certain degree of convexity.",
"Since the FLC can be computed and stored into a table by the pre-preprocessing modules, the system and process according to the present invention (e.g., the HPN) would need to only check such table.",
"For the filtering purposes, it is preferable to create two lookup matrices—IsRequired[c1, c2] and IsLegal[c1, 1, c2], where c1 and c2 range over character class indices, and 1 is the respective FLC.",
"IsRequired[c1, c2] generates a value “TRUE” if a ligature is required between the two character classes c1 and c2 in a continuous writing data, and “FALSE” otherwise.",
"IsLegal[c1, 1, c2] is set to “TRUE” if the FLC 1 is a permissible ligature between c1 and c2, and “FALSE” otherwise.",
"The quantity of IsRequired[c1, c2] can be used when the segments of c1 and c2 touch one another, thus requiring no ligature there between.",
"If IsRequired[c1, c2] returns “TRUE” in this case, that indicates that c1 followed by c2 is mis-hypothesized because they require a ligature in a continuous formation.",
"When two characters c1 and c2 are separated by an interval labeled with the FLC 1, the matrix entry IsLegal[c1, 1, c2] can be looked up by the system and process according to the present invention so as to determine if the ligature is formed in a permissible manner.",
"Thus, if the hypothesis being considered by the HPN for the extension with such context is not consistent with the ligature models, such an instance can be blocked from further propagation.",
"The value can be entered into the matrix entries by training performed in the samples, and using the observed probability quantity therefrom (instead of the Boolean values).",
"It is also possible to make such entries manually.",
"D. Examples and Experimental Results when Modeling Ligatures FIGS.",
"20A-20C show two examples of the possible use of the matrices IsRequired[c1, c2] and IsLegal[c1, 1, c2].",
"In particular, FIG.",
"20A illustrates an incorrect segmentation of the handwritten letter “g” into the letters “o” and “j.” The string “oj”, when written continuously, generally utilizes a ligature between the two characters.",
"This is because without using the ligature for this handwritten combination, the output string would have a shape of the letter “g.” Using the look-up matrices described above, the result of IsRequired[“o”, “j”] for this combination would be “TRUE”.",
"Because the segmentation provides no room for a ligature, the system and process of the present invention can likely detect that the possible combination “oj” has been interpreted incorrectly and remove such possibility.",
"FIGS.",
"20B and 20C illustrate the segmentation and interpretation of the handwritten word “pie”.",
"In particular, FIG.",
"20B shows an incorrect segmentation of the handwritten word “pie” into “jie.” However, as can be seen from the figure, the direction and the convexity of the hypothetical ligature between “j” and “i” is not likely to be a possible pattern.",
"Therefore, the FALSE output will be generated at the entry IsLegal[“j”, 1, “i”], where 1 is the FLC of the ligature.",
"E. Visual Bigram Modeling The ligature modeling procedure of the present invention is one exemplary procedure for the hypothesis filtering model using a visual context.",
"With the visual bigram modeling, the geometric characteristics of the character hypotheses are compared to determine the consistency with one another.",
"The variability of the relative geometric information (e.g., the relative size and positioning of a character unit in comparison with its neighbors) can be modeled, and the fitness of a hypothesis can be evaluated according to the modeling techniques.",
"A single character can be highly ambiguous, while its identity can be more evident when provided in a context.",
"For example, FIG.",
"21 shows that the handwritten word “go” can be confused with the combination “90” when the individual characters are evaluated.",
"However, if the relative size and the positioning of the second character hypothesis are taken into account in relation with the first character hypothesis, it becomes easier to establish that the word “go” is the more likely interpretation of the handwritten data.",
"F. Modeling Visual Bigram Information According to the exemplary embodiment of the present invention, when the feature vector is extracted for a particular character segment by the local level filtering procedure as described in further detail above, the bounding box of the segment is also determined.",
"For example, a visual bigram <c1, c2> can be a pair of two consecutive character hypotheses c1 and c2, along with the information of their respective bounding boxes.",
"Given the visual bigram <c1, c2>, let topi be the top-most y-coordinate of a bounding box 785 of the hypothesis ci in FIG.",
"21 (e.g., the bounding box 785 and a point 781 of the letter “g”); bottomi be the bottom-most y-coordinate of the bounding box of the hypothesis ci (e.g., a point 782 of the letter “g” in FIG.",
"21); and hi=topi−bottomi, that is the height of the bounding box 785 of ci (e.g., a distance-790 in FIG.",
"21).",
"The combined height of the visual bigram <c1, c2> is defined as H(<c1, c2>)=max (top1−top2)−min (bottom1−bottom2).",
"Next, three functions of the visual bigram <c1, c2> can be defined as follows: Height difference ratio : HDR ( < c 1 , c 2 > ) = h 1 - h 2 H ( < c 1 , c 2 > ) ; Top difference ratio : TDR ( < c 1 , c 2 > ) = top 1 - top 2 H ( < c 1 , c 2 > ) ; and Bottom difference ratio : BDR ( < c 1 , c 2 > ) = bottom 1 - bottom 2 H ( < c 1 , c 2 > ) .",
"In one example, a model MH can measure the fitness of the height difference ratio of an input class visual bigram <c1, c2>.",
"For lowercase English alphabet, each ci ranges over 26 letter classes, and a procedure would utilize 26×26=676 bigram classes for two hypotheses (e.g., two characters).",
"According to the exemplary embodiment of the present invention, it is preferable to utilize the relative size and positioning between the characters.",
"Thus, by categorizing the letters into groups according to this criteria, the number of required bigram classes can be greatly reduced.",
"Therefore, it is preferable to consider the type of strokes made for each character or letter.",
"In one exemplary implementation of the present invention, the three types of handwritten sub-strokes can be used—“ascender”, “descender” and “none”.",
"The ascender can be a sub-stroke extending beyond a predetermined upper-baseline of the lowercase letters.",
"The descender may be a sub-stroke extending below a predetermined lower-baseline of the lowercase letters.",
"The last type of stroke, i.e., “none”, does not extend above the upper-baseline and below the lower-baseline of the lowercase letters.",
"Accordingly, in a preferred embodiment of the present invention, all lowercase letters can be divided into three groups: a first group having the ascenders, a second group having the descenders, and a third and last group having neither, i.e., being the third class.",
"The table below shows the three categories, possible names and exemplary corresponding member letters for the classes.",
"Ascender or descender Type name Letter Members Ascender A b, d, f, h, k, l, t Descender D d, f, g, j, p, q, y, z None N a, c, e, i, m, n, o, r, s, u, v, w, x In this manner, the 26-letter classes have been reduced to just 3 classes of “A”, “D” and “N” representing the “ascender” group, the “descender” group and the “none” group respectively.",
"Therefore, only 9 bigram classes would have to be utilized between two characters (or hypotheses) as provided by: { A , D , N } × { A , D , N } : < A , A > , < A , D > , < A , N > , < D , A > , < D , D > , < D , N > , < N , A > , < N , D > , < N , N > .",
"A model MH can be generated for 9 bigram classes as provided above, instead of 676 without using the above described classifications.",
"FIG.",
"22 shows the exemplary flow diagram of the steps utilized for determining MH(<c1, c2>) for the class <11, 12> (with 11 and 12 being the classes of “A”, “D” and “N” described above).",
"In this figure, The Height Difference Ratio of two consecutive character hypotheses—HDR(<c1, c2>)—is determined in step 810.Then, the HDR(<c1, c2>) is compared the parameters of the corresponding character class <11, 12> in step 815; and The confidence value of HDR(<c1, c2>) is generated as being that of the corresponding character class <11, 12>.",
"The models MT and MB (measuring the fitness of the top-difference ratio and the bottom difference ratio, respectively) can be calculated similarly to the calculation for the model MH.",
"Therefore, the score for the character combination of the visual bigram <c1, c2>, using the modeling of the visual bigram information can be summarized in the following form: VBScore(<c1, c2>)=kH·MH(<c1, c2>)+kT·MT(<c1, c2>)+kB·MB(<c1, c2>), where kH, kT and kB are coefficients or weights assigned to the corresponding models.",
"G. Training Visual Bigram Model In order to compute the score VBScore(<c1, c2>) for the model M(<c1, c2>) which is MH, MT, and/or MB, it is preferable to compare the related difference ratio of the visual bigram <c1, c2> with the parameters of the model.",
"This can be achieved by a look-up into a table storing a distribution histogram of the ratio.",
"The parameters of the visual bigram model (“VBM”) can be trained by constructing the model distributions from the visual bigram samples.",
"As an example, the training of the HDR model for the bigram class of <A, A> can be considered as follows.",
"The set of the HDR values for <A, A> class samples are collected first.",
"Also, S=<s1, s2, .",
".",
".",
", sk> can be a sorted list of the HDR values.",
"Thus, the interval [s1, sk] can be divided into N equal length sub-intervals.",
"There are ten bins established for each such sub-interval so as to count the number of si values that fit inside the sub-interval.",
"After this counting procedure is completed, the sequence of the bins may preferably form the histogram of the distribution of the HDR values.",
"Thereafter, the histogram can be processed by applying a Gaussian smoothing technique thereto to form the trained histogram.",
"After the training procedure, the quantity in a particular histogram slot represents a likelihood of the HDR values that fall within such slot.",
"The training procedure applied to the rest of the models MT and MB proceeds in a similar manner.",
"The above-described training procedure can be iterated and repeated on each of the 9 bigram classes so as to obtain a total of 27 histograms as a result of this procedure.",
"FIG.",
"23 shows a flow diagram for establishing a score for the model according to an exemplary embodiment of the present invention.",
"In particular, after the histogram is established for the model M (as described above), the model score on the inputted visual bigram of character hypotheses <c1, c2> is determined as follows: determine the model M's difference ratio R from the visual bigram <c1, c2> in step 830; compute a histogram slot index k from the difference ratio R in step 835.In particular, this index k can be determined as follows: k=[R-min)/slotsize], where min is the lower-bound of the first histogram slot interval, and slotsize is the interval length of the histogram slots; and output the histogram of the model M. H. Computing the Class Model Coefficients Intuitively, the coefficient of the model (e.g., kH, kT, and kB shown above) measures the amount of contribution that the information originating from the model provides in determining the fitness of the input with the suggested class interpretation.",
"According to an exemplary embodiment of the present invention, these coefficients can be determined by computing the amount of discrepancy of the distribution, as compared to the same type of the models of different classes.",
"If the discrepancy of a particular model is greater than the discrepancies in other models of the same class, that particular model should provide a larger coefficient.",
"This is because the current model provides more information in measuring the fitness of the input data.",
"FIG.",
"24 shows a flow diagram of an exemplary embodiment according to the present invention for ascertaining a distribution discrepancy between two bigram classes.",
"For example, for two bigram classes T1 and T2, and the model M, the distribution discrepancy of the class T1's model M from the class T2's model M can be labeled as D(M, T1, T2).",
"The exemplary steps for determining the distribution discrepancy D(M, T1, T2) are as follows: The scales of distribution of the models M of the respective of bigram classes T1 and T2 are aligned by extending the corresponding histograms HISTT1 and HIST2 to HIST′T1 and HIST′T2, so that HIST′T1 and HIST′T2 have the same range of intervals as HISTT1 and HISTT2 (step 850).",
"The discrepancy DSUM is computed in step 855 as follows (by iterating over i) DSUM := { HIST T 2 ′ [ i ] - HIST T 1 ′ [ i ] if HIST T 1 ′ [ i ] < HIST T 2 ′ [ i ] HIST T 1 ′ [ i ] - HIST T 2 ′ [ i ] else Then, in step 860, the distribution discrepancy DSUM is output.",
"It is preferable to utilize step 850 for determining DSUM because the bigram classes T1 and T2 usually have different distributions that correspond to different real value intervals.",
"Thus, for a given class T, DM(T) can be assigned to be the sum of D(M, T, *)'s over all other classes.",
"This procedure can then be repeated on other models of the bigram classes T. Also, DH(T), DT(T) and DB(T) can be assigned as being the three discrepancy quantities for the respective class T's models MH, MT and MB, respectively.",
"Then, the coefficients for the respective class T can be as follows: kH=DH(T)/(DH(T)+DT(T)+DB(T)) kT=DT(T)/(DH(T)+DT(T)+DB(T)) kB=DB(T)/(DH(T)+DT(T)+DB(T)).",
"The three coefficients are computed for each of the 9 bigram classes, so the total of 27 coefficients are computed for each model of each bigram class, using the above procedure.",
"One having ordinary skill in the art would clearly recognize that many other applications of the embodiments of the system and process for handwriting recognition according to the present invention.",
"Indeed, the present invention is in no way limited to the exemplary applications and embodiments thereof described above."
]
] | Patent_10416319 |
[
[
"Sucking atomizer for gasoline-oxygen cutting-welding",
"A jet-inducing atomizer for gasoline-oxygen cutting-welding comprises a main body, a diffusion tube, a preheating oxygen fine-tube and a gasoline fine-tube.",
"The diffusion tube and the preheating oxygen fine-tube are provided at the center of the main body co-axially, and communicated with each other.",
"The gasoline fine-tube enters the main body from one side of the main body, and is communicated with the diffusion tube.",
"The diffusion tube is a conical tube.",
"The coarse end of the conical tube is coincidence with the end face of the body.",
"The fine end of the conical tube is connected with the preheating oxygen fine-tube.",
"The advantages of the gasoline-oxygen cutting machine are as follows: the operation is easy as the gasoline tank needs not be pressurized; there is no leak of gasoline or jet of gasoline in leaking manner during operation.",
"The atomized mixture of gasoline and oxygen is immediately jetted while the oxygen valve is opened, and gasoline is immediately shut off while the oxygen valve is closed.",
"Therefore, safety of operation is increased and the cost is reduced."
],
[
"1.A jet-inducing atomizer for gasoline-oxygen cutting-welding comprising a main body of the atomizer, a diffusion tube, a preheating oxygen fine-tube and a gasoline fine-tube, the diffusion tube and the preheating oxygen fine-tube being provided at the center of the main body co-axially, and communicated with each other, the gasoline fine-tube entering the main body from one side of the main body, and being communicated with the diffusion tube, the diffusion tube being a conical tube, the coarse end of the conical tube being coincident with the end face of the body, the fine end of the conical tube being connected with the preheating oxygen fine-tube, the taper α1 of the conical tube equaling to 5°-10°, the length 10 of the conical tube being 2-5 tines of length 11 of the preheating oxygen fine-tube, i.e.",
"10=(2-5) 11, the length 11 of the preheating oxygen fine-tube being 3-5 mm, the distance 12 between the intersection point of the gasoline fine-tube with the diffusion tube and the connection point of the diffusion tube and the preheating oxygen tube equaling to 2-8 nun.",
"2.The atomizer as cited in claim 1, wherein the angle α2 between the gasoline fine-tube and the perpendicular surface of the diffusion tube equals to 0-45°.",
"3.The atomizer as cited in claim 1, wherein the diameter d1 of the preheating oxygen fine-tube equals to 0.5-2.5 mm.",
"4.The atomizer as cited in claim 1, wherein the diameter d2 of the small end of the diffusion tube equals to 1.5-3 mm.",
"5.The atomizer as cited in claim 1, wherein the diameter d3 of the gasoline fine tube equals to 0.5-2 mm."
],
[
"<SOH> BACKGROUND OF INVENTION <EOH>At present, a series of gasoline-oxygen cutting machines invented by the present applicant can cut steel materials having various thickness.",
"Said cutting machines consist of a cutting nozzle, a special cutting torch and a pressure-stabilized gasoline supply tank, etc.",
"(c.f.",
"ZL95211651.0, ZL94247487.2 and ZL97250604.7).",
"The cutting machines according to said patents have the following deficiencies: when gasoline passes from the gasoline tank to the torch and nozzle by compressed air, the atomizing of gasoline in the cutting torch is not perfect; gasoline often leaks from the outlet of the nozzle while the operation is not appropriate; and gasoline may leak or jet in leaking manner, thus causing unsafety in case the rubber tube connecting the gasoline tank and the cutting torch breaks."
],
[
"<SOH> SUMMARY OF INVENTION <EOH>The object of invention is to provide a jet-inducing atomizer for gasoline -oxygen cutting-welding.",
"Based on the principle of hydrodynamics, the atomizer is mounted in the cutting torch, gasoline is sucked from the gasoline tank by an oxygen jet stream under high speed, and is atomized in the cutting torch and the cutting nozzle, and mixed with oxygen, and the mixture is then jetted from the cutting nozzle for ignition to obtain a cutting flame.",
"A Jet-inducing atomizer for gasoline-oxygen cutting-welding according to the present invention comprises a main body of the atomizer, a diffusion tube, a preheating oxygen fine-tube and a gasoline fine-tube.",
"The diffusion tube and the preheating oxygen fine-tube are provided at the center of the main body co-axially, and communicated with each other.",
"The gasoline fine-tube enters the main body from one side of the main body, and is communicated with the diffusion tube.",
"The diffusion tube is a conical tube.",
"The coarse end of the conical tube is coincident with the end face of the body.",
"The fine end of the conical tube is connected with the preheating oxygen fine-tube.",
"The taper α 1 of the conical tube equals to 5°-10°.",
"The length 1 0 of the conical tube is 2-5 times of the length 1 1 of the preheating oxygen fine-tube, i.e.",
"1 0 =(2-5) 1.The length 1 1 of the preheating oxygen fine-tube is 3-5 mm.",
"The distance 1 2 between the intersection point of the gasoline fine-tube with the diffusion tube and the connection point of the diffusion tube with the preheating oxygen tube equals to 2-8 mm.",
"In the atomizer as mentioned above, the angle α 2 between the gasoline fine-tube and the perpendicular surface of the diffusion tube equals to 0-450.The diameter d 1 of the preheating oxygen fine-tube equals to 0.5-2.5 mm.",
"The diameter d 2 of the small end of the diffusion tube equals to 1.5-3 mm.",
"The diameter d 3 of the gasoline fine tube equals to 0.5-2 mm.",
"The atomizer of the present invention is fixed at the head of the cutting torch or in the preheating oxygen tube.",
"After the preheating oxygen tube is opened, gasoline is sucked into the cutting torch and atomized to obtain cutting flame which is combusted sufficiently, has concentrated flame and is easy to be adjusted.",
"The obvious advantages of the gasoline-oxygen cutting machine according to the invention are as follows: the operation is easy as the gasoline tank needs not be pressurized; there is no leak of gasoline or jet of gasoline in leaking manner during operation.",
"The atomized compound of gasoline and oxygen is immediately jetted while the oxygen valve is opened, and gasoline is immediately shut off while the oxygen valve is closed.",
"Therefore, safety of operation is increased and the cost is reduced."
],
[
"FIELD OF INVENTION The present invention relates to a jet-inducing atomizer for gasoline -oxygen cutting-welding, and belongs to machining field.",
"BACKGROUND OF INVENTION At present, a series of gasoline-oxygen cutting machines invented by the present applicant can cut steel materials having various thickness.",
"Said cutting machines consist of a cutting nozzle, a special cutting torch and a pressure-stabilized gasoline supply tank, etc.",
"(c.f.",
"ZL95211651.0, ZL94247487.2 and ZL97250604.7).",
"The cutting machines according to said patents have the following deficiencies: when gasoline passes from the gasoline tank to the torch and nozzle by compressed air, the atomizing of gasoline in the cutting torch is not perfect; gasoline often leaks from the outlet of the nozzle while the operation is not appropriate; and gasoline may leak or jet in leaking manner, thus causing unsafety in case the rubber tube connecting the gasoline tank and the cutting torch breaks.",
"SUMMARY OF INVENTION The object of invention is to provide a jet-inducing atomizer for gasoline -oxygen cutting-welding.",
"Based on the principle of hydrodynamics, the atomizer is mounted in the cutting torch, gasoline is sucked from the gasoline tank by an oxygen jet stream under high speed, and is atomized in the cutting torch and the cutting nozzle, and mixed with oxygen, and the mixture is then jetted from the cutting nozzle for ignition to obtain a cutting flame.",
"A Jet-inducing atomizer for gasoline-oxygen cutting-welding according to the present invention comprises a main body of the atomizer, a diffusion tube, a preheating oxygen fine-tube and a gasoline fine-tube.",
"The diffusion tube and the preheating oxygen fine-tube are provided at the center of the main body co-axially, and communicated with each other.",
"The gasoline fine-tube enters the main body from one side of the main body, and is communicated with the diffusion tube.",
"The diffusion tube is a conical tube.",
"The coarse end of the conical tube is coincident with the end face of the body.",
"The fine end of the conical tube is connected with the preheating oxygen fine-tube.",
"The taper α1 of the conical tube equals to 5°-10°.",
"The length 10 of the conical tube is 2-5 times of the length 11 of the preheating oxygen fine-tube, i.e.",
"10=(2-5) 1.The length 11 of the preheating oxygen fine-tube is 3-5 mm.",
"The distance 12 between the intersection point of the gasoline fine-tube with the diffusion tube and the connection point of the diffusion tube with the preheating oxygen tube equals to 2-8 mm.",
"In the atomizer as mentioned above, the angle α2 between the gasoline fine-tube and the perpendicular surface of the diffusion tube equals to 0-450.The diameter d1 of the preheating oxygen fine-tube equals to 0.5-2.5 mm.",
"The diameter d2 of the small end of the diffusion tube equals to 1.5-3 mm.",
"The diameter d3 of the gasoline fine tube equals to 0.5-2 mm.",
"The atomizer of the present invention is fixed at the head of the cutting torch or in the preheating oxygen tube.",
"After the preheating oxygen tube is opened, gasoline is sucked into the cutting torch and atomized to obtain cutting flame which is combusted sufficiently, has concentrated flame and is easy to be adjusted.",
"The obvious advantages of the gasoline-oxygen cutting machine according to the invention are as follows: the operation is easy as the gasoline tank needs not be pressurized; there is no leak of gasoline or jet of gasoline in leaking manner during operation.",
"The atomized compound of gasoline and oxygen is immediately jetted while the oxygen valve is opened, and gasoline is immediately shut off while the oxygen valve is closed.",
"Therefore, safety of operation is increased and the cost is reduced.",
"DESCRIPTION OF DRAWINGS FIG.",
"1 is a schematic view of structure of the jet-inducing atomizer for gasoline-oxygen cutting-welding according to the invention; FIGS.",
"2-4 are schematic views of three kinds of structures of gasoline-oxygen cutting torch used with the jet-inducing atomizer for gasoline oxygen cutting-welding according to the invention; PREFERRED EMBODIMENTS OF INVENTION The invention will be described in details with reference to the drawings.",
"In FIG.",
"1, the reference numeral 1 denotes a main body of the Jet-inducing atomizer; 2—a gasoline inlet; 3—a preheating oxygen inlet; 4—a preheating oxygen fine-tube; 5—a gasoline fine-tube; 6—a diffusion tube.",
"FIGS.",
"2-4 are schematic views of three kinds of structures of gasoline-oxygen cutting torch used with the jet-inducing atomizer for gasoline-oxygen cutting-welding.",
"In FIGS.",
"2-4, the reference numeral 7 denotes a gasoline feeding tube; 8—an oxygen feeding tube; 9—a gasoline fine-adjusting valve; 10—a main body of a cutting torch; 11—a cutting oxygen tube; 12—a connector of the cutting nozzle; 13—a cutting nozzle; 14—a preheating oxygen valve; 15—a cutting oxygen valve; and 16—a preheating oxygen tube.",
"As shown in FIG.",
"1, the jet-inducing atomizer for gasoline-oxygen cutting-welding according to the invention comprises a main body 1 of the atomizer, a diffusion tube 6, a preheating oxygen fine-tube 4 and a gasoline fine-tube 5.The diffusion tube 6 and the preheating oxygen fine-tube 4 are provided at the center of the main body 1 co-axially, and communicated with each other.",
"The gasoline fine-tube 5 enters the main body from one side of the main body, and is communicated with the diffusion tube 6.The diffusion tube is a conical tube.",
"The coarse end of the conical tube is coincident with the end face of the main body.",
"The fine end of the conical tube is connected with the preheating oxygen fine-tube.",
"The taper α1 of the conical tube equals to 5°-10°.",
"The length 10 of the conical tube is 2-5 times of length 11 of the preheating oxygen fine-tube, i.e.",
"10=(2-5) 11.The length 11 of the preheating oxygen fine-tube is 3-5 mm.",
"The distance 12 between the intersection point of the gasoline fine-tube with the diffusion tube and the connection point of the diffusion tube and the preheating oxygen tube equals to 2-8 mm.",
"The operating principle of the atomizer of the invention is as follows: the oxygen flow is accelerated while the preheating oxygen enters the fine-tube 4 from the inlet 3, the oxygen flow is then enters the diffusing tube and produces a negative pressure at the outlet of the gasoline tube 5, and the suction force is increased to suck the gasoline into the diffusion tube 6 to co-act with the oxygen flow under high speed.",
"The gasoline is atomized and mixed with oxygen.",
"The mixture is jetted from the diffusing tube to enter the cutting nozzle for igniting.",
"The parameters of one embodiment of the invention are as follows: the diameter of the preheating oxygen fine-tube is 0.8 mm; the length of the preheating fine-tube is 4 mm; the diameter of the gasoline fine-tube is 1.5 mm; the diameter of the small end of the diffusion tube is 2 mm; the length of the diffusion tube is 12 mm; the taper of the diffusion tube is 6°; and the distance between the outlet of the gasoline fine-tube entering the diffusion tube and the outlet of the preheating oxygen fine-tube is 3 mm.",
"As shown in FIG.",
"2, the atomizer is provided in the connector of the cutting nozzle 12.A solid metal rod having a diameter of 1 mm less than the inner diameter of the gasoline feeding tube is fixed in the gasoline feeding tube 7 to feed gasoline into the gasoline fine-tube via an inclined opening on the connector 12.The preheating oxygen tube 16 is communicated with the inlet of oxygen of the atomizer.",
"The outlet of the diffusion tube is communicated with the chamber in the head of the cutting nozzle 13.In this embodiment, the diameter of the preheating oxygen fine-tube is 0.8 mm; the diameter of the gasoline fine-tube is 1.5 mm; the diameter of the small end of the diffusion tube is 2 mm; the taper of the diffusion tube is 6°; and the length of the diffusion tube is 12 mm.",
"As shown in FIG.",
"3, the atomizer is fixed in the preheating tube 16.The oxygen inlet is connected with the preheating oxygen tube in the main body of the cutting torch.",
"The gasoline feeding tube 7 feeds the gasoline into the gasoline fine-tube.",
"The outlet of the diffusion tube is communicated with the chamber at the upper part of the cutting nozzle 13.In this embodiment, the diameter of the preheating oxygen fine-tube 4 is 0.8 mm; the length of the preheating oxygen fine-tube is 3 mm; the diameter of the gasoline fine-tube 5 is 1.5 mm; the diameter of the diffusion tube 6 is 2 mm; and the length of the diffusion tube is 12 mm.",
"As shown in FIG.",
"4, the atomizer of the invention is used for a distributor head of a semi-automatic cutting machine.",
"The atomizer is provided at the connection of the preheating oxygen tube 16 and the connector of the cutting torch.",
"The gasoline fine-tube is communicated with the gasoline feeding tube 7.The preheating oxygen fine-tube is communicated with the preheating oxygen tube 16.The outlet of the diffusion tube is communicated with the chamber at the head of the cutting nozzle 13.In this embodiment, the diameter of the preheating oxygen fine-tube 4 is 0.9 mm; the length of the preheating oxygen fine-tube 4 is 3 mm; the diameter of the gasoline fine-tube 5 is 1.5 mm; the taper of the diffusion tube is 6°; the diameter of the small end of the diffusion tube is 2 mm; and the length of the diffusion tube is 12 mm.",
"Industrial Application The present invention can be used for cutting, such as cutting the steel materials having various thickness."
]
] | Patent_10416374 |
[
[
"Deflection fitting for the safety belt of a motor vehicle",
"A deflection fitting for the safety belt of a motor vehicle consists of a belt guide (17, 18) which surrounds the safety belt (7) and a pivot bearing (13) about which said belt guide is pivoted, about a pivot pin (14) and parallel to the direction in which the belt passes through.",
"Said pivot hearing is fixed to the vehicle or to a part which is connected thereto.",
"The belt guide consists of two chain links (17, 18) which can pivot independently of each other in the pivot bearing (13), respectively, and which are located some distance apart from each other in the direction in which the belt (7) passes through.",
"This configuration enables easy adjustment of the belt guide, even when the angles of the strands (8, 9) of the belt (7) deviate considerably from the standard."
],
[
"1.Deflecting fitting for the safety belt of a motor vehicle, said fitting consisting of a belt guide (17, 18) which encloses the safety belt (7) and of a swivel bearing (13) about which said belt guide can be swivelled, wherein the swivel bearing (13) is fastened to the vehicle or to a part connected thereto and wherein the belt guide (17, 18) can be swivelled about an axis parallel to the direction of running through of the belt (7; 36), characterised in that the belt guide (17, 18) consists of two chain links (17, 18) which can each be swivelled, independently of one another, in the swivel bearing (13) and which are disposed at some distance from one another in the direction of running through of the belt (7).",
"2.Deflecting fitting according to claim 1, characterised in that each chain link (17, 18) possesses a bearing eye (19, 20) through which a swivel bolt (14) passes, and that a bush (15) is provided between the two bearing eyes.",
"3.Deflecting fitting according to claim 2, characterised in that the swivel bolt (14) is fastened by means of the bush (15) to the vehicle or to a part connected thereto.",
"4.Deflecting fitting according to claim 2 or 3, characterised in that the bush (15) is fastened to a safety shackle (12) and is provided with a longitudinal slit (30), said safety shackle (12) possessing an intercepting arm (31) on which the safety belt (7) rests if the bush (15) is torn open.",
"5.Deflecting fitting according to claim 4, characterised in that the safety shackle (12) is fastened to the backrest (2) of a seat.",
"6.Motor vehicle seat having a deflecting fitting (6) according to one of the preceding claims."
],
[
"The invention relates to a deflecting fitting for the safety belt of a motor vehicle, said fitting consisting of a belt guide which encloses the safety belt and of a swivel bearing about which said belt guide can be swivelled about an axis parallel to the direction of running through of the belt, wherein the swivel bearing is fastened to the vehicle or to a part connected thereto.",
"Deflecting fittings are used in all possible arrangements of safety belts which extend over the shoulder of the driver or passenger wearing them.",
"Said fittings ensure that the belt, which is fastened by one end at the side of, below, behind or next to the passenger, is guided over the shoulder in question at all times, that is to say irrespective of the sitting position.",
"Deflecting fittings are therefore attached, at or above shoulder height, to the B or C pillar of the vehicle or to the backrest of the seat in question; see, for example, DE 198 14 845 A1.Their main component is a belt guide which is a closed, flattened chain link which encompasses the belt.",
"Said chain link consists of two parallel straight parts, over one of which the belt runs downwards and which are connected by a tightly curved part.",
"If the strands of the belt which run upwards and downwards are oriented perpendicularly to the straight part of the chain link and are not twisted, the belt runs downwards without any problems.",
"If the angles of the two strands deviate from the perpendicular, the chain link tends to adjust itself according to the line bisecting the angle of the strands.",
"As a result of this, however, the quality of the guidance declines, particularly if adjustment of the chain link is hindered by friction, and on account of the small distance from the swivel bearing.",
"Under these circumstances, the belt shifts into the tightly curved part of the chain link and thus becomes folded or completely turned over.",
"This causes particular problems in the case of deflecting fittings which are fastened to the seat rest and in which the angular deviation from the perpendicular is, as a rule, greater.",
"From the generic DE-A 26 01 171, a deflecting fitting is known which has two guide members which can be swivelled, independently of one another, about an axis of swivelling and are disposed at a distance from one another, said guide members being formed by a run-through eye and a swivel arm which is provided with a deflecting eye at its free end.",
"The direction in which the belt runs through between the guide members extends, in a manner corresponding to the swivel arm, approximately parallel to the side wall of the vehicle and thereby perpendicular to the axis of swivelling.",
"As a result of this, however, a relatively large angle of splay of the belt occurs in the deflecting region.",
"U.S. Pat.",
"No.",
"5,385,370 indicates two so-called “D-rings” as the chain links, which can be moved in a swivelling manner, of a deflecting fitting.",
"However, these are offset in relation to one another at the lateral distance between the openings of the rings and are associated with different belts, so that there are problems, as initially mentioned, in the quality of guidance for the individual belt.",
"It is therefore the aim of the invention to provide a deflecting fitting which does not suffer from these disadvantages.",
"The intention is that it should adjust itself easily, even in the event of large angular deviations from the perpendicular and of fairly large angles of torsion.",
"According to the invention, this is achieved through the fact that the belt guide can be swivelled about an axis parallel to the direction of running through of the belt and consists of two chain links which can each be swivelled, independently of one another, in the swivel bearing and which are disposed at some distance from one another.",
"Since the two chain links are capable of twisting in relation to one another, larger angular deviations are possible without defective running of the belt, the small clearance of the axis of swivelling even proving to be an advantage.",
"The distance between the two chain links allows the belt to twist slightly.",
"In the process, the outer braids of the belt are subjected to somewhat greater tension, which makes it still more difficult for the belt to creep up into the sharply curved part of the chain links.",
"In one advantageous form of embodiment, each chain link possesses a bearing eye through which the swivel bolt passes, and a bush is provided between the two bearing eyes.",
"The bush serves as a bearing, so that the chain link is able to adjust itself with little friction, and said bush also serves as a spacer between the two chain links.",
"The bush may be constructed in such a way that the swivel bolt is fastened, by means of the bush, to the vehicle or to a part connected thereto.",
"The swivel bolt thus serves only for mounting the two chain links with equally little friction, and fastening via the bush brings about central stressing of said swivel bolt.",
"In one advantageous further development, the bush is fastened to a safety shackle and is interrupted in its peripheral direction, said safety shackle possessing an intercepting arm on which the safety belt rests if the bush is torn open.",
"In addition to its retaining function, the safety shackle thus also acts as a graduated, second safety device if the stress on the bush exceeds a limit value in the event of a collision.",
"The swivel bolt with the two chain links is then released and the belt is supported on the intercepting arm, which may be of heavier dimensions.",
"Finally, the invention may be used with special advantage if the safety shackle is fastened to the backrest of a seat.",
"The invention therefore also provides a motor vehicle seat having a deflecting fitting according to one of the preceding claims.",
"The invention will be described and explained below with the aid of illustrations, in which: FIG.",
"1: represents a seat having a deflecting fitting according to the invention, from the front; FIG.",
"2: represents, in enlarged form, the detail II in FIG.",
"1; FIG.",
"3: represents, enlarged still further, the section AA in FIG.",
"2; and FIG.",
"4: is a representation like that in FIG.",
"2, but in an extreme position.",
"FIG.",
"1 shows, in part, a bench seat in a motor vehicle.",
"Said seat consists of a seating surface 1 and a backrest 2, with a seat frame 3.A belt reel 4 is fastened, behind the seat and therefore in a manner shown in broken lines, to the said seat frame, a belt lock 5 is fastened next to the seating surface 1 and a deflecting fitting 6 is fastened above the seat.",
"The belt reel 4 contains the usual winding-on and clamping mechanism.",
"In this instance, the deflecting fitting 6 is provided on the backrest 2 at the top, above the shoulder of a passenger, who is not represented, but said fitting could also be fastened to the side wall of the vehicle, which side wall is likewise not represented.",
"For the sake of simplicity, a seat belt 7 is represented only in the form of a shoulder belt.",
"It then consists of a first (upward-running) strand 8, which extends from the belt reel 4 to the deflecting fitting 6, and of a second strand 9, the downward-running strand, which extends from the deflecting fitting 6 to the belt lock 5.If the belt in question is a three-point one, a fastening point 10, to which the safety belt is then conveyed onwards, is provided on the other side of the passenger.",
"The second strand 9 then forms a loop which extends from the belt lock 5 to the said fastening point 10.7* designates a three-point belt of this kind in the inoperative position.",
"FIGS.",
"2 and 3 show the deflecting fitting 6 in detail.",
"Fastened, for example welded on, to the seat frame 3 is a safety shackle 12 from which the belt guide is suspended in a swivel bearing 13.Said swivel bearing 13 is formed by a swivel bolt 14 which is mounted in a bush 15 by means of bearing shells 16 which act as radial and step bearings.",
"Said bush 15 is fixedly connected, for instance welded, to the safety shackle 12.According to the invention, the belt guide is formed by two chain links 17, 18 which can be swivelled, independently of one another, about the swivel bolt 14.Said chain links possess bearing eyes 19, 20 through which the swivel bolt 14 passes.",
"For fixing it in position axially, the swivel bolt 14 possesses a head 22 and, at the other end, a washer 21 which is followed, for example, by a split pin which is not represented.",
"In addition, a metal diverting plate 23 is also provided on the swivel bolt.",
"On its outer periphery, the bush 15 has a slit 30, as a result of which it opens under extreme load, in the event of a collision, and the safety belt 7, together with the belt guide, falls onto an intercepting arm 31 belonging to the safety shackle 12, a fact which is drawn in in broken lines in FIG.",
"3 and designated by 7′.",
"The chain links 17, 18 are closed steel rings which consist of two straight parts 32 and two sharply curved parts 33, and are thus similar to links which have been pressed together in an ordinary chain.",
"The action of the chain links 17, 18, which can be swivelled independently of one another, will now be explained with the aid of FIG.",
"4, which shows the belt guide in an extreme case in which the two strands 8, 9 of the safety belt 7 form a considerable angle 37 with one another.",
"A belt guide having a single chain link according to the prior art would adjust itself to the line 34 bisecting the angle.",
"The belt would then run obliquely through the chain link, namely at an oblique angle amounting to about half the angle 37.As a result of this, the belt easily arrives in the sharply curved part 33 of the chain link, climbs up it and is folded or completely twisted.",
"Owing to the two chain links 17, 18, which can be swivelled independently of one another, and to their distance from one another in the longitudinal direction of the belt, said two chain links 17, 18 can adjust themselves in such a way that the oblique angle 35 of the upward-running strand 8 of the belt 7 in the chain link 17, and likewise that of the downward-running strand 9 in the chain link 18 is substantially smaller.",
"The part 36 of the safety belt 7 between the two chain links 17, 18 (see also FIG.",
"3) is slightly twisted in the process.",
"The consequence of the small oblique angle is that the belt 7 is no longer able to creep upwards at the sharply curved parts 33 of the chain links 17, 18.The same action also occurs if the deflecting fitting 6 is fastened to the vehicle body, for instance to a B pillar which is not represented, in spite of said deflecting fitting being disposed in a manner rotated about a right angle."
]
] | Patent_10416605 |
[
[
"Method and system for tubing a borehole in single diameter",
"The invention concerns a method for casing a borehole which consists of successively lowering an unlimited plurality of strings of casing having a common diameter, the next one through the preceding one, to be installed one beneath the other and being sealingly connected.",
"Thus a next casing (6) is lowered folded through the preceding casing (5) and the expansion process consists either in injecting pressurized fluid through the drillstring (54) towards the next casing (6) so as to re-inflate it, or the next casing (6) is a cylindrical pipe having a diameter smaller than the internal diameter of the preceding casing (5) and the expansion process consists in forcing a cylindrical gauge (52) from the top end (26) to the bottom bell (28) of the next casing (6) so as to increase its diameter to make it equal to the diameter of the preceding casing (5).",
"The method can use a cementing shoe (34) and a metal-to-metal seal (29) between the casing string."
],
[
"1.Method for casing a well of the type in which, after each drilling phase to a predetermined depth using a drillstring from a preceding drilling phase, a string of casing (6, 6′) is run into the well, each successive casing being lowered through the previously run casing (5, 5′) and the top end (26) from the next casing being connected in a sealed way with said preceding casing (5, 5′); the said method being characterized in that it comprises the following steps: a) run, through a preceding casing (5, 5′) having a common diameter, a next casing (6, 6′) into the well, in a form whose largest transverse dimension is smaller than the internal diameter of the said preceding casing (5, 5′), the top end (26) of the said next casing being secured to the drillstring (54) b) position the top end (26) of the said next casing (6, 6′) in the vicinity of the bottom bell (28) of the preceding casing (5, 5′), c) expansion of the diameter of the said next casing (6, 6′) so that the diameter of the said next casing (6, 6′) is equal to the said common diameter, and d) establishing a sealed connection between the top end (26) of said next casing (6, 6′) and bottom end (28) of said preceding casing (5, 5′).",
"2.Method according to claim 1, in which the said next casing (6, 6′) is lowered, folded up, through the said preceding casing (5, 5′) and the expansion of the stage d) consists of injecting a pressurized fluid through the drillstring (54) to said next casing (6, 6′) so as to first, re-inflate it then second, force a gauge from the top end (26) to the bottom end (28) of the next casing (6, 6′) so that this at last gains a cylindrical form with a diameter equal to the said common diameter.",
"3.Method according to claim 1, in which the said next casing (6, 6′) is a cylindrical pipe having a diameter smaller than the internal diameter of said preceding casing (5, 5′) and the expansion of the stage d) consists in forcing an expansion mandrel from the top end (26) to the bottom end (28) of the next casing (6, 6′) so as to increase its diameter to make it equal to the said common diameter.",
"4.Method according to claim 1, in which the bottom end (28) of said preceding casing (5, 5′) is lowered, folded up during the descent, then inflated by injection of a pressurized fluid through the drillstring (54) after the step b) in order to gain a widened internal diameter equal or higher than the common diameter.",
"5.Method according to claim 4, in which the said positioning of the step b) of the said top end (26) of the said next casing (6, 6′) consists in hanging the top hanger (26) of the said next casing (6, 6′) from said bottom bell (28) of said preceding casing (5, 5′).",
"6.Method according to claim 5, in which the said top hanger (26) of the said next casing (6, 6′) is hung by collet fingers (30) which allows the expansion of said top end (26) as well as the passage of the return fluid in the annulus space (122) between the casing and the wellbore.",
"7.Method according to claim 4, in which said top end (26) of said next casing (6, 6′) is expanded by forcing a gauge/expansion mandrel (52, 52′) through said top end (26) of said next casing (6, 6′).",
"8.Method according to claim 4, in which a partially deformable cementing shoe (34) seals the lower end of said bottom bell (28) of said preceding casing (5, 5′).",
"9.Method according to claim 8, in which said cementing shoe (34) comprises a rigid cylindrical lower end with a smaller diameter than the internal diameter of said preceding casing (5, 5′) and this said lower end is offset compared to the axis of the said preceding casing (5, 5′) when the said bottom bell (28) of said preceding casing (5, 5′) is re-inflated.",
"10.Method according to claim 8, in which the drillable part of the said cementing shoe (34) is in aluminum so as to be easily milled with conventional well drillbit.",
"11.Method according to claim 8, in said next casing (6, 6′) is cemented in said well by pumping of a cement slurry through said drillstring (54) and through said cementing shoe (34), the fluid return of annulus space (122) formed between said wellbore and said next casing (6, 6′) flows between said top end (26) of the said next casing (6, 6′) and said bottom bell (28) of said preceding casing (5, 5′).",
"12.Method according to claim 4, in which the said sealed connection is carried out by one or more plastic deformations or swedging of rings located on said top end (26) of the said next casing (6, 6′) so that external surface of each ring cooperates in a sealing way with enlarged interior surface of said bottom bell (28) of said preceding casing (5, 5′).",
"13.Method according to claim 12, in which enlarged interior surface of said bottom bell (28) of said preceding casing (5, 5′) or the said external surface of the plastically deformed ring comprises circular grooves (27 or 29) increasing the contact pressure to achieve a metal-to-metal type sealing.",
"14.Method according to claim 15, in which the said surfaces cooperating to carry out said sealed connection comprises grooves with the same pitch (27, 29) and a soft metal is laid out in bottom of groove to allow the interpenetration of the grooves in order to improve tolerance to debris and scratches.",
"15.System of well casing including the adapted means to implement the steps of the method according tone of the preceding claims."
],
[
"<SOH> BACKGROUND ART <EOH>Oil and gas wells are typically drilled by installing a conductor pipe to first depth, then drilling the well to a second depth.",
"Normally, a string of casing is made up by coupling together sections of pipe, each being about forty feet long, and lowering the string through the conductor pipe to the second depth.",
"Cement is then pumped down the casing, which flows back up the annulus between the casing and the open borehole.",
"Drilling is resumed to a third depth and the process is repeated with another smaller diameter nested casing.",
"An even smaller diameter string of casing may be installed to a fourth depth.",
"Casing serves to support the borehole wall and to prevent undesired outflow of drilling fluid into the formation or inflow of fluid from the formation into the borehole from strata other than the target production strata.",
"The nested arrangement of successively smaller casing strings requires a relatively large borehole at the upper part of the wellbore due to the thickness of the casing and couplings and also due to the minimum clearance necessary between casing to displace cement in the annulus space.",
"Large diameter wells are more expensive to drill since they request larger drillbit, more mud and more cutting disposal.",
"Also, a casing with a large diameter has a lower operating pressure for the same wall thickness than a smaller diameter casing.",
"Consequently, each casing may have to cover the preceding one up to the wellhead to enhance the well pressure capability, as the well goes deeper.",
"Whenever pressure is not critical for the production casing, which is the last casing lowered and in consequence the smallest in diameter, the last casing is run as liner of the preceding casing and thus instead of extending it up to the wellhead, the top of a liner is suspended from the inside surface of the preceding casing, generally at an elevation right above the shoe of the preceding casing.",
"A certain length of casing is saved, also increasing the higher section of the well, but since the liners are always formed by the assembly of pipes of a diameter lower than the preceding casing, a bore restriction on a substantial length between the production zone and the wellhead still exists.",
"Lastly, today when a well is drilled in a unknown zone (exploratory drilling) it is necessary to increase the amount of casing in order to be able to face the possibility to have to case the well before having reached the targeted depths.",
"To take care of this kind of difficulty, an extra casing is considered which requires to start to drill from the surface with the next size above what would be usually needed, with a substantial increase in the cost of exploratory drilling."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a partial sectional perspective view for a lining folded up according to the first embodiment of the invention.",
"FIG.",
"2 is a partial sectional perspective view for an expansible casing according to the second embodiment of the invention.",
"FIG.",
"3 is a partial sectional perspective view for the final casing according to the first or the second embodiment of the invention with the top of the casing forged in the foot of the preceding casing and the shoe drilled.",
"The FIGS.",
"4 a to 4 c are sectional views of the casing folded up, from the running tool to the shoe, in the lowering phase configuration according to the first embodiment of the invention.",
"The FIG.",
"4 d is a sectional view of the shoe in the lowering phase configuration according to the second embodiment of the invention.",
"The FIGS.",
"5 a and 5 b are sectional views of the running tool and shoe, of the FIGS.",
"4 a - 4 c after the folded up lining and the foot of lining were re-inflated according to the first embodiment of the invention.",
"The FIGS.",
"6 a and 6 b are sectional views of the running tool and shoe of the FIG.",
"4 d , after the bottom head of casing was re-inflated, of the expansible casing according to the second embodiment of the invention.",
"The FIG.",
"7 a is a sectional view of a portion of the running tool of the FIGS.",
"4 a and 4 b when it is pushed downwards to expand the top of the casing according to the first embodiment of the invention.",
"The FIG.",
"7 b is a sectional view of a portion of the running tool of the FIGS.",
"4 a and 4 b when it is pushed downwards to expand the top of the casing according to the second embodiment of the invention.",
"The FIG.",
"8 a is a sectional view of the gauge detached from the running tool gauging the re-inflated casing according to the first embodiment of the invention.",
"The FIG.",
"8 b is a sectional view of the expanding tool detached from the running tool expanding the expandable casing according to the second embodiment of the invention.",
"The FIG.",
"9 is a sectional view illustrating the expansion mandrel located at the shoe and reconnected with the running tool according to the second embodiment of the invention.",
"The FIG.",
"10 is a sectional view illustrating the running tool swaging the casing head according to the first or the second embodiment of the invention.",
"The FIG.",
"11 is an enlarged partial sectional view of the swaging area of FIG.",
"10 .",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD This invention relates in general to installing well casing in oil and gas wells and in particular to a method of running collapsed or expansible casing into the well through a preceding casing of a given diameter and rounding the collapsed casing into a cylindrical configuration or expanding the expansible casing into the same given diameter.",
"BACKGROUND ART Oil and gas wells are typically drilled by installing a conductor pipe to first depth, then drilling the well to a second depth.",
"Normally, a string of casing is made up by coupling together sections of pipe, each being about forty feet long, and lowering the string through the conductor pipe to the second depth.",
"Cement is then pumped down the casing, which flows back up the annulus between the casing and the open borehole.",
"Drilling is resumed to a third depth and the process is repeated with another smaller diameter nested casing.",
"An even smaller diameter string of casing may be installed to a fourth depth.",
"Casing serves to support the borehole wall and to prevent undesired outflow of drilling fluid into the formation or inflow of fluid from the formation into the borehole from strata other than the target production strata.",
"The nested arrangement of successively smaller casing strings requires a relatively large borehole at the upper part of the wellbore due to the thickness of the casing and couplings and also due to the minimum clearance necessary between casing to displace cement in the annulus space.",
"Large diameter wells are more expensive to drill since they request larger drillbit, more mud and more cutting disposal.",
"Also, a casing with a large diameter has a lower operating pressure for the same wall thickness than a smaller diameter casing.",
"Consequently, each casing may have to cover the preceding one up to the wellhead to enhance the well pressure capability, as the well goes deeper.",
"Whenever pressure is not critical for the production casing, which is the last casing lowered and in consequence the smallest in diameter, the last casing is run as liner of the preceding casing and thus instead of extending it up to the wellhead, the top of a liner is suspended from the inside surface of the preceding casing, generally at an elevation right above the shoe of the preceding casing.",
"A certain length of casing is saved, also increasing the higher section of the well, but since the liners are always formed by the assembly of pipes of a diameter lower than the preceding casing, a bore restriction on a substantial length between the production zone and the wellhead still exists.",
"Lastly, today when a well is drilled in a unknown zone (exploratory drilling) it is necessary to increase the amount of casing in order to be able to face the possibility to have to case the well before having reached the targeted depths.",
"To take care of this kind of difficulty, an extra casing is considered which requires to start to drill from the surface with the next size above what would be usually needed, with a substantial increase in the cost of exploratory drilling.",
"DISCLOSURE OF THE INVENTION Consequently, the aim of the invention is to offer an economic method to case a well in which an unlimited plurality of casings of the same diameter are descended successively, the next through the preceding, to be installed one below the other and to be connected in a sealed manner.",
"Another goal of the invention is to reduce cutting volume and to allow the driller a large freedom on the number of casings to be run down to deliver the well objectives in full safety.",
"The object of the invention is thus a method to case a well in which, after each drilling phase to a predetermined depth using a drillpipe, a casing is lowered into the well with each successive casing being lowered down through the preceding casing and the top of the next casing being connected in a sealed manner to the preceding casing.",
"This method includes the step of: lowered into the well through the preceding casing having a common diameter, the next casing in a form whose greatest transverse dimension is smaller than the internal diameter of the preceding casing with the top of the next casing being suspended to the drillstring, positioning the top of the next casing near the foot of the preceding casing, expanding the diameter of the next casing so that the diameter of the next casing is equal to the common diameter, establishing a sealed connection between the top of the next casing and the foot of the preceding casing.",
"According to a first embodiment of the invention, the next casing is lowered folded up through the preceding casing and the step of expansion consists in sending fluid under pressure through the drillstring down the next casing so as to re-inflate it, then an expansion mandrel is forced from the top to the foot of the next casing so that the casing has a cylindrical form whose diameter is equal to the common diameter.",
"According to a second embodiment of the invention, the next casing is a cylindrical pipe having a diameter smaller than the internal diameter of the preceding casing and the stage of expansion consists in forcing an expansion mandrel from the head to the foot of the next casing so as to increase its diameter to make it equal to the common diameter.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a partial sectional perspective view for a lining folded up according to the first embodiment of the invention.",
"FIG.",
"2 is a partial sectional perspective view for an expansible casing according to the second embodiment of the invention.",
"FIG.",
"3 is a partial sectional perspective view for the final casing according to the first or the second embodiment of the invention with the top of the casing forged in the foot of the preceding casing and the shoe drilled.",
"The FIGS.",
"4a to 4c are sectional views of the casing folded up, from the running tool to the shoe, in the lowering phase configuration according to the first embodiment of the invention.",
"The FIG.",
"4d is a sectional view of the shoe in the lowering phase configuration according to the second embodiment of the invention.",
"The FIGS.",
"5a and 5b are sectional views of the running tool and shoe, of the FIGS.",
"4a-4c after the folded up lining and the foot of lining were re-inflated according to the first embodiment of the invention.",
"The FIGS.",
"6a and 6b are sectional views of the running tool and shoe of the FIG.",
"4d, after the bottom head of casing was re-inflated, of the expansible casing according to the second embodiment of the invention.",
"The FIG.",
"7a is a sectional view of a portion of the running tool of the FIGS.",
"4a and 4b when it is pushed downwards to expand the top of the casing according to the first embodiment of the invention.",
"The FIG.",
"7b is a sectional view of a portion of the running tool of the FIGS.",
"4a and 4b when it is pushed downwards to expand the top of the casing according to the second embodiment of the invention.",
"The FIG.",
"8a is a sectional view of the gauge detached from the running tool gauging the re-inflated casing according to the first embodiment of the invention.",
"The FIG.",
"8b is a sectional view of the expanding tool detached from the running tool expanding the expandable casing according to the second embodiment of the invention.",
"The FIG.",
"9 is a sectional view illustrating the expansion mandrel located at the shoe and reconnected with the running tool according to the second embodiment of the invention.",
"The FIG.",
"10 is a sectional view illustrating the running tool swaging the casing head according to the first or the second embodiment of the invention.",
"The FIG.",
"11 is an enlarged partial sectional view of the swaging area of FIG.",
"10.BEST MODE FOR CARRYING OUT THE INVENTION Referring on FIG.",
"1, string 6 is formed of multiple sections of collapsible casing 8.The string 6 is comprised of a top end 26 connected by joints of casing 8 to a bottom end 28 also collapsed and a cement shoe 34 only partially collapsed.",
"The top end 26 can act as a casing hanger and includes collet fingers 30 for this purpose.",
"The top hanger 26 of casing also comprises circumferential triangular grooves 27 on its external surface.",
"Preferably, a soft metal coating is laid out in triangular bottom of groove 27 to facilitate sealing.",
"The peaks of the triangular grooves make it possible to increase the contact pressure to obtain metal-to-metal sealing.",
"A profile with interior groove 31 is laid out on the internal diameter top hanger 26 to allow the attachment to the running tool for installation 50 (FIG.",
"4b).",
"Casing 8 connected to the top end 26 can extend in casing 6 for several thousand meters.",
"The bottom end 28 comprises of an internal recess profile 32 that is designed to mate with the collet fingers 30 of the top hanger 26 of the next string (not shown) that will be suspended below.",
"Bottom bell 28 has internal circumferential wickers 29 (FIG.",
"9) that similarly mate with the wickers 27 of the top hanger 26 of the next string (not shown) that will be suspended below.",
"Cement shoe 34 is offset from the axis of the string 6 and comprised of a check valve 35 with a top valve seat 38; a bottom valve seat 36 held in place by a shear pin 37 and sealed to the cement shoe 34 by seals; a movable ball 39; and a fluid passage 40 fluidly linked to and below the check valve 35.The shoe which is partially collapsed can be made of aluminium in order to be easily milled by conventional drillbits.",
"FIG.",
"1 shows string 6 and the bottom bell 28 in a collapsed configuration.",
"Top hanger 26 and cement shoe 34 do not collapse.",
"Top hanger 26 is later first cylindrically expanded (FIG.",
"7a) to allow the running tool and drift tool to go through and is finally locally expanded in a swage connection with the bottom bell 28 of a previously installed string of casing.",
"FIG.",
"2 shows the same configuration of string as FIG.",
"1 except that the string consists of expandable pipes 8′ and not collapsed 8′ but the same expandable top hanger 26 and bottom bell 28 describes on FIG.",
"1 are used.",
"Referring to FIG.",
"3, the top hanger 26 has engaged the previously run, rounded and cemented string 5 with collet fingers 30.Wickers 27 have been deformed to sealingly mate with the wickers 29 of the preceding string 5.Casing 8 has been rounded.",
"Referring to FIGS.",
"4a and 4b, a running tool 50 is used to install a string of collapsible casing 6 in a well 1.The running tool 50 includes a detachable gauge 52 that is shown mounted to the bottom of the running tool 50.The top of the running tool 50 is mechanically and fluidly connected to the drill pipe 54, which extends to the surface.",
"There is an annulus 58 for return of fluids to the surface located between the drill pipe 54 and a string 5 of previously installed casing.",
"An annular sliding valve 60 is located at the top end of the running tool 50.When fluid is pumped down the drill pipe 54, it flows through sliding valve 60 and into the running tool 50.Sliding valve 60 is sealed within a chamber in running tool 54 and initially held in place by a shear pin 64.While in the upper position shown in FIG.",
"4a, sliding valve 60 blocks a bypass passage 66 that leads to two piston chambers 73.An upper fluid return passage 72 is fluidly connected to drill pipe annulus 58.Return passage 72 extends through an upper body 68 of the running tool 50.A sleeve 71 having a pair of piston elements 74 is axially movable relative to upper body 68, and there are multiple seals between the upper body 68 and the piston elements 74.There are multiple annular chambers 73 between the upper body 68 and the piston elements 74.A forging or swaging ring 78 is located on the lower side of the lower piston element 74, and attached to the upper body 68 by attachment elements 80.Sleeve 71 terminates at the upper portion of swaging ring 78 by an actuating cone.",
"An upper cup tester 82 and a lower cup tester 84 provide a seal between the upper running tool body 68 and the previously installed and cemented casing 5.A middle running tool body 86 is located beneath and secured to upper body 68.A lower running tool body 118 is located beneath the middle running tool body 86.The detachable gauge 52 is located beneath the lower running tool body 118.The detachable gauge 52 has a threaded receptacle secured to lower tool body by a threaded ratchet ring 110 located on the lower running tool body 118.An engaging member 114 is mounted to detachable gauge 52 for engaging grooved profile 31 in top hanger 26 of the string of casing 6.Detachable gauge 52 has a drifting element 128.A downward flow passage 88 runs the length of the running tool 50 and the detachable gauge 52.Return fluid flows up an annulus 122 that initially exists between the new casing string 6 and the previously installed casing string 5.Annulus 122 extends between the detachable gauge 52 and the installed casing string 5 bypass the drift tool cup tester 89 through a bottom check valve 98 and communicates with a port leading to a middle check valve 94 just prior to entering the lower fluid passage 120.The lower check valve 98 comprises a ball 99 and the middle check valve 94 comprises a ball 96.Check valve 94 is located at the lower end of a return flow passage 120.The returning fluid through passage 120 encounters an upper check valve 90 prior to entering an upper return fluid passage 72.The upper check valve 90 is comprised of a ball 92 and is connected by a port to the annulus 122 surrounding running tool 50.Upper check valve 90 is located above lower cup tester 84 while middle check valve 94 is located below.",
"Upper check valve 90 will allow inward flow from annulus 122 into passage 72 but not an outward flow.",
"Upper flow passage 72 leads to the drill pipe annulus 58.The downward flow passage 88 encounters a lower check valve 100 after entering the detachable gauge 52.The lower check valve 100 is comprised of a ball 102 within a portion of fluid passage 88.An axially movable mandrel 103 is carried in lower running tool body 118 and detachable gauge 52, as shown in FIG.",
"4b.",
"Mandrel 103 has an upper pointed end that extends through a passage and actuates ball 96.A lower pointed end extends into a passage in detachable gauge 52 and actuates lower ball 102.A coil spring 105 urges mandrel 103 downward.",
"In the position shown in FIG.",
"4b, the upper end of mandrel 103 prevents ball 96 from moving to the right to a blocking or seated position in check valve 94.The lower end of mandrel 103 prevents ball 102 from moving to the left to a blocking position in check valve 100.FIG.",
"4C shows cement shoe 34 in a collapsed configuration and lowered into an under-reamed section of the well.",
"While being lowered on drill pipe 54, casing string 6 will be supported by engaging member 114 on detachable gauge 52, which engages grooved profile 31.This position is shown in FIG.",
"4b, with running tool 50 and casing 6 being lowered through previously installed casing string 5.When reaching the desired depth, collet fingers 30 of top hanger 26 of casing string 6 will snap into grooved profile 32 in bottom bell 28 at the lower end of previously installed casing string 5.This engagement is shown in FIG.",
"5a.",
"This engagement stops further downward movement of drill pipe 54 and casing string 6.Then, casing string 6 and the upper portion of cement shoe 34 are inflated by applying fluid pressure from the surface.",
"FIGS.",
"5a and 5b show casing 6 and cement shoe 34 after being inflated.",
"A fluid, such as water or drilling fluid, is pumped down drill pipe 54.The fluid flows through valve 60 (FIG.",
"4a) down passage 88.The fluid flows through check valve 102 and discharges into casing string 6.At the lower end of casing string 6, the high pressure fluid flows into cement shoe 34 and is initially blocked from flowing out passage 40 by check valve 39, which is initially held in the upper position of FIG.",
"4c by shear pin 37 and seat 36.The fluid pressure causes casing 6 and bottom bell 28 to round by inflation.",
"When a selected pressure is reached, shear pin 37 shears, allowing seat 36 to move downward to the position shown in FIG.",
"5b.",
"This maximum pressure is selected to assure that casing string 6 and bottom bell 28 are enough rounded.",
"The fluid can now discharge from passage 40 of casing shoe 34 into the well.",
"When casing 6 inflates, well fluid initially surrounding the string of casing 6 will be displaced.",
"Referring to FIG.",
"5a, the fluid flows through annulus 122 in the overlap between the previously installed casing 5 and the string 6 being installed.",
"Because of lower cup tester 84 engaging previously installed casing string 5, the displaced fluid cannot flow upward around running tool 50.The fluid flows through the lateral port of check valve 94, and from check valve 94 into return flow passage 120 (FIG.",
"5a).",
"The fluid flows from return flow passage 120 into upper return passage 72 (FIG.",
"4a), and from there into annulus 58 surrounding drill pipe 54 to return to the surface.",
"After inflating, as shown in FIGS.",
"5a and 5b, detachable gauge 52 will be pumped downward from running tool 50 to cement shoe 34 to round out imperfections in casing string 6.This is handled by closing a blowout preventer (not shown) at the surface around drill pipe 54 and applying pressure to annulus 58 surrounding drill pipe 54.The pressure acts against upper cup tester 82 (FIG.",
"4a), causing upper body 68 to move downward with drill pipe 54 a short distance into casing string 6.Engaging member 114 releases from groove 116 and moves downward in casing string 6 as shown in FIG.",
"7a.",
"Collet 30 (FIG.",
"5a) continues to support the weight of casing string 6.The upper cup tester 82 of running tool 50 will still be above the lower end previously installed casing string 4, and the lower cup tester 84 will be now located in the top hanger 26 of lower string 6.Once at the desired position, the operator stops pumping fluid on drill pipe annulus 58.Then drill pipe 54 is rotated to the right.",
"Detachable gauge 52 has left-hand threads connecting it to ratchet ring 110 of lower running tool body 118, and detachable gauge 52 also has anti-rotation engaging members (not shown) that frictionally engage the inner wall of casing string 6.Running tool 50 rotates with drill pipe 54, unscrewing detachable gauge 52, as shown in FIG.",
"14a.",
"When detachable gauge 52 separates from lower tool body 118, mandrel 103 remains with lower tool body 118.Mandrel spring 105 pushes mandrel 103 downward, allowing ball 96 to move to a seated position, blocking upward flow through check valve 94.Also, the absence of mandrel 103 in check valve 100 allows ball 102 to close passage 88 leading downward from check valve 100.The closure of check valves 94 and 100 and bottom cup tester 89 with lower cup tester 84 allow fluid pressure to drive down detachable gauge 52.Once unscrewed, the operator applies internal fluid pressure to drill pipe 54.The pressure flows through passage 88 and out the lower end of lower body 118.This pressure enters port 140 driving down actuating sleeve 142 which push slips 150 over the conical profile of tool body 118 against the inner wall of installed casing string 5 to prevent running tool 50 to move upward.",
"Helicoidal spring 148 prevented the locking of slips 150 during lowering of casing 6 through casing 5.This pressure acts also against the cup tester 89 of detachable gauge 52, forcing it downward.",
"Drifting elements 128 will round out casing string 6 to the desired configuration as detachable gauge 52 moves downward in lower casing string 6.At the lower end of casing string 6, detachable gauge 52 will enter cement shoe 34, as shown in FIG.",
"9 for the second embodiment.",
"The lower concentric end of detachable gauge 52 enters a receptacle in cement shoe 34 directly above ball 39.Then the operator stops pumping fluid through drill pipe 54.The operator then lowers running tool 50 on drill pipe 54 until lower body 118 reengages with detachable gauge 52, as shown in FIG.",
"9.Mandrel 103 will stab back into the passage in detachable gauge 52, opening check valve 100.The upper end of mandrel 103 pushes ball 96 to the left, opening check valve 94.The split ratchet ring 110 allows detachable gauge 52 to reconnect to running tool 50 by a straight downward stabbing movement.",
"The operator then pumps cement downward through drill pipe 54, which flows through running tool passage 88 and check valve 39 of cement shoe 34.The cement flows up the annulus surrounding lower casing string 6.Well fluids displaced by the cement return through annulus 122 (FIG.",
"4b) and check valve port 104 and back up return flow passage 120.Ball 39 of the cement shoe 34 prevents cement to back flow up running tool 50.While the cement is curing, the operator pulls running tool 50 back up to the upper end of lower casing string 6 to swage top hanger 26 to the bottom bell 28 of previously installed casing string 5.Detachable gauge 52 will continue to remain in engagement with running tool 50.The swaging is illustrated in FIGS.",
"10 and 11.The operator drops a ball 132 through drill pipe 54, which lands on sliding valve 60, closing passage 88.Pumping downward through drill pipe 54 causes shear pin 64 to shear, allowing sliding valve 60 to move downward.",
"This opens bypass passage 66 that leads to the two piston chambers 73.The pressure acts against pistons 74, causing sleeve 71 and upper body 68 to move apart from each other.",
"This causes forging ring 78 to be cammed outward, deforming top hanger 26 into sealing engagement with bottom bell 28.Wickers 27 and 29 engage each other for sealing, aided by a soft inlay of metal.",
"The operator then relieves the swaging pressure and pressure tests the sealing capability using upper cup tester 82.This is done by closing the blowout preventer around drill pipe 54 and applying pressure to drill pipe annulus 58.The lower tester cup 82 prevents flow downward past running tool 50.If the connection leaks, the pressure will fail to hold.",
"If there is no leakage, the operator pulls running tool 50 from casing string 5.After installation, casing strings 5 and 6 will have the same inner and outer diameters.",
"If the well is to be drilled deeper, casing shoe 34 will be drilled out as it is made of a frangible metal as aluminum.",
"The offset position reduces the chances of it rotating in unison with a rotating drill bit.",
"It also includes a transition cone 33 which has a revolution axis inclined to the axis of the well to prevent drilling through the wall of the cone 33 around the circumference at the same time.",
"In the second embodiment, referring to FIG.",
"2, the casing 8′ is not collapsed, rather it is cylindrical when run into the well.",
"Casing 8′ can be expanded to obtain the same dimension than casing 8 when rounded.",
"Casing 8′ can be made up of sections approximately 12 meters in length that are secured by threaded connectors while being run, as in conventional casing.",
"However, expandable casing 8′ has a smaller diameter while being run so as to pass through previously set string of the same dimension but after being expanded.",
"Casing 8′ is expanded to the same diameter as the upper string or strings of casing after reaching the desired depth.",
"For example, casing 8′ may have an outer diameter of 6¼ inch while running and an expanded outer diameter of 7 inch.",
"The string of casing 8′ will shrink in length while expanding.",
"The same running tool 50, expandable top hanger 26, and collapsible bottom bell 28, and cement shoe 34 are employed.",
"The same numbers will be used in the second embodiment, except for the prime symbol when referring to the casing.",
"The assembly in FIG.",
"8 initially will be run in with a string 6′ of casing 8′ that is in a cylindrical configuration but is expandible.",
"Because lower bell 28 is of a larger diameter than the previously installed string of casing, it will be collapsed in the same manner as the first embodiment shown in FIG.",
"1.The sections 8′ of casing string 6′ are screwed together at the surface and attached to running tool 50 with top hanger 26 (FIG.",
"4a).",
"Running tool 50 is lowered on drill pipe 54 until reaching the desired depth.",
"FIG.",
"4d shows collapsed bottom bell 28 and cement shoe 34 attached to unexpanded casing string 6′ as it is lowered through previously run casing 5′.",
"As illustrated in FIG.",
"6a, collet fingers 30 of the top hanger 26 will snap into grooved profile 32 of the bottom bell 28 of the previously installed string 5′.",
"The operator applies pressure to drill pipe 54 which causes bottom bell 28 of string 6′ to inflate as shown in FIG.",
"6b.",
"The fluid pressure transmits through running tool 50 in the same manner as the first embodiment.",
"Running tool 50 is then pushed down a short distance to the position shown in FIG.",
"7b, with expanding elements 128′, and a lower expanding element 130′ with a smaller outside diameter than the upper drifting element 128′ located within casing string 6′.",
"By locating two or more expanding elements having different expanding diameters, we prevent early striction to occur on the expanding cone triggered by small defects.",
"This downward movement occurs by applying pressure to the drill pipe annulus 58 (FIG.",
"6a), which acts against lower cup tester 82 to push the entire assembly downward.",
"As drift elements 128, 130 entered lower string 6′, they began expanding the string of casing 6′ to the desired diameter.",
"The operator then unscrews detachable expansion mandrel 52′ in the same manner as the first embodiment the detachable gauge 52.The operator pumps down drill pipe 54 to push detachable expansion tool 52′ downward relative to running tool 50, as shown in FIG.",
"8b.",
"This also is performed in the same manner as in the first embodiment except that seal 132 is used to drive the detachable tool down instead cup tester 89.Smaller seals can be used since expansible casing has a better internal surface offering less friction than conventional casing as it is used for collapsible casing.",
"As detachable expansion mandrel 52′ moves downward, it expands casing string 6′ to the desired diameter.",
"There will be some displaced fluid that was located in the annulus surrounding casing 6′.",
"This displaced fluid flows through annulus 122 (FIG.",
"4b), check valve 94, returns fluid passages 120 and 72 to drill pipe annulus 58 (FIG.",
"4a) When detachable expansion mandrel 52′ reaches cement shoe 34, the operator lowers running tool 50 on drill pipe 54 until running tool 50 reengages with detachable expansion mandrel 52′.",
"This is done in the same manner as in the first embodiment, and the assembly will appear as shown in FIG.",
"9.The operator pumps cement down drill pipe 54, which flows out passage 40 and up the annulus surrounding casing string 6′ in the same manner as the first embodiment.",
"After cementing, the operator pulls running tool 50 back up to top hanger 26 and swages it into bottom bell 28 of casing string 5′ in the same manner as in the first embodiment.",
"This procedure is shown in FIGS.",
"10 and 11.The operator tests and drills out in the same manner as in the first embodiment.",
"As one can realize, a mono-diameter casing system can be obtained with either collapsible casing or expansible casing.",
"The diameter reduction for installation through the preceding installed casing of the same dimension can be obtained from either technologies collapsible or expansible.",
"In some case it might be advantageous to combine both technologies by first collapsing a 6⅝″ casing (168 mm), to an overall diameter of 6 inches (152 mm), and then instead drifting it with the same diameter of the original pipe expanding it to a 7 inch diameter (178 mm), therefore being able to use the heavy wall capability and low cold work of the collapsible technology with the good geometry of the expansible technology.",
"Despite the fact that the casing strings are made of coupled straight joints in the description, continuous casing on reel can also be used to obtain a monodiameter well casing system."
]
] | Patent_10416773 |
[
[
"Exoenzyme toxin of aeromonas salmonicida, and uses thereof",
"A protein toxin named Aeromonas salmonicida exoenzyme T (AcxT), which belongs to the family of ADP-ribosylating toxins, is disclosed as is a Calcium (or other cation concentration) dependent promoter of A. salmonicida.",
"Also disclosed are diagnostic, preventive, and therapeutic techniques, including the preparation of bacterin vaccines based on AexT for inducing immunity against A. salmonicida infections."
],
[
"1-29.",
"(canceled) 30.An isolated polypeptide comprising at least one selected epitope or epitopic region of AexT.",
"31.An isolated nucleic acid fragment encoding the polypeptide of claim 30.32.An isolated nucleic acid fragment comprising SEQ ID NO:1, or the complement thereof.",
"33.A method for reducing the susceptibility of fish to infection by a virulent strain of A. salmonicida comprising the intraperitoneal, intramuscular, intradermal, intracellular, spray, immersion, or oral administration to said fish of a composition comprising an immunogenic amount of at least one epitope or epitopic region of AexT, a natural or genetically modified variant thereof, or an antigenic peptide derived or synthesized thereof.",
"34.The use of the polypeptide of claim 30 in a vaccine for reducing the susceptibility of fish to infection by a virulent strain of A. Salmonicida.",
"35.The use of the nucleic acid fragment of claim 31 in a vaccine for reducing the susceptibility of fish to infection by a virulent strain of A. salmonicida.",
"36.The use of the nucleic acid fragment of claim 32 in a vaccine for reducing the susceptibility of fish to infection by a virulent strain of A. salmonicida.",
"37.A method for the inducement of AexT production in A. salmonicida comprising the growth of A. salmonicida on Ca2+ or other cation depleted medium.",
"38.A bacterin vaccine for the immunization of fish comprising killed or inactivated A. salmonicida cells in which expression of the polypeptide of claim 31 had been induced prior to the inactivation, or in which A. salmonicida has been manipulated (using recombinant or other means) to constitutively express said polypeptide prior to inactivation.",
"39.A method for reducing the susceptibility or a poikilothermic fish to infection by a virulent strain of A. salmonicida comprising the intraperitoneal, intramuscular, intradermal, intracellular, spray, immersion, or oral administration to said fish of an immunogenic amount of a composition comprising the nucleic acid fragment as claimed in claim 31, fragments, synthetic oligonucleotides, or DNA sequence homologues thereof.",
"40.A method for reducing the susceptibility or a poikilothermic fish to infection by a virulent strain of A. salmonicida comprising the intraperitoneal, intramuscular, intradermal, intracellular, spray, immersion, or oral administration to said fish of an immunogenic amount of a composition comprising the nucleic acid fragment as claimed in claim 32, fragments, synthetic oligonucleotides, or DNA sequence homologues thereof."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The fish disease furunculosis derived its name from the characteristic lesions observed as furuncles formed on the surface of fish as a result of infection with Aeromonas salmonicida .",
"This pathogen causes most severe losses in production farms of salmon and trout, and leads to the use of large amounts of antibiotics in closed and open waters for therapy of fununculosis.",
"In order to develop efficient strategies to prevent A. salmonicida outbreaks, it is essential to know the main mechanisms of pathogenicity of A. salmonicida.",
"Several potential virulence factors of A. salmonicida have been described thus far.",
"They include the surface array-layer protein, the hemolysins ASH1, ASH3, ASH4, H-lysin, salmolysin, the serine protease AspA and the Glycerophospholipid:Cholesterol Acyltransferase (GCAT) complexed with lipopolysaccharide (LPS).",
"While there are many reports on potential virulence factors of A. salmonicida , in particular hemolysins, little is known about their activity and their role in pathogenesis.",
"Many of them seem not to play a primary role in pathogenesis, since deletion mutants of GCAT and aspA genes showed neither of them to be essential for acute A. salmonicida -induced furunculosis.",
"AspA however is essential for pro-GCAT processing in broth cultures and might also be involved in activation of other secreted enzymes or toxins.",
"Various attempts have been made to develop vaccines to prevent A. salmonicida infections mainly on the basis of killed cells (bacterins).",
"Current vaccines achieve some level of protection.",
"However, the nature of the antigens in efficient vaccines is not well defined.",
"Significant differences of protein patterns are seen in cultures of A. salmonicida grown in vivo by an intraperitoneal implant technique in rainbow trout compared to cultures grown in vitro in culture medium.",
"Such differences are thought to be the reasons of variable efficacy of former furunculosis vaccines due to a lack of appropriate antigens in certain vaccine preparations.",
"Several pathogenic bacteria use ADP-ribosylation as a key mechanism to modify properties of host cell proteins, thus to modulate their function and induce disease.",
"Hence ADP-ribosylation of eukaryotic regulatory proteins is the underlying pathogenic mechanism of a heterogeneous family of bacterial protein toxins.",
"ADP-ribosylating toxins are broadly distributed among highly pathogenic bacteria and are the primary cause of various severe human diseases such as diphtheria, cholera and pertussis.",
"Among them, the ADP-ribosyltransferase toxin called exoenzyme S (ExoS) of Pseudomonas aeruginosa is one of the most prominent representatives.",
"It is secreted via a type III-dependent secretion mechanism.",
"Type II secretion systems generally have the potential to recognize receptors on target cells, induce biosynthesis of the corresponding toxins, and finally inject these bacterial toxins directly into the host cells without secretion to the medium.",
"Recently, it was shown that ExoS is a bifunctional toxin containing an N-terminal part resembling the Yersiniae YopE toxin which catalyses rho-dependent actin depolymerisation, and a C-terminal ADP-ribosylating domain.",
"Unique to most bacterial toxins, the ADP-ribosylating toxin ExoS does not have a rigid target protein specificity and ribosylates a number of target proteins including IgG3, apolipoprotein A-I, vimentin and several members of the Ras superfamily.",
"Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS (factor activating exoenzyme S)-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.",
"FAS is a member of the 14-3-3 family of proteins that regulate the activity of several eukaryotic enzymes.",
"Prior to this invention, no analogues to ExoS have been found in bacteria other than P. aeruginosa."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>A protein toxin named Aeromonas salmonicida exoenzyme T (AexT), which belongs to the family of ADP-ribosylating toxins, was identified and characterized in Aeromonas salmonicida , the etiological agent of furunculosis in fish.",
"This discovery has enabled the development of diagnostic, preventative, and therapeutic techniques, including the preparation of traditional or recombinant vaccines based on AexT for inducing immunity against A. salmonicida infections, and including the identification and characterization by known methods of homologous toxins and promoters in other Aeromonas species or other bacterial genera.",
"Also identified and characterized was the Calcium- (or other cation concentration-) dependent promoter of A. salmonicida .",
"This novel promoter is useful for regulating the expression of proteins in recombinant expression systems.",
"In one embodiment, the invention comprises an isolated nucleic acid segment (SEQ ID NO:1) encoding a 50 kDa exoenzyme of A. salmonicida .",
"In another embodiment, the invention comprises a nucleic acid segment that encodes a protein having the amino acid sequence of SEQ ID NO:2, including variants that retain either biological activity or immunogenicity or both.",
"Due to the degeneracy of the genetic code and the possible presence of flanking nucleic acid fragments outside of the coding region, it will be understood that many different nucleic acid sequences may encode the amino acid sequence of SEQ ID NO:2 and variants, and that all such sequences would be encompassed within the present invention.",
"In a further embodiment, a method of producing, protecting, capturing, or preserving AexT toxin by growing A. salmonicida on Ca 2+ or other cations depleted medium is provided.",
"This provides a means of preparing a vaccine that is much more effective than currently available vaccines against A. salmonicida .",
"In another embodiment, the invention relates to the use of AexT as an immunogen and to the use of AexT in a recombinant or traditional vaccine to reduce the incidence of infection by A. salmonicida.",
"In another embodiment, the invention comprises an improved bacterin vaccine in which the production of AexT has been induced prior to inactivation of the A. salmonicida cells, or in which A. salmonicida has been manipulated (using recombinant or other means) to constitutively express AexT prior to inactivation.",
"In a further embodiment, the invention provides a means of diagnosing A. salmonicida , or other bacteria found to contain AexT homologues, by the detection of the AexT protein or the homologous proteins.",
"Also, the invention provides a toxin that in itself may have therapeutic activity in certain unrelated disease states, or for treatment of certain conditions in man or animals.",
"In a further embodiment, the invention comprises an isolated nucleic acid segment (SEQ ID NO:3) encoding the promoter sequence of the gene encoding the AexT protein.",
"This promoter is regulated by Calcium, and possibly by other cations as well as other undefined sensory signals, and is useful for regulating the expression of proteins in recombinant expression systems.",
"The gene aexT encoding the toxin AexT, was identified by broad range toxin gene probes.",
"It was cloned and characterized by sequence analysis.",
"The cloned gene was expressed, and purified AexT was obtained by recombinant gene technology in E. coli .",
"AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of Yersiniae sp.",
"Recombinant AexT possesses enzymatic ADP-ribosyltransferase activity.",
"Monospecific polyclonal antibodies directed against purified recombinant AexT cross-react with ExoS and ExoT of P. aeruginosa .",
"Secretion of AexT from freshly isolated strains of A. salmonicida requires medium depleted of free Ca 2+ ions (or other cations) or necessitates contact with fish cells, as demonstrated with cultivated rainbow trout gonad cells.",
"These cells undergo significant morphological changes upon infection through the toxic activity of AexT.",
"The dependence on fish cells or on Ca 2+ (or other cation) restricted conditions for the expression and secretion of the AexT protein toxin by A. salmonicida indicates that regulation of expression of the aexT gene and secretion of AexT is coupled to a type III secretion system.",
"The induction of AexT biosynthesis in A. salmonicida is regulated through contact with target cells via a sensory process similar to that found in Yersiniae sp.",
"as indicated by the orfX gene flanking the aexT gene.",
"The orfX shows high similarity to the gene for specific Yop chaperon E (sycE) of Yersiniae .",
"SycE serves as a secretion signal and is part of the type III secretion pathway for secretion of YopE.",
"Cultures of the A. salmonicida type strain ATCC 33658 T , which were passaged in vitro for a large number of generations, and which seem to have lost virulence, do not produce significant amounts of AexT and do not affect rainbow trout gonad cells morphologically upon infection in spite of the presence of the aexT gene, in contrast to a fresh field isolate of A. salmonicida .",
"The ADP-ribosylating toxin AexT is a determinative virulence factor of A. salmonicida and is expected to provide new insights in basic mechanisms of virulence of this pathogen, and potentially a protective antigen for vaccination against furunculosis.",
"Sequence Listing The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids.",
"Only one strand of each nucleotide acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.",
"Aeromonas salmonicida gene for aexT complete coding DNA sequence.",
"ATGCAGA TTCAAGCAAA CACCGTCGGC Seq ID 1 ACACAGGCCG TCGCTCACCA CAGTGATGCC ACGACCGGAG TTGGCCGGAT GGGTCAGATG GAGGCGCGTC AGGTCGCCAC CGGACAAGAT GCGATCCTGC TGGGCAGTCG CAGCGAACCG CAAAAAGGGC AGGGGCTGCT CTCGCGACTG GGGGCCCAGC TGGCCCGCCC GTTCGTGGCC ATCAAAGAGT GGATCAGCAA CCTGCTGGGG ACGGACAAGC GTGCCGCTGC GCCGAAGGCG CAAACCGCCG TTTCCCCCGA GGATCTTCAG CGACTGATGA AGCAGGCTGC ATTTGGTAGC TCGCTGGGTG GCTTCGCCAA GGCGGACGTG TTGAACAACA TCACAGGCGA ACAATTGGGC AAGGATCACG CCAGTCTGGC GACCGGCAAT GGCCCCCTGC GCTCTCTCTG CACCGCGTTG CAGGCCGTTG TCATAGGATC TCAGCAACCG CAACTCCGGG AGTTGGCTAC CGGGCTGCTG GCCCGCCCCA TCGCCGGTAT CCCGCTCCAG CAGTGGGGGT CGGTAGGCGG CAAGGTGACC GAGCTGCTCA CCAGCGCCCC CCCCGAACTG TTGAAGGAGG CTATGAGCCA GCTACACACC GCGATGGGTG AAGTTGCCGA CCTGCAGCGC GCTGTAAAGG CAGAAGTTGC TGGCGAACCG GCGCGAAGCG CGACCACAGC GGCCGCTGTG GCGCCGCTCC AAAGCGGTGA GAGCGAAGTT AACGTTGAGC CTGCCGACAA GGCGCTGGCA GAGGGCTTGC AGGAGCAATT CGGCCTGGAG GCCGAGCAAT ATCTGGGTGA ACAGCCCCAC GGTACTTACA GCGATGCTGA AGTGATGGCG CTTGGGCTCT ACACCAACGG CGAATACCAG CACCTGAATC GCTCGCTGCG TCAGGAAAAG CAGCTGGATG CAGGGCAAGC GTTGATCGAT CAGGGTATGT CCACCGCTTT TGAGAAAAGT ACCCCCACCG AGCAGTTGAT CAAGACCTTC CGCGGTACCC ACGGCGGCGA TGCGTTCAAC GAGGTGGCAG AGGGGCAAGT CGGTCATGAT GTCGCTTATC TTTCCACCTC TCGGGATCCC AAGGTGGCAA CCAACTTTGG TGGTTCAGGC TCCATATCCA CGATATTTGG CCGCTCGGGG ATCGATGTCA GCGACATATC CGTTGAAGGT GACGAGCAGG AGATCCTCTA TAACAAAGAG ACTGATATGC GGGTATTGCT CAGTGCCAAA GATGAACGGG GCGTCACCCG GCGGGTACTG GAAGAGGCCT CGCTGGGGGA ACAAAGCGGC CACAGCAAGG GGCTGCTGGA CGGGCTGGAT CTGGCAAGAG GAGCGGGCGG TGCCGACAAG CCGCAAGAGC AAGATATCCG TCTGAAGATG CGCGGGCTCG ATTTGGCGTG A Aeromonas salmonicida protein sequence for the AexT protein 1 MQIQANTVGT QAVAHHSDAT TGVGRMGQME ARQVATGQDA ILLGSRSEPQ Seq ID 2 51 KGQGLLSRLG AQLARPFVAI KEWISNLLGT DKRAAAPKAQ TAVSPEDLQR 101 LMKQAAFGSS LGGFAKADVL NNITGEQLGK DHASLATGNG PLRSLCTALQ 151 AVVIGSQQPQ LRELATGLLA RPIAGIPLQQ WGSVGGKVTE LLTSAPPELL 201 KEAMSQLHTA MGEVADLQRA VKAEVAGEPA RSATTAAAVA PLQSGESEVN 251 VEPADKALAE GLQEQFGLEA EQYLGEQPHG TYSDAEVMAL GLYTNGEYQH 301 LNRSLRQEKQ LDAGQALIDQ GMSTAFEKST PTEQLIKTFR GTHGGDAFNE 351 VAEGQVGHDV AYLSTSRDPK VATNFGGSGS ISTIFGRSGI DVSDISVEGD 401 EQEILYNKET DMRVLLSAKD ERGVTRRVLE EASLGEQSGH SKGLLDGLDL 451 ARGAGGADKP QEQDIRLKMR GLDLA* Aeromonas salmonicida promoter sequence for the AexT gene TGATG GCTCCAGATT GATGATGGCG Seq ID 3 CCATTAGAGC AGGTCGCCGC CAGCGGCACT GTTAATGGTG GCTCTCATTT TTTAGCTTTT CGGTCAGCAG GATGGCGCGC CGCGCTCAGT ACAAAAATCG CGACCAATCC CGATAGTCCC TGTTGATACC CTCTCCTAGA CTGGCGGCGA AACATCACAA GAAGACAATC ATC Aeromonas salmonicida protein sequence for the OrfX protein 1 MNSLYHAAIH QLFLSLSLPQ PQQEESVTSL QIGELTCHLT EHPADYLLMF Seq ID 4 51 TRLEVASGAQ AAAQNLFSQD PCKPVLGFDP DDLTPVLWSR QPLQQLDRAQ 101 IHHQVEQLVS AADELSRW* Aeromonas salmonicida gene for orfX complete coding DNA sequence TT ACCACCTGCT TAGCTCGTCA GCGGCAGAGA Seq ID 5 CCAGTTGCTC CAGCTGGTGA TGGATCTGGG CGCGATCCAG CTGCTGCAAC GGCTGGCGAC TCCACAACAC CGGCGTCAGA TCGTCGGGGT CAAAACCCAG AACGGGTTTG CAAGGGTCCT GACTAAACAG GTTTTGCGCG GCGGCCTGGG CGCCGCTAGC TACCTCAAGA CGGGTAAACA TCAGCAGATA GTCGGCTGGG TGCTCGGTCA GGTGGCAGGT CAGTTCGCCG ATCTGCAGGC TGGTGACGCT TTCCTCCTGC TGCGGCTGAG GAAGCGAGAG GGAGAGAAAC AGCTGGTGGA TGGCGGCGTG ATAAAGAGAG TTCAT"
],
[
"FIELD OF THE INVENTION This invention relates to bacterial toxins and to bacterial promoters, and in particular to a newly identified and characterized toxin and promoter of Aeromonas salmonicida.",
"The invention also encompasses methods for the production or accumulation of the A. salmonicida toxin, as well as the diagnostic, therapeutic, and preventative use (including in particular the preparation of traditional or recombinant protein or DNA vaccines) of the A. salmonicida toxin.",
"Also encompassed are methods for improving A. salmonicida bacterin vaccines by controlling expression of the toxin.",
"BACKGROUND OF THE INVENTION The fish disease furunculosis derived its name from the characteristic lesions observed as furuncles formed on the surface of fish as a result of infection with Aeromonas salmonicida.",
"This pathogen causes most severe losses in production farms of salmon and trout, and leads to the use of large amounts of antibiotics in closed and open waters for therapy of fununculosis.",
"In order to develop efficient strategies to prevent A. salmonicida outbreaks, it is essential to know the main mechanisms of pathogenicity of A. salmonicida.",
"Several potential virulence factors of A. salmonicida have been described thus far.",
"They include the surface array-layer protein, the hemolysins ASH1, ASH3, ASH4, H-lysin, salmolysin, the serine protease AspA and the Glycerophospholipid:Cholesterol Acyltransferase (GCAT) complexed with lipopolysaccharide (LPS).",
"While there are many reports on potential virulence factors of A. salmonicida, in particular hemolysins, little is known about their activity and their role in pathogenesis.",
"Many of them seem not to play a primary role in pathogenesis, since deletion mutants of GCAT and aspA genes showed neither of them to be essential for acute A. salmonicida-induced furunculosis.",
"AspA however is essential for pro-GCAT processing in broth cultures and might also be involved in activation of other secreted enzymes or toxins.",
"Various attempts have been made to develop vaccines to prevent A. salmonicida infections mainly on the basis of killed cells (bacterins).",
"Current vaccines achieve some level of protection.",
"However, the nature of the antigens in efficient vaccines is not well defined.",
"Significant differences of protein patterns are seen in cultures of A. salmonicida grown in vivo by an intraperitoneal implant technique in rainbow trout compared to cultures grown in vitro in culture medium.",
"Such differences are thought to be the reasons of variable efficacy of former furunculosis vaccines due to a lack of appropriate antigens in certain vaccine preparations.",
"Several pathogenic bacteria use ADP-ribosylation as a key mechanism to modify properties of host cell proteins, thus to modulate their function and induce disease.",
"Hence ADP-ribosylation of eukaryotic regulatory proteins is the underlying pathogenic mechanism of a heterogeneous family of bacterial protein toxins.",
"ADP-ribosylating toxins are broadly distributed among highly pathogenic bacteria and are the primary cause of various severe human diseases such as diphtheria, cholera and pertussis.",
"Among them, the ADP-ribosyltransferase toxin called exoenzyme S (ExoS) of Pseudomonas aeruginosa is one of the most prominent representatives.",
"It is secreted via a type III-dependent secretion mechanism.",
"Type II secretion systems generally have the potential to recognize receptors on target cells, induce biosynthesis of the corresponding toxins, and finally inject these bacterial toxins directly into the host cells without secretion to the medium.",
"Recently, it was shown that ExoS is a bifunctional toxin containing an N-terminal part resembling the Yersiniae YopE toxin which catalyses rho-dependent actin depolymerisation, and a C-terminal ADP-ribosylating domain.",
"Unique to most bacterial toxins, the ADP-ribosylating toxin ExoS does not have a rigid target protein specificity and ribosylates a number of target proteins including IgG3, apolipoprotein A-I, vimentin and several members of the Ras superfamily.",
"Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS (factor activating exoenzyme S)-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.",
"FAS is a member of the 14-3-3 family of proteins that regulate the activity of several eukaryotic enzymes.",
"Prior to this invention, no analogues to ExoS have been found in bacteria other than P. aeruginosa.",
"SUMMARY OF THE INVENTION A protein toxin named Aeromonas salmonicida exoenzyme T (AexT), which belongs to the family of ADP-ribosylating toxins, was identified and characterized in Aeromonas salmonicida, the etiological agent of furunculosis in fish.",
"This discovery has enabled the development of diagnostic, preventative, and therapeutic techniques, including the preparation of traditional or recombinant vaccines based on AexT for inducing immunity against A. salmonicida infections, and including the identification and characterization by known methods of homologous toxins and promoters in other Aeromonas species or other bacterial genera.",
"Also identified and characterized was the Calcium- (or other cation concentration-) dependent promoter of A. salmonicida.",
"This novel promoter is useful for regulating the expression of proteins in recombinant expression systems.",
"In one embodiment, the invention comprises an isolated nucleic acid segment (SEQ ID NO:1) encoding a 50 kDa exoenzyme of A. salmonicida.",
"In another embodiment, the invention comprises a nucleic acid segment that encodes a protein having the amino acid sequence of SEQ ID NO:2, including variants that retain either biological activity or immunogenicity or both.",
"Due to the degeneracy of the genetic code and the possible presence of flanking nucleic acid fragments outside of the coding region, it will be understood that many different nucleic acid sequences may encode the amino acid sequence of SEQ ID NO:2 and variants, and that all such sequences would be encompassed within the present invention.",
"In a further embodiment, a method of producing, protecting, capturing, or preserving AexT toxin by growing A. salmonicida on Ca2+ or other cations depleted medium is provided.",
"This provides a means of preparing a vaccine that is much more effective than currently available vaccines against A. salmonicida.",
"In another embodiment, the invention relates to the use of AexT as an immunogen and to the use of AexT in a recombinant or traditional vaccine to reduce the incidence of infection by A. salmonicida.",
"In another embodiment, the invention comprises an improved bacterin vaccine in which the production of AexT has been induced prior to inactivation of the A. salmonicida cells, or in which A. salmonicida has been manipulated (using recombinant or other means) to constitutively express AexT prior to inactivation.",
"In a further embodiment, the invention provides a means of diagnosing A. salmonicida, or other bacteria found to contain AexT homologues, by the detection of the AexT protein or the homologous proteins.",
"Also, the invention provides a toxin that in itself may have therapeutic activity in certain unrelated disease states, or for treatment of certain conditions in man or animals.",
"In a further embodiment, the invention comprises an isolated nucleic acid segment (SEQ ID NO:3) encoding the promoter sequence of the gene encoding the AexT protein.",
"This promoter is regulated by Calcium, and possibly by other cations as well as other undefined sensory signals, and is useful for regulating the expression of proteins in recombinant expression systems.",
"The gene aexT encoding the toxin AexT, was identified by broad range toxin gene probes.",
"It was cloned and characterized by sequence analysis.",
"The cloned gene was expressed, and purified AexT was obtained by recombinant gene technology in E. coli.",
"AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of Yersiniae sp.",
"Recombinant AexT possesses enzymatic ADP-ribosyltransferase activity.",
"Monospecific polyclonal antibodies directed against purified recombinant AexT cross-react with ExoS and ExoT of P. aeruginosa.",
"Secretion of AexT from freshly isolated strains of A. salmonicida requires medium depleted of free Ca2+ ions (or other cations) or necessitates contact with fish cells, as demonstrated with cultivated rainbow trout gonad cells.",
"These cells undergo significant morphological changes upon infection through the toxic activity of AexT.",
"The dependence on fish cells or on Ca2+ (or other cation) restricted conditions for the expression and secretion of the AexT protein toxin by A. salmonicida indicates that regulation of expression of the aexT gene and secretion of AexT is coupled to a type III secretion system.",
"The induction of AexT biosynthesis in A. salmonicida is regulated through contact with target cells via a sensory process similar to that found in Yersiniae sp.",
"as indicated by the orfX gene flanking the aexT gene.",
"The orfX shows high similarity to the gene for specific Yop chaperon E (sycE) of Yersiniae.",
"SycE serves as a secretion signal and is part of the type III secretion pathway for secretion of YopE.",
"Cultures of the A. salmonicida type strain ATCC 33658T, which were passaged in vitro for a large number of generations, and which seem to have lost virulence, do not produce significant amounts of AexT and do not affect rainbow trout gonad cells morphologically upon infection in spite of the presence of the aexT gene, in contrast to a fresh field isolate of A. salmonicida.",
"The ADP-ribosylating toxin AexT is a determinative virulence factor of A. salmonicida and is expected to provide new insights in basic mechanisms of virulence of this pathogen, and potentially a protective antigen for vaccination against furunculosis.",
"Sequence Listing The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids.",
"Only one strand of each nucleotide acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.",
"Aeromonas salmonicida gene for aexT complete coding DNA sequence.",
"ATGCAGA TTCAAGCAAA CACCGTCGGC Seq ID 1 ACACAGGCCG TCGCTCACCA CAGTGATGCC ACGACCGGAG TTGGCCGGAT GGGTCAGATG GAGGCGCGTC AGGTCGCCAC CGGACAAGAT GCGATCCTGC TGGGCAGTCG CAGCGAACCG CAAAAAGGGC AGGGGCTGCT CTCGCGACTG GGGGCCCAGC TGGCCCGCCC GTTCGTGGCC ATCAAAGAGT GGATCAGCAA CCTGCTGGGG ACGGACAAGC GTGCCGCTGC GCCGAAGGCG CAAACCGCCG TTTCCCCCGA GGATCTTCAG CGACTGATGA AGCAGGCTGC ATTTGGTAGC TCGCTGGGTG GCTTCGCCAA GGCGGACGTG TTGAACAACA TCACAGGCGA ACAATTGGGC AAGGATCACG CCAGTCTGGC GACCGGCAAT GGCCCCCTGC GCTCTCTCTG CACCGCGTTG CAGGCCGTTG TCATAGGATC TCAGCAACCG CAACTCCGGG AGTTGGCTAC CGGGCTGCTG GCCCGCCCCA TCGCCGGTAT CCCGCTCCAG CAGTGGGGGT CGGTAGGCGG CAAGGTGACC GAGCTGCTCA CCAGCGCCCC CCCCGAACTG TTGAAGGAGG CTATGAGCCA GCTACACACC GCGATGGGTG AAGTTGCCGA CCTGCAGCGC GCTGTAAAGG CAGAAGTTGC TGGCGAACCG GCGCGAAGCG CGACCACAGC GGCCGCTGTG GCGCCGCTCC AAAGCGGTGA GAGCGAAGTT AACGTTGAGC CTGCCGACAA GGCGCTGGCA GAGGGCTTGC AGGAGCAATT CGGCCTGGAG GCCGAGCAAT ATCTGGGTGA ACAGCCCCAC GGTACTTACA GCGATGCTGA AGTGATGGCG CTTGGGCTCT ACACCAACGG CGAATACCAG CACCTGAATC GCTCGCTGCG TCAGGAAAAG CAGCTGGATG CAGGGCAAGC GTTGATCGAT CAGGGTATGT CCACCGCTTT TGAGAAAAGT ACCCCCACCG AGCAGTTGAT CAAGACCTTC CGCGGTACCC ACGGCGGCGA TGCGTTCAAC GAGGTGGCAG AGGGGCAAGT CGGTCATGAT GTCGCTTATC TTTCCACCTC TCGGGATCCC AAGGTGGCAA CCAACTTTGG TGGTTCAGGC TCCATATCCA CGATATTTGG CCGCTCGGGG ATCGATGTCA GCGACATATC CGTTGAAGGT GACGAGCAGG AGATCCTCTA TAACAAAGAG ACTGATATGC GGGTATTGCT CAGTGCCAAA GATGAACGGG GCGTCACCCG GCGGGTACTG GAAGAGGCCT CGCTGGGGGA ACAAAGCGGC CACAGCAAGG GGCTGCTGGA CGGGCTGGAT CTGGCAAGAG GAGCGGGCGG TGCCGACAAG CCGCAAGAGC AAGATATCCG TCTGAAGATG CGCGGGCTCG ATTTGGCGTG A Aeromonas salmonicida protein sequence for the AexT protein 1 MQIQANTVGT QAVAHHSDAT TGVGRMGQME ARQVATGQDA ILLGSRSEPQ Seq ID 2 51 KGQGLLSRLG AQLARPFVAI KEWISNLLGT DKRAAAPKAQ TAVSPEDLQR 101 LMKQAAFGSS LGGFAKADVL NNITGEQLGK DHASLATGNG PLRSLCTALQ 151 AVVIGSQQPQ LRELATGLLA RPIAGIPLQQ WGSVGGKVTE LLTSAPPELL 201 KEAMSQLHTA MGEVADLQRA VKAEVAGEPA RSATTAAAVA PLQSGESEVN 251 VEPADKALAE GLQEQFGLEA EQYLGEQPHG TYSDAEVMAL GLYTNGEYQH 301 LNRSLRQEKQ LDAGQALIDQ GMSTAFEKST PTEQLIKTFR GTHGGDAFNE 351 VAEGQVGHDV AYLSTSRDPK VATNFGGSGS ISTIFGRSGI DVSDISVEGD 401 EQEILYNKET DMRVLLSAKD ERGVTRRVLE EASLGEQSGH SKGLLDGLDL 451 ARGAGGADKP QEQDIRLKMR GLDLA* Aeromonas salmonicida promoter sequence for the AexT gene TGATG GCTCCAGATT GATGATGGCG Seq ID 3 CCATTAGAGC AGGTCGCCGC CAGCGGCACT GTTAATGGTG GCTCTCATTT TTTAGCTTTT CGGTCAGCAG GATGGCGCGC CGCGCTCAGT ACAAAAATCG CGACCAATCC CGATAGTCCC TGTTGATACC CTCTCCTAGA CTGGCGGCGA AACATCACAA GAAGACAATC ATC Aeromonas salmonicida protein sequence for the OrfX protein 1 MNSLYHAAIH QLFLSLSLPQ PQQEESVTSL QIGELTCHLT EHPADYLLMF Seq ID 4 51 TRLEVASGAQ AAAQNLFSQD PCKPVLGFDP DDLTPVLWSR QPLQQLDRAQ 101 IHHQVEQLVS AADELSRW* Aeromonas salmonicida gene for orfX complete coding DNA sequence TT ACCACCTGCT TAGCTCGTCA GCGGCAGAGA Seq ID 5 CCAGTTGCTC CAGCTGGTGA TGGATCTGGG CGCGATCCAG CTGCTGCAAC GGCTGGCGAC TCCACAACAC CGGCGTCAGA TCGTCGGGGT CAAAACCCAG AACGGGTTTG CAAGGGTCCT GACTAAACAG GTTTTGCGCG GCGGCCTGGG CGCCGCTAGC TACCTCAAGA CGGGTAAACA TCAGCAGATA GTCGGCTGGG TGCTCGGTCA GGTGGCAGGT CAGTTCGCCG ATCTGCAGGC TGGTGACGCT TTCCTCCTGC TGCGGCTGAG GAAGCGAGAG GGAGAGAAAC AGCTGGTGGA TGGCGGCGTG ATAAAGAGAG TTCAT BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1.Genetic map of the genes encoding AexT and the ORFX of A. salmonicida in alignment with the corresponding genes of P. aeruginosa and Y. pestis.",
"Maps were constructed from EMBL/GenBank Accession no L27629 for P. aeruginosa ExoS, L46800 for P. aeruginosa ExoT and from AF053946 for Y. pestis.",
"Due to the high homology found within the virulence plasmids of Y. pestis, Y. pseudotuberculosis and Y. enterecolitica, AF053946 also represents these Yersiniae sp.",
"Boxes with arrowheads indicate ORFs.",
"Numbers indicate corresponding amino acid positions.",
"The putative biglutamic acid active site is dashed and the alignment with other ADP-ribosylating toxins is shown at the bottom.",
"Black boxes indicate the transcription activator (ExsA) binding site and black triangles indicate consensus sequences for the transcription promoter, containing −10 and −35 -boxes.",
"Abbreviations used: AS, A. salmonicida; PA, P. aeruginosa; YP, Y. pestis; CP, Clostridium perfringens; EC, E. coli; VC, V. cholera; AexT, Aeromonas exoenzyme T; ExoS, exoenzyme S; ExoT, exoenzyme T; SycE, specific Yop chaperone E; YopE, Yersinia outer protein E; IOTA, IOTA toxin; LT, heat labile toxin; CT, cholera toxin.",
"Scale on top is given in kb.",
"FIG.",
"2.Immunoblot reacted with rabbit serum raised against nitrilotriacetic acid-purified recombinant AexT-His (Lane 5).",
"Equal amounts of supernatants derived from A. salmonicida strains were mixed with SDS-loading buffer and loaded onto a 10% SDS-polyacrylamide gel.",
"A. salmonicida strains JF2267 (Lane 1 and 3) and ATCC 33658 (Lane 2 and 4) were grown in standard media (Lane 1 and 2) or in Calcium depleted media containing 10 mM NTA (Lane 3 and 4).",
"The molecular masses of the prestained broad range protein markers (BioLabs, Std.)",
"are indicated in kDa to the left.",
"FIG.",
"3.Infection of fish cells with A. salmonicida.",
"RTG-2 cells were exposed to 100 μl PBS containing no cells (A), A. salmonicida ATCC 33658 (B) and A. salmonicida JF2267 (C).",
"Bacteria in panel C seem to be attached to cells and cell debris whereas bacteria in panel B are equally distributed over the whole surface and were observed to be floating in the medium.",
"Pictures were taken after 24 hrs of infection under a phase contrast microscope.",
"DETAILED DESCRIPTION OF THE INVENTION I. Definitions: Epitope: An epitope refers to an immunologically active region of an immunogen (most often a protein, but sometimes also a polysaccharide or lipid or other molecule) that binds to specific membrane receptors for antigen on lymphocytes or to secreted antibodies.",
"To generate an immune response to a foreign antigen, lymphocytes and antibodies recognize these specific regions (epitopes) of the antigen rather than the entire molecule.",
"B cell epitope: The region of an immunogen which is recognized by B cells when it binds to their membrane bound antibody.",
"The B cells which recognize that particular region then proliferate and secrete antibody molecules which are specific for that region of the immunogen.",
"B cell epitopes tend to be highly accessible regions on the exposed surface of the immunogen.",
"Stimulation of the immune system by B cell epitopes results in “humoral” immunity.",
"T cell epitope: The region (epitope) of an immunogen which is recognized by a receptor on T cells after being processed and presented on the surface of an antigen presenting cell (APC) in the context of a major histocompatability complex (MHC) class I or II molecule.",
"T cells can be split into two distinct groups, T helper cells (Th) and T cytotoxic cells (Tc).",
"T helper cells recognize epitopes bound to MHC class II molecules whereas T cytotoxic cells recognize epitopes bound to MHC class I molecules.",
"T helper cells can be further subdivided into two classes, Th1 and Th2, Th1 being responsible for stimulation of cell-mediated immunity and Th2 cells stimulating the humoral arm of the immune system.",
"When a given T cell recognizes the epitope-MHC complex at the surface of the APC it becomes stimulated and proliferates, leading to the production of a large number of T cells with receptors specific for the stimulating epitope.",
"Stimulation of the immune system by T cell epitopes normally results in “cell-mediated” immunity.",
"Attenuated Bacterial Vaccine: This refers to bacterial strains which have lost their pathogenicity while retaining their capacity for transient growth within an inoculated host.",
"Because of their capacity for transient growth, such vaccines provide prolonged immune-system exposure to the individual epitopes on the attenuated organisms, resulting in increased immunogenicity and memory-cell production, which sometimes eliminates the need for repeated booster injections.",
"The ability of many attenuated vaccines to replicate within host cells makes them very suitable to induce a cell-mediated immunity.",
"Typically, bacterial strains are made attenuated by introducing multiple defined gene mutations into the chromosome thereby impairing growth in vivo.",
"Recombinant Vector Vaccine: This refers to the introduction of genes (or pieces of genes) encoding major antigens (or epitopes) from especially virulent pathogens into attenuated viruses or bacteria.",
"The attenuated organism serves as a vector, replicating within the host and expressing the gene product of the pathogen.",
"Traditional Vaccine: A traditional vaccine is a preparation yielding protection from disease based on: an inactivated whole pathogen; an attenuated live pathogen; a closely related organism (live or dead) sharing protective epitopes; a toxin; a chemically modified or heated toxin; or a purified or partially purified protein from the pathogen or a closely related organism.",
"Sequence Identity: Identity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of identical residues shared between the sequences.",
"Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are.",
"Sequence Similarity: Similarity between two amino acid sequences is expressed in terms of the level of sequence conservation, including shared identical residues and those residues which differ but which share a similar size, polarity, charge or hydrophobicity.",
"Sequence similarity is typically expressed in terms of percentage similarity; the higher the percentage, the more similar the two sequences are.",
"Recombinant: A recombinant nucleic acid is one that has a sequence that is not normally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence.",
"This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.",
"Oligonucleotide (oligo): A linear polymer sequence of up to approximately 100 nucleotide bases in length.",
"Probes and primers: Nucleic acid probes and primers may readily be prepared based on the amino acid and DNA sequence provided by this invention.",
"A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.",
"Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.",
"Methods for labelling and guidance in the choice of labels appropriate for various purposes are well known in the art.",
"Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length.",
"Primers may be annealed to a complementary target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme.",
"Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.",
"Methods for preparing and using probes and primers are well known in the art.",
"PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as DNAStar Lasergene software.",
"One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length.",
"Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides.",
"Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.",
"Isolated: An “isolated” biological component (such as nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.",
"Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods.",
"The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.",
"An “isolated” bacterial strain or colony is purified away from other colonies and yields a pure culture without any contaminants upon plating on selective media.",
"Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.",
"A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.",
"A vector may also include one or more selectable marker genes and other genetic elements known in the art.",
"A “temperature-sensitive” vector is one which replicates normally at a low growth temperature (i.e., 28 C) and will not replicate at a higher growth temperature (i.e., 42 C) due to mutations at or near the origin of replication.",
"An “imperfectly segregating” vector is one which is not stably inherited by new daughter cells at the time of cell division in the absence of selection pressure due to mutations within the vector sequence.",
"Host Cell: Refers to those cells capable of growth in culture and capable of expressing AexT protein and/or AexT fusion protein.",
"The host cells of the present invention encompass cells in in vitro culture and include prokaryotic and eukaryotic cells, including insect cells.",
"A host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.",
"Expression from certain promoters can be elevated in the presence of certain inducers (i.e.",
"temperature, small inducer molecules such as Beta-galactosides for controlling expression of T7 or lac promoters or variants thereof).",
"The preferred host cell for the cloning and expression of the AexT protein and AexT fusion protein is a prokaryotic cell.",
"An example of a prokaryotic cell useful for cloning and expression of the AexT protein of the present invention is E. coli BL21 (DE3).",
"Cell Culture: Refers to the growth of eukaryotic (non-bacterial) cells in a complex culture medium generally consisting of vitamins, buffers, salts, animal serum, and other nutrients.",
"Fusion Partner: Any DNA sequence cloned in frame to the 5′ or 3′ end of an ORF that results in transcription and translation of amino acid sequence added to the N- or C-terminus of the original protein.",
"Fusion Protein: The term fusion protein used herein refers to the joining together of at least two proteins, an AexT protein and a second protein.",
"In some embodiments of the present invention, the second protein may be fused or joined to a third protein.",
"In the present invention, examples of second proteins include any polypeptide that facilitates the following: expression, secretion, purification, condensation, precipitation, or any property which facilitates concentration or purification.",
"Promoter: A sequence of DNA to which the enzyme RNA polymerase binds (directly or through an intermediary protein, RNA polymerase binding protein) before initiation of transcription of the DNA into RNA.",
"A promoter allows expression of a protein from the DNA coding sequence.",
"Variant: Any molecule in which the amino acid sequence, glycosylation, phosphorylation, and/or lipidation pattern, or any other feature of a naturally occurring molecule which has been modified covalently or non-covalently and is intended to include mutants.",
"Some of the variants falling within this invention possess amino acid substitutions, deletions, and/or insertions provided that the final construct possesses the desired ability of AexT.",
"Amino acid substitutions in AexT may be made on a basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.",
"Also included within the definition of variant are those proteins having additional amino acids at one or more of the C-terminal, N-terminal, and within the naturally occurring AexT sequence as long as the variant protein retains biological activity or the capability to act as an antigen and hence as a vaccine.",
"Original Residue Conservative Substitutions Ala ser Arg lys Asn gln; his Asp glu Gln asn Glu asp Gly pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu Table 1: More substantial changes in functional or other features may be obtained by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.",
"The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.",
"Variant peptides having one or more of these more substantial changes may also be employed in the invention, provided that biological activity or immunogenicity of AexT is retained.",
"More extensive amino acid changes may also be engineered into variant AexT peptides.",
"These variant peptides will typically be characterized by possession of at least 40% sequence identity counted over the full length alignment with the amino acid sequence of their respective naturally occurring sequences using the alignment programs described below.",
"In addition, these variant peptides will retain either biological activity or immunogenicity or both.",
"Confirmation that an AexT peptide has biological activity may be achieved using the assay system described herein.",
"Confirmation that an AexT peptide has immunogenic effect may be achieved by studies showing protection.",
"Following confirmation that the AexT peptide has the desired activity or immunogenic effect, a nucleic acid molecule encoding the AexT peptide may be readily produced using standard molecular biology techniques.",
"Where appropriate, the selection of the open reading frame will take into account codon usage bias of the bacterial, plant or other eukaryotic species in which the AexT peptide is to be expressed.",
"Inclusion body: Intracellularly confined, insoluble, protein-containing particles of bacterial cells comprised of either homologous or heterologous proteins.",
"These particles are the reservoirs and consequence of overproduction of bacterial recombinant proteins.",
"Inclusion bodies can be purified or semi-purified and used directly as protein antigens or can be solubilized by various procedures and used as soluble protein antigen preparations.",
"Alignment programs: Methods for aligning sequences for comparison purposes are well known in the art.",
"Various programs and alignment algorithms are described in Smith and Waterman (1981), Needleman and Wunsch (1970), Pearson and Lipman (1988), Higgins and Sharp (1988, 1989), Corpet et al.",
"(1988), Huang et al.",
"(1992), Pearson et al.",
"(1994).",
"Altschul et al.",
"(1990) presents a detailed consideration of sequence alignment methods.",
"The National Centre of Biotechnology Information (NCBI) Basic Local Alignment Search Tool BLAST; Altschul et al, 1990) is available from several sources, including the NCBI (Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN, TBLASTX.",
"BLAST can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/.",
"A description of how to determine sequence identity using this program is available at http://www.ncbi.nln.nih.gov/BLAST/blast_help.html.",
"For comparisons of amino acid sequences of greater than 30 amino acids, the “BLAST 2 sequences” function in the BLAST program is employed using the BLASTP program with the default BLOSUM62 matrix set to default parameters, (open gap 11, extension gap 1 penalties).",
"When aligning short peptides (fewer than 30 amino acids), the alignment should be performed sing the “Blast 2 Sequences” function employing the BLASTP program with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).",
"Proteins having even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.",
"II.",
"Selection and Creation of Nucleic Acid Sequences Encoding the 50 kD AexT Protein, and use of AexT in recombinant and bacterin vaccines: a.",
"Growth and Purification of Aeromonas sp.",
"and Plasmid Cloning Vectors.",
"Aeromonas strains (Table 1) were routinely cultured on blood agar plates (Trypticase soy agar supplemented with 0.1% CaCl2 and 5% sheep blood) at 37° C. except for A. salmonicida which were grown at 20° C. A. salmonicida strain JF2267 was freshly isolated from an arctic char (Salvelinus alpinus) with typical furunculosis symptoms.",
"The strain was identified as A. salmonicida by a routine diagnostic agglutination test using rabbit anti-Aeromonas salmonicida specific antiserum and by sequence analysis of the rrs (16S rRNA) genes (EMBL/GenBank Accession No.",
"AF200329) as described by Kuhnert et al.",
"Escherichia coli strains XL1-blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB laclqZ M15 Tn10 (Tetr)]c), and BL21l(DE3) (F'dcm ompT hsdS(rB−mB−) gal (DE3)) were used for cloning and expression of cloned genes respectively.",
"Plasmid pBluescriptIISK-(Stratagene, La Jolla, Calif.) was used as cloning vector.",
"Plasmid pETHIS-1 is a T7 promoter based expression vector and allows addition of poly-histidine tails at the N-terminal or at both N- and C-terminal ends of proteins (nucleotide sequence accession no.",
"AF012911).",
"E. coli strains were grown at 37° C. in Luria-Bertani broth (LB) supplemented when necessary with ampicillin (50 μg/ml) for selection and maintenance of recombinant plasmids.",
"For blue-white selection with pBluescriptIISK-125 μM X-Gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) and 1 mM IPTG (isopropyl-D-thiogalacto-pyranoside) were added.",
"P. aeruginosa ATCC 27853 was grown for 8 hrs on an LB-plate at 20° C. In order to induce ExoS and ExoT secretion cells were then incubated 18 hrs at 20° C. in 20 ml TSB (2.75 g/100 ml Trypticase-Soy broth without dextrose, 1% Glycerol, 0.1 M L-Glutamic acid, pH=7.3) supplemented with 10 mM NTA (Nitrilo-triacetic acid (Titriplex I) pH7.3) for chelation of Ca2+ ions.",
"Subsequently 5 mM PMSF were added and the culture was centrifuged for 15 min at 4′000 rpm.",
"This protein fraction was used for further analyses on Western blots and for activity assays.",
"A. salmonicida ATCC 33658T was cultured at 20° C. in various media consisting of TSB supplemented with either 10 mM CaCl2, or 0.01 to 1 mM EDTA, or 1 mM EDTA+1 mM PMSF, or 10 mM NTA, or 100 mM NTA, or 0.1 mM FeIII-Cl3, or 0.1 mM FeII-Cl2, or 0.1 mM EDDA (Ethylendiamine di(o-hydroxy-phinyl-acetic-acid)), or 20 mM Na-oxalate.",
"A temperature shift experiment was performed by cultivating A. salmonicida ATCC 33658T in TSB at 20° C. to an OD600 of 0.1 followed by further incubation over night at 32° C. Additionally various other A. salmonicida strains were cultured using the same conditions as described for P. aeruginosa.",
"Culture supernatants and total cell protein extracts were analysed on western blots.",
"b. PCR, Cloning and Preparation of Gene Probes for ADP-Ribosylating Toxins.",
"PCR were carried out with a DNA thermal cycler (GeneAmp 9600; PE Biosystems, Norwalk, Conn.) in 50 μl reaction mixes containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.25 μM forward and reverse primers, 0.5 units Taq polymerase, and 5 ng template DNA.",
"The DNAs were amplified for 35 cycles with 30 s denaturation at 94° C., 30 s annealing at corresponding temperatures (Table 2), and 1 min extension at 72° C. For fragments above 1 kb, the extension time was extended by 1 min per kb.",
"When DNA fragments were produced by PCR for subsequent cloning and expression, the expand long template PCR kit (Roche Molecular Biochemicals, Rotkreuz, Switzerland) containing polymerase with proof-reading capacity was used instead of Taq polymerase.",
"In addition, an extension step of 7 min at 72° C. was added at the end of the last cycle in order to ensure full length synthesis of the different fragments.",
"For the production of DIG-labelled probes PCR mixtures were supplemented with 40 μM digoxigenin-11-dUTP (Roche Molecular Biochemicals).",
"DNA fragment (called REXOS) corresponding to the catalytic portion of the P. aeruginosa exoS gene was amplified with the primer-pair REXOS-L and REXOS-R (Table 2) both containing EcoRI restriction site linkers.",
"When genomic DNA of P. aeruginosa ATCC 27853 was used as template for PCR, 10% dimethyl sulfoxide were added.",
"PCR-fragments were purified with the QIAquick PCR purification kit (QIAGEN, Basel, Switzerland).",
"Plasmid pBluescriptIISK- and purified PCR fragments were digested with EcoRI and ligated for 2 hrs at room temperature before transformation of E. coli K-12 strain XL1-blue.",
"After blue-white screening a white colony was selected and the prepared plasmid was sequenced in order to exclude cloning artefacts.",
"To get pure, plasmid-contaminant free probes, the cloned exoS derived fragment (REXOS) was excised with EcoRI, purified twice over agarose gels with the Jet-Sorb kit (Genomed GmbH, Bad Oeynhausen, Germany), and then used as template for PCR with the primers REXOS-L and REXOS-R (Table 2) for production of the DIG-labelled probe REXOS.",
"A DNA fragment (called RASEXOS) corresponding to the putative catalytic portion of A. salmonicida aexT gene was amplified with the primer-pair RASEXOS-L and RASEXOS-R (Table 2) and DIG-labelled.",
"Genomic DNA derived from A. salmonicida ATCC 33658T (type strain) served as template.",
"All cloning procedures were essentially performed using standard protocols.",
"DNA was extracted by the method of Pitcher et al.",
"and manipulated using conventional methods.",
"The CaCl2 method was used for preparation of competent cells.",
"Sequencing reactions were performed with a Taq Dye Deoxy Terminator cycle sequencing kit (PE Biosystems) and reaction products were analysed on an ABI Prism 310 genetic analyser (PE Biosystems).",
"Amplification of a DNA fragment containing the intergenic orfX-aexT region with the putative promoter region of aexT was performed by PCR using the primer pair BASEXOS693 and BASEXOS-250 (Table 2), genomic DNA of either the A. salmonicida ATCC 22658T or JF2267 strain as template and the Pwo DNA Polymerase (Roche Molecular Biochemicals).",
"The intergenic region of the two genes orfX and aexT was subsequently sequenced with primer BASEXOS-250.c.",
"Construction of A. salmonicida Lambda Phage—Gen library.",
"Genomic DNA (0.1 μg) from A. salmonicida ATCC 33658T was digested partially with the restriction enzyme Sau3a to get fragments in the 3 to 4 kb range which were ligated to ZapExpress digested with BamHI (Pharmacia LKB, Biotechnology AB, Uppsala, Sweden) and packed into Lambda.",
"A fresh culture of 200 μl of E. coli XL1-blue MRF' (Pharmacia LYB) was resuspended in 10 mM MgSO4 at an OD600 of 1.0 and infected with the packed phages during 20 min at 37° C. Five ml Top Agarose (LB-broth supplemented with 0.7% Agarose) supplemented with IPTG and X-Gal were added, gently mixed and immediately poured onto an LB-Agar plate.",
"Plates were incubated over night at 37° C. and plaques were lifted on nylon filters and screened using DIG-labelled probes.",
"Positive plaques were cut out and stored over night at 4° C. in 0.5 ml SM-buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris pH7.5 and 0.01% gelatine) containing 20 μl chloroform.",
"The in vivo excision of plasmids from selected phagemid plagues was done according the instructions of the ZAP express kit (Pharmacia LKB).",
"Colonies with plasmids containing cloned fragments were isolated and mini-preps (Quiagen) were performed for plasmid purification.",
"d. Sequence Data Analyses.",
"Sequence alignment and editing were done with the software Sequencher (Gene Codes Corporation, Ann Arbor, Mich.).",
"Sequence comparisons were done as described by Altschul et al.",
"and sequences were aligned with the Wiscosin Package (Genetics Computer Group, Inc. [GCG], Madison, Wis.).",
"The theoretical isoelectric pH (pI) and molecular masses of protein were calculated with the GCG software.",
"e. Southern Blot Analyses.",
"Southern blotting was done by alkaline transfer onto positively charged nylon membranes (Roche Molecular Biochemicals) with an LKB 2016 VacuGene vacuum blotting pump (Pharmacia LKB).",
"Gels were depurinated for 10 min in 0.25 M HCl, and subsequent transfer was performed with 0.4 M NaOH.",
"After blotting, filters were baked for 30 min at 80° C. under vacuum.",
"After at least 1 h of prehybridisation, hybridisation was carried out in 5×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% blocking reagent (Roche Molecular Biochemicals)-0.1% N-lauroylsarcosine sodium salt-0.02% sodium dodecyl sulphate (SDS) at 68° C. over night, using DIG-labelled DNA fragments as probe.",
"Filters were washed under nonstringent conditions twice for 5 min each with 50 ml of 2×SSC-0.1% SDS per 100 cm2 at room temperature (20° C.), followed by medium-stringency washing twice for 15 min each with 50 ml of 0.2×SSC-0.1% SDS per 100 cm2 at 20° C. The filters were then processed with phosphatase-labelled anti-DIG antibody according to the producer's protocol.",
"Signals were produced with chemiluminescent substrate (CDP-Star; Roche Molecular Biochemicals) or with the chromogene substrate NBT-BCIP.",
"Luminescence was detected on X-ray films.",
"f. Expression of His-Tailed Fusion Proteins.",
"E. coli BL21 (DE3) cells harboring recombinant pETHIS-1 plasmids with cloned genes were inoculated in 50 ml of LB-ampicillin at 37° C. to an OD600 of 0.3 and induced by addition of 0.2 mM IPTG (final conc.)",
"and subsequent growth for 3 hrs.",
"The cells were sedimented by centrifugation at 4'000 rpm, resuspended in 5 ml of buffer pH7.9 containing 10 mM Tris-HCl, 1 M Urea, 250 mM NaCl, 2.5 mM Imidazole, 3 M Guanidium HCl, 0.2 mM PMSF and sonicated with a microtip for 20 min at 50% and 1-s interval in a Branson Sonifier 250 (Branson Ulatrasonics, Danbury, Conn.).",
"This sonicated fraction was directly loaded onto a prewashed 1.25-ml-bed-volume Ni-NTA column (Quiagen) and washed once more with 5 ml binding buffer (2 M Urea, 20 mM Tris, 500 mM NaCl, 5 mM Imidazole, 60 mM Guanidium HCl pH7,9).",
"addition of the poly-histidine proteins was performed with a 40-ml binding buffer-to-elution buffer (2 M Urea, 20 mM Tris, 500 mM NaCl, 500 mM Imidazole, 60 mM Guanidium HCl, pH7.9) gradient with a flow rate of 0.25 ml/min and collection of fractions of 1 ml with a HiLoad system (Pharmacia LKB).",
"The fractions were analysed on SDS-10% acrylamide gels.",
"Those containing the purified fusion protein were pooled and dialysed over night against 5 liters of 0.85% NaCl.",
"g. Immunisation of Rabbits with Purified Proteins.",
"Purified and dialysed recombinant protein solution (100 μg/ml) was mixed 1:1 with complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.) and 2 ml of the emulsion were then injected subcutaneously to a rabbit.",
"The rabbit was booster immunised with the same amount of protein emulsified with Freund's incomplete adjuvant 21 days later.",
"On day 45 after the first immunisation, the rabbit was bled, and blood serum was prepared and stored at −20° C. h. Infection of Fish Cell Cultures with A. salmonicida.",
"Rainbow trout (Oncorhynchus mykiss) gonad cells (RTG-2, ATCC CCL-55) were grown in 75 cm2 tissue culture flasks (Techno plastic products AG, Trasadingen, Switzerland) at 22° C. in minimum essential medium (GibcoBRL Life Technologies, Basel, Switzerland) supplemented with 2 mM L-glutamine (GibcoBRL), 1× non essential amino acids (GibcoBRL), 3 g/l sodium bicarbonate and 10% foetal bovine serum.",
"Three days before infection the cells were trypsinised and 4 million cells were seeded into a 25 cm2 tissue culture flask.",
"Monolayered RTG-2 cells were infected at a multiplicity of infection of 10:1 (bacteria:fish cells) with cells of A. salmonicida cultures resuspended in phosphate buffered saline (PBS) pH7.4.As control 100 μl of pure PBS pH7.4 were added.",
"After 24 hrs of infection at 15° C. the fish cells were photographed under a green filtered phase contrast microscope (Axiovert 100, Zeiss, Jena, Germany).",
"Detachment of the cells from the flask was obtained by shaking them by hand.",
"The suspended cells were centrifuged for 5 min at 4'000 rpm.",
"Lysis of the fish cells was performed in 100 μl distilled water with two subsequent freeze thawing steps and verified by microscopy.",
"The lysed fish cells were used for further analyses on Western blots and for activity assays.",
"i. SDS-PAGE and Immunoblot Analyses.",
"Proteins were separated by SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli and transferred to a nitrocellulose membrane (Bio-Rad laboratories, Hercules, Calif).",
"For immunoblotting, Western blots were blocked with 1% milk buffer for 30 min and then incubated with the rabbit antiserum (1:1500) or with sera (1:100) derived from diseased fish in milk buffer overnight at 4° C. After a thorough wash with water, the appropriate phosphatase-labelled conjugate (Goat anti-Rabbit IgG (H+L) [cat.",
"no.",
"075-15061 or Goat anti-Trout Immunoglobulin [cat.",
"no.",
"05-29-05], Kirkegaard & Perry, Gaithersburg, Md.)",
"diluted 1:2000 or 1:500 respectively in milk buffer was added, and the reaction was visualised 90 min later by incubation with BCIP-NBT as the substrate.",
"j. ADP-Ribosyltransferase Assays.",
"ADP-ribosyltransferase assays contained 100 μM 14C-NAD (specific activity: 6 Ci/mol) and 0.2 M NaAc pH6 in a total of 20 μl.",
"Either 0.1 mM soy bean trypsin inhibitor (SBTI, Roche Molecular Biochemicals) and 50 μg ml1 wheat germ extract (Promega Corporation, Madison, Wis.) as source of FAS or 4 μl (approximately 200,000 cells) of non infected RTG2 fish cells were added as substrate.",
"The reaction was started by adding 4 μl aliquots of supernatants of either P. aeruginosa ATCC 27853, A. salmonicida ATCC 33658 or A. salmonicida JF2267.An aliquot of pure growth medium was used for background determination.",
"The reaction was performed at 20° C. for 1 hr and stopped by addition of 500 μl 10% trichloro acetic acid (TCA).",
"The mixtures were blotted onto filters (GS 0.22 μm, Millipore, Bedford, Mass.)",
"using a vacuum pump and washed 5 times with 0.75 ml 10% TCA.",
"The filters were air dried and scintillation liquid Emulsifier scintillator plus, Packard instrument company, Meriden, Conn.) was added.",
"Scintillation was detected as counts per minute (CPM) on a liquid scintillation counter (Wallac 1410, Pharmacia, Dübendorf, Switzerland).",
"Experiments were performed in triplicate and scintillation was counted three times per experiment.",
"Background counts were subtracted and results with their standard deviations are given in CPM (Table 3).",
"Due to high background ADP-ribosyltransferase activity of the fish cells the activity of AexT in infected fish cells could not be measured.",
"EXAMPLES The following examples are included to demonstrate preferred embodiments of the invention, and it will be appreciated by those skilled in the art, in light of this disclosure, that many changes can be made in the specific embodiments disclosed without departing from the scope of the invention.",
"1.Cloning and sequence analyses of aexT and its promoter.",
"Analyses of different Aeromonas sp.",
"with broad-range ADP-ribosylating toxin probes revealed a signal for a potential ADP-ribosyltransferase gene for A. salmonicida with probe REXOS which is derived from the catalytic domain of ExoS.",
"This probe was used to screen a Lambda Phage gene library of A. salmonicida ATCC 33658T.",
"Three positive overlapping clones were found and joined together to a continuous DNA fragment of 2260 bp in length.",
"The derived DNA sequence of this fragment revealed a complete ORF of 1428 bp showing high similarity with ExoT of P. aeruginosa.",
"In analogy to ExoT, it was called Aeromonas exoenzyme T (AexT) and its corresponding gene aexT.",
"The cloned fragment contains a further open reading frame, named ORFX which shows similarity to the sycE gene of Yersinia sp.",
"and to ORF1 which precedes exoS of P. aeruginosa (FIG.",
"1).",
"ORFX is preceded by a RBS and followed by a putative rho-independent transcription termination site.",
"The sequenced DNA fragment encoding AexT and ORFX showed a high G+C content of 60%, which is above the average G+C content of A. salmonicida of 55%.",
"The ORF representing aexT contains an ATG initiation codon and TGA stop codon.",
"The 87 bp preceding the ATG show 71% identical nucleotide positions to the sequence preceding exoS and exoT in P. aeruginosa.",
"The putative ribosomal binding site (RBS), AGAAG, is positioned 10 bp upstream of the ATG.",
"The putative promoter sequences −10 box (TAGACT) and the canonical −35 box (CCGATA) of aexT are located at the same positions as those for exoS and exoT.",
"Upstream of the promoter −10 and −35 box sequences there is a consensus binding site (TACAAAAA) similar to the one found upstream of exoS and exoT which is known in P. aeruginosa to be bound by the transcriptional regulator ExsA.",
"An inverted repeat (AACGGACACCCtcGGGTGTCCGTT) is located 25 bp downstream of the stop codon of the aexT gene.",
"It has the same stem sequence (CGGACAC) as inverted repeats of the putative transcription termination sites of exoS and exoT.",
"2.Structural analyses of the AexT.",
"The amino acid sequence for AexT was deduced from the nucleotide sequences using the universal genetic code.",
"AexT has a calculated pI of 5.13 and a molecular mass of 50.1 kDa Blast searches revealed similarity of AexT with ExoT and ExoS over the whole length.",
"In addition similarity with the YopE cytotoxin of Yersinia pseudotuberculosis (EMBL/GenBank Accession No.",
"P08008), Y. pestis (Acc.",
"No.",
"P31493) and Y. enterocolitica (Acc.",
"No.",
"M34280) was found within the N-terminal 210 amino acids of AexT (FIG.",
"1).",
"Gap comparisons with the amino acid sequence of AexT with ExoT and ExoS revealed AexT to be identical in 62.8% with ExoT (57.9% with ExoS) and similar in 67.5% with ExoT (62.8% with ExoS) of the positions (FIG.",
"1) with a gap of 25 aa in length, which separates the N-terminal from the C-terminal domain.",
"Gap comparisons of ExoT with ExoS showed them to be identical in 75.1% and similar in 77.7% of the positions.",
"The N-terminal domain of AexT revealed 33.5% identical and 37.4% and similar amino acid positions compared to the cytotoxin YopE of Y. pseudotuberculosis and 26.8% identical and 32.8% similar amino acids to YopE of Y. pestis (FIG.",
"1).",
"The biglutamic acid active site (GDEQEILYNK) found for various ADP-ribosylating toxins is also conserved within the C-terminal domain of AexT (FIG.",
"1).",
"3.Specificity of aexT genes for A. salmonicida.",
"Southern blot analyses of genomic DNA of various Aeromonas sp.",
"(Table 1) with DIG labelled probe for aexT(RASEXOS) revealed a single copy of aexT with a size estimated to be approximately 3 kb for all A. salmonicida strains tested (Table 1).",
"None of the other analyzed Aeromonas strains showed hybridisation signals with the aexT probe under these hybridisation conditions, butt this does not rule out the possibility that other strains of Aeromonas or other bacterial genera may possess homologues of aexT.",
"4.Production and characterisation of recombinant AexT.",
"In order to characterize biochemically the AexT protein and to produce polyclonal, monospecific antibodies directed against AexT, we have expressed poly-histidine tailed AexT, named AexT-His, in recombinant E. coli K-12 strains.",
"The entire coding part inclusive the stop codon of the aexT gene was amplified by PCR using primers BASEXOSH8L and BASEXOSH8R and genomic DNA of A. salmonicida as template.",
"The purified PCR product was digested with restriction enzymes EcoRI and SpeI and cloned into EcoRI and SpeI digested vector pETIHS-1 to obtain plasmid pJFFASAexT-His, encoding N-terminally poly-histidine tailed AexT (AexT-His) under the control of the T7 promoter.",
"For the expression of the aexT-His gene, plasmid pJFFASAexT-His was transformed into E. coli strain BL21(DE 3) as described in Materials and Methods.",
"Biochemical analysis of purified and renatured recombinant AexT-His revealed that it possessed ADP-ribosylating activity (Table 3).",
"Monospecific polyclonal antibodies against AexT were obtained by immunisation of a rabbit with purified AexT-His protein as described for other poly-histidine tailed proteins.",
"Anti-AexT antibodies reacted on immunoblots with purified AexT-His.",
"It also cross reacted with the 49 kDa and 53 kDa proteins in supernatant from a culture of P. aeruginosa ATCC 27853, representing the ExoS and ExoT protein toxins as expected from sequence similarities with AexT.",
"5.Expression of AexT in A. salmonicida.",
"The expression of AexT by A. salmonicida type strain ATCC 33658T, which seems to have lost its pathogenicity, and of A. salmonicida field strain JF2267, which was freshly isolated from a diseased arctic char and which still possesses its virulence, was assessed by immunoblots with anti AexT-His antibodies.",
"Neither supernatants nor the cell pellets of the type strain ATCC 3365T grown under various conditions showed any specific reactions on immunoblots with monospecific, polyclonal anti-AexT antibodies.",
"In contrast supernatants and cell pellets of A. salmonicida strain JF2267 grown in TSB supplemented with 10 mM NTA reacted on immunoblots with anti-AexT-His antibodies (FIG.",
"2).",
"When NTA was omitted in the growth media, AexT protein in strain A. salmonicida JF2267 only was found in the cell pellet but not in supernatants.",
"When A. salmonicida strain JF2267 was analysed during infection of RTG-2 fish cells, a specific reaction on immunoblots using anti-AexT-His antibodies with a 56 kDa protein corresponding to AexT was found.",
"This indicates that strain JF2267 required contact with fish cells or depletion of Ca2+ ions (or other cations) to induce the production or protection of AexT.",
"However, no AexT could be detected for A. salmonicida type strain ATCC 33658 under the same conditions (FIG.",
"2).",
"ADP-ribosyltransferase activity was determined in culture supernatants of A. salmonicida strains and as control of P. aeruginosa, grown under Ca2+ depleted conditions.",
"A. salmonicida field strain JF2267 showed ADP-ribosyltransferase values slightly above background and no activity could be measured in A. salmonicida ATCC 33658T, while P. aeruginosa showed high activity (Table 3).",
"ADP-ribosyltransferase could not be determined in infected fish cells due to high background activity.",
"Infection of RTG-2 cells with freshly cultured A. salmonicida strain JF2267 caused a toxic effect showing characteristic cell rounding, detachment and lysis of cells within 24 hours (FIG.",
"3C) whereas the cells infected with A. salmonicida type strain ATCC 33658T (FIG.",
"3B) or the control cells incubated with pure PBS (FIG.",
"3A) showed no morphological changes at all.",
"Infection of fish cell culture with A. salmonicida JF2267 also induced the production of the AexT protein which reacted with anti-AexT-His antiserum.",
"Similar morphological changes have been reported for cells infected with ExoS producing P. aeruginosa.",
"The sera raised against AexT-His also showed cross-reactivity with ExoS and ExoT produced by P. aeruginosa.",
"Similar cross-reactivity was found for anti-exoenzyme S IgG which reacted with both ExoS and ExoT.",
"The fact that AexT is produced specifically in contact with fish cells (FIG.",
"3C) or in Ca2+ depleted medium (FIG.",
"2), which is believed to act as pseudo-trigger to induce aexT, suggests the protein to be produced specifically during infection and hence to play an important role in pathogenicity.",
"Interestingly, A. salmonicida type strain ATCC 33658T does not affect the morphology of RTG-2 cells.",
"It seems to have lost the ability of producing cell contact induced AexT probably due to repeated passages on growth medium.",
"In order to determine whether the significant differences in AexT production and toxic effect between A. salmonicida isolate JF2267 and type strain ATCC 33658 could be due to mutations within the putative promoter regions of their respective aexT genes, the intergenic regions between orfX and aexT were sequenced and found to be identical.",
"Thus, the alteration responsible for the loss of AexT production in the type strain seems to reside outside the aexT operon.",
"Nevertheless, both A. salmonicida strains ATCC 33658T and JF2267 have the same haemolytic activity as estimated on blood agar plates implying that the toxic effect for RTG-2 cells is not due to the A. salmonicida haemolysins but rather to production of AexT in strain JF2267.The loss of expression of aexT as observed in A. salmonicida type strain ATCC 33658 is a frequent event in this species, and may explain the currently observed variations in virulence and also differences in efficacy of protection of whole cell antigen vaccines.",
"6.Recombinant AexT Vaccine Trial (See Appendix A) 7.Testing of different A. salmonicida bacterin vaccines in Altantic salmon (Salmo salar) (See Appendix B) The current data indicate that AexT of A. salmonicida is a determinative virulence factor of this pathogen.",
"While particular elements, embodiments and applications of the present invention have been shown and described, it will be understood, of course, that the invention is not limited thereto, since modifications may be made by those skilled in the applicable technologies, particularly in light of the foregoing description The appended claims include within their ambit such modifications and variants of the exemplary embodiments of the invention described herein as would be apparent to those skilled in the applicable technologies.",
"TABLE 1 Aeromonas strains used aexT positiveb/ Species Straina strains tested A. salmonicida ATCC 33658T 1/1 A. salmonicida JF2267c 1/1 A. salmonicida field isolates 10/10 A. bestiarum CDC 9533-76 0/1 A. bestiarum field isolates 0/2 A. caviae ATCC 15468 0/1 A. caviae field isolates 0/3 A. encheleia DSM 11577 0/1 A. eucrenophila NCMB 74 0/1 A. eucrenophila field isolate 0/1 A. hydrophila ATCC 7966 0/1 A .",
"hydrophila field isolates 0/15 A. jandaei ATCC 49568 0/1 A. media ATCC 33907 0/1 A. schubertii ATCC 43700 0/1 A. schubertii field isolate 0/1 A. sobria CIP 7433 0/1 A. trota ATCC 49657 0/1 A. trota field isolate 0/1 A. veronii ATCC 35624 0/1 A. veronii field isolates 0/4 aATCC, American Type Culture Collection, Rockville, Md.",
"; JF, Joachim Frey, University of Berne, Switzerland; CDC, Center for Disease Control, Atlanta, Georgia; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; NCMB, National Collection of Marine Bacteria, Aberdeen, Scotland; CIP, Collection of the Institut Pasteur, Paris, France.",
"bDetermined by Southern blotting using DIG labelled RASEXOS as probe.",
"cJF2267 was isolated freshly from an arctic char with typical symptoms of furunculosis.",
"Identification was done phenotypically and by 16s rDNA gene sequencing.",
"TABLE 2 Oligonucleotide primers Annealing temp Name Sequencea 5′ to 3′ Position ° C. EXOS-L cgcgaattcACTGGCTGGGCAAACTG 1128-1144b 52 EXOS-R cgcgaattCCCGCTGACATCGATTC 2034-2019b 52 RASEXOS-L GGCGCTTGGGCTCTACAC 1537-1554c 60 RASEXOS-R GAGCCCGCGCATCTTCAG 2089-2072c 60 BASEXOSH8L cgcgaattCGGCGAAACATCACAAGA 645-662c 59 BASEXOSH8R ggactagTCCCGCCAGCATAAAAAAC 2165-2147c 59 BASEXOS693 AGGCTCAACGTTAACTTCGC 1432-1413 58 BASEXOS-250 AGAGGGAGAGAAACAGCTGG 427-446 58 aLowercase letters indicate nucleotides added to create restriction enzyme recognition sites (underlined) for cloning.",
"bBased on nucleotide sequence L27629 of P. aeruginosa cBased on nucleotide sequence of A. salmonicida TABLE 3 Determination of ADP-ribosyltransferase activity cpm A) std.",
"dev.C) A. salmonicida ATCC 33658T, culture supernatant 0 ±10 JF2267, culture supernatant 5 ±12 AexT-His 123 ±11 P. aeruginosa ATCC 27853 culture supernatant (ExoS + ExoT) 4286B) ±125 A) Mean values corrected for background.",
"Experiments were performed in triple and scintillation was measured three times per experiment.",
"B)The proportion of ADP-ribosyltransferase activity of ExoT is estimated to be approximately 0.2% corresponding to 8 cpm under these conditions.",
"C)Standard deviation in cpm Appendix A Recombinant AexT Vaccine Trial Materials: VACCINE FORMULATIONS: 1.The AexT vaccine was formulated using recombinant, Histidine-tagged AexT resuspended in 10 mM phosphate buffer, pH 7.0, to 200 μg/mL.",
"Four parts of this protein solution were mixed with one part oil adjuvant for a final AexT concentration of 150 μg/mL.",
"The dose for testing was 0.1 mL, or 15 μg/fish.",
"2.The commercial comparator vaccine was serial 4-13 of the vaccine MultiVacc4 (Bayotek International Ltd.) 3.The placebo (control) vaccine consisted of phosphate buffered saline(PBS) (10 mM phosphate, 150 mM NaCl, pH 7.2).",
"4.All vaccines were maintained at 4° C. until use.",
"Methods TRIAL DESIGN: Fish (rainbow trout Oncorhynchus mykiss) that have been determined to be pathogen free and are at least 15 g in size are held for at least one-week pre vaccination for acclimation purposes.",
"During the acclimation period the fish are offered 1% body weight in salmonid fish food every day, however they are denied food 24 hours pre and post-vaccination.",
"At least 50 fish are vaccinated 0.1 mL of AexT vaccine via intra-peritoneal (IP) injection, or 0.2 mL of the commercial vaccine MultiVacc4.At the same time a group of at least 50 fish from the same stock are mock vaccinated with 0.1 mL of PBS.",
"Vaccinated fish are then held for a period of at least 350-degree days to allow specific immune response generation in an acclimation tank with a continuous flow of water at a temperature of 12-13° C. The fish are offered 1% body weight in salmonid fish food daily until 24 hours pre-challenge and post-challenge.",
"After at least 350-degree days post vaccination 50 fish per group were challenged by IP injection with a pre-determined concentration of virulent Aeromonas salmonicida.",
"The dosage depends on the source of the fish and the water temperature (this is determined empirically immediately prior to challenge of test fish).",
"The identical procedure is performed with the placebo vaccinated control fish.",
"The fish are observed daily for mortality for 21 days post challenge and the cause of mortality assessed and examined to ensure that mortality is attributed to the challenge organism.",
"After 24 hours post-challenge the fish are again offered 1% body weight in salmonid fish feed daily.",
"Tanks are maintained with a continuous flow of water at a temperature of 12-13° C. For a challenge series to be considered satisfactory; all challenge groups must meet the following criteria: 1.At least 70% of the non-immunized controls must die within 21 days of challenge.",
"2.A relative percent survival (RPS) of no less than 25% must be achieved for the challenge disease before a vaccine is considered even partially efficacious for this disease.",
"RPS=[1-(% mortality vaccinates/% mortality controls)]×100 Specificity of Immunity was Confirmed by Challenge with Vibrio anguillarum Developed from: The Rules Governing Medicinal Products in the European Union, Volume VII, Guidelines for the testing of veterinary medicinal products.",
"1994.Specific Requirements for the Production and Control of Live and Inactivated Vaccines Intended for Fish.",
"Section 3.2.Potency.",
"RESULTS Group % Mortality RPS PBS 82 — AexT 37 55 MultiVacc4 30 63 1.There was a strong challenge with 82% control mortalities.",
"Vibrio anguillarum immersion challenge shows that the AexT protects specifically against A. salmonicida (93% mortality in AexT vaccinates compared to 15% for commercial vibrio vaccine vaccinates).",
"Challenged survivors of the A sal challenge (and salines) with Vibdio anguillarum type 1.The challenge organism used was Vibrio anguillarum serotype 01 at an O.D.",
"of 0.5 (˜8.0×10E8 CFU/mL).",
"This indicates that the immune response is specific.",
"Appendix B Testing of different Aeromonas salmonicida bacterin vaccines in Atlantic salmon (Salmo salar) Purpose: Determination of the efficacy of Aeromonas salmonicida bacterins produced by different methods in Atlantic salmon (Salmo salar), and a correlation between protection and AexT production.",
"Materials: Methods: A. salmonicida Vaccine Preparations: 1.Bacterin Preparations: A standard A. salmonicida vaccine master working seed from Microtek International (1998) Ltd., MSW26, was used for all vaccine preparations.",
"The starter culture for each fermentation was derived from 25 mL of Tryptic Soy Broth inoculated with a single I mL frozen (−80° C.) aliquot of MSW26 followed by incubation with shaking (18° C. at 100 rpm) for 36 hours.",
"This primary starter culture was used to inoculate a 10 L fermenter a. Bacterin 1: Bacterin 1 was prepared by fermented culture incubated at 20° C. ±2° C., with sufficient agitation and aeration to maintain the dissolved oxygen (DO2) at above approximately 25%.",
"The pH is maintained between 6.5 and 7.5 (pH controlled by the addition of a NH4OH solution and aqueous KOH).",
"The media is Tryptic soy broth with glucose at 1%.",
"During fermentation a concentrated sterile solution of glucose is fed into the fermenter.",
"Glucose is fed to maintain a glucose concentration of between 1.0 g/l to 10.0 g/l.",
"Concentrated, sterile solutions of TSB without glucose are also fed at 1-2% of the culture volume into the fermenter.",
"The TSB without glucose is fed at OD650nm of between 2 and 3, again at OD650nm of between 4 and 5.The fermenter culture is fed periodically with a concentrated sterile solution of glucose to maintain a glucose concentration of between 1.0 g/l to 10.0 g/l until the OD650nm reaches approximately 8.0.The culture is fed with a concentrated solution of TSB without glucose to an OD650nm of approximately 10-12.The pH is maintained between 6.5 and 7.5 (pH controlled by the automatic addition of KOH and H2SO4).",
"The A. salmonicida culture is inactivated by the addition of 7.0 mL/l±0.7 mL/l formalin.",
"Aeration to the fermenter is stopped.",
"The inactivated culture is agitated in the fermenter for a period of 1 hour at 20° C.±2° C. The inactivated bacterial culture will then be pumped or gravity fed directly from the fermenter into holding vessels and stirred for a further 24 hour inactivation step.",
"The Inactivated bacterial culture was then held at 4° C. for further processing and formulation.",
"b. Bacterin 2 and 3: Bacterins 2 and 3 were prepared as duplicates by fermented culture incubated at 20° C.±2° C., with sufficient agitation and aeration to maintain the dissolved oxygen (DO2) at approximately 15-25%.",
"The pH is maintained between 6.9 and 7.1 (pH controlled by the addition of a NH4OH solution and H2SO4).",
"The media is Tryptone (1.5%), Yeast extract (0.5%), Glycerol (1%), NaCl (0.5%), Glutamate (100 mM), and Citrate (20 mM).",
"During fermentation a concentrated sterile solution of Tryptone (15%), Yeast extract (5%), NaCl (0.5%), Glycerol (10%), Glutamate (100 mM), and Citrate (20 mM) is fed into the fermenter.",
"The fermenter culture is fed continuously with this concentrated sterile solution to maintain a stable pO2.The resulting A. salmonicida culture is inactivated by the addition of 7.0 mL/l±0.7 mL/I formalin.",
"Aeration to the fermenter is stopped.",
"The inactivated culture is agitated in the fermenter for a period of 1 hour at 20° C.±2° C. The inactivated bacterial culture will then be pumped or gravity fed directly from the fermenter into holding vessels and stirred for a further 24 hour inactivation step.",
"The inactivated bacterial culture was then held at 4° C. for further processing and formulation.",
"2.Vaccine Formulations: Vaccines were formulated using washed cells (performed by centrifugation at 1500×g) resuspended in 10 mM phosphate buffer, pH 7.0, to 10 O.D.650.Each test vaccine based on the bacterins 1 through 3 were formulated by mixing 1 part adjuvant with 5 parts washed bacterin cells and 4 parts 10 mM phosphate pH 7.All vaccines were maintained at 4° C. until use.",
"3.Western Immunoblotting: Prior to electrophoresis bacterin samples containing equivalent cellular mass were solublized by mixing with equal amounts of sample loading buffer followed by boiling for 5 minutes.",
"The prepared samples were then separated on a 12% polyacrylamide gel by the discontinuous gel method of Laemmli (1970).",
"Two identical gels were prepared.",
"One gel was stained for total protein with Coomassie Blue and dried on cellulose film.",
"The proteins from the second gel were electrophoretically transferred to nitrocellulose membrane as described by Towbin et al.",
"(1979).",
"Following transfer, the membrane was blocked with phosphate-buffered saline (pH 7.2) and 0.1% Tween-20 (PBS-Tween) with 2% BSA.",
"The blot was probed with polyclonal rabbit anti-AexT at a 1:500 dilution in PBS-Tween+1% BSA for 2 h and washed with PBS-Tween.",
"Alkaline phosphatase-conjugated polyclonal goat anti-rabbit antibody (Caltag) was applied to the membrane at a 1:2500 dilution in TBBT for 2 h, washed In TBBT.",
"The transblot was then developed using NBT and BCIP as a colour reagent system at pH 9.4.A.",
"sal{dot over (m)}onicida Immunity: Fish (rainbow trout Oncorhynchus mykiss and/or Atlantic.",
"salmon Salmo salar) that have been determined to be pathogen free and are at least 15 g in size are held for at least one-week pre vaccination for acclimation purposes.",
"During the acclimation period the fish are offered 1% body weight in salmonid fish food every day, however they are denied food 24 hours pre and post-vaccination.",
"At least 50 fish are vaccinated 0.2 cc of vaccine via intra-peritoneal (IP) injection, at the same time a group of at least 50 fish from the same stock are mock vaccinated with 0.2 cc of saline.",
"Vaccinated fish are then held for a period of at least 350-degree days to allow specific immune response generation in an acclimation tank with a continuous flow of water at a temperature of 12-13° C. The fish are offered 1% body weight in salmonid fish food daily until 24 hours pre-challenge and post-challenge.",
"After at least 350-degree days post vaccination 50 fish are challenged by immersion in, or IP injection with, a pre-determined concentration of virulent Aeromonas salmonicida.",
"The dosage depends on the source of the fish and the water temperature (this is determined empirically immediately prior to challenge of test fish).",
"The identical procedure is performed with the mock-vaccinated control fish.",
"The fish are observed daily for mortality for 21 days post challenge and the cause of mortality assessed and examined to ensure that mortality is attributed to the challenge organism.",
"After 24 hours post-challenge the fish are again offered 1% body weight in salmonid fish feed daily.",
"Tanks are maintained with a continuous flow of water at a temperature of 12-13° C. For a challenge series to be considered satisfactory; all challenge groups must meet the following criteria: 1.At least 70% of the non-immunized controls must die within 21 days of challenge.",
"2.A relative percent survival (RPS) of no less than 25% must be achieved for the challenge disease before a vaccine is considered even partially efficacious for this disease.",
"RPS=[1-(% mortality vaccinates/% mortality controls)]×100 A. sal Vaccinate Challenge 50 RBT of approx.",
"12 g were vaccinated with 2 vaccine candidates.",
"Saline controls were additionally vaccinated.",
"350 degree days post-vaccination, all groups were challenged with A. salmonicida Challenge Culture Information OD650 nm = 0.201 = 1.0E+08 cfu/mL Used 0.1 mL of 3.3E+05 cfu/mL (washed cells) per fish A. sal: washed in 0.85% saline, final titre of 3.3E+05 cfu/mL Number of RBT remaining TANKS C1 C2 B1 B2 Date Days Saline Bacterin 1 no AexT Bacterin 2 + AexT Bacterin 3 + AexT 26-Apr-01 0 50 50 45 50 27-Apr-01 1 50 50 45 50 28-Apr-01 2 50 50 45 50 29-Apr-01 3 28 48 45 49 30-Apr-01 4 9 36 45 46 01-May-01 5 8 31 45 45 02-May-01 6 4 26 44 44 03-May-01 7 4 26 43 43 04-May-01 8 4 26 42 42 05-May-01 9 4 21 41 41 06-May-01 10 4 21 41 41 07-May-01 11 3 21 41 41 08-May-01 12 2 21 41 41 09-May-01 13 2 21 41 41 10-May-01 14 2 21 41 41 11-May-01 15 2 21 41 41 12-May-01 16 2 21 41 41 13-May-01 17 2 21 41 41 14-May-01 18 2 21 41 41 15-May-01 19 2 21 41 41 16-May-01 20 2 21 41 41 17-May-01 21 2 21 41 41 Gross % survival 4.00 46.00 91.11 82.00 Gross % mortality 96.00 54.00 8.89 18.00 Gross RPS 0 44 91 81"
]
] | Patent_10416981 |
[
[
"Rapid curing anaerobic compositions",
"A curable composition having enhanced cure speed which includes a curable component, a cure system and an additive for enhancing cure speed.",
"The additive includes an alkali metal halide salt and the reaction product of: (a) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (b) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene glycol ether units terminated at one end with an ether group and at the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (c) a monohydroxy compound, a diisocyanate and a polyamine.",
"Alternatively, in place of or in combination with the cure speed enhancing additive, an aromatic substituted (meth)acryl functionalized component may be incorporated into the curable component to achieve enhanced cure speed."
],
[
"1.A curable composition comprising: (a) a (meth)acryl functionalized curable component; (b) a cure system for said curable component; (c) an additive which comprises an alkali metal cation and the reaction product of: (i) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (ii) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene glycol ether units terminated at one end with an ether group and at the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (iii) a monohydroxy compound, a diisocyanate and a polyamine.",
"2.The composition of claim 1 which cures under anaerobic conditions.",
"3.The composition of claim 2, wherein said cation is introduced as an alkali metal salt.",
"4.The composition of claim 3, wherein said alkali metal salt is an alkali metal halide.",
"5.The composition of claim 4, wherein said alkali metal salt is selected from the group consisting of lithium chloride, lithium iodide, lithium bromide, sodium chloride, sodium iodide, sodium bromide, potassium chloride, potassium bromide, potassium iodide and combinations thereof.",
"6.The composition of claim 1, wherein said reaction product is the reaction product of a polyisocyanate and a member selected from the group consisting of an alkylalkenyloxy alcohol, an amine, acid-terminated polyethylene glycol ethers and combinations thereof.",
"7.The composition of claim 1, wherein said cure system comprises a peroxy, perester or peracid.",
"8.The composition of claim 1, wherein said (meth)acryl functionalized curable component includes a reactive monomer having the structure: wherein R5 may be H, CH3 or CH2CH3; R6 is (CH3)n, and n may be an integer from 1-4; R6 may also be substituted with —OH or halide when n is 2-4; and R7 may be a substituted or unsubstituted aromatic group.",
"9.The composition of claim 1, wherein said (meth)acryl functionalized curable component includes a phenoxy ethyl (meth)acrylate monomer.",
"10.The composition of claim 1, wherein said polyisocyanate is selected from the group consisting of toluene diisocyanate, 4,4′-diphenyl diisocyanate, 4,4′-diphenylene diisocyanate, phenyl diisocyanate, methylene diisocyanate and combinations thereof.",
"11.The composition of claim 1, wherein said additive is in a solvent solution.",
"12.The composition of claim 11, wherein said solvent is selected from a group consisting of 1-methyl-2-pyrrolidone, tetrahydrofuran, diethylether, and combinations thereof.",
"13.The composition of claim 1, wherein said additive is present in amounts of about 0.1 to about 2.0% by weight of the total composition.",
"14.A method of increasing the cure speed of a (meth)acryl functionalized monomer composition comprising incorporating into said composition an additive comprising a metal salt and the reaction product of: (a) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (b) a phosgene and a material selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (c) a monohydroxy compound, a diisocyanate and a polyamine.",
"15.The method of claim 14, wherein said additive is a solution of an alkali metal salt and said reaction product is the reaction product of a polyfunctional isocyanate and an alkylalkenyloxy alcohol.",
"16.The method of claim 15, wherein said polyfunctional isocyanate is selected from the group consisting of toluene diisocyanate, 4,4′-diphenyl diisocyanate, 4,4′-diphenylene diisocyanate, phenyl diisocyanate, methylene diisocyanate and combinations thereof.",
"17.The method of claim 14, wherein said additive is present in amounts of about 0.1% to about 2.0% by weight of the total composition.",
"18.The method of claim 14, wherein said (meth)acryl functionalized monomer composition is curable under anaerobically curable conditions.",
"19.An anaerobically curable composition comprising: (a) a (meth)acrylate ester monomer, at least a portion of which includes an aromatic substituted phenoxy (meth)acrylate; (b) a peroxide; and (c) a cure speed accelerator comprising an alkali metal cation and the reaction product of: (i) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (ii) a phosgene and a compound having 3 to 7 polyethylene ether units terminated at one end with an ether group and at the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (iii) a monohdyroxy compound, a diisocyanate and a polyamine, wherein said composition exhibits increased cure speed and strength as compared to the same composition without said cure speed accelerator.",
"20.The composition of claim 19, wherein said aromatic substituted (meth)acrylate is represented by the formula: wherein R5 may be H, CH3, CH2CH3; R6 may be (CH3)n, and n may be an integer from 1-4 R6 may also be substituted with —OH or halide when n is 2-4; and R7 may be a substituted or unsubstituted aromatic group.",
"21.The composition of claim 19, wherein said aromatic substituted (meth)acrylate comprises a mono(meth)acrylate.",
"22.The composition of claim 19, wherein said aromatic substituted (meth)acrylate comprises phenoxy (meth)acrylate.",
"23.An anaerobically curable composition comprising: (a) a (meth)acryl functionalized curable component, at least a portion of which includes an aromatic substituted (meth)acrylate; and (b) a cure system for said curable component.",
"24.The composition of claim 23, wherein said aromatic substituted (meth)acrylate is represented by the formula: wherein R5 may be H, CH3, CH2CH3; R6 may be (CH3)n, and n may an integer from 1-4; R6 may also be substituted with —OH or halide when n is 2-4; and R7 may be a substituted or unsubstituted aromatic group.",
"25.The composition of claim 23, wherein said aromatic substituted (meth)acrylate comprises phenoxy (meth)acrylate.",
"26.A method of enhancing the cure speed of an anaerobically curable composition comprising the step of incorporating into said anaerobically curable composition a reactive monomer comprising an aromatic substituted (meth)acryl functionalized component.",
"27.The composition of claim 19, wherein said composition is in the form of a threadlocker composition.",
"28.The composition of claim 22 wherein the cure speed accelerator is the reaction product of a monohydroxy compound, a diisocyanate and a polyamine."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention relates to anaerobically curable compositions, their use and method of preparation.",
"More particularly, this invention relates to anaerobically curable compositions which exhibit rapid curing irrespective of the type of surface, thereby rendering them surface-insensitive.",
"2.Brief Description of Related Technology Anaerobic adhesive compositions are well known for their ability to remain in a liquid, unpolymerized state in the presence of oxygen and to cure to a solid state upon the exclusion of oxygen.",
"Early work on anaerobic adhesive compositions concentrated on developing a cure system which improved the speed and/or bond strength of the adhesive composition.",
"Various cure systems for anaerobic adhesive compositions have been developed to efficiently perform the redox reaction, which is the basis for anaerobic chemistry.",
"Hydroperoxides were found to serve as a catalyst for the generation of a free radical.",
"For example, U.S. Pat.",
"No.",
"2,895,950 to Krieble, discloses the inclusion of hydroperoxides in amounts of 0.01 to 10% by weight in anaerobic adhesive compositions to achieve faster cure times.",
"Amines, used in amounts up to about 10 percent by weight, are also disclosed in the '950 patent as accelerators to generate free radicals of the peroxide.",
"In many thread-locking applications, it is desirable for the composition to have a relatively low viscosity to permit penetration and wicking into the thread-engaging spaces.",
"Viscosities which are too high to exhibit sufficient flow characteristics, may not sufficiently fill the voids in this area.",
"Many anaerobic threadlocking applications, such as in the electronics industry, require compositions which require relatively low strength to permit disassembly and replacement parts.",
"Low strength anaerobic compositions, typically have less reactive monomer content than those anaerobic compositions where reaction products exhibit higher strength.",
"As a result, the cure speed of low strength anaerobic compositions is slower than that of higher strength anaerobic compositions containing a greater level of reactive monomer component.",
"On surfaces, such as stainless steel, zinc, dichromate, and cadmium, which are considered “slow” or relatively inactive materials for anaerobic cure, primer compositions have been considered necessary for quick fixture and cure times required of many applications.",
"The cure speed on these materials is significantly slower when compared to typical ferrous-containing surfaces, regardless of the reactive monomer content.",
"To enhance the cure speed of anaerobic compositions, and particularly low and medium strength threadlocking compositions, various surface preparations and primers have been used.",
"While such primers are often very effective, their use requires the additional step of applying the primer with a certain degree of care to prevent migration and contamination of surrounding parts.",
"This requires considerable attention in applications where small and/or sensitive components are involved.",
"Additionally, many primers contain undesirable solvents which may be harmful to component parts and/or the surrounding environment.",
"Currently, the addition of viscosity modifiers, such as thickeners or fillers, are added to control the viscosity and curb the tendency of adhesive to migrate.",
"This is usually accomplished, however, at the loss of a certain amount of cure strength, since the addition of viscosity modifiers usually replaces a portion of the reactive monomer component.",
"Other (meth)acrylate-based curable compositions have used a combination of uv curable components to achieve the desired cure properties.",
"For example, Japanese Laid-Open Patent Publication Nos.",
"10-130601 and 10-130602 reportedly assigned to Nippon Kayakuco, Ltd., disclose uv curing adhesive compositions for use as protective coatings on optical discs which employ a uv curable ethylenically compound, such as a (meth)acrylate compound, a photoinitiator, an organic peroxide and an amino accelerator compound.",
"Among the disclosed curable compounds are 2-hydroxyethyl (meth)acrylate, benzyl (meth)acrylate, isobornyl (meth)acrylate and phenyl oxyethyl (meth)acrylate.",
"Another example of a uv curing (meth)acrylate-based composition is disclosed in U.S. Pat.",
"No.",
"5,908,873, which contains an aliphatic urethane acrylate oligomer, a reactive (meth)acrylate monomer, a release agent and a photoinitiator.",
"This composition is used to affix coated and inked optical fibers in a ribbon configuration.",
"There is a need for curable compositions which can fulfill applications requiring a range of cure strengths, i.e., low to high strength, and cure more rapidly without the use of surface treatments, such as primers.",
"There is also a need for a curable composition that can achieve rapid cure on inactive of surfaces and reach a high percentage of its final cure strength in a relatively short time."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides curable compositions, such as anaerobic adhesive compositions, which exhibit enhanced cure speed due to the unique combination of components.",
"Enhanced cure speed can be achieved even on surfaces considered inactive, such as non-ferrous surfaces.",
"Additionally, the present invention provides compositions which can achieve enhanced cure speed without the need for surface preparations, such as primers.",
"Compositions within the scope of the present invention have particular application as threadlockers and can be formulated into low, medium and high strength anaerobic threadlockers which are fast curing on inactive surfaces.",
"In addition, compositions within the scope of the present invention have particular application as anaerobic impregnation sealants.",
"In one aspect of the invention, the inventive compositions include: (a) a (meth)acryl functionalized curable component; (b) a cure system therefor; (c) an additive which includes an alkali metal cation and the reaction product of: (i) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (ii) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene glycol ether units terminated at one end with an ether group and the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (iii) a monohydroxy compound, a diisocyanate and a polyamine.",
"In another aspect of the present invention there is included a composition having enhanced cure speed which includes the aforementioned components, where at least a portion of the (meth)acryl functionalized curable component includes a phenoxy (meth)acrylate.",
"In yet another aspect of the present invention, there is included a composition having enhanced cure speed which includes: (a) a (meth)acryl functionalized curable component, at least a portion of which includes an aromatic substituted (meth)acrylate; and (b) a cure system therefor.",
"The aromatic substituent may be selected from a wide variety of substituted and unsubstituted groups such as alkaryl, aryloxy, alkaryloxy and the like.",
"In still another aspect of the present invention, there is included in anaerobically curable composition which includes: (a) a polyfunctional (meth)acrylate ester component; (b) a peroxide; and (c) a cure speed accelerator which includes intermolecular polyethylene glycol ether oligomers coordinated with an alkali metal cation, where the composition exhibits increased cure speed and strength on substrates, and especially non-ferrous substrates, as compared to the same composition without the cure speed accelerator.",
"In still another aspect of the present invention, there is included a method of increasing the cure speed of a (meth)acryl functionalized monomer composition.",
"This method includes incorporating into the composition an additive including an alkali metal cation and the reaction product of: (a) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (b) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene glycol ether units terminated at one end with an ether group and the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (c) a monohydroxy compound, a diisocyanate and a polyamine.",
"The invention will be further described below.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to anaerobically curable compositions, their use and method of preparation.",
"More particularly, this invention relates to anaerobically curable compositions which exhibit rapid curing irrespective of the type of surface, thereby rendering them surface-insensitive.",
"2.Brief Description of Related Technology Anaerobic adhesive compositions are well known for their ability to remain in a liquid, unpolymerized state in the presence of oxygen and to cure to a solid state upon the exclusion of oxygen.",
"Early work on anaerobic adhesive compositions concentrated on developing a cure system which improved the speed and/or bond strength of the adhesive composition.",
"Various cure systems for anaerobic adhesive compositions have been developed to efficiently perform the redox reaction, which is the basis for anaerobic chemistry.",
"Hydroperoxides were found to serve as a catalyst for the generation of a free radical.",
"For example, U.S. Pat.",
"No.",
"2,895,950 to Krieble, discloses the inclusion of hydroperoxides in amounts of 0.01 to 10% by weight in anaerobic adhesive compositions to achieve faster cure times.",
"Amines, used in amounts up to about 10 percent by weight, are also disclosed in the '950 patent as accelerators to generate free radicals of the peroxide.",
"In many thread-locking applications, it is desirable for the composition to have a relatively low viscosity to permit penetration and wicking into the thread-engaging spaces.",
"Viscosities which are too high to exhibit sufficient flow characteristics, may not sufficiently fill the voids in this area.",
"Many anaerobic threadlocking applications, such as in the electronics industry, require compositions which require relatively low strength to permit disassembly and replacement parts.",
"Low strength anaerobic compositions, typically have less reactive monomer content than those anaerobic compositions where reaction products exhibit higher strength.",
"As a result, the cure speed of low strength anaerobic compositions is slower than that of higher strength anaerobic compositions containing a greater level of reactive monomer component.",
"On surfaces, such as stainless steel, zinc, dichromate, and cadmium, which are considered “slow” or relatively inactive materials for anaerobic cure, primer compositions have been considered necessary for quick fixture and cure times required of many applications.",
"The cure speed on these materials is significantly slower when compared to typical ferrous-containing surfaces, regardless of the reactive monomer content.",
"To enhance the cure speed of anaerobic compositions, and particularly low and medium strength threadlocking compositions, various surface preparations and primers have been used.",
"While such primers are often very effective, their use requires the additional step of applying the primer with a certain degree of care to prevent migration and contamination of surrounding parts.",
"This requires considerable attention in applications where small and/or sensitive components are involved.",
"Additionally, many primers contain undesirable solvents which may be harmful to component parts and/or the surrounding environment.",
"Currently, the addition of viscosity modifiers, such as thickeners or fillers, are added to control the viscosity and curb the tendency of adhesive to migrate.",
"This is usually accomplished, however, at the loss of a certain amount of cure strength, since the addition of viscosity modifiers usually replaces a portion of the reactive monomer component.",
"Other (meth)acrylate-based curable compositions have used a combination of uv curable components to achieve the desired cure properties.",
"For example, Japanese Laid-Open Patent Publication Nos.",
"10-130601 and 10-130602 reportedly assigned to Nippon Kayakuco, Ltd., disclose uv curing adhesive compositions for use as protective coatings on optical discs which employ a uv curable ethylenically compound, such as a (meth)acrylate compound, a photoinitiator, an organic peroxide and an amino accelerator compound.",
"Among the disclosed curable compounds are 2-hydroxyethyl (meth)acrylate, benzyl (meth)acrylate, isobornyl (meth)acrylate and phenyl oxyethyl (meth)acrylate.",
"Another example of a uv curing (meth)acrylate-based composition is disclosed in U.S. Pat.",
"No.",
"5,908,873, which contains an aliphatic urethane acrylate oligomer, a reactive (meth)acrylate monomer, a release agent and a photoinitiator.",
"This composition is used to affix coated and inked optical fibers in a ribbon configuration.",
"There is a need for curable compositions which can fulfill applications requiring a range of cure strengths, i.e., low to high strength, and cure more rapidly without the use of surface treatments, such as primers.",
"There is also a need for a curable composition that can achieve rapid cure on inactive of surfaces and reach a high percentage of its final cure strength in a relatively short time.",
"SUMMARY OF THE INVENTION The present invention provides curable compositions, such as anaerobic adhesive compositions, which exhibit enhanced cure speed due to the unique combination of components.",
"Enhanced cure speed can be achieved even on surfaces considered inactive, such as non-ferrous surfaces.",
"Additionally, the present invention provides compositions which can achieve enhanced cure speed without the need for surface preparations, such as primers.",
"Compositions within the scope of the present invention have particular application as threadlockers and can be formulated into low, medium and high strength anaerobic threadlockers which are fast curing on inactive surfaces.",
"In addition, compositions within the scope of the present invention have particular application as anaerobic impregnation sealants.",
"In one aspect of the invention, the inventive compositions include: (a) a (meth)acryl functionalized curable component; (b) a cure system therefor; (c) an additive which includes an alkali metal cation and the reaction product of: (i) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (ii) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene glycol ether units terminated at one end with an ether group and the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (iii) a monohydroxy compound, a diisocyanate and a polyamine.",
"In another aspect of the present invention there is included a composition having enhanced cure speed which includes the aforementioned components, where at least a portion of the (meth)acryl functionalized curable component includes a phenoxy (meth)acrylate.",
"In yet another aspect of the present invention, there is included a composition having enhanced cure speed which includes: (a) a (meth)acryl functionalized curable component, at least a portion of which includes an aromatic substituted (meth)acrylate; and (b) a cure system therefor.",
"The aromatic substituent may be selected from a wide variety of substituted and unsubstituted groups such as alkaryl, aryloxy, alkaryloxy and the like.",
"In still another aspect of the present invention, there is included in anaerobically curable composition which includes: (a) a polyfunctional (meth)acrylate ester component; (b) a peroxide; and (c) a cure speed accelerator which includes intermolecular polyethylene glycol ether oligomers coordinated with an alkali metal cation, where the composition exhibits increased cure speed and strength on substrates, and especially non-ferrous substrates, as compared to the same composition without the cure speed accelerator.",
"In still another aspect of the present invention, there is included a method of increasing the cure speed of a (meth)acryl functionalized monomer composition.",
"This method includes incorporating into the composition an additive including an alkali metal cation and the reaction product of: (a) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (b) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene glycol ether units terminated at one end with an ether group and the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (c) a monohydroxy compound, a diisocyanate and a polyamine.",
"The invention will be further described below.",
"DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions which can be cured anaerobically and which have particular usefulness as threadlockers.",
"The curable component includes a polyfunctional (meth)acrylate ester monomer.",
"Such monomers may be chosen from any of those commonly used in the art.",
"For example, useful monomers include, without limitation, those corresponding to the following general formula: wherein R1is selected from hydrogen, halogen, and lower alkyl of 1-4 carbon atoms; R2 is selected from the group consisting of hydrogen, —OH and R3 is a radical selected from hydrogen, lower alkyl of 1-4 carbon atoms and hydroxyalkyl of 1-4 carbon atoms; and m is an integer equal to at least 1, desirably from 1-20 and more desirably from 1 to 4; n is an integer equal to at least 1, desirably 1 to 20; and p is 0 or 1.Typical of these monomers are mono-, di-, tri- tetra- and polyethylene glycol di(meth)acrylate and the corresponding diacrylates; di(pentamethylene glycol) di(meth)acrylate; tetraethylene glycol di(chloroacrylate); diglycerol diacrylate; diglycerol tetra(meth)acrylate; butylene glycol di(meth)acrylate; neopentyl glycol diacrylate; and trimethylpropane triacrylate.",
"Particularly useful polymerizable crosslinkable components are ethoxylated trimethylolpropane triacrylate, trimethylol propane tri(meth)acrylate, dipentaerythritol monohydroxypentacrylate, pentaerythritol triacrylate, ethoxylated trimethylolpropane triacrylate, 1,6-hexanedioldiacrylate, neopentyl glycoldiacrylate, pentaerythritol tetraacrylate, 1,2-butylene glycoldiacrylate, trimethylolpropane ethoxylate tri(meth)acrylate, glycerol propoxylate tri(meth)acrylate, trimethylolpropane tri(meth)acrylate, dipentaerythritol monohydroxy penta(meth)acrylate, tri(propylene glycol) di(meth)acrylate, neopentylglycol propoxylate di(meth)acrylate, 1,4-butanediol di(meth)acrylate, polyethyleneglycol di(meth)acrylate (PEGMA), triethyleneglycol di(meth)acrylate, butylene glycol di(meth)acrylate, ethoxylated bisphenol A di(meth)acrylate and combinations thereof.",
"Other useful monomers include those acrylates derived from bisphenol-A, such as bisphenol-A di(meth)acrylate, hydrogenated bisphenol-A di(meth)acrylate, and ethoxylated bisphenol-A di(meth)acrylate (EBIPMA).",
"While di- and other polyacrylate esters are used for producing a cross-linked product, monofunctional acrylate esters (esters containing one acrylate group) also may be additionally incorporated.",
"These materials are often incorporated as reactive diluents which are capable of copolymerizing with the various other polymerizable materials.",
"When dealing with monofunctional acrylate esters, it is desirable to use an ester which has a relatively polar alcoholic moiety.",
"Such materials are less volatile than low molecular weight alkyl esters and, more importantly, the polar group tends to provide intermolecular attraction during and after cure, thus producing more desirable cure properties, as well as a more durable sealant or adhesive.",
"Particularly desirable are the polar groups selected from labile hydrogen, heterocyclic ring, hydroxy, amino, cyano, and halogen polar groups.",
"Useful examples of compounds within this category include cyclohexyl (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, hydroxyethyl acrylate, hydroxypropyl (meth)acrylate, t-butylaminoethyl (meth)acrylate, cyanoethylacrylate, and chloroethyl (meth)acrylate.",
"Other unsaturated reactive diluents, such as styrene and acrylonitrile, can also be used.",
"It is desirable that aromatic reactive diluents be incorporated into the composition.",
"Certain aromatic reactive diluents have been formed to further promote the cure speed of the final compositions.",
"(Meth)acryl functionalized components having an aromatic substituent have been found to enhance cure speed.",
"In particular, phenoxy ethyl (meth)acrylate has been found to increase cure speed alone and in combination with the aforementioned inventive additive (c).",
"Useful aromatic-containing reactive diluents include, without limitation, those compounds corresponding to the structure: wherein R5 may be H, CH3, CH2CH3; R6 may be (CH2)n, and n may be an integer from 1-4; R6 may also be substituted with —OH or halide when n is 2-4; and R7 may be a substituted or unsubstituted aromatic group such as phenyl, benzyl or a fused aromatic ring, such as napthalene, anthracene, and the like.",
"R7 may also be an alkaryl, alkaryloxy or aryloxy group.",
"The curable component is generally present in amounts sufficient to achieve the desired cure strength.",
"This will depend, in part, on the intended application of the final product.",
"Useful amounts may be in the range of about 10% to about 90% by weight of the total composition, and desirably about 20% to about 50% by weight.",
"In instances where a portion of the curable component includes an aromatic substituted (meth)acryl component, the aromatic substituted (meth)acryl component may be present in amounts of about 0.5% to about 40% by weight of the total composition, and desireably in amounts of about 10% to about 30%.",
"The cure system used in the present invention may be chosen from a variety of materials useful for achieving cure.",
"Anaerobically curable compositions typically employ a free radical initiator to serve as a free radical generating source which initiates free radical curing of the polymerizable monomer component.",
"Accelerators may be employed to enhance the speed at which the peroxide free radical is generated.",
"Reducing agents for the initiator, such as saccharin, may also be employed.",
"Free radical initiators such as peroxy, perester and peracid compounds may be employed.",
"Illustrative of peroxy initiators are the diacyl peroxides such as benzoyl peroxide; dialkyl peroxides such as di-tert-butyl peroxide; ketone peroxides such as methylethyl ketone peroxide; and peresters which readily hydrolyze, e.g., tert-butyl peracetate, tert-butyl perbenzoate and di-tert-butyl diperphthalate.",
"A particularly useful class of peroxy initiators are the organic hydroperoxides such as cumene hydroperoxide (CHP), methylethyl ketone hydroperoxide and tert-butyl hydroperoxide (TBH).",
"Of these, cumene hydroperoxide is especially desirable.",
"The initiators may be used in amounts of about 0.01% to about 10% by weight of the total composition, and desirably in amounts of about 0.1% to about 3% by weight of the total composition.",
"Another useful class of initiators includes carbonyl-containing ultraviolet-activated free-radical generators, such as acetophenones, benzophenones and the benzoin ethers.",
"Initiator combinations may also be employed.",
"Useful accelerators for the present invention include compounds having the following formula: where R8 is selected from alkyl from 2 to 6 carbon atoms, cycloalkyl, aryl, alkenyl, and cycloalkenyl and R9 is selected from hydrogen, alkyl, cycloalkyl, alkenyl and cycloalkenyl, aryl, alkoxy, aryloxy, carbonyl, amino, and the following groups: where R10 is selected from alkyl groups containing one to about 10 carbon atoms, alkenyl groups containing two to about 10 carbon atoms, and aryl groups containing up to about 10 carbon atoms.",
"Examples of useful accelerator compounds include 1-acetyl-2-phenyl hydrazine, 1-acetyl-2(p-tolyl) para-toluene sulfonyl hydrazide, 1-formyl-2-phenyl hydrazine and combinations thereof Stabilizers and inhibitors may also be employed as well as chelating agents to control and prevent premature peroxide decomposition and polymerization.",
"Among those useful inhibitors include phenols such as hydroquinone and quinones.",
"Chelating agents may be used to remove trace amounts of metal contaminants.",
"An example of a useful chelating agent is the tetrasodium salt of ethylenediamine tetraacetic acid (EDTA).",
"Other agents such as thickeners, plasticizers, fillers, elastomers, thermoplastics, and other well-known additives may be incorporated where functionally desirable.",
"In addition to a curable component and a cure system, the present invention includes an additive (c), which serves to control the rheology of the composition, as well as increase the composition's cure speed.",
"As previously stated, the additive includes a combination of an alkali metal cation and the reaction product of (a) a polyfunctional isocyanate and a member selected from the group consisting of an hydroxy and an amine; or (b) a phosgene or phosgene derivative, and a compound having 3 to 7 polyethylene ether units terminated at one end with an ether group and at the other end with a reactive functional group selected from the group consisting of an amine, an amide, a thiol and an alcohol; or (c) a monohydroxy compound, a diisocyanate and a polyamine.",
"When the reaction product described in (c) is employed it is generally formed by first reacting a monohydroxy compound with a diisocyanate to form a mono-isocyanate adduct, and subsequently reacting the mono-isocyanate reaction product with a polyamine in the presence of an alkali metal salt and aprotic solvent, as described in U.S. Pat.",
"No.",
"4,314,924, the disclosure of which is hereby expressly incorporated herein by reference.",
"A commercially available version of the reaction product described in (c) is believed to be BYK-410, from BYK-Chemie, Wallingford, Conn. BYK-Chemie describes this reaction product as a urea urethane.",
"Useful isocyanates for forming the reaction product(s) of the additive include polyisocyanates such as phenyl diisocyanate, toluene diisocyanate, 4,4′-diphenyl diisocyanate, 4,4′-diphenylene methane diisocyanate, dianisidine diisocyanate, 1, 5-naphthalene diisocyanate, 4,4′-diphenyl ether diisocyanate, p-phenylene diisocyanate, 4,4′-dicyclo-hexylmethane diisocyanate, 1,3-bis-(isocyanatomethyl) cyclohexane, cyclohexylene diisocyanate, tetrachlorophenylene diisocyanate, 2,6-diethyl-p-phenylenediisocyanate, and 3,5-diethyl-4,4′-diisocyanatodiphenylmethane.",
"Still other polyisocyanates that may be used are polyisocyanates obtained by reacting polyamines containing terminal, primary and secondary amine groups or polyhydric alcohols, for example, the alkane, cycloalkane, alkene and cycloalkane polyols such as glycerol, ethylene glycol, bisphenol-A, 4,4′-dihydroxy-phenyldimethylmethane-substituted bisphenol-A, and the like, with an excess of any of the above-described isocyanates.",
"Useful alcohols for reacting with the polyisocyanates also include polyethyl glycol ethers having 3-7 ethylene oxide repeating units and one end terminated with an ether or an ester, polyether alcohols, polyester alcohols, as well as alcohols based on polybutadiene.",
"The specific type of alcohol chosen and the molecular weight range can be varied to achieve the desired effect.",
"Generally, monohydroxy compounds, straight or branched chain aliphatic or cyclic primary or secondary alcohols containing C5-25, and alkoxylated derivatives of these monohydroxy compounds are useful.",
"Phosgene and phosgene derivatives, such as bischloroformates, may be used to make the reaction product of the additive (c).",
"These compounds are reacted with a nitrogen-containing compound, such as an amine, an amide or a thiol to form the adduct.",
"Phosgenes and phosgene derivatives may also be reacted with an alcohol to form the reaction product.",
"Amines which can be reacted with phosgene or phosgene derivatives to make the reaction product include those which conform to the general formula R11—NH2, where R11 is aliphatic or aromatic.",
"Desirable aliphatic amines include polyethylene glycol ether amines.",
"Desirable aromatic amines include those having polyethylene glycol ether substitution on the aromatic ring.",
"For example, commercially available amines sold under the tradename JEFFAMINE by Huntsman Corporation, Houston, Texas, may be employed.",
"Examples include JEFFAMINE D-230, JEFFAMINE D400, JEFFAMINE D-2000, JEFFAMINE T-403, JEFFAMINE ED-600, JEFFAMINE ED-900, JEFFAMINE ED-2001, JEFFAMINE EDR-148, JEFFAMINE XTJ-509, JEFFAMINE T-3000, JEFFAMINE T-5000, and combinations thereof.",
"The JEFFAMINE D series are diamine based products and may be represented by: (CAS Registry No.",
"904610-0) where x is about 2.6 (for JEFFAMINE D-230), 5.6 (for JEFFAMINE D-400) and 33.1 (for JEFFAMINE D-2000), respectively.",
"The JEFFAMINE T series are trifunctional amine products based on propylene oxide and may be represented by: where x, y and z are set forth below in Table A.",
"TABLE A JEFFAMINE Approx.",
"Mole Product Initiator (A) Mol.",
"Wt.",
"PO T-403 Trimethylolpropane 440 5-6 T-3000 Glycerine 3,000 50 T-5000 Glycerine 5,000 85 More specifically, the JEFFAMINE T-403 product is a trifunctional amine and may be represented by: where x+y+z is 5.3.",
"(CAS Registry No.",
"39423-51-3) The JEFFAMINE ED series are polyether diamine-based products and may be represented by: where a, b and c are set forth below in Table B.",
"TABLE B JEFFAMINE Approx.",
"Value Approx.",
"Product B a + c Mol.",
"Wt.",
"ED-600 8.5 2.5 600 ED-900 15.5 2.5 900 ED-2001 40.5 2.5 2,000 Amides useful for reacting with the phosgene or phosgene derivatives include those which correspond to the following formula: where R12 may be an aliphatic or aromatic, substituted or unsubstituted, hydrocarbon or heterohydrocarbon, substituted or unsubstituted, having C1-36.Alcohols useful in forming the reaction product with the phosgene or phosgene derivatives include those described above.",
"The alkali metal cations are usually provided in the form of a halide salt.",
"For example, sodium, potassium and lithium halide salts are useful.",
"In particular, sodium chloride, sodium iodide, sodium bromide, potassium chloride, potassium iodide, potassium bromide, lithium chloride, lithium iodide, lithium bromide and combinations thereof may be employed.",
"The reaction products of additive (c) of the present invention are usually present in and added to the composition with an alkali metal salt, in a solvent carrier.",
"The solvents are desirably polar aprotic solvents in which the reaction to form the reaction product was carried out.",
"For example, N-methyl pyrrolidone, dimethylsulfoxide, hexamethylphosphoric acid triamide, N,N-dimethylformamide, N,N,N′,N′-tetramethylurea, N,Ndimethylacetamide, N-butylpyrrolidone, tetrahydrofuran and diethylether may be employed.",
"One particularly desirable additive is the combination of a lithium salt and a reaction product which is formed by reacting a monohydroxy compound with a diisocyanate compound to form a mono-isocyanate first adduct, which is subsequently reacted with a polyamine in the presence of lithium chloride and 1-methy-2-pyrrolidone to form a second adduct.",
"A commercially available additive of this sort is sold by BYK Chemie, Wallingford, Conn. under the trade name BYK 410.This commercially available additive is described by BYK-Chemie product literature as being a urea urethane having a minor amount of lithium chloride present in a 1-methyl-2 pyrrolidone solvent.",
"Desirably, the additive is present in the inventive compositions in amounts of about 0.1% to about 2.0% and more desirably in amounts of about 0.5% to about 0.7% by weight of the total composition.",
"Without wishing to be bound by any one theory, it is believed that the additive of the present invention allows for intermolecular attraction, which forms a loosely held network This network may temporarily be broken or disturbed, but quickly reforms due to intermolecular forces.",
"It is believed that the alkali metal coordinates with polar groups on the reaction product(s) of the additive, thereby “lining-up” polar chains which are in proximity to each other.",
"Such intermolecular alignment of the additive creates a network, which helps control the rheological properties of the compositions.",
"Moreover, the alkali metal ions are believed to enhance cure speed.",
"Polymerization accelerators may also be included in the present composition.",
"For example, organic imides (e.g., benzoic sulfimide) and primary, secondary or tertiary amines, and inhibitors or stabilizers of the quinone or hydroquinone families may be employed.",
"The accelerators are generally employed in amounts of less than 10% by weight, and the inhibitors are conventionally employed in amounts of about 10 to about 1,000 parts per million (ppm).",
"The anaerobic compositions of the present invention have the advantage of long-term stability and the ability to cure at room temperature upon the exclusion of oxygen, such as when placed between the mating threads of a nut and a bolt or between the juxtaposed surfaces of a bearing and a shaft.",
"The anaerobic cure speed can be further enhanced by application of heat, e.g., up to about 150° C. The compositions of the present may also include various additional components such as viscosity modifiers, pigments and coloring agents, plasticizers and the like in amounts suitable to achieve their intended purpose.",
"While described illustratively below in terms of threadlocking compositions, compositions within the scope of this invention may be employed as anaerobic sealants, a general description which may be found in U.S. Pat.",
"No.",
"3,672,942 (Neumann); U.S. Pat.",
"No.",
"3,969,552 (Malifsky); U.S. Pat.",
"No.",
"Re 32,240 (De Marco); and U.S. Pat.",
"No.",
"4,632,945 (Garcia) the disclosures of which are hereby incorporated herein by reference.",
"EXAMPLES Anaerobically curable threadlocking compositions, set forth below in Table I, were prepared.",
"Composition A is a control and does not include the inventive additive.",
"Compositions B-D are representative of an aspect of the inventive compositions which include the additive.",
"Composition E is representative of inventive compositions which achieve enhanced cure speed by the inclusion of an aromatic substituted reactive (meth)acrylate (2-phenoxy methacrylate) along with the curable component without the additive.",
"Composites F-I are representative of inventive compositions which employ both the additive and the aromatic substituted reactive (meth)acrylate.",
"The compositions were tested for their cure speed, as measured by the threadlocking strength (breakloose/prevail [in.-lbs.])",
"developed on various metal substrates (nuts and bolts) at room temperature for a given cure time.",
"The compositions were applied in equal amounts to the threads of the bolts and mated with the nut using a pretorque of 5 newton-meters (NM) (about 44 in.-lbs.).",
"The compositions were allowed to cure at room temperature for 1 hour and 24 hours.",
"The results of these tests are shown below in Table II.",
"TABLE I COMPOSITION (% by weight) COMPONENT A B C D E F G H I PEGMA* 69.05 69.55 68.05 67.55 39.05 38.55 39.03 38.05 37.55 2-phenoxy ethyl 0.00 0.00 0.00 0.00 30.00 30.00 30.00 30.00 30.00 (meth)acrylate Additive** 0.00 0.50 1.00 1.50 0.00 0.50 0.65 1.00 1.50 Plasticizer 23.00 23.00 23.00 23.00 23.00 23.00 23.00 23.00 23.00 initiator reducing agent 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Accelerator 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 free radical initiator 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Stabilizers 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 Fillers 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 coloring agents 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 *Poly(ethylene glycol)di(meth)acrylate **Urea urethane and lithium chloride in 1-methyl-pyrrolidone solvent, commercially available as BYK-410 from BYK-Chemie, Wallingford, CT. TABLE II EFFECTS OF ADDITIVE ON CURE STRENGTH IN 1 HR AND 24 HRS (Breakloose/Prevail [in.-lbs.])",
"1 hr RTC 24 hr RTC COMPOSITION DgrdsStl ReoilStl Zinc StnlssStl DgrsdStl ReoilStl Zinc StnlssStl A* 127/28 88/9 64/4 104/34 233/75 189/34 199/51 134/40 B 159/54 95/22 59/5 120/28 241/80 221/52 207/50 154/41 C 169/61 92/18 68/9 121/27 260/79 225/49 200/51 156/38 D 181/57 117/28 69/9 125/27 260/67 210/40 191/20 173/36 E 157/39 135/27 82/8 117/30 191/45 182/32 167/32 127/34 F 164/61 113/25 86/10 133/36 201/53 207/54 166/28 146/30 H 170/52 121/34 83/11 130/34 210/52 206/43 174/30 149/29 I 162/41 138/40 97/13 119/29 213/46 211/47 165/27 141/36 *Control RTC stands for Room Temperature Cure DgrdsStl stands for Degreased Steel ReoilStl stands for Reoiled Steel StnlssStl stands for Stainless Steel As indicated from the results in Table II, the inventive compositions B-D containing the additive showed significant cure speed improvement as measured by strength developed after 1 hour room temperature cure, when compared to composition A (control).",
"The most significant strength measurement is breakloose, since this indicates the amount of torque (force) necessary to initially move the nut bonded to the bolt.",
"In nearly every test, the amount of strength increased as the amount of additive increased, regardless of the type of metal substrate used.",
"Similar results are seen when inventive compositions E, F, H and I are compared with composition A (control).",
"Additional anaerobically curable threadlocking compositions were prepared as set forth in Table III.",
"These compositions included a combination of ethoxylated bisphenol A dimethacrylate and poly(ethylene glycol) dimethacrylate monomers.",
"Composition J was a control and contained neither the additive nor an aromatic substituted methacrylate.",
"Compositions K and L are representative of an aspect of the invention which employs aromatic-substituted methacrylates, i.e.",
"3-phenoxy-2-hydroxypropyl methacrylate and 2-phenoxy ethyl methacrylate, respectively, to enhance cure speed.",
"TABLE III Threadlocker Compositions (% by weight) Composition Component J K L Ethoxylated bisphenol A dimethacrylate 47.05 47.05 47.05 Poly(ethylene glycol) dimethacrylate 30.00 0.00 0.00 3-phenoxy-2-hydroxypropyl methacrylate 0.00 30.00 0.00 2-phenoxy ethyl methacrylate 0.00 0.00 30.00 Plasticizer 15.00 15.00 15.00 Initiator reducing agent 1.00 1.00 1.00 Accelerator 0.10 0.10 0.10 Free radical initiator 1.00 1.00 1.00 Stabilizer 0.70 0.70 0.70 Fillers 5.00 5.00 5.00 Coloring agents 0.15 0.15 0.15 Additive** 0.00 0.00 0.00 **Without additive BYK 410, (Ure Urethane and lithium chloride in 4-methyl-Pynolidone) J = Control The compositions were tested for their cure speed, as measured by the threadlocking strength (breakloose/prevail [in.-lbs.])",
"developed on various metal substrates (nuts and bolts) at room temperature for a given cure time.",
"The compositions were applied in equal amounts to the threads of the bolds and mated with the nut using a pretorque of 5 newton-meters (NM) (about 44 in.",
"-lbs.).",
"The compositions were allowed to cure for 1 hour and 24 hours.",
"The results are shown in Table IV.",
"TABLE IV Effects of monomers on cure strength in 1 hr and 24 hrs (breakloose/Prevail [in.lbs] Com- 1 hr RTC Br/Pv 24 hr RTC Br/Pv position Zinc StnlssStl DgrsdStl ReoilStl Zinc StnlssStl J 59/2 60/11 318/173 225/176 170/140 90/112 K 53/2 71/30 357/173 226/181 210/119 120/114 L 68/2 54/3 305/147 268/158 183/122 98/81 When compared to Control J, inventive composition K demonstrated a significant increase in breakloose strength on stainless steel after 1 hr.",
"room temperature cure, as well as higher breakloose strengths after 24 hour room temperature cure.",
"Inventive composition L demonstrated a significant increase in breakloose strength on zinc after 1 hr.",
"room temperature cure and comparable or enhanced overall breakloose strength after 24 hrs.",
"The examples set forth above serve to illustrate the present invention, but are in no way intended to limit the spirit and scope thereof, which are defined by the claims."
]
] | Patent_10432199 |
[
[
"Mewthod and device for analysing the surface of a substrate",
"The object of the invention is a process for scanning a surface of a substrate (2), which process consists in taking at least one reflected image of at least one test pattern (1) on the said surface and extracting by digital processing local phases in two directions, characterized in that variations in local slopes are calculated by digital processing from the local phases in order to deduce therefrom variations in curvature or variations in altitude of the said surface."
],
[
"1-27.",
"(canceled) 28.Process for scanning a surface of a substrate, comprising: taking at least one reflected image of at least one test pattern on the surface; extracting by digital processing local phases in two directions; and calculating variations in local slopes by digital processing from the local phases to deduce therefrom variations in curvature or variations in altitude of the surface.",
"29.Process for scanning a surface of a substrate according to claim 28, wherein the at least one reflected image is taken after reflected on a reflective surface.",
"30.Process for scanning a surface of a substrate according claim 28, wherein the digital processing comprises superimposing the at least one reflected image of the at least one test pattern onto a reference test pattern to obtain a moiré from which the extracting of the local phases is realized.",
"31.Process for scanning a surface of a substrate according to claim 28, wherein the extracting of the local phases is realized by a phase stepping.",
"32.Process for scanning a surface of a substrate according to claim 28, wherein in the digital processing the extracted local phases are compared to reference phases, phase variations are deduced therefrom, which are converted by a sensitivity factor to form variations in local slopes.",
"33.Process for scanning a surface of a substrate according to claim 32, wherein the variations in curvature are obtained by the digital processing involving derivation of the variations in local slopes.",
"34.Process for scanning a surface of a substrate according to claim 32, wherein the variations in altitude are obtained by the digital processing involving integration of the variation in local slopes.",
"35.Process for scanning a surface of a substrate according to claim 28, further comprising placing the substrate in a position suitable for facilitating the digital processing.",
"36.Process for scanning a surface of a substrate according to claim 35, wherein the substrate is placed by a mechanical positioning device.",
"37.Process for scanning a surface of a substrate according to claim 28, wherein the taking of the at least one reflected image is effected in a course of movement of the substrate.",
"38.Process for scanning a surface of a substrate according to claim 28, wherein the taking of the at least reflected image is effected on a transparent substrate in a course of deformation.",
"39.Process for scanning a surface of a substrate according to claim 28, wherein the at least one test pattern is reflected over a whole of a surface of the substrate.",
"40.Process for scanning a surface of a substrate according to claim 28,, wherein the at least one reflected image is taken instantaneously.",
"41.Process for scanning a surface of a substrate according to claim 28, wherein a design of the test pattern is deformed in at least one direction.",
"42.Process for scanning a surface of a substrate according to claim 28, wherein the substrate is shaped and transparent.",
"43.Process for scanning a surface of a substrate according to claim 37, wherein the taking of the at least one reflected image is effected on a passing substrate.",
"44.Process for scanning a surface of a substrate according to claim 43, wherein the substrate is passed on a conveyor and the substrate is focused in two directions forming a plane of the conveyor.",
"45.Device for realizing the process according to claim 28, comprising at least one camera, at least one flash-type pulse lighting, at least one test pattern, and digital image-processing means, wherein the processing means are configured to generate a moiré.",
"46.Device according to claim 45, wherein the digital image-processing means comprises algorithms for phase extraction calculation, for conversion calculation, and for derivation or integration calculation.",
"47.Device according to claim 45, wherein the camera is positioned above a conveyor.",
"48.Device according to claim 47, further comprising mechanical means provided for focusing the substrate in two directions forming a plane of the conveyor.",
"49.Device according to claim 45, wherein the device is positioned within a warm chamber.",
"50.Device according to claim 45, wherein an optical axis of a lens of the camera is normal to a surface of the substrate at a center of a field of observation when a single test pattern is used.",
"51.Use of a device according to claim 45 for scanning defects of a window.",
"52.Use of a device according to claim 45 for measuring a variation in overall three-dimensional shape of a window.",
"53.Use of a device according to claim 45 for checking inherent flatness of a window.",
"54.Use of a device according to claim 45, for checking a window intended for realizing a visual display screen."
],
[
"The invention relates to a process and a device for scanning a surface of a shaped substrate, the said scan particularly allowing the detection of local defects and the three-dimensional measurement of the shape of a substrate having a reflective surface.",
"Although not limited to such applications, the invention will be more particularly described with reference to scanning of the surface of windows and even more specifically to the scanning of curved windows intended for the automotive industry.",
"Current specifications by vehicle manufacturers are encouraging the glass industry to realize laminated side windows constituted, in particular, by two panes of glass of reduced thickness compared with a toughened monolithic window.",
"The realization of such panes of glass of reduced thickness is delicate and can lead to surface defects.",
"These defects can become very troublesome after assembly to form a laminated window, since they lead to optical distortion phenomena, accentuated owing to the connection to a second pane of glass, and also to delamination problems.",
"The presence of such optical defects generally results in the windows being rejected, since they are unacceptable.",
"The windows being already laminated, they are difficult to recycle and the costs of production become unacceptable.",
"There is therefore a desire to proceed to detect such defects as early as possible in the production chain and particularly prior to realizing the assembly of a laminated window.",
"The methods which are normally used consist in observing the laminated window in terms of transmission or reflection according to standardized methods, such as by visual observation outside the production line and after the assembly of the laminated window.",
"As previously explained, such a check is too late and has an adverse effect upon production costs.",
"In addition, industrial methods exist for checking a reflective surface, by which surface defects can be detected by reflection measurements of the reflection of a regular design.",
"These methods are shown to be limited in terms of their applications and the reliability of their results.",
"Indeed, in the case, particularly, of curved windows, these types of scanning methods display weaknesses owing, in particular, to the adopted principle for the reflection measurement.",
"Apart from the defects, it is worth making sure that the geometry of the window conforms to that which is desired or at least falls within a fixed envelope of tolerance.",
"The checking method is mechanical; sensors take punctual measurements so as to compare them to those of a template consistent with the idle time.",
"Now, this method constitutes a labour cost, in particular for the positioning of the sensors relative to each new window geometry to be checked, as well as a cost of supplies for the measurements, particularly the need for a distinct template for each window geometry, costs in which a reduction would be increasingly welcome.",
"The inventors have thus set themselves the task of designing a method for scanning a surface, particularly a reflective surface, which does not have the drawbacks of the methods previously stated and, in particular, by which a reflective surface of a shaped substrate can be scanned in a precise and repetitive manner and by which, in particular, the costs of checking the consistency of windows on a production line are reduced.",
"This object has been achieved according to the invention by the use of a process for scanning a surface of a substrate, which process consists in taking at least one reflected image of at least one test pattern on the said surface and extracting by digital processing local phases in two directions, characterized in that variations in local slopes are calculated by digital processing from the local phases in order to deduce therefrom variations in curvature or variations in altitude of the said surface.",
"Thus, a first type of surface scan leads from the phase extraction to the resultant deduction of variations in local slopes, which variations, by means of a calculation by derivation, allow variations in local curvatures to be defined.",
"Such a scan allows the presence of defects on the surface of a substrate to be determined.",
"A second type of surface scan leads from the phase extraction to the resultant deduction of variations in local slopes, which variations, by means of a calculation by integration, allow altitude deviations to be defined in order to establish the variation in overall three-dimensional shape of the substrate.",
"The image is taken reflected on a reflective surface.",
"It is important that the test pattern or test patterns, a plurality being able to be used in the case of a complex surface, should be reflected over the whole of the surface of the substrate.",
"Preferably, the reflected image is taken instantaneously.",
"The scanning method according to the invention envisages the adoption of a test pattern, the design of which can be deformed, whereby an image of the said test pattern can be obtained which is substantially less distorted than that of an ordinary test pattern owing to the curvature of a substrate when a rounded substrate is scanned.",
"The invention can thus advantageously be applied to the scanning of rounded substrates, such as curved windows.",
"The scan being usually effected on the convex surface of such curved windows, the process according to the invention envisages the use of a test pattern, the size of which is dictated by that of the window, bearing in mind that the captured image is that of the test pattern on a convex surface.",
"According to one embodiment of the invention, a real or physical test pattern is envisaged, the size of which, as previously stated, is dictated by the size of the substrate and more particularly by its shape.",
"Such a test pattern is more particularly suitable for taking an image on reflective surfaces.",
"According to another embodiment of the invention, a virtual test pattern is used; this is constituted in this case, for example, by a projected test pattern, the image of which is preferably taken reflected on the substrate.",
"A preferred embodiment of the invention consists in using a flat test pattern, the design of which is deformed.",
"Another embodiment envisages a rounded test pattern; in this latter case, the design, which would be non-deformed if the test pattern were flat, is once again deformed such as envisaged by the invention.",
"It is further possible to envisage a combination of these two embodiments, by realizing a rounded test pattern comprising a design which would already be deformed if the test pattern were flat.",
"According to a first embodiment of the invention, the test pattern is designed such as to compensate rigorously for the curvature of the scanned substrate.",
"Such an embodiment is industrially conceivable in the case of a scanning of identical substrates.",
"Yet particularly in the case of curved windows intended for fitting in automotive vehicles, the production series relate to limited numbers of windows, whereas the number of windows which are different, and hence possess different curvatures, is very large.",
"According to a second embodiment, the inventors thus envisage a deformation of the design of the test pattern compatible with the scanning of several models of substrates having different curvatures.",
"Tests have shown that the choice of a deformation of the design which does not allow strict compensation of the rounding of the substrate yielded precise and reliable scanning results; indeed, the deformation of the design of the test pattern according to the invention allows the at least partial compensation of the deformation of the image taken on the shaped substrate and thus allows satisfactory scanning in the zones of curvature and better scanning of the peripheral zones of the substrate.",
"An advantageous realization of the invention envisages scanning of the substrate in dynamic state, that is to say in motion or in the course of deformation.",
"According to one embodiment of this realization, the scanning is effected on a substrate moving past on a conveyor.",
"Compared with methods which require the substrate to be immobilized, the invention has the advantage of being able to be used to scan moving substrates.",
"It therefore additionally offers an advantage in terms of productivity and production costs, owing to the speed of its execution.",
"In order to ensure the reproducibility of the scan, the substrate in travel on a conveyor is advantageously placed by mechanical positioning means in a position suitable for facilitating the digital processing, in particular for best realizing a comparison of the reflected image of the test pattern with a reference test pattern.",
"Also, prior to carrying out the scan, the passing substrates are positioned identically in the two directions of the plane formed by the conveyor.",
"The processing of the image according to the invention comprises several stages, in particular a stage of superimposing the reflected image of the test pattern onto a reference test pattern in such a way as to obtain a moiré, the phase extraction stage being realized, for example, according to a “phase stepping” method.",
"This method is described in the article “Design of algorithms for phase measurements by the use of phase stepping”, signed by Yves Surrel and published in Applied Optics Vol.",
"35, No.",
"1, dated 1 Jan. 1996.Compared with other processing methods, of the Fourier transform type, such image processing operations according to the invention yield, in particular, precise and repetitive scanning results for the whole of the surface of the substrate.",
"Indeed, the other image-processing methods, particularly using Fourier transform, lead to unsatisfactory results for the peripheral zones of the substrate.",
"The stage following the extraction of local phases consists in comparing these phases to reference phases, deducing therefrom phase variations and correcting these phase variations through the use of a sensitivity factor s, which is heavily dependent upon the observation conditions and the measurement tools used, such as to calculate the variations in local slopes.",
"The scan which is thus obtained according to the invention allows, in particular, a check to be made of the surface state of a substrate and, in addition, an overall scan to be made which allows measurement of the rounding of the substrate, that is to say the variation in three-dimensional shape of the substrate.",
"The process thus described according to the invention allows the productivity and production costs of, in particular, curved windows to be further improved; indeed, the current requirements in relation to curving within the automotive industry are very demanding and call for rigorous checking.",
"The methods currently used generally call for an off-line checking apparatus.",
"The apparatuses allow either a checking of the periphery or, increasingly, a checking of the whole of the surface, required in order to meet the aesthetic requirements.",
"The necessary apparatuses are therefore either mechanical sensors, which have a not insignificant cost, or a three-dimensional checking machine, in the most demanding cases, which makes use of a marble.",
"In this latter case, the apparatus is dedicated to a single type of window and gives rise to a very considerable cost; in addition, the measurement times are very long.",
"It is evident that the process according to the invention yields satisfactory results at reduced costs and allows much more rapid checking.",
"This latter advantage is considerable, since, should a defect or non-fulfilment of the required rounding become apparent, it will allow rapid intervention in the production process.",
"Such intervention will allow, on the one hand, the quantity of defective products, and hence material losses, to be limited and will consequently allow an improvement in both production rates and productivity.",
"Compared with customary methods, the scanning process according to the invention, which allows the rounding to be checked, also has the advantage of carrying out a scan without contact and without any preparation of pre-treatment of the surface to be scanned.",
"That procures a certain advantage in the production of a thin glass pane, that is to say having a thickness less than 2.5 mm.",
"Indeed, such panes of glass are very sensitive to contact throughout the production phase and, if subjected to a contact, can find their rounding damaged during the checking.",
"This process can equally well be applied to the checking of the rounding for toughened monolithic glasses, such as for the side windows or backlights of vehicles.",
"The invention has a further advantage in the checking of thin glass panes; these thin glass panes can be intended, for example, for fitting in automotive vehicles to form side windows.",
"For this type of use, the thin glass panes are put together in pairs using a plastics film, made of polyvinyl butyral (PVB) for example, to form a laminated window.",
"The scanning process according to the invention, which consists in obtaining an image of a glass pane, will allow the quality of the laminated window to be predicted through the use of mathematical modelling and, in particular, numerical calculation; indeed, the knowledge according to the process of the image of two panes of glass intended to be put together to form a laminated window will yield information regarding the optical quality of the laminated window.",
"The process according to the invention thus allows laminated windows which are unacceptable in terms of their optical quality to be detected prior to their assembly.",
"Amongst other advantages, the invention thus prevents all recycling problems arising from the presence of two materials and it further improves, of course, the productivity of the production process for such laminated windows.",
"According to another, dynamic state embodiment, the scanning process according to the invention is effected on a substrate in the course of deformation.",
"According to such an application of the process according to the invention, it is possible to check the rounding of the substrate during the course of its deformation.",
"When a glass pane is curved, for example, instantaneous scans of the type permitted by the invention, successively repeated, will allow the deformation of the window to be checked.",
"More particularly, should complex shapes be sought, such scans will allow the process used to be optimized; in these cases, indeed, the curving processes which are used can be realized in several stages and the knowledge of the deformation of the window during the process can thus allow the moment of passage from one stage to another to be optimized.",
"The knowledge of the deformation can further allow the improved use of heating means during one or other of the stages.",
"Still in the case of the rounding of a substrate being checked in the course of its deformation, the invention advantageously envisages this checking being carried out with reference to the concave face of the substrate, in particular when the said substrate is supported by mechanical tools during its deformation, for example by frames or rollers in the case of windows.",
"Preferably, when conditions allow, the scan according to the invention will be carried out on the convex face of the substrate.",
"The invention likewise proposes a device for realizing the process according to the invention.",
"The device according to the invention comprises at least one camera, a flash-type pulse lighting, digital image-processing means and at least one test pattern, the projection of which covers the whole of the surface of the substrate, the design of the test pattern being able to be deformed in at least one direction.",
"The camera is advantageously a digital camera; such a camera allows the digitalization to be made at the level of the CCD pick-up.",
"That has the advantage that no analogue signal is transported by cables within which it can be damaged and, furthermore, deteriorations in the signal due to an analogue/digital conversion are likewise avoided.",
"The pulse-type lighting is envisaged and is suitable for obtaining a clear image of the substrate in travel or in the course of deformation; advantageously, a laser or laser diode type lighting is used.",
"The luminosity is chosen, moreover, so as to illuminate the whole of the surface of the test pattern in a homogeneous manner.",
"The digital processing means are fit to generate a moiré and comprise algorithms for phase extraction calculation, for conversion calculation and for derivation or integration calculation.",
"The device according to the invention, when it relates to the scanning of a moving substrate, is advantageously used above a conveyor.",
"This conveyor is preferably designed to offer a dark surface and thus to enhance the contrast of the image captured by the camera; the conveyor is constituted, for example, by a belt conveyor, of the carpet type, the colour of which is advantageously dark and preferably black.",
"The conveyor has a very precise inherent flatness in order to define a precise supporting plane for the substrate to be measured.",
"According to a preferred embodiment of the invention in the case of a substrate moving past on a conveyor, the camera is placed above the test pattern.",
"Preferably, also, the optical axis of the camera lens is normal to the surface of the substrate at the centre of the field of observation when a single test pattern is used; in other words, the direction of observation of the camera is normal to the surface of the substrate at the centre of the field of observation.",
"According to such an embodiment and in the case of a virtual test pattern, the optical axis of the projector is parallel and close to the optical axis of the camera.",
"Before the scanning station, mechanical positioning means are advantageously provided on the conveyor, which allow the substrate to be positioned in the two directions formed by the plane of the conveyor in a position suitable for facilitating the digital processing.",
"These means are all means known to the person skilled in the art, for example systems of limit stops and/or guide rails.",
"The device according to the invention, when it relates to the scanning of a substrate in the course of deformation, is advantageously placed in a warm chamber, for example in the case of the curving of a window realized in just such a warm chamber.",
"According to other processes for the curving of windows, the device is used at ambient temperature, The device thus described according to the invention can therefore be used to scan a surface, particularly a reflective surface, of a substrate, which is in particular shaped and preferably transparent.",
"The invention first of all allows the state of such a surface to be checked and hence the defects to be scanned.",
"In the case of windows, for example, such a check can thus allow the origin of the defects to be understood and for these to be remedied by appropriate action upon the device or upon the production or forming process.",
"Where laminated windows are realized, moreover, the scanning of the surface of each of the monolithic windows will allow the optical quality of the laminated window to be predicted.",
"The operation corresponds in this case to a numerical calculation based on the two images obtained and to an appropriate statistical processing.",
"The invention can additionally allow measurement of the variation in overall three-dimensional shape of a shaped substrate.",
"The scan, which can be made in dynamic state, allows either the shape of the obtained substrate to be checked or the evolution of the shape to be checked when the scan is done in the course of deformation.",
"The invention can also allow the inherent flatness of a substrate to be measured.",
"Indeed, the knowledge of the scan according to the invention made of the surface of the substrate, which brings defects to light and yields its three-dimensional configuration, allows the inherent flatness of the said substrate to be checked for applications of flat substrates.",
"Such checking of the inherent flatness is, for example, useful for window applications, for example in the building trade field.",
"This checking of the inherent flatness becomes very important for other applications of windows, in particular in the field of visual display screens.",
"Indeed, for this type of application, the inherent flatness must be very strict, in particular in order to satisfy the manipulations to which the window is subjected during all the treatments effected for the purpose of realizing a screen; in order to prevent it being damaged during manipulations for leading the window from one treatment to another, the said window is transported by suction devices which limit the contacts and the assaults of tools on the glass.",
"Manipulations of this type by the use of suction call for a perfect inherent flatness of the window in order to prevent any risk of fall of the transported window.",
"The above-stated visual display screens are all types of screens which have to be subjected to treatments such as coating applications, engravings, etc.",
"and are, for example, plasma screens, field emission displays (FED), micropoint screens.",
"A checking of the inherent flatness of a window can likewise be sought for the realization of vacuum windows or flat lamps, for which a limited and regular space is sought between two glass panes.",
"It is evident that, for these different applications, the knowledge of the inherent flatness of a glass pane or window can allow a defective product to be scrapped before it is actually used in the make-up of the final product and thus leads to a considerable reduction in production costs by enhancing the production yields in the final stages of development of these products.",
"In addition, where substrates are transported by suction, wherein falls of the windows are able to block a production line, the prior scrapping of substrates not offering sufficient inherent flatness to satisfy the process brings improved productivity.",
"A final advantage of the scanning method according to the invention for checking the inherent flatness of a substrate is that, as in the preceding cases, it can be carried out rapidly and, moreover, on substrates in travel, for example on a conveyor, and therefore without risk of lowering the production rates.",
"Other details and advantageous characteristics of the invention will emerge below from the description of illustrative embodiments of the invention with reference to FIGS.",
"1 and 2, in which FIG.",
"1 shows a diagram illustrating the scanning principle, FIG.",
"2 shows a diagram representing an application of the invention for checking window glazing.",
"The figures are not represented to scale in order to make them easier to understand.",
"FIG.",
"1 represents a device illustrating the measurement principle according to the invention, resulting in an instantaneous image being taken of a test pattern 1 reflected on a substrate 2.The image of the test pattern is, as previously stated, advantageously obtained with the aid of a CCD camera 3 in order to avoid all risks of damage to the captured signal.",
"The test pattern 1 used is advantageously a flat, physical test pattern, the design of which can, for example, be deformed in that it contains shaped lines.",
"The instantaneous image is advantageously obtained by the use of a flash-type lighting system 4; this is constituted, for example, by a laser diode.",
"The test pattern 1 according to the invention, particularly where a curved substrate 2 is scanned, has a design which is deformed such that the image of the test pattern captured in reflection on the said substrate displays quasi-parallel lines, these being strictly parallel given that the deformation of the design of the test pattern compensates for the rounding of the substrate.",
"As previously stated, a deformation of the test pattern design is advantageously chosen which will produce satisfactory results in terms of scans for a range of substrates having different curvatures.",
"Moreover, the measurement is preferably realized on the convex face of the substrate, particularly in order to obtain a better contrast, and consequently calls for a test pattern of relatively large size such that its reflected image covers the whole of the surface of the substrate to enable the whole of the surface to be scanned.",
"The image captured by the camera 3 is transmitted to a computer 5 in order to be processed, in a number of stages, by digital processing means.",
"First of all, the image of the reflected test pattern taken by the camera is digitalized, it is superimposed onto a reference test pattern to form a moiré, the reference test pattern being able, for example, to be constituted by the pixels of the camera.",
"To each pixel of the digitalized image is attached an elementary area of the surface of the substrate.",
"Starting from the moiré and using an algorithm for calculation according to the “phase stepping” method, for example, a map is extracted of the local phases relating to the elementary areas of the surface of the substrate.",
"Next, the local phases are compared to memorized reference phases deriving from a measurement of a reference sample or from a calculation produced by CAD (computer-aided design), in order to deliver phase variations.",
"From the phase variations, the variations in slopes are calculated by means of a sensitivity factor s, which is heavily dependent upon the observation conditions and the measurement tools used.",
"The variations in local slopes are next used in a calculation by derivation algorithm or in a calculation by integration algorithm in order respectively to show the presence of surface defects 6, 7, the knowledge of which is obtained precisely in two directions, or to measure the variation in three-dimensional shape of the substrate.",
"In the case of a flat substrate, it is similarly possible to check the inherent flatness of the entirety of the surface of the said substrate.",
"FIG.",
"2 represents a diagram of an installation which allows a scan according to the invention of the surface of a window 8 in travel on a conveyor 9.The window 8 which arrives on the conveyor is first of all reorientated in a direction perpendicular to its direction of advancement on the conveyor 9, with the aid of two guides 10, 11.These guides 10, 11 will allow the window 8 to be orientated in the desired direction, the latter having already possessed an approximate orientation imposed when it was deposited onto the conveyor.",
"The trajectory of the window 8 is next interrupted by two limit stops 12 supported by an arm 13, the position of which can be altered in a vertical direction.",
"When the window 8 comes into contact with a first limit stop 12, it is blocked at this point and the conveyor causes the said window to pivot until it comes into contact with the second limit stop 12; the window then finds itself reorientated, in its direction of advancement, in a predefined position suitable for best effecting the comparison of the reflected image of the screen pattern with the reference screen pattern.",
"The device for lifting the arm 13 is next actuated such that the limit stops 12 no longer obstruct the passage of the window 8, which thus continues its travel on the conveyor 9.The device for lifting the arm 13 is advantageously actuated automatically as soon as the window 8 is in contact with the two limit stops 12.Moreover, the ascent of the limit stops 12 is designed to be very quick such that it does not risk damaging the window 8, which has its convex surface upwards and is therefore higher in its centre than on the edge which was in contact with the limit stops 12.The properly positioned window 8 then passes under a detector 14, for example an optical detector, which will determine the moment of triggering of the flash attached to the capture of an image of the window 8.This triggering of the flash occurs as soon as the window 8 is located beneath the test pattern 15, such that the image captured by the camera 16 of the test pattern 15 reflected on the window covers the entirety of the convex surface of the said window 8.The captured signal is next transmitted to a computer, in which it is processed according to the algorithms for “phase stepping”, conversion and derivation or integration calculation.",
"Thus, the scanning method of the invention allows large surfaces of m2 proportions, such as a window, to be scanned in dynamic state and with very good resolution in the order of 10 μm.",
"The results obtained lead to possible detection of the defects or to a knowledge of the rounding of the window.",
"This information can then be used to intervene in stages in the window production process and to remedy the defects.",
"In other cases and particularly for the realization of laminated windows, the results allow the quality of the window to be known in advance and possibly for the window not to be made if the optical properties are not satisfactory.",
"In the case of a window-curving process, the results obtained according to the invention in the course of realization of a curved window will allow better monitoring of the process and improvement of the production yield.",
"Where the inherent flatness of a window, intended for the realization of a visual display screen, is checked, the results obtained according to the invention will also bring about improved rates of production of the said screens, in that those windows whose inherent flatness is not satisfactory are scrapped before they find themselves on the production lines for these screens.",
"The device described above has been adopted by way of example, but the tools used can be more numerous, for example several cameras and test patterns, in particular where it is a case of scanning complex shapes having, for example, different curvatures.",
"These will be studied using several reflected images of a plurality of test patterns recorded by several cameras, the whole of the surface of the substrate having to be covered by reflected test patterns."
]
] | Patent_10432269 |
[
[
"Radio receiver",
"A radio receiver (30) comprises an ADC (13) including a clip counter.",
"The power of digitised signals provided by the ADC (13) is estimated by a power estimator (31), and an ideal gain value is computed from the power so estimated by a gain computation device (32).",
"Gain computation signals are fed to a gain control input of an amplifier (11) via an LPF (33).",
"A saturation detector (34) is connected to a clip counter output of the ADC (13), and to a control input of the LPF (33).",
"The saturation detector (34) is arranged when saturation of the ADC is detected to reduce the gain setting value by at least two steps, by which the gain of the amplifier is immediately reduced.",
"A detector detects the Doppler frequency of signals received and accordingly determines the size of the drop in amplification which is effected when saturation of the ADC (13) is detected.",
"The gain reduction may be 3 dB under very low Doppler shift conditions and 12 dB under very high Doppler shift conditions."
],
[
"1.A radio receiver comprising: a downconverter; a controllable gain amplifier connected to receive signals from the downconverter, the gain of the amplifier being controllable to adopt any of a plurality of discrete values in a series of steps; an analogue-to-digital converter, arranged to sample signals provided by the amplifier; and a monitor arranged to monitor signals provided by the analogue-to-digital converter and to reduce the gain of the amplifier by at least two steps in response to a predetermined level of saturation of the analogue-to-digital converter being detected.",
"2.A receiver as claimed in claim 1, in which the monitor is arranged to reduce the gain of the amplifier by an amount in the range 3 dB to 12 dB when the predetermined level of saturation is detected.",
"3.A receiver as claimed in claim 1, in which the monitor is arranged to reduce the gain of the amplifier by an amount dependent on the fading characteristics of the channel over which a received signal is transmitted.",
"4.A receiver as claimed in claim 3, in which the fading characteristics are estimated by a detector arranged to detect the Doppler frequency of the received signal.",
"5.A radio receiver comprising, in sequence: a downconverter; a controllable gain amplifier; an analogue-to-digital converter (ADC); a gain computation device arranged to provide a gain setting signal on the basis of the output signal of the ADC; and a filter having a gain setting memory device, the filter being arranged to filter the gain setting signal and to provide the filtered signal to a gain setting input of the controllable gain amplifier; and a monitor arranged to monitor signals provided by the ADC and to detect a predetermined level of saturation of the ADC thereform; wherein the output of the gain setting memory device is connected to its input and the gain setting memory device is in a first operating condition when the monitor detects said predetermined level of saturation, whereby the gain setting memory device reduces the gain of the amplifier by a predetermined amount.",
"6.A radio receiver as claimed in claim 5, wherein the gain of the amplifier is controllable to adopt any of a plurality of discrete values in a series of steps, and the gain setting memory device replaces the gain of the amplifier by at least two steps when the gain setting memory device is in the first operation condition.",
"7.A radio receiver as claimed in claim 5, wherein the gain setting memory device is in a second operating condition when the monitor does not detect said predetermined level of saturation.",
"8.A radio receiver according to claim 5, in which the reduction of the gain setting signal effects an amplifier gain reduction of between 3 dB and 12 dB.",
"9.A radio receiver according to claim 5, in which the extent of reduction of the gain setting signal is dependent on the fading characteristics of the channel over which a signal is received.",
"10.A radio receiver according to claim 9, in which the fading characteristics are estimated by a detector arranged to detect the Doppler frequency of the received signal.",
"11.A receiver as claimed in claim 2, in which the monitor is arranged to reduce the gain of the amplifier by an amount dependent on the fading characteristics of the channel over which a received signal is transmitted.",
"12.A radio receiver as claimed in claim 6, wherein the gain setting memory device is in a second operating condition when the monitor does not detect said predetermined level of saturation.",
"13.A radio receiver according to claim 6, in which the reduction of the gain setting signal effects an amplifier gain reduction of between 3 dB and 12 dB.",
"14.A radio receiver according to claim 7, in which the reduction of the gain setting signal effects an amplifier gain reduction of between 3 dB and 12 dB.",
"15.A radio receiver according to claim 6, in which the extent of reduction of the gain setting signal is dependent on the fading characteristics of the channel over which a signal is received.",
"16.A radio receiver according to claim 7, in which the extent of reduction of the gain setting signal is dependent on the fading characteristics of the channel over which a signal is received.",
"Page 5 17.A radio receiver according to claim 8, in which the extent of reduction of the gain setting signal is dependent on the fading characteristics of the channel over which a signal is received."
],
[
"This invention relates to a radio receiver and in particular, although not exclusively, to a code division multiple access radio receiver.",
"A known form of code division multiple access (CDMA) radio receiver 10 is shown in FIG.",
"1.Referring to FIG.",
"1, the radio receiver 10 includes a controllable gain amplifier 11 interposed between a radio circuit 12 including a downconverter (not shown), and an analogue-to-digital converter (ADC) 13.The radio circuit 12 receives radio frequency signals from an antenna 14.Digital signals are provided by the ADC 13 on an output 15, from which the signals are extracted for processing.",
"A power estimator 16 is also connected to the output of the ADC 13.The power estimator 16 examines the digital signals, and provides an output signal indicative of the power of the received signal at regular intervals, typically 30,000 times a second or so.",
"The power estimator 16 may be implemented in software or in hardware.",
"Since the power of the received signals depends to some extent on the information that is modulated onto it, the instantaneous output of the power estimator 16 is not truly indicative of the strength of the signal being received.",
"It is the signal strength which is of interest although, since this is not directly measurable, it is estimated from power estimations.",
"To avoid information-dependent changes in the power estimation having an effect on the signal strength estimation, it is usual to include a low-pass filter (LPF) 17 downstream of the power estimator 16.The LPF 17 may be a relatively simple device, in hardware or in software, which averages the signals provided by the power estimator 16 over time.",
"The average signals are provided on an output 18, from where they are fed to the input of a gain controller 19, which sets the gain setting of the controllable gain amplifier 11.The gain of the amplifier 11 is controllable to adopt any of a number of discrete regular steps of 0.5 dB from 10 dB to 80 dB.",
"It is usual to detect the averaged signals at the output, to compare the signals to a threshold level at regular intervals, and to increment or to decrement the gain of the amplifier depending whether the averaged signal is lower than or greater than the threshold respectively.",
"The aim is to keep the output of the amplifier 11 at a level at which the ADC 13 can work well.",
"In accordance with a first aspect of this invention there is provided a radio receiver comprising: a downconverter; a controllable gain amplifier connected to receive signals from the downconverter, the gain of the amplifier being controllable to adopt any of a plurality of discrete values in a series of steps; an analogue-to-digital converter, arranged to sample signals provided by the amplifier; and a monitor arranged to monitor signals provided by the analogue-to-digital converter and to reduce the gain of the amplifier by at least two of the steps if a predetermined level of saturation of the analogue-to-digital converter is detected.",
"A receiver constructed according to this aspect of the invention can offer improved performance, especially in fast fading channel environments.",
"There are two influencing factors.",
"Firstly, the invention does not need a low-pass filter in the path leading to amplifier gain reduction, which eliminates a cause of delay (filters necessarily delay signals).",
"Secondly, the amplifier gain may be reduced by more than a single step for each control interval, which provides advantages since it is possible for the strength of a received signal to rise at a rate greater than can be compensated for by conventional one-step incremental/decremental radio receivers.",
"Preferably, the monitor is arranged to reduce the gain of the amplifier by an amount dependent on the fading characteristics of the channel over which a received signal is transmitted.",
"This allows the construction of an adaptive radio receiver which reduces the amplifier gain by an amount which is appropriate for the channel.",
"In accordance with a second aspect of the invention, there is provided a radio receiver comprising, in sequence: a downconverter; a controllable gain amplifier an analogue-to-digital converter (ADC); a gain computation device, arranged to provide a gain setting signal on the basis of the sampled signal; and a filter device having a memory, the filter device being arranged to filter the gain setting signal and to provide the filtered signal to a gain setting input of the controllable gain amplifier, representation of the filtered gain setting signal being stored in the memory; a monitor arranged to monitor signals provided by the ADC and to detect a predetermined level of saturation of the ADC therefrom; and means to reduce the gain setting signal in response to the predetermined level of saturation being detected.",
"Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, of which: FIG.",
"1 shows schematically a prior art CDMA radio receiver; and FIG.",
"2 shows schematically a CDMA radio receiver according to a first embodiment of this invention; FIG.",
"3 shows schematically a CDMA radio receiver according to a second embodiment of this invention; and FIG.",
"4 shows schematically the low pass filter (LPF) of FIG.",
"3 in detail.",
"Referring to the drawings, FIG.",
"2 shows a radio receiver 20.Reference numerals have been retained from FIG.",
"1 for like elements.",
"Although not shown, the receiver 20 includes the power estimator 16 and the LPF 17 of FIG.",
"1 for conventional control of the gain of the amplifier 11, i.e.",
"in a single-step incremental/decremental manner.",
"The receiver 20 also includes a monitor 21, which is arranged to monitor the signals provided by the ADC 13.During normal operation, i.e.",
"where the channel has sufficiently low fading characteristics, operation is as described above with reference to FIG.",
"1.Where the channel fading characteristics are not so low, the ADC 13 may occasionally saturate, i.e.",
"the signal level at its input becomes equal to, or greater than, the maximum level that it can faithfully sample.",
"When the monitor 21 detects a predetermined level of saturation, from examination of the output of the ADC 13 for a number of digital signals corresponding to the maximum level, it provides a signal pulse on an output 22, which is connected to the gain controller 19.This signal pulse causes the gain of the amplifier 11 to be reduced by 6 dB, or twelve steps, instantly, although the reduction may be anywhere between 3 dB and 12 dB.",
"The ADC 13 subsequently receives signals from the amplifier 11, which signals are at a suitable level for analogue-to-digital conversion within the operating range of the ADC 13.Following this gain reduction, gain control is effected by the power estimator 16, the gain controller 19 and the LPF 17 until saturation of the ADC 13 is again detected.",
"Reducing the gain of the amplifier 11 in this way presents significant disadvantages.",
"In particular, one or two fifteenths of a frame of data may be corrupted to the extent that it is not recoverable.",
"Also, where the radio receiver 20 includes a rake receiver (a receiver in which plural rays are detected and subsequently combined), signal tracking for fingers of the rake receiver is not possible for a short but significant period of time.",
"However, the inventors feel that the advantages outweigh these disadvantages, the main advantage being that the negative effects of ADC saturation are substantially avoided.",
"As will be appreciated, these negative effects include high noise levels in the signals provided at the output 15, and interference with the power control algorithm.",
"The latter may on occasion cause the transmitter power to be increased, further increasing the problem, so its avoidance is an advantage.",
"In a preferred embodiment (not shown) a detector detects the Doppler frequency of signals received, in a conventional manner, and accordingly determines the size of the drop in amplification which is effected when saturation of the ADC 13 is detected.",
"The larger the Doppler shift, the faster changing the channel fading characteristics are assumed to be and hence the larger the drop in gain.",
"It is expected that the gain may be dropped by 3 dB for very low Doppler shift conditions and by 12 dB for very high Doppler shift conditions in a typical radiotelephone receiver.",
"To detect saturation of the ADC 13, the monitor 21 typically examines the output of the ADC and counts the number of samples for which the ADC clips the maximum signal level which the ADC can provide.",
"The count is made for clips in both the positive and negative directions.",
"The number of clips are counted over an update period of 10,000 chips, which is the interval between updates of the gain of the amplifier 11, and the number of clips detected is compared to a threshold.",
"In this embodiment, the threshold is 1,000 chips, or 10% of the number of chips in an update period.",
"However, the threshold selected for a particular implementation depends on particularly the length of the update period and the resolution of the ADC 13.Alternatively, the ADC 13 may be designed so as to include its own clip detector, so that it is only clip counting and thresholding which is performed by the monitor 21.The monitor 21 may be called a saturation detector.",
"FIG.",
"3 shows a preferred CDMA radio receiver 30 according to the invention.",
"Referring to FIG.",
"3, the radio receiver 30 comprises an ADC 13 including a clip counter.",
"The power of digitised signals provided by the ADC 13 is estimated by a power estimator 31, and an ideal gain value is computed from the power so estimated by a gain computation device 32 in a known manner.",
"Gain computation signals are fed to a gain control input of the amplifier 11 via an LPF 33, which is shown in FIG.",
"4, described below.",
"A saturation detector 34 is connected to a clip counter output of the ADC 13, and to a control input of the LPF 33.The saturation detector 34 provides a logic “one” signal on its output when saturation of the AGC is determined in the manner described above with reference to FIG.",
"3, and a logic “zero” signal otherwise.",
"The LPF 33 is arranged, on receipt of a logic “one” signal from the saturation detector 34, to reduce the gain setting value by at least two steps, by which the gain of the amplifier is immediately reduced.",
"The LPF 33 is shown in more detail in FIG.",
"4.Referring to FIG.",
"4, the LPF 33 comprises a first input 35 which is connected to the output of the gain computation device 32, a second input 36 which is connected to the saturation detector 34, an output 37, first and second adders 38, 39, a controllable switch 40, a gain setting memory device 41 and first to third bit shifting devices 42 to 44.In normal operation, i.e.",
"when the saturation detector 34 provides a logic “zero” output, the switch 40 rests as shown in the figure.",
"In this condition, a gain setting output of the memory device 41 is connected to its own input via the first and second adders 38, 39.The gain setting number is first reduced by subtraction, in the first adder 38, of a fraction of the gain setting number, which fraction is provided by the first bit shifter 42.The first bit shifter 42, as with the second bit shifter 43, shifts the binary gain setting number to the right by n1 bits, effectively dividing the gain setting number by 2n1.The resulting number is then increased, in the second adder 39, by an amount equal to the gain setting signal provided by the gain computation device 32 divided by 2n1.The resulting number is then passed, via the switch 40, to the input of the AGC setting memory device 41 to set the gain setting number for the next gain setting period.",
"In steady state conditions, therefore, the output 37 shows a gain setting number which is equal to that of the input of the gain computation device 32.Where this input signal varies, the LPF 33 serves as a low pass filter, averaging its input signals to provide a smoothed output.",
"When the input signal changes rapidly, the LPF 33 does not react instantly, since its architecture introduces a delay, as is conventional with low pass filters.",
"The extent of the delay and the other main characteristics of the LPF 33 are determined by the value of n1 and the length of the gain setting number.",
"When a logic “one” signal is received at the second input 36, indicative of an ADC saturation condition, the controllable switch 40 is switched over.",
"In this position, the input of the AGC setting memory device 41 is connected to an output of the third bit shifter 44, which has its input connected to the output of the AGC setting memory device.",
"Accordingly, in this condition, the AGC setting memory device 41 receives at its input a gain setting number which is equal to its output gain setting number divided by 2n2.This effects a dramatic decrease in the gain setting number over a single gain setting period, which results in an immediate decrease in the gain setting of the amplifier 11.The value of n2 is chosen, having regard to the length of the gain setting number, to result in the amplifier gain being reduced by an amount in the range 3 dB to 12 dB.",
"Preferably, the value of n2 is dynamically controllable, and is determined on the basis of a detected Doppler frequency of signals received; as is discussed above in relation to the FIG.",
"2 embodiment."
]
] | Patent_10432312 |
[
[
"Information storage apparatus and electronic device in which information storage apparatus is installed",
"There are disclosed an information storage apparatus and an electronic device mounting the information storage apparatus, wherein when the electronic device mounting an information storage element utilizing a magnetized direction control of a ferromagnetic material, for example a MRAM, receives something strong magnetic field, an erroneous storing of the information is prevented by preventing an influence of the magnetic field to a storage layer.",
"In an information storage apparatus (1) equipped with an information storage element (12) for storing information by utilizing a magneto-resistive effect, a resin material (13) used when the information storage element (12) is mounted is the one being mixed up a high-permeability material, or the information storage element (12) is the one formed with a high-permeability material film or a thin film including a high-permeability material on a part or an entire front surface thereof."
],
[
"1.An information storage apparatus including an information storage element for storing information by utilizing a magneto-resistive effect, said information storage apparatus is characterized in which: a resin material used when said information storage element is mounted includes a high-permeability material.",
"2.The information storage apparatus as cited in claim 1, characterized in which: said resin material is formed at least a part or entire surface of said information storage element.",
"3.An information storage apparatus including an information storage element for storing information by utilizing a magneto-resistive effect, said information storage apparatus is characterized in which: said information storage element is formed with a high-permeability material film, or a thin film including a high-permeability material on a part or an entire surface of a front surface thereof.",
"4.The information storage apparatus as cited in claim 3, characterized in which: said thin film including the high-permeability material includes a resin film including a high-permeability material.",
"5.An information storage apparatus including an information storage element for storing information by utilizing a magneto-resistive effect, said information storage apparatus is characterized in which: a substrate on which said information storage element is mounted is formed by mixing up a high-permeability material.",
"6.An information storage apparatus including an information storage element for storing information by utilizing a magneto-resistive effect, said information storage apparatus is characterized in which: a substrate on which said information storage element is mounted is formed with a high-permeability material film or a thin film including a high-permeability material on a part or an entire surface of a front surface thereof.",
"7.The information storage apparatus as cited in claim 6, characterized in which: said thin film including the high-permeability material includes a resin film including a high-permeability material.",
"8.An information storage apparatus including an information storage element for storing information by utilizing a magneto-resistive effect, said information storage apparatus is characterized in which: a radiator of said information storage apparatus includes at least a high-permeability material.",
"9.The information storage apparatus as cited in claim 6, characterized in which: a part or all of said radiator is formed with a high-permeability material.",
"10.An information storage apparatus including an information storage element for storing information by utilizing a magneto-resistive effect, said information storage apparatus is characterized in which: a radiator of said information storage apparatus includes at least a high-permeability material.",
"11.The information storage apparatus as cited in claim 10, characterized in which: said thin film including said high-permeability material is made of a resin film including a high-permeability material.",
"12.An electronic device mounting an information storage apparatus which is equipped with an information storage element for storing information utilizing a magneto-resistive effect; said electronic device is characterized in which: a housing of said electronic device is at least made of the one including a high-permeability material.",
"13.An electronic device mounting an information storage apparatus equipped with an information storage element for storing information by utilizing magneto-resistive effect, said electronic device mounting an information storage apparatus is characterized in which a housing of said electronic device is formed with a high-permeability material film or a thin film including a high-permeability material at least a part or entire surface of a front surface and a back surface thereof.",
"14.The electronic device mounting the information storage apparatus as cited in claim 13, characterized in which: said thin film including said high-permeability material is made of a resin film including a high-permeability material."
],
[
"<SOH> BACKGROUND TECHNOLOGY <EOH>With the rapid spread of a communication device, and particularly a small device for personal use such as a mobile terminal, further high performance such as high integration high speed, low power consumption and the like is required to a memory element and logic element constructing this.",
"Particularly, a non-volatile memory is thought to be absolutely essential for ubiquitous era.",
"Even in the case where a shortage of power source, something trouble, or a server and a network are cut their connections due to failure, such non-volatile memory is able to protect important personal information.",
"Further, recently, a portable device is so designed as to reduce power consumption as much as possible by carrying out a standby condition to un-necessary circuit block, and if it is possible to realize such non-volatile memory that is able to combine with a work memory and a large sized storage memory, then it becomes possible to reduce the waste of power consumption and memory.",
"Further, so-called [Instant-on] function that is able to activate at once when the power is made on becomes possible if a high speed large sized non-volatile memory is realized.",
"As a non-volatile memory, a semiconductor flash memory and FRAM (Ferro-electric Random Access Memory) using Ferro-electric substance are known.",
"However, it is difficult to apply a high integration to a flash memory due to its complex configuration, and further it has a defect that the access time is only around 100 ns.",
"On the contrary, it has been pointed out the problem that a FRAM has the possible re-writable number of from 10 12 times to 10 14 times and has a low durability so that it is difficult to completely replace with a static random access memory or a dynamic random access memory.",
"Further, it is also pointed out such problem that a microfabrication of a Ferro-electric capacitor is difficult.",
"It is a magnetic memory that is so-called MRAM (Magnetic Random Access Memory) having an attention as a non-volatile memory having no such defects, and is described in the article [Wang et al., IEEE Trans.",
"Magn.",
"33 (1997) p4498], and it also has been keen attention owing to an improvement of characteristics in recent TMR (Tunnel Magneto-resistance) material.",
"The MRAM is easy to apply a high integration as it has a simple configuration, and it is expected that the number of re-writing number becomes large because it carries out the storage by the rotation of a magnetic moment.",
"Further, an extremely high speed is expected as to access time, and already, the operation with 100 Mhz has been reported in the article [R. Scheuerlein et al, ISSCC Digest of Papers (February 2000) p128-129].",
"Further it has been greatly improved in the recent year where it becomes possible to get high power due to TMR (Tunnel Magnetic Resistance) effect.",
"As described above, although the MRAM has advantages in the easiness of high speed and high integration, its writing is carried out with a generated magnetic field by flowing a current through a writing bit line and a writing word line provided near the TMR element.",
"A reverse magnetic field in a storage layer of the TMR element is necessary to be about 20 Oe to 200 Oe, although it depends on material, and the current at this time becomes several mA to several 10 mA.",
"This causes the increase of the consuming current and becomes a large problem when carrying out a low power consumption of a portable device.",
"Accordingly, a development of a material and a configuration that makes the reverse magnetic field lower has been promoted.",
"In addition, a floating magnetic field in a natural world is several Oe, and by the reduction of the reverse magnetic field, a magnetic noise margin becomes small, and accordingly, this easily invite errors, on the contrary, by the effects of the magnetic noises inside the element and outside the element.",
"When the MRAM utilizing a magnetized direction control of a ferromagnetic material is installed in a plenty of electronic devices instead of the DRAM in the near future, and when a something strong magnetic field is generated in the vicinity of the MRAM, it is possible to store erroneous data by the rotation of the magnetization in the storage layer due to the strong magnetic field.",
"In order to put into practical use the information storage element utilizing the magnetized direction control of the ferromagnetic material like MRAM, for example, it is necessary to shield the MRAM so as to avoid any effect from the external magnetic field."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a schematic constructive sectional view of a first mode for carrying out an information storage apparatus of the present invention.",
"FIG.",
"2 is a schematic constructive sectional view of a second mode for carrying out an information storage apparatus of the present invention.",
"FIG.",
"3 is a schematic constructive sectional view of a forth mode for carrying out an information storage apparatus of the present invention.",
"FIG.",
"4 is a schematic constructive sectional view of showing another embodiment of the forth mode for carrying out in FIG.",
"4 .",
"FIGS.",
"5A and 5B are external views of an electronic device of a mode for carrying out the present invention, and FIG.",
"5C is a partial sectional view.",
"FIG.",
"6 is a partial sectional view showing a mode for carrying out related to an electronic device of the present invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD This invention relates to an information storage apparatus and an electronic device in which the information storage apparatus is mounted, and more particularly to an information storage apparatus equipped with information storage elements for storing information utilizing a magneto-resistive effect and applied a magnetic shield, and to an electronic device in which the information storage apparatus is mounted.",
"BACKGROUND TECHNOLOGY With the rapid spread of a communication device, and particularly a small device for personal use such as a mobile terminal, further high performance such as high integration high speed, low power consumption and the like is required to a memory element and logic element constructing this.",
"Particularly, a non-volatile memory is thought to be absolutely essential for ubiquitous era.",
"Even in the case where a shortage of power source, something trouble, or a server and a network are cut their connections due to failure, such non-volatile memory is able to protect important personal information.",
"Further, recently, a portable device is so designed as to reduce power consumption as much as possible by carrying out a standby condition to un-necessary circuit block, and if it is possible to realize such non-volatile memory that is able to combine with a work memory and a large sized storage memory, then it becomes possible to reduce the waste of power consumption and memory.",
"Further, so-called [Instant-on] function that is able to activate at once when the power is made on becomes possible if a high speed large sized non-volatile memory is realized.",
"As a non-volatile memory, a semiconductor flash memory and FRAM (Ferro-electric Random Access Memory) using Ferro-electric substance are known.",
"However, it is difficult to apply a high integration to a flash memory due to its complex configuration, and further it has a defect that the access time is only around 100 ns.",
"On the contrary, it has been pointed out the problem that a FRAM has the possible re-writable number of from 1012 times to 1014 times and has a low durability so that it is difficult to completely replace with a static random access memory or a dynamic random access memory.",
"Further, it is also pointed out such problem that a microfabrication of a Ferro-electric capacitor is difficult.",
"It is a magnetic memory that is so-called MRAM (Magnetic Random Access Memory) having an attention as a non-volatile memory having no such defects, and is described in the article [Wang et al., IEEE Trans.",
"Magn.",
"33 (1997) p4498], and it also has been keen attention owing to an improvement of characteristics in recent TMR (Tunnel Magneto-resistance) material.",
"The MRAM is easy to apply a high integration as it has a simple configuration, and it is expected that the number of re-writing number becomes large because it carries out the storage by the rotation of a magnetic moment.",
"Further, an extremely high speed is expected as to access time, and already, the operation with 100 Mhz has been reported in the article [R. Scheuerlein et al, ISSCC Digest of Papers (February 2000) p128-129].",
"Further it has been greatly improved in the recent year where it becomes possible to get high power due to TMR (Tunnel Magnetic Resistance) effect.",
"As described above, although the MRAM has advantages in the easiness of high speed and high integration, its writing is carried out with a generated magnetic field by flowing a current through a writing bit line and a writing word line provided near the TMR element.",
"A reverse magnetic field in a storage layer of the TMR element is necessary to be about 20 Oe to 200 Oe, although it depends on material, and the current at this time becomes several mA to several 10 mA.",
"This causes the increase of the consuming current and becomes a large problem when carrying out a low power consumption of a portable device.",
"Accordingly, a development of a material and a configuration that makes the reverse magnetic field lower has been promoted.",
"In addition, a floating magnetic field in a natural world is several Oe, and by the reduction of the reverse magnetic field, a magnetic noise margin becomes small, and accordingly, this easily invite errors, on the contrary, by the effects of the magnetic noises inside the element and outside the element.",
"When the MRAM utilizing a magnetized direction control of a ferromagnetic material is installed in a plenty of electronic devices instead of the DRAM in the near future, and when a something strong magnetic field is generated in the vicinity of the MRAM, it is possible to store erroneous data by the rotation of the magnetization in the storage layer due to the strong magnetic field.",
"In order to put into practical use the information storage element utilizing the magnetized direction control of the ferromagnetic material like MRAM, for example, it is necessary to shield the MRAM so as to avoid any effect from the external magnetic field.",
"DISCLOSURE OF THE INVENTION As above described, an information storage apparatus and electronic device mounted with the its information storage apparatus of the present invention is equipped with one of resin including high-permeability material, a high-permeability material film, a thin film including a high-permeability material, a substrate formed with a high-permeability material, and a substrate formed with resin including a high-permeability material, so that it is possible to shield the information storage apparatus from any external magnetic field.",
"Accordingly, it is possible to improve a reliability of the electronic device that includes the information storage apparatus, and the electronic device is able to be put into a practical use.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a schematic constructive sectional view of a first mode for carrying out an information storage apparatus of the present invention.",
"FIG.",
"2 is a schematic constructive sectional view of a second mode for carrying out an information storage apparatus of the present invention.",
"FIG.",
"3 is a schematic constructive sectional view of a forth mode for carrying out an information storage apparatus of the present invention.",
"FIG.",
"4 is a schematic constructive sectional view of showing another embodiment of the forth mode for carrying out in FIG.",
"4.FIGS.",
"5A and 5B are external views of an electronic device of a mode for carrying out the present invention, and FIG.",
"5C is a partial sectional view.",
"FIG.",
"6 is a partial sectional view showing a mode for carrying out related to an electronic device of the present invention.",
"BEST MODE FOR CARRYING OUT THE INVENTION A first mode for carrying out an information storage apparatus of the present invention is explained with reference to a schematic constructive partial sectional view of FIG.",
"1.In FIG.",
"1, a MRAM integrated circuit is employed as an information storage element that memorizes information utilizing a magneto-resistive effect, and is depicted a state before mounting in a package.",
"As shown in FIG.",
"1, on the substrate 11, there is mounted an information storage element 12.The information storage element 12 is electrically connected with a terminal (not shown in the figure) formed on the substrate 11 and a lead 14.Further on the substrate 11, a resin material 13 covering the information storage element 12 is formed, and the information storage element 12 is sealed by the resin material 13 and the substrate 11.The above mentioned resin material film 13 is made by dispersing powder of a high-permeability material having a magnetic permeability of 3.5 or more to a polyimide film by 20 wt.",
"% or more, and 95 wt.",
"% or less.",
"When a containing ratio of the above high-permeability material becomes less than 20 wt.",
"%, it becomes difficult to obtain an adequate magnetic shield effect.",
"Further, if the containing ratio of the high-permeability material exceeds 95 wt.",
"%, it becomes difficult to disperse it into the resin.",
"Accordingly, the containing ratio of the high-permeability material relative to the resin is set as mentioned above.",
"As the high-permeability material, a permalloy, an alphenol of the synthesized magnetic material, a sendust, a high density ferrite of a ferrite material, a single crystal ferrite, a hot-press ferrite, a general sintered ferrite, a magnetic pure iron, a silicon steel, or the like can be used.",
"Further the above-mentioned resin material 13 may be formed, although not shown in the figure, on an opposite side (back surface side) relative to the side where the information storage element 12 of the substrate 11 is mounted.",
"Or, as shown in the above-mentioned FIG.",
"1, the resin material 13 may be provided as to cover the information storage element 12, and may also be formed at the back surface side of the substrate 11.The resin material 13 including the above mentioned high-permeability material is formed by mixing up the power of the high-permeability material into the melted resin within a solvent, and by coating.",
"It is preferable to cure the resin by baking in order to evaporate the solvent in the resin.",
"Thus configured information storage apparatus 1 not only protects the information storage element 12 by the resin material 13, but also is possible to magnetically shield an upside of the information storage element 12 utilizing the magnetized direction control of the ferromagnetic material.",
"Further, in the configuration where the resin material 13 is provided at the back surface of the substrate 11, it is possible to magnetically shield the substrate 11 side of the information storage element 12.Next, a second mode for carrying out the information storage apparatus according to the present invention is explained with reference to a schematic constructive partial sectional view in FIG.",
"2.In FIG.",
"2, a MRAM integrated circuit is used as an information storage element for storing information by utilizing the magneto-resistive effect, and is shown a state before it is installed in a package.",
"As shown in FIG.",
"2, on the substrate 11, there is mounted an information storage element 12.The information storage element 12 is electrically connected with a terminal (not shown in the figure) formed on the substrate 11 and a lead 14.Further, on the substrate 11, a protective film 15 covering the information storage element 12 is formed, and the information storage element 12 is sealed by the protective film 15 and the substrate 11.At a front surface of the protective film 15, a high-permeability material film 16 is formed.",
"A film thickness thereof is set to be the thickness suitable for functioning a magnetic protection (magnetic shield) effect.",
"For example, if the high-permeability material film 16 is formed with a permalloy, the thickness thereof is set to be from 0.1 μm to 1000 μm.",
"As the high-permeability material, a thin film of an alphenol of the synthesized magnetic material, a sendust, a high density ferrite of a ferrite material, a single crystal ferrite, a hot-press ferrite, a general sintered ferrite, a magnetic pure iron, a silicon steel, or the like may be employed in addition to the permalloy.",
"Further, a resin film including a high-permeability material may be formed instead of the high-permeability material film 16.The resin film as mentioned above may be made by dispersing a powder of a high-permeability material having a magnetic permeability of 3.5 or more to a polyimide film by the amount of 20 wt.",
"% or more and 95 wt.",
"% or less.",
"When a containing ratio of the above high-permeability material becomes less than 20 wt.",
"%, it becomes difficult to obtain an adequate magnetic shield effect.",
"Further, if the content of the high-permeability material exceeds 95 wt.",
"%, it becomes difficult to disperse it into the resin.",
"Accordingly, the content of the high-permeability material relative to the resin is set as mentioned above.",
"A powder of a high-permeability material used for the above resin film may be employed the powder similar to that explained before.",
"The above high-permeability material film 16 may be formed by, for example, a film forming technology based on a PVD (Physical Vapor Deposition) method such as a sputtering, a deposition method or the like.",
"The resin material including the above mentioned high-permeability material is formed by mixing up the powder of the high-permeability material into the melted resin by a solvent, and by coating.",
"It is preferable to cure the resin by baking in order to evaporate the solvent in the resin.",
"Further the above mentioned high-permeability material film 16 may be formed, although not shown in the figure, on an opposite side (back surface side) relative to the side where the information storage element 12 of the substrate 11 is mounted.",
"Or, as shown in the previous FIG.",
"2, the high-permeability material film 16 is provided as to cover a front surface of the protective film 15 which covers the information storage element 12, and may be also formed at the back surface side of the substrate 11.Thus configured information storage apparatus 2 protects the information storage element 12 by the protective film 15, and also is possible to magnetically shield the upside of the information storage element 12 by the high-permeability material film 16 (or the resin film including the high-permeability material).",
"Further in the configuration where the high-permeability material film 16 (or resin film including the high-permeability material) is provided at the back surface of the substrate 11, it is possible to magnetically shield the substrate 11 side of the information storage element 12.Next, a third mode for carrying out related to the information storage apparatus of the present invention is explained here-in-after.",
"In the information storage apparatuses 1 and 2 shown in the FIG.",
"1 and FIG.",
"2, it is possible to form the substrate 11 with the material in which a high-permeability material is mixed.",
"The resin substrate as mentioned above may be made by dispersing a powder of a high-permeability material having a magnetic permeability of 3.5 or more in an insulating organic resin such as a polystyrol, a bakelite, a polyimide, or the like by the amount of 20 wt.",
"% or more and 95 wt.",
"% or less.",
"When the containing ratio of the above high-permeability material becomes less than 20 wt.",
"%, it becomes difficult to obtain adequate magnetic shield effect.",
"Further, if the content of the high-permeability material exceeds 95 wt.",
"%, it becomes difficult to disperse it into the resin.",
"Accordingly, the content of the high-permeability material relative to the resin is set as mentioned above.",
"The above substrate 11 is formed by mixing up the powder of a high-permeability material in the resin dissolved by a solvent, forming, and curing.",
"As the above-mentioned high-permeability material, a high-permeability material similar to the one explained in the above is possible to be employed.",
"In the information storage apparatus configured as above, it is possible to magnetically shield the substrate 11 side of the information storage element 12 by constructing the substrate 11 with the one including the high-permeability material.",
"Next, a forth mode for carrying out related to the information storage apparatus of the present invention is explained with reference to a schematic constructive partial sectional view in FIG.",
"3, here-in-after.",
"FIG.",
"3 shows MRAM integrated circuit is employed as an information storage element for storing information by utilizing the magneto-resistive effect in a state before installed in a package.",
"As shown in FIG.",
"3, the information storage apparatus 12 is mounted on the substrate 11 made of a BGA (Ball Grid Array) substrate.",
"A radiator (heat sink) 17 is mounted on the information storage apparatus 12, and has a function of radiating heat of the information storage element 12.The above mentioned radiator (heat sink) 17 may be formed by including a high-permeability material.",
"For example, the radiator 17 is formed by a permalloy.",
"Or, the above mentioned radiator 17 may be formed by the one including a high-permeability material.",
"Or, the radiator 17 is to be the one made by forming a high-permeability material film on its front surface.",
"For example, in the radiator 17, a main body thereof is made of an aluminum or a copper having a high heat conductance, and the one where the front surface thereof is coated by the high-permeability material film.",
"Or, the radiator 17 is to be the one formed of a thin film including a high-permeability material.",
"For example, as a thin film including such high-permeability material, there is a resin film in which a powder of a high-permeability material is mixed up as explained before.",
"As the above-mentioned high-permeability material, a material similar to the one explained as above is able to be employed.",
"Next, another embodiment related to the forth mode for carrying out is explained with FIG.",
"4.As shown in FIG.",
"4, the information storage element 12 is mounted on the substrate 11 made of a BGA (Ball Grid Array) substrate.",
"A radiator (heat spreader) 18 is formed on the information storage element 12, and has a function of radiating a heat of the information storage element 12.The radiator (heat spreader) 18 is made of a high-permeability material.",
"For example, the radiator 18 is formed with a permalloy.",
"Or, the radiator 18 is made of the one including a high-permeability material on its front surface.",
"Or, the radiator 18 is to be the one made by forming a high-permeability material film on its front surface.",
"For example, in the radiator 18, a main body thereof is made of an aluminum or a copper having a high heat conductance, and the one where the front surface thereof is coated by the high-permeability material film.",
"Or, the radiator 18 is to be the one formed of a thin film including a high-permeability material.",
"For example, as a thin film including such high-permeability material, there is a resin film in which a powder of a high-permeability material is mixed up as explained before.",
"As the above-mentioned high-permeability material, a material similar to the one explained as above is able to be employed.",
"In the information storage apparatus 3 configured as above, it is possible to magnetically shield an upside of the information storage element 12 with the radiator 17 (or 18).",
"One example of an electronic device mounting an information storage apparatus having an information storage element for storing information by utilizing the magneto-resistive effect is explained.",
"FIG.",
"5A shows an external view of a notebook type personal computer, and FIG.",
"5B shows an external view of a mobile phone.",
"In addition, FIG.",
"5C shows a partial sectional construction of the housing of the partial sectional construction, and a printed circuit board installed in the housing and mounting the information storage apparatus.",
"In an electronic device such as a notebook type personal computer 31, a mobile phone 35 or the like as shown in above-mentioned FIG.",
"5A and FIG.",
"5B, a printed circuit board 21 mounting the information storage apparatus 1 for storing information by utilizing the magneto-resistive effect is protected by the housing 41.The above-mentioned housing 41 is itself formed with a high-permeability material.",
"As the high-permeability material to be used in the above-mentioned housing 41, a permalloy, an alphenol of the synthesized magnetic material, a sendust, a high density ferrite of a ferrite material, a single crystal ferrite, a hot-press ferrite, a general sintered ferrite, a magnetic pure iron, a silicon steel, or the like may be employed.",
"Or, the above mentioned housing 41 is to be the one including at least a high-permeability material.",
"For example, it is made of a resin including a powder of the high-permeability material.",
"The resin as mentioned above may be made by dispersing a powder of a high-permeability material having a magnetic permeability of 3.5 or more in a resin such as an ABS resin, an AS resin, a PBT resin, a polyimide resin, a polyamide resin, an epoxy resin, a methacryl resin, or the like by the amount of 20 wt.",
"% or more and 95 wt.",
"% or less.",
"When a containing ratio of the above high-permeability material becomes less than 20 wt.",
"%, it becomes difficult to obtain an adequate magnetic shield effect.",
"Further, if the content of the high-permeability material exceeds 90 wt.",
"%, it becomes impossible to maintain the strength of the resin.",
"Accordingly, the content of the high-permeability material relative to the resin is set as mentioned above.",
"As the powder of the high-permeability material to be used in the above-mentioned housing 41, a powder of a permalloy, an alphenol of the synthesized magnetic material, a sendust, high density ferrite of a ferrite material, a single crystal ferrite, a hot-press ferrite, a general sintered ferrite, a magnetic pure iron, a silicon steel, or the like may be employed.",
"As shown in a partial sectional view in FIG.",
"6, inside of the housing 41, there is installed the printed circuit board 21 on which an electronic part such as the information storage apparatus 1 for storing information by utilizing the magneto-resistive effect is mounted, for example.",
"A high-permeability material film (or a thin film containing a high-permeability material) may be formed on a periphery of this information storage apparatus 1.Further, the high-permeability material film (or a thin film containing a high-permeability material) may be formed above inner surface of the housing 41 of the information storage apparatus 1.In the electronic device as above configured, a high-permeability material film (or a thin film containing a high-permeability material) is formed on either outer surface or inner surface of the housing 41, so that it is possible to magnetically shield the information storage apparatus 1.Further, a high-permeability material film (or a thin film containing a high-permeability material) is formed on a periphery of the information storage apparatus 1, so that it is possible to magnetically shield the information storage apparatus 1.It is possible to simultaneously carry out several ways of the magnetic shield explained in each mode of carrying out in the above.",
"By carrying out plural ways of the magnetic shield, it is possible to carry out the magnetic shield more effectively.",
"In the above-described each mode for carrying out, an electronic device mounting an information storage apparatus for storing information utilizing a magneto-resistive effect is explained, and also by applying a thin film including a high-permeability material film or a high-permeability material to an electronic device to which a DRAM is mounted, it is possible to obtain such magnetic shield effect, for example.",
"The resin including the high-permeability material, the high-permeability material film, the thin film including the high-permeability material, or the like are formed as described below.",
"For example, when a part material surrounding the information storage element is a resin based material, the above mentioned part material is formed by mixing up a powder of a high-permeability material (permalloy, for example) into a liquid or a liquid conditioned resin, and by forming it.",
"If the part material surrounding the information storage element is a ceramic based material, the above mentioned part material is formed by mixing up a powder of a high-permeability material (permalloy, for example) into a ceramic powder material, and by sintering them.",
"Along a front surface of the part material surrounding the information storage element, the high-permeability material is possible to be formed to be a thin film form by a film forming technology such as a sputtering, a deposition method, or the like.",
"Or, it may be a method for spray-coating a powder of a high-permeability material.",
"The above-mentioned various forming methods are possible to be applied to all of the above-explained high-permeability materials.",
"There are many types of high-permeability material for shielding the information storage element as mentioned above.",
"A magnetic shield effect is proportional to a magnetic permeability of these high-permeability materials.",
"Therefore, when the external magnetic field is small and the magnetic saturation of the material is not a matter of problem, a larger magnetic shield effect is obtained by using a high-permeability material.",
"On the contrary, when the external magnetic field is large, the reduction of a magnetic permeability is concerned, so that it is effective to use a material having a high saturation flux density among high-permeability materials, for example, the material such as a magnetic pure iron, a silicon steel, or the like.",
"APLICABILITY IN THE INDUSTRY The present invention is an information storage apparatus and an electronic device mounting the information storage apparatus being carried out in order to solve the above-mentioned problems.",
"In the above-mentioned information storage apparatus, the information storage element is magnetically shielded from an external magnetic field by utilizing a resin including a high-permeability material or a thin film including a high-permeability material so that the improvement in the reliability of the information storage apparatus is expected.",
"In the information storage apparatus, the information storage element is magnetically shielded from an external magnetic field by utilizing a substrate including a high-permeability material, or a substrate being formed thereon a high-permeability material film or a thin film including a high-permeability material, so that the improvement in the reliability of the information storage apparatus is expected.",
"In the above mentioned information storage apparatus, the information storage element is magnetically shielded from an external magnetic field by utilizing a radiator including a high-permeability material, or the one on which a high-permeability material film is formed or a thin film including a high-permeability material is used at front surface, so that the improvement in the reliability of the information storage apparatus is expected.",
"In the electronic device mounting the above mentioned information storage apparatus, the information storage element is magnetically shielded from an external magnetic field by using the housing of the electronic device including a high-permeability material, or the housing which is formed with a high-permeability material film or a thin film including a high-permeability material on at least a part or entire surface of front and or back surface thereof, so that the improvement in the reliability of the electronic device is expected."
]
] | Patent_10432330 |
[
[
"Fabrication of integrated circuit",
"A method of fabricating an integrated device on a chip comprising first and second features (A, B), the second feature, B, having greater dimension and/or being of coarser design than the first feature A.",
"The method involves the steps of: depositing a resist onto the chip, the resist being of a type that forms a thinner deposit on larger or coarser features than on smaller or finer features; treating the resist in dependence upon the thickness thereof to render it susceptible to a subsequent etching step, the thicker areas of resist being treated for a longer period of time or by a more intense treatment than the thinner areas of resist; and etching the treated areas of the resist to form a mask for use in the fabrication of said first and second features (A, B), on the chip."
],
[
"1.A method of fabricating an integrated device on a chip comprising first and second features the second feature having greater dimensions and/or being of coarser design than the first feature, the method involving the steps of: depositing a resist onto the chip, the resist being of a type that forms a thinner deposit on larger or coarser features than on smaller or finer features; treating the resist in dependence upon the thickness thereof to render it susceptible to a subsequent etching step, the thicker areas of resist being treated for a longer period of time or by a more intense treatment than the thinner areas of resist; and etching the treated areas of the resist to form a mask for use in fabrication of said first and second features on the chip.",
"2-20.(Cancelled).",
"21.The method as claimed in claim 1 wherein a first etch step is used to at least partially define a first mask, and a second etch step is subsequently used to define further the first and second features through the mask formed from said treated areas of resist.",
"22.The method as claimed in claim 21 wherein the first mask comprises at least one relatively narrow etch window for fabrication of the first feature and at least one relatively wide etch window for fabrication of the second feature.",
"23.The method as claimed in claim 21 wherein the first etch step comprises a dry etch process.",
"24.The method as claimed in claim 21 wherein the first etch step comprises etching through a mask formed in an oxide layer.",
"25.The method as claimed in claim 24 wherein said treatment comprises exposing selected areas of resist to ultra-violet light.",
"26.The method as claimed in claim 25 wherein said selected areas of resist are etched away following exposure to ultra-violet light.",
"27.The method as claimed in claim 25 wherein said resist comprises a resin which is applied in liquid form.",
"28.The method as claimed in claim 1 wherein two areas of the resist are defined, a first area which includes said first feature and a second area which includes said second feature, the first area being subjected to said treatment for a longer period of time and/or by a more intense treatment than said second area, the integrated circuit to be formed comprising a component which extends from the first area to the second area wherein at least one substantially non-functional feature is formed on the component at a position where the first and second areas meet.",
"29.The method as claimed in claim 28 wherein said component is a rib waveguide and the substantially non-functional feature comprises a projection formed on each side of the waveguide at the position where the first and second areas meet.",
"30.The method as claimed in claim 1 wherein the integrated device comprises an optical device formed in silicon.",
"31.The method as claimed in claim 30 wherein the device is formed on a silicon-on-insulator chip.",
"32.The method as claimed in claim 1 wherein said second feature has greater dimensions in a direction perpendicular to the plane of the chip than said first feature.",
"33.The method as claimed in claim 32 wherein the first feature has a dimension, from a top surface thereof to the base thereof, in a direction perpendicular to the plane of the chip, of at least 2 microns.",
"34.The method as claimed in claim 1 wherein the first feature comprises a tapered rib waveguide.",
"35.The method as claimed in claim 1 wherein the second feature comprises a rib waveguide having substantially parallel sides.",
"36.An integrated optical device fabricated by a method as claimed in claim 1, the device comprising, two areas, a first area which includes said first feature and second area which includes said second feature, and comprising a component which extends from the first area to the second area wherein at least one substantially non-functional feature is formed on the component at a position where the first and second areas meet.",
"37.An integrated optical device as claimed in claim 36 wherein said component comprises a rib waveguide and said non-functional feature comprises a projection formed on each side of the waveguide."
],
[
"<SOH> BACKGROUND ART <EOH>The fabrication of integrated circuits, e.g.",
"comprising optical waveguides in silicon, is well established using conventional semi-conductor technologies such as wet and dry etching.",
"However, some devices require a mixture of relatively coarse and relatively fine features to be formed on the same chip.",
"This can pose problems in the fabrication of the device because of the physical properties inherent in the masking materials commonly used.",
"This problem is exemplified by the masking procedure associated with making a fine taper of the type used to provide a low loss optical coupling between an optical fibre and a silicon waveguide, e.g.",
"of the type described in U.S. Pat.",
"No.",
"6,108,478."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>The invention will now be further described merely by way of example, with reference to the accompanying drawings, in which: FIG.",
"1 is a perspective view of an integrated optical device according to one embodiment of the invention; FIG.",
"2 is a plan view of the device shown in FIG.",
"1 ; FIGS.",
"3 and 4 illustrate a series of steps in the fabrication of a first and second areas, respectively, of a device such as that shown in FIGS.",
"1 and 2 .",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD This invention relates to a method of fabricating an integrated circuit and to an integrated optical circuit fabricated thereby.",
"BACKGROUND ART The fabrication of integrated circuits, e.g.",
"comprising optical waveguides in silicon, is well established using conventional semi-conductor technologies such as wet and dry etching.",
"However, some devices require a mixture of relatively coarse and relatively fine features to be formed on the same chip.",
"This can pose problems in the fabrication of the device because of the physical properties inherent in the masking materials commonly used.",
"This problem is exemplified by the masking procedure associated with making a fine taper of the type used to provide a low loss optical coupling between an optical fibre and a silicon waveguide, e.g.",
"of the type described in U.S. Pat.",
"No.",
"6,108,478.DISCLOSURE OF INVENTION This invention seeks to provide a method of fabricating an integrated circuit, and a device fabricated thereby, which helps reduce such problems.",
"A method of fabricating an integrated device on a chip comprising first and second features the second feature having greater dimensions and/or being of coarser design than the first feature, the method involving the steps of: depositing a resist onto the chip, the resist being of a type that forms a thinner deposit on larger or coarser features than on smaller or finer features; treating the resist in dependence upon the thickness thereof to render it susceptible to a subsequent etching step, the thicker areas of resist being treated for a longer period of time or by a more intense treatment than the thinner areas of resist; and etching the treated areas of the resist to form a mask for use in fabrication of said first and second features on the chip.",
"According to a second aspect of the invention, there is provided an integrated optical device fabricated by the above method, the device comprising, two areas, a first area which includes said first feature and a second area which includes said second feature, and comprising a component which extends from the first area to the second area wherein at least one substantially non-functional feature is formed on the component at a position where the first and second areas meet.",
"Other preferred and optional features of the invention will be apparent from the following description and the subsidiary claims of the specification.",
"BRIEF DESCRIPTION OF DRAWINGS The invention will now be further described merely by way of example, with reference to the accompanying drawings, in which: FIG.",
"1 is a perspective view of an integrated optical device according to one embodiment of the invention; FIG.",
"2 is a plan view of the device shown in FIG.",
"1; FIGS.",
"3 and 4 illustrate a series of steps in the fabrication of a first and second areas, respectively, of a device such as that shown in FIGS.",
"1 and 2.BEST MODE OF CARRYING OUT THE INVENTION The integrated optical device shown in FIGS.",
"1 and 2 is fabricated on a silicon-on-insulator (SOI) chip, comprising a layer of silicon 1 separated from a substrate 2, which may also be of silicon, by an insulating layer 3, typically of silicon dioxide.",
"The device illustrated comprises a rib waveguide 4 with a tapered waveguide structure at one end thereof comprising a wedge shaped portion 5 formed on top of a tapering portion 4A of the rib waveguide 4.These latter two components 4, and 4A, are defined between trenches 6 and 7 etched in the silicon layer 1.A tapered waveguide structure somewhat similar to that shown is further described in U.S. Pat.",
"No.",
"6,108,478, the disclosure of which is incorporated herein.",
"The rib of the rib waveguide 4 typically has a height R (measured from the bottom of the trenches 6 and 7 to the top surface of the rib) of about 1.5 microns.",
"The wedge shaped portion 5 typically has a height W (measured from the top surface of the rib to the top surface of the wedge shaped portion) of about 5 microns but, in some cases, may be 8 microns or more, and a length of about 1000 microns.",
"The trenches 6 and 7 typically have a width of about 60 microns and the layer of silicon 1 between the bottom of the trenches 6 and 7 and the oxide layer 3 is typically about 2.8 microns thick.",
"Such a wedge shaped structure is used to provide a low loss coupling between an optical fibre (not shown) and the rib waveguide 4 as discussed in U.S. Pat.",
"No.",
"6,108,478.Fabrication of such a structure in which the total etch depth from the upper surface of the silicon layer 1 to the bottom of the trenches 6 and 7 may typically be in the range 6 to 10 microns presents difficulties as the accuracy with which features can be formed decreases with the increase in etch depth.",
"One solution to this difficulty is described in GB9924098.8 (publication No.",
"GB2355312A) the disclosure of which is also incorporated herein.",
"The fabrication process described herein provides an alternative solution to these difficulties.",
"A device such as that shown in FIGS.",
"1 and 2 is preferably etched in two-stages, a first stage in which the wedge shaped portion 5 is defined and the positions of the trenches 6 and 7 defined and a second stage in which the trenches 6 and 7 are etched to form the rib waveguide 4.Such a two-stage process will be described further with reference to FIGS.",
"3 and 4 in which FIG.",
"3 illustrates the steps in relation to a first area A of the device encompassing the wedge shaped structure at the end of the waveguide 4 and FIG.",
"4 illustrates the step in relation to a second area B of the device encompassing the part of the waveguide 4 having parallel sides.",
"The boundary between areas A and B is shown by the dashed line in FIGS.",
"1 and 2.In the first step illustrated by FIGS.",
"3(a) and 4(a), the surface of the silicon layer 1 is covered in appropriate regions by an oxide layer 8.Then, as shown in FIGS.",
"3(b) and 4(b), the oxide layer 8 is used as a mask for dry etching to define the wedge shaped portion 5 (shown in FIG.",
"3(b)) and the trenches 6 and 7, but only to a depth corresponding to the top surface of the rib of the rib waveguide 4.Then, as shown in FIGS.",
"3(c) and 4(c), an optical resist 9, e.g.",
"a photosensitive material such as Novalak™ resin, in liquid form, is spread across the device by a conventional spinning process and then baked.",
"Such a resist has a tendency to deposit as a thicker layer in fine, narrow features, than it does in coarse, wide features.",
"Thus, it deposits in a thicker layer in corners and crevices of the relatively narrow portions of trenches 6 and 7 on each side of the wedge shaped portion 5 in area A of the device (FIG.",
"3(c)) and in a thinner layer in the wider, flatter areas of the trench formed in area B of the device (FIG.",
"4(c)).",
"Areas of the resist 9 which are to be removed are then exposed to ultra violet (UV) light through a mask (not shown) in the areas being marked UV in FIGS.",
"3(c) and 4(c).",
"The areas of resist exposed to UV light are then removed by a leaching chemical as shown in FIGS.",
"3(d) and 4(d) to expose areas of the silicon layer 1 at the bottom of the trenches 6 and 7 formed in step (b).",
"These exposed areas of the silicon layer 1 are then etched by a conventional, typically dry, etch process as shown in FIGS.",
"3(e) and 4(e) to complete the etching of trenches 6 and 7 and thus form both portions 4 and 4A of the rib waveguide.",
"As shown in FIGS.",
"3(e) and 4(e), the areas of resist exposed to UV are approximately central within the trenches 6 and 7 where the thickness of the resist is fairly uniform and the areas are narrower than the width of the trenches.",
"This avoids the need to expose the very thick parts of the photoresist at the sides of the trenches 6 and 7.This results in steps 1A being left at the outer edges of the trenches 6 and 7 of similar height to the steps on the inner sides of the trenches 6 and 7 which form the tapered part 4A of the rib waveguide.",
"The steps 1A are also shown in FIG.",
"1.The remaining parts of the resist 9 are then removed by conventional means, e.g.",
"by a wet chemical etch or plasma strip, as illustrated in FIGS.",
"3(f) and 4(f) to leave a structure as shown in FIGS.",
"1 and 2.The device may then be subject to further process steps, e.g.",
"covering with an oxide layer, but these steps are not relevant to the present invention so will not be described further.",
"As indicated above, the resist 9 tends to deposit as a thicker layer in area A of the device that it does in area B (the difference is shown exaggerated in FIGS.",
"3(c) and 4(c).",
"To achieve satisfactory treatment of the resist so the relevant parts thereof are removed in step (d), the thicker areas of resist 9 in area A need to be subjected to UV light for a longer period of time than the thinner areas of resist 9 in area B.",
"Inadequate exposure of the resist would result in inadequate removal of resist in step (d) whereas over exposure of the resist results in a too wide an area of resist being removed as a result of stray (e.g.",
"reflected and refracted) UV light causing undesirable reaction in the resist adjacent the area intended to be removed.",
"Furthermore, if a thin area of exposed resist is treated with more leaching chemical than necessary to remove the exposed area, non exposed resist will start to be leached causing inaccurate and unreliable results.",
"The thicker area of resist may be subjected to a longer exposure time to UV light than the thinner areas, as mentioned above.",
"Alternatively, or additionally, the thicker areas of resist may be subjected to a more intense exposure to UV light that the thinner areas.",
"Thus, the manufacture of a device such as that shown in FIGS.",
"1 and 2 involves a multi-stage exposure process so that some areas are exposed more than other areas.",
"In the example described in relation to FIGS.",
"2 and 3, a two stage process in used: area A being subjected to a greater exposure of UV light than area B.",
"This may be achieved by exposing areas A and B separately or by exposing both areas together, and then subjecting area A, but not area B, to further exposure.",
"However, in more complex products, a greater number of areas may require differential exposure and a greater number of exposure stages may be used.",
"The use of such differential exposures depending on the thickness of the resist thus enables product quality to be improved and the usable chip yield to be increased.",
"The technique described above can be applied to any form of integrated circuit comprising both relatively small or fine features and relatively large or coarse features.",
"This is particularly the case in a device in which the coarse features have dimensions perpendicular to the plane of the chip of 5 or more microns.",
"However, it may also be applicable to devices in which the coarse features have dimensions perpendicular to the plane of the chip of at least 2 or 3 microns.",
"In practice, the dimensions of an area of resist to be exposed to UV light needs to be adjusted to allow for shrinkage of the resist which occurs after the exposure, developing and baking steps.",
"However, it is found that the degree of shrinkage varies with the thickness of the resist; the shrinkage tending to be greater with thicker layers of resist.",
"However, the degree of shrinkage is not easily predictable.",
"As a result of this, at the position where two areas subjected to different exposures meet, a discontinuity, e.g.",
"a small step or notch, tend to form due to the differential shrinkage of the resist either side of the boundary.",
"Such a discontinuity is undesirable as its size may be variable and it may cause a significant perturbation in operation of the device being fabricated.",
"For instance, in the case of an optical rib waveguide which extends from one area to the other area, a discontinuity in the width of the waveguide may arise where the waveguide crosses the boundary between the two areas.",
"Such a discontinuity or step in the side faces of a rib waveguide may give rise to a significant loss of light from the waveguide.",
"To overcome this problem, the initial mask may be designed to provide a substantially non-functional feature, which does not give rise to any significant perturbation of the operation of the device, at the boundary between the areas subjected to different exposures.",
"In the case of a rib waveguide discussed above, a small projection 15 may be provided on each side of the rib 4 at the point where the rib waveguide crosses the boundary between areas A and B, as shown in FIGS.",
"1 and 2.Such small projections have no significant effect upon an optical signal transmitted along the waveguide.",
"Thus, a perturbation in the size of the projections at the boundary between the two areas also has no significant effect upon the optical signal.",
"As shown in FIGS.",
"1 and 2, the boundary between areas A and B also coincides with the junction between the tapered section 4A of the rib waveguide in area A with the section 4 having parallel sides in area B.",
"The projections 15 are thus formed at this point.",
"Preferably, the projections 15 have a depth the same as that of the waveguide 4, as shown, a width of about 1.5 microns and extend along the boundary between areas A and B substantially perpendicular to the sides of the waveguide 4, for a distance of about 5 microns.",
"The projections 15 thus allows for small differences in shrinkage rate between two exposed areas by providing a transition or grading feature at the boundary between the two areas which does not cause significant light loss in the finished device.",
"It will be appreciated that the need to provide non-functional features, such as the projections 15, at the boundary between areas of the chip subjected to different exposures, will depend on the nature of the device and the nature of components which cross the boundary.",
"Thus, such features will not always be required and may be omitted where the perturbation caused by a discontinuity may be relatively small."
]
] | Patent_10432526 |
[
[
"Differential gear for vehicle",
"There is disclosed an automotive differential device for transferring a rotational drive force from an automotive engine to a drive axle.",
"This differential device includes: a differential gear mechanism equipped with a pair of side gears, three pinions meshing with the side gears, three pinion shafts pivotably supporting the three pinions, and a shaft supporting member supporting inner ends of the three pinion shafts; and a case member with an integral structure housing the differential gear mechanism.",
"This case member includes a flange section receiving the rotational drive force by way of a ring gear, and a plurality of openings for mounting the side gears and pinions.",
"The three pinions are oriented so that their axes form 120 degree phase angles, and the three openings are formed at three equidistant positions along the perimeter wall section of the case member."
],
[
"1.In an automotive differential device transferring a rotational drive force from an automotive engine to a drive axle, an automotive differential device comprising: a differential gear mechanism equipped with a pair of side gears, three pinions meshing with said side gears, three pinion shafts pivotably supporting said three pinions, respectively, and a shaft supporting member supporting inner ends of said three pinion shafts; and a case member with an integral structure housing said differential gear mechanism, said case member including a flange section receiving said rotational drive force by way of a ring gear, and a plurality of openings for introducing said side gears and pinions.",
"2.An automotive differential device as described in claim 1 wherein: said three pinions are disposed-so that axes thereof form 120 degree phase angles; and said plurality of openings is three openings formed at three equidistant positions along a perimeter wall section of said case member.",
"3.An automotive differential device as described in claim 2 wherein: said three openings are two first openings for introducing said three pinions and one second opening for consecutively introducing said two side gears.",
"4.An automotive differential device as described in claim 3 wherein: said first opening is a roughly D-shaped opening with a straight section on a side toward said flange; and said second opening is an opening with a shape different from that of said first opening, said shape being a special shape allowing said side gear to be introduced while in an inclined state when said side gear is mounted.",
"5.An automotive differential device as described in claim 4 wherein said flange section is secured to a separate ring gear using a plurality of bolts.",
"6.An automotive differential device as described in claim 4 wherein a ring gear is formed integral with said flange section."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to a differential device for automobiles.",
"More specifically, in the present invention, three pinions are disposed on a differential gear mechanism, a case member is formed as an integral structure, improved performance is provided in the differential gear mechanism, and production costs are reduced.",
"A differential device for automobiles generally includes: a differential gear mechanism including left and right side gears and multiple pinions; and a case member housing the differential gear mechanism.",
"Two types of differential devices are often 2-pinion type and 4-pinion type.",
"In a 2-pinion differential gear mechanism, the structure is simple but the load on the pinions is high, leading to high wear on the pinion that shortens the lifetime.",
"In a 4-pinion differential gear mechanism, the structure is somewhat more complicated but the load on the pinions is low and the pinions have a longer lifetime.",
"The case member includes a main case section for housing the differential gear mechanism and a flange section.",
"A ring gear is secured to this flange section using multiple bolts, and rotational drive force from the engine is transferred to the flange section by way of the ring gear.",
"In some cases, this ring gear is formed integrally with the flange section.",
"In a differential device equipped with a 2-pinion differential gear mechanism described in Japanese utility model registration number 3071694, a case member is formed integrally, and openings are formed facing each other on both sides of the case member to allow assembly of the side gears and pinions.",
"The pinions are disposed inside the perimeter walls on which the openings are not formed, and one shared pinion shaft is supported on the perimeter wall.",
"In a case structure for a differential device as described in Japanese laid-open patent publication number Hei 11-72158, a case member for housing a 2-pinion differential gear mechanism is formed from a ring-shaped main case body integral with a ring gear, and a pair of cover members, each one being secured to one axial end of the main case body.",
"In the case of a differential device equipped with a 4-pinion differential gear mechanism, the pinion shafts must be supported at four uniformly spaced positions along the perimeter of the wall of the case member.",
"This makes forming the openings described above more difficult.",
"Thus, the case member is generally formed as a two-part structure (divided structure).",
"Forming the case member as a two-part structure increases the number of parts for the case member as well as requiring expensive machining operations, thus increasing production costs.",
"While production costs for the case member can be reduced by using a 2-pinion differential gear mechanism and an integrally formed case member, the use of larger side gears and pinions will require larger openings, leading to disadvantages relating to the rigidity and strength of the case member.",
"Also, the pinions experience a high load, resulting in pinion wear and short lifetime.",
"The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements."
],
[
"<SOH> OBJECTS AND SUMMARY OF THE INVENTION <EOH>The object of the present invention is to provide an automotive differential device that reduces pinion load to improve durability and reduce production costs for the case member.",
"In an automotive differential device transferring a rotational drive force from an automotive engine to a drive axle, the present invention provides an automotive differential device including: a differential gear mechanism equipped with a pair of side gears, three pinions meshing with the side gears, three pinion shafts pivotably supporting the three pinions, respectively, and a shaft supporting member supporting inner ends of the three pinion shafts; and a case member with an integral structure housing the differential gear mechanism.",
"The case member includes a flange section receiving the rotational drive force by way of a ring gear, and a plurality of openings for introducing the side gears and pinions.",
"By providing three pinions in the differential gear mechanism, the load on the pinions can be reduced, durability can be improved, and the adoption of a high power engine can be possible.",
"By providing a shaft support member for supporting the inner ends of the three pinion shafts, the inner ends of the three pinion shafts can be reliably supported.",
"Since the case member is formed as an integral structure, the production costs for the case member can be reduced.",
"Since multiple openings are formed in the case member to introduce and mount the side gears and pinions, the side gears and pinions can be introduced and mounted in a reliable manner from these openings.",
"Preferred alternative examples will be described.",
"The three pinions are disposed so that axes thereof form 120 degree phase angles, and the plurality of openings is three openings formed at three equidistant positions along a perimeter wall section of the case member.",
"Thus, shaft openings for supporting the pinion shafts can be formed on wall sections other than the three openings formed on the perimeter wall of the case member.",
"The three openings are two first openings for introducing the three pinions and one second opening for consecutively introducing the two side gears.",
"The first opening is a roughly D-shaped opening with a straight section on a side toward the flange.",
"The second opening is an opening with a shape different from that of the first opening, the shape being a special shape allowing the side gear to be introduced while in an inclined state when the side gear is mounted.",
"The flange section is secured to a separate ring gear using a plurality of bolts.",
"A ring gear is formed integral with the flange section."
],
[
"BACKGROUND OF THE INVENTION The present invention relates to a differential device for automobiles.",
"More specifically, in the present invention, three pinions are disposed on a differential gear mechanism, a case member is formed as an integral structure, improved performance is provided in the differential gear mechanism, and production costs are reduced.",
"A differential device for automobiles generally includes: a differential gear mechanism including left and right side gears and multiple pinions; and a case member housing the differential gear mechanism.",
"Two types of differential devices are often 2-pinion type and 4-pinion type.",
"In a 2-pinion differential gear mechanism, the structure is simple but the load on the pinions is high, leading to high wear on the pinion that shortens the lifetime.",
"In a 4-pinion differential gear mechanism, the structure is somewhat more complicated but the load on the pinions is low and the pinions have a longer lifetime.",
"The case member includes a main case section for housing the differential gear mechanism and a flange section.",
"A ring gear is secured to this flange section using multiple bolts, and rotational drive force from the engine is transferred to the flange section by way of the ring gear.",
"In some cases, this ring gear is formed integrally with the flange section.",
"In a differential device equipped with a 2-pinion differential gear mechanism described in Japanese utility model registration number 3071694, a case member is formed integrally, and openings are formed facing each other on both sides of the case member to allow assembly of the side gears and pinions.",
"The pinions are disposed inside the perimeter walls on which the openings are not formed, and one shared pinion shaft is supported on the perimeter wall.",
"In a case structure for a differential device as described in Japanese laid-open patent publication number Hei 11-72158, a case member for housing a 2-pinion differential gear mechanism is formed from a ring-shaped main case body integral with a ring gear, and a pair of cover members, each one being secured to one axial end of the main case body.",
"In the case of a differential device equipped with a 4-pinion differential gear mechanism, the pinion shafts must be supported at four uniformly spaced positions along the perimeter of the wall of the case member.",
"This makes forming the openings described above more difficult.",
"Thus, the case member is generally formed as a two-part structure (divided structure).",
"Forming the case member as a two-part structure increases the number of parts for the case member as well as requiring expensive machining operations, thus increasing production costs.",
"While production costs for the case member can be reduced by using a 2-pinion differential gear mechanism and an integrally formed case member, the use of larger side gears and pinions will require larger openings, leading to disadvantages relating to the rigidity and strength of the case member.",
"Also, the pinions experience a high load, resulting in pinion wear and short lifetime.",
"The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements.",
"OBJECTS AND SUMMARY OF THE INVENTION The object of the present invention is to provide an automotive differential device that reduces pinion load to improve durability and reduce production costs for the case member.",
"In an automotive differential device transferring a rotational drive force from an automotive engine to a drive axle, the present invention provides an automotive differential device including: a differential gear mechanism equipped with a pair of side gears, three pinions meshing with the side gears, three pinion shafts pivotably supporting the three pinions, respectively, and a shaft supporting member supporting inner ends of the three pinion shafts; and a case member with an integral structure housing the differential gear mechanism.",
"The case member includes a flange section receiving the rotational drive force by way of a ring gear, and a plurality of openings for introducing the side gears and pinions.",
"By providing three pinions in the differential gear mechanism, the load on the pinions can be reduced, durability can be improved, and the adoption of a high power engine can be possible.",
"By providing a shaft support member for supporting the inner ends of the three pinion shafts, the inner ends of the three pinion shafts can be reliably supported.",
"Since the case member is formed as an integral structure, the production costs for the case member can be reduced.",
"Since multiple openings are formed in the case member to introduce and mount the side gears and pinions, the side gears and pinions can be introduced and mounted in a reliable manner from these openings.",
"Preferred alternative examples will be described.",
"The three pinions are disposed so that axes thereof form 120 degree phase angles, and the plurality of openings is three openings formed at three equidistant positions along a perimeter wall section of the case member.",
"Thus, shaft openings for supporting the pinion shafts can be formed on wall sections other than the three openings formed on the perimeter wall of the case member.",
"The three openings are two first openings for introducing the three pinions and one second opening for consecutively introducing the two side gears.",
"The first opening is a roughly D-shaped opening with a straight section on a side toward the flange.",
"The second opening is an opening with a shape different from that of the first opening, the shape being a special shape allowing the side gear to be introduced while in an inclined state when the side gear is mounted.",
"The flange section is secured to a separate ring gear using a plurality of bolts.",
"A ring gear is formed integral with the flange section.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a cross-sectional view of an automotive differential device according to an embodiment.",
"FIG.",
"2 is a cross-sectional view along the II-II line in FIG.",
"1.FIG.",
"3 is a perspective view of a case member.",
"FIG.",
"4 is a side-view of a case member when seen from the direction of arrow IV in FIG.",
"3.FIG.",
"5 is a bottom-view of a case member as seen from arrow V in FIG.",
"3.FIG.",
"6 is a cross-sectional view along the VI-VI line FIG.",
"4.FIG.",
"7 is a view illustrating how a left side gear is assembled from a second opening.",
"FIG.",
"8 is a view illustrating how a right side gear is assembled from a second opening.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS This embodiment of the present invention is an example of an automotive differential device for transferring rotational drive force from a front-wheel drive automotive engine to a drive axle.",
"As shown in FIG.",
"1 and FIG.",
"2, an automotive differential device 1 includes: a differential gear mechanism 2; a case member 3 formed with an integral structure and housing the differential gear mechanism 2; a ring gear 6 secured with multiple bolts 5 to a flange section 4 of the case member 3; and a differential case 7.The differential gear mechanism 2 includes: a pair of side gears 9 connected respectively to ends of left and right drive axles 8 so that they cannot rotate relative to the drive axles; three pinions 10 meshing with these side gears 9; three pinion shafts 11 pivotably supporting the three pinions 10, respectively; a shaft support member 12 supporting the inner ends of the three pinion shafts 11; and a thrust washer 13 mounted between the case member 3 and the side gears 9.The three pinions 10 are disposed so that axes thereof form 120 degree phase angles, and are rotatably supported by pinion shafts 11.Spring pins 15 are mounted on the ends of the pinion shafts 11 and the main case body 14, restricting the pinion shafts 11 to prevent them from disengaging.",
"The shaft support member 12 is formed as a short cylinder on which are disposed three support openings 16 positioned at three equidistant positions.",
"The inner ends of the three pinion shafts 11 are passed through and supported by these support openings 16.The ring gear 6 described above is formed separately from the flange section 4.This ring gear 6 is secured to the flange section 4 with multiple bolts 5, and the rotational drive force from the engine is applied to this ring gear 6.The case member 3 includes: a main case body 14; the flange section 4 receiving the rotational drive force from the engine by way of the ring gear 6; and three openings 20, 21, 22 for introducing the side gears 9 and the pinions 10.The three openings 20, 21, 22 are formed on the perimeter wall 14a of the main case body 14 at three equidistant positions around the axes of the pair of side gears 9.The three openings 20, 21, 22 include two first openings 20, 21 for introducing the three pinions 10 and one second opening 22 for consecutively introducing the two side gears 9.As shown in FIG.",
"3 through FIG.",
"7, when viewed from the side, the first openings 20, 21 form roughly D-shaped openings having roughly identical widths along the perimeter direction and the axial direction, with the side toward the flange 4 being straight.",
"In FIG.",
"3 and FIG.",
"4, the two first openings 20, 21 are formed at the upper section of the main case body 14, and the widths along the perimeter direction of the first openings 20, 21 are approximately 20% of the total perimeter of the perimeter wall 14a of the main case body 14.As shown in FIG.",
"5, FIG.",
"6, and FIG.",
"7, the second opening 22 is formed as an opening having a shape that is different from those of the first openings 20, 21.The second opening 22 is formed with a special shape that allows the side gear 9 to be mounted while in an inclined orientation (inclined state).",
"The second opening 22 is roughly pentagonal, resembling a rectangle connected to the base side of an isosceles triangle having a 120 degree vertex angle.",
"The sloped sides of the isosceles triangle are near the flange section 4, and the angle formed between the bottom side of the pentagon and the axis in FIG.",
"5 is roughly 60 degrees.",
"In order to facilitate mounting of the side gear 9, a cavity 22a that connects to the second opening 22 is formed near the inside of the flange section 4.Three pin holes 23 are formed on three strength perimeter walls 14b formed by the perimeter wall 14a of the main case body 14 between the first and second openings 20, 21, 22.The phase angle of the axes of the three pin holes 23 is 120 degree.",
"As shown in FIG.",
"1, the differential case 7 is a structure in which two separable differential case sections are secured using multiple bolts near the ring gear 6.The differential case 7 holds lubrication oil and surrounds the outer perimeter of the ring gear 6 and the case member 3 housing the differential gear mechanism 2.A bearing 26a is mounted between a ring section 25a at the left end of the differential case 7 and the main case body 14.A bearing 26b is mounted between a ring section 25b at the right end of the differential case 7 and the main case body 14.An oil seal 27a is mounted between the ring section 25a and an axle 8, and an oil seal 27b is mounted between the ring section 25b and an axle 8.The case member 3 described above is cast from ductile iron or the like.",
"The pinions 10 and the ring gear 6 are formed from standard materials used to form gear and the like (chromium molybdenum steel, nickel-chrome molybdenum steel, or the like), and carburizing is performed.",
"Next, a method for mounting the differential gear mechanism 2 in the case member 3 will be described briefly.",
"As shown in FIG.",
"7, the left side gear 9a and the thrust washer 13 are tilted approximately 30 degrees and mounted in the main case body 14 from the second opening 22 to the position shown in FIG.",
"1.Next, as shown in FIG.",
"8, the right side gear 9b and the thrust washer 13 are tilted approximately 30 degrees and assembled in the main case body 14 so that they are assembled in the position shown in FIG.",
"1.Next, two pinions 10 are mounted from the two first openings 20, 21, respectively.",
"With two side gears 9 and two pinions 10 rotated approximately 120 degrees clockwise or counter-clockwise, the shaft supporting member 12 is mounted from the first opening 20.Next, the remaining one pinion 10 is mounted from the first opening 20 so that it forms a 120 degree phase angle relative to the other two pinions 10.Next, the two side gears 9, the three pinions 10, and the shaft supporting member 12 are rotated approximately 60 degrees clockwise or counterclockwise.",
"The pin holes 30 of the three pinions 10 are aligned with the three pin holes 23 of the strength perimeter walls 14b, and the three pin holes 16 of the shaft supporting member 12 are aligned with the pin holes 30 of the three pinions 10.Next, for each pinion 10, a pinion shaft 11 is mounted in the pin hole 23 of the strength perimeter wall 14b, the pin hole 30 of the pinion 10, and the pin hole 16 of the shaft supporting member 12, and three spring pins 15 are mounted.",
"The differential case 7 and the bearing 26a, 26b is assembled using any method known to those in the art.",
"Next, the operations and advantages of the automotive differential device 1 will be described.",
"Since the differential gear mechanism 2 uses three pinions 10, the load to the pinions 10 can be reduced and the durability of the pinions 10 and the side gear 9 can be improved.",
"Furthermore, higher rotational drive forces from the engine to the input ring gear 6 can be handled.",
"Since the shaft supporting member 12 supports the inner ends of the three pinion shafts 11, the inner ends of the three pinion shafts 11 can be supported in a reliable manner.",
"Since the case member 3 is formed as an integral structure, the production costs for the case member 3 can be reduced.",
"Since the case member 3 is formed with two first openings 20, 21 for introducing the three pinions 10 and a single second opening 22 for consecutively introducing the two side gears 9, the side gears 9 and the pinions 10 can be mounted easily and reliably.",
"Since the first and second openings 20, 21, 22 are formed at three equidistant positions along the perimeter wall 14a of the main case body 14 and the three pinions 10 are disposed so that their axes form 120 degree phase angles, the pin holes 23 can be formed to support the three pinion shafts 11 on the three strength perimeter walls 14b of the perimeter wall 14a of the main case body 3, and the pinion shafts 11 can be mounted in the pin holes 23 to support the pinions 10.Examples of partial modifications of the above embodiment will be described.",
"1) The shapes of the first and second openings 20, 21, 22 are just examples and the present invention is not restricted in the shapes shown in the figures.",
"2) The ring gear 6 can be formed integral with the flange section 4.In this case, it would be preferable for the case member 3 to be formed from a high-quality cast material such as austempered cast iron.",
"The material used is not restricted to this, however.",
"3) The automotive differential device of this embodiment was described in a sample implementation as a differential device for a front-wheel drive automobile.",
"However, the present invention can be implemented in a similar manner for a differential device in rear-wheel drive automobiles.",
"In a differential device for rear-wheel drive automobiles, the ring gear would be a helical gear rather than a spur gear.",
"Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims."
]
] | Patent_10432539 |
[
[
"Hinge arrangment for a cabinet door",
"A hinge arrangement for a cabinet door, comprising: a pair of hinge seats (10), which are affixed to one of the parts defined by the door (P) and the cabinet (G); hinge pins (20), which are affixed to the other of said parts and which are each designed to be fitted into a respective hinge seat (10) when the respective side of the door is closed; a locking slide (30), which is mounted to the hinge seats (10), and which may be displaced in opposite directions, as a function of the door opening operation, in order to lock the hinge pin (20) inside the respective hinge seat (10) at the non-opened side of the door, said hinge arrangement further comprising a first stop means (15) incorporated to each hinge seat (10), and a second stop means (25), which is fixed and slightly spaced in relation to each hinge pin (20), said first and second stop means (15, 25) being arranged so that, after the door (P) has been opened by a certain degree from one side, the first stop means (15) of the other side is angularly displaced, in order to cooperate with the second stop means (25), avoiding said hinge pin (20) from being liberated from its hinge seat (10), regardless of the positioning of the locking slide (30)."
],
[
"1.A hinge arrangement for a cabinet door, comprising: a pair of hinge seats (10), which are affixed close to each of the upper and lower ends of one of the parts defined by the door (P) and the cabinet (G), symmetrically in relation to the center line of said parts, in their right and left end portions, respectively; hinge pins (20), which are affixed to the other of said parts and which are each designed to be fitted into a respective hinge seat (10) when the respective side of the door is closed; a locking slide (30), which is mounted to each of the upper and lower ends of the part that carries the hinge seats (10), and which may be displaced in opposite directions from a median position, said locking slide being provided, at its opposite ends, with cam portions (31), which are arranged in order that, in the initial phase in which the door (P) is opened by one of its sides, the hinge pin (20) of said side, acting on the adjacent cam portion (31), causes the displacement of the locking slide (30) in order to lock the hinge pin (20) of the other side of the door inside the respective hinge seat (10), characterized in that it comprises a first stop means (15) incorporated to each hinge seat (10), and a second stop means (25), which is fixed and slightly spaced in relation to each hinge pin (20), said first and second stop means (15, 25) being arranged so that, after the door (P) has been opened by a certain degree from one side, the first stop means (15) of the other side is angularly displaced around the respective hinge pin (20), from an inoperative position, in which it allows the liberation of said hinge pin (20) from its hinge seat (10), to an operative position, in which it begins to cooperate with the second stop means (25), avoiding said hinge pin (20) from being liberated from its hinge seat (10), regardless of the positioning of the locking slide (30).",
"2.The hinge arrangement according to claim 1, characterized in that, after a certain additional rotation for opening the door (P) from said side, the first stop means (15) of the other side also begins to cooperate with the respective hinge pin (20), avoiding the latter from being liberated from its hinge seat (10).",
"3.The hinge arrangement according to any of the claims 1 or 2, characterized in that the first stop means (15) is defined by a semi-circular projection incorporated to each hinge seat, the second stop means (25) being defined by a semi-circular wall, whose inner radius of curvature is larger than the external radius of curvature of the first stop means (15), which is concentric and spaced from a respective hinge pin (20) by a distance slightly larger than the radial thickness of the first stop means (15).",
"4.The hinge arrangement according to claim 3, characterized in that the first stop means (15) is internally coincident with the contour of the portion of the hinge seat (10) that retains a respective hinge pin (20) in the closed position of the door (P).",
"5.The hinge arrangement according to claim 3, characterized in that the hinge seat (10) takes the form of a slot, and in that, when the door (P) is in the closed position, the first and the second stop means (15, 25) have the same axis of symmetry coinciding with the axis of the slot of the hinge seat (10), the chord of the second stop means (25) cutting the end portions of the first stop means (15), which is kept substantially concentric in relation to both the hinge pin (20) and the second stop means (25).",
"6.The hinge arrangement according to claim 5, characterized in that the first stop means (15) is angularly displaced when the opposite side of the door (P) is opened, in order to be progressively inserted between the hinge pin (20) and the second stop means (25) as the door is progressively opened.",
"7.The hinge arrangement according to any of the claims 1 to 6, characterized in that each end of a locking slide (30) has a ramp (36), which is provided in the V shaped cut (33) so that, upon contacting a hinge pin (20) when the respective side of the door (P) is closed, it forces the locking slide (30) to move to the other side.",
"8.The hinge arrangement according to claim 1, characterized in that it further comprises support elements (60), each defined by a rolling element (61), which is rotatively and partially lodged in a frame (62) to be affixed to one of the parts defined by the door (P) and the cabinet (G), in order to cooperate with a surface of the other of said parts, when the door (P) is conducted to the closing position, guaranteeing the correct leveling of the door (P) in the closing operation, regardless of the amount of products that said door is supporting internally.",
"9.The hinge arrangement according to claim 8, characterized in that the rolling element (61) is defined by a ball.",
"10.The hinge arrangement according to any of the claims 1 to 7, characterized in that at least one pair of hinge seats (10), horizontally leveled, are formed on a seating plate (1), while the respective pair of hinge pins (20) are incorporated in a support plate (2).",
"11.The hinge arrangement according to claim 10, characterized in that the seating plate (1) carries a plurality of rollers (18), which are free rotating and with the contour of their cylindrical surface slightly projecting outwardly from the free external face of the seating plate (1), the support plate (2) having a front edge in the form of a ramp, in order to receive the rollers (18) when the door (P) is closed and to conduct the latter to the correct leveling before the closing operation is completed."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>It is known from the prior art the use of a hinge arrangement for the cabinet door of a refrigeration appliance, which allows said door to be opened and closed from any one of the sides thereof, comprising: four hinge seats, usually in the form of slots provided in plates, which are each horizontally affixed close to one of the upper and lower ends of the door or of the cabinet and close to the right and left portions thereof.",
"In each of said slots are fitted, when the door is angularly horizontally displaced, a vertical hinge pin affixed to the other of said parts defined by the door and the cabinet, upon closing the respective side of the door.",
"In order to guarantee that the hinge pins of the door side opposite to that selected by the user for opening the door remain retained inside the respective hinge seats, journalling and defining the articulation for the opening/closing rotation of the door, there is provided a locking slide, usually in the form of an elongated bar, which is guided on that part defined by the door or the cabinet which sustains the hinge seats, so that said locking slide may be linearly displaced in opposite directions, from a median closed door position, towards either of the sides of the door or the cabinet.",
"The opposite ends of the locking slide are provided with respective cam portions, which are arranged so that the locking slide be displaced by the hinge pin of the door side selected to be opened towards the other side, in order to lock the hinge pin of said other side inside the respective hinge seat.",
"Thus, when door opening is initiated from any of the sides, the locking slide is automatically and immediately forced to move to the other side, locking the hinge pin of said other side and preventing the door from being displaced from the cabinet while it is kept in the open position.",
"The locking slide is medianly provided with a marginal V shaped cut, in order to work jointly with a positioning pin, which is mounted so as to be pressed when contacting the other part defined by either the door or the cabinet, against the V shaped cut, upon the door reaching a position immediately anterior to the closing thereof.",
"In such situation, the positioning pin presses one of the inclined walls of the V shaped cut, forcing the locking slide to the median position and allowing, with the door completely closed, the hinge pins to be adequately locked in their respective hinge seats by the cam portions in the opposite ends of the locking slide, preventing the door from being detached from the cabinet.",
"In this type of construction mentioned above, the locking of the hinge pins is assured by the locking slide in the open door condition and it is not possible for the door to fall, since the opening thereof by any of the sides may only be achieved as a function of the dimensioning of the locking slide, with the latter being displaced to the other side, to a mandatory condition in which the hinge pin is locked to said other side.",
"However, when the door is in the open condition on one side, with the locking slide displaced to the other side, it is possible for the user to manually press the positioning pin, which is mounted to the part defined by either the door or the cabinet that carries the locking slide, towards the V shaped cut of the latter, in order to cause the displacement of the locking slide to the median position, from which the door may be pulled outwardly from the hinged side, so that the hinge pin of this side acts on the adjacent cam portion of the locking slide, forcing it to move towards the already opened side and fully liberating the door from its connection with the cabinet.",
"The completely liberated door condition mentioned above is only possible in case the user deliberately and unduly acts on the positioning pin, manually forcing it against the locking slide.",
"However, in case said condition occurs inadvertently or by action of children, the door may fall, seriously hurting the user and causing material damage.",
"It should be noted that, even in the constructions in which the positioning pin is affixed to the door provided with the hinge pins, it is possible for the user to insert any kind of pin in the orifice of the other part that gives access to the V shaped cut of the locking slide, forcing the latter to the median position, from which it may be displaced by the hinge pin itself when the respective side of the door is forced to open."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The object above is achieved by the provision of a hinge arrangement for a cabinet door, comprising: a pair of hinge seats, which are affixed close to each of the upper and lower ends of one of the parts defined by the door and the cabinet, symmetrically in relation to the center line of said parts, in their right and left end portions, respectively; hinge pins, which are affixed to the other of said parts defined by the door and cabinet and which are each designed to be fitted into a respective hinge seat when the respective side of the door is closed; a locking slide, which is mounted to each of the upper and lower ends of the part defined by the door or the cabinet that carries the hinge seats, and which may be displaced in opposite directions from a median position, said locking slide being provided, at its opposite ends, with cam portions, which are arranged in order that, in the initial phase in which the door is opened by one of its sides, the hinge pin of said side, acting on the adjacent cam portion, causes the displacement of the locking slide in order to lock the hinge pin of the other side of the door inside the respective hinge seat.",
"According to the invention, the hinge arrangement further comprises a first stop means incorporated to each hinge seat, and a second stop means, which is fixed and slightly spaced in relation to each hinge pin, said first and second stop means being arranged so that, after the door has been opened by a certain degree from one side, the first stop means of the other side is angularly displaced around the respective hinge pin, from an inoperative position, in which it allows the liberation of said hinge pin from its hinge seat, to an operative position, in which it cooperates with the second stop means, avoiding said hinge pin from being liberated from its hinge seat, regardless of the locking slide being positioned in its median position.",
"With the construction cited above, after the door has been opened from one of its sides, by an angle sufficient to allow the user to access the positioning pin or the orifice that gives access to the V shaped cut of the locking slide, the stop means of the other side of the door are correspondingly angularly displaced relative to each other, from an inoperative position, in which they allow the adjacent hinge pins to be liberated from the fitting condition in the respective hinge seats, to an operative condition, in which they begin to mutually cooperate, locking the respective hinge pins in the respective hinge seats, even if the locking slide is unduly displaced to its median position, from which said non-opened side of the door may be detached from the cabinet if pulled by the user."
],
[
"FIELD OF THE INVENTION The present invention refers to a hinge arrangement, which allows opening and closing the cabinet door of refrigeration appliances or similar products, from either the right side or the left side of the door.",
"BACKGROUND OF THE INVENTION It is known from the prior art the use of a hinge arrangement for the cabinet door of a refrigeration appliance, which allows said door to be opened and closed from any one of the sides thereof, comprising: four hinge seats, usually in the form of slots provided in plates, which are each horizontally affixed close to one of the upper and lower ends of the door or of the cabinet and close to the right and left portions thereof.",
"In each of said slots are fitted, when the door is angularly horizontally displaced, a vertical hinge pin affixed to the other of said parts defined by the door and the cabinet, upon closing the respective side of the door.",
"In order to guarantee that the hinge pins of the door side opposite to that selected by the user for opening the door remain retained inside the respective hinge seats, journalling and defining the articulation for the opening/closing rotation of the door, there is provided a locking slide, usually in the form of an elongated bar, which is guided on that part defined by the door or the cabinet which sustains the hinge seats, so that said locking slide may be linearly displaced in opposite directions, from a median closed door position, towards either of the sides of the door or the cabinet.",
"The opposite ends of the locking slide are provided with respective cam portions, which are arranged so that the locking slide be displaced by the hinge pin of the door side selected to be opened towards the other side, in order to lock the hinge pin of said other side inside the respective hinge seat.",
"Thus, when door opening is initiated from any of the sides, the locking slide is automatically and immediately forced to move to the other side, locking the hinge pin of said other side and preventing the door from being displaced from the cabinet while it is kept in the open position.",
"The locking slide is medianly provided with a marginal V shaped cut, in order to work jointly with a positioning pin, which is mounted so as to be pressed when contacting the other part defined by either the door or the cabinet, against the V shaped cut, upon the door reaching a position immediately anterior to the closing thereof.",
"In such situation, the positioning pin presses one of the inclined walls of the V shaped cut, forcing the locking slide to the median position and allowing, with the door completely closed, the hinge pins to be adequately locked in their respective hinge seats by the cam portions in the opposite ends of the locking slide, preventing the door from being detached from the cabinet.",
"In this type of construction mentioned above, the locking of the hinge pins is assured by the locking slide in the open door condition and it is not possible for the door to fall, since the opening thereof by any of the sides may only be achieved as a function of the dimensioning of the locking slide, with the latter being displaced to the other side, to a mandatory condition in which the hinge pin is locked to said other side.",
"However, when the door is in the open condition on one side, with the locking slide displaced to the other side, it is possible for the user to manually press the positioning pin, which is mounted to the part defined by either the door or the cabinet that carries the locking slide, towards the V shaped cut of the latter, in order to cause the displacement of the locking slide to the median position, from which the door may be pulled outwardly from the hinged side, so that the hinge pin of this side acts on the adjacent cam portion of the locking slide, forcing it to move towards the already opened side and fully liberating the door from its connection with the cabinet.",
"The completely liberated door condition mentioned above is only possible in case the user deliberately and unduly acts on the positioning pin, manually forcing it against the locking slide.",
"However, in case said condition occurs inadvertently or by action of children, the door may fall, seriously hurting the user and causing material damage.",
"It should be noted that, even in the constructions in which the positioning pin is affixed to the door provided with the hinge pins, it is possible for the user to insert any kind of pin in the orifice of the other part that gives access to the V shaped cut of the locking slide, forcing the latter to the median position, from which it may be displaced by the hinge pin itself when the respective side of the door is forced to open.",
"OBJECT OF THE INVENTION It is a general object of the invention to provide a hinge arrangement for a cabinet door of the type considered herein and presenting, by means of a very simple construction, a safety device for preventing the door from being detached from the cabinet by deliberately or inadvertently displacing the locking slide to the median position, after the door has been opened by one of its sides.",
"SUMMARY OF THE INVENTION The object above is achieved by the provision of a hinge arrangement for a cabinet door, comprising: a pair of hinge seats, which are affixed close to each of the upper and lower ends of one of the parts defined by the door and the cabinet, symmetrically in relation to the center line of said parts, in their right and left end portions, respectively; hinge pins, which are affixed to the other of said parts defined by the door and cabinet and which are each designed to be fitted into a respective hinge seat when the respective side of the door is closed; a locking slide, which is mounted to each of the upper and lower ends of the part defined by the door or the cabinet that carries the hinge seats, and which may be displaced in opposite directions from a median position, said locking slide being provided, at its opposite ends, with cam portions, which are arranged in order that, in the initial phase in which the door is opened by one of its sides, the hinge pin of said side, acting on the adjacent cam portion, causes the displacement of the locking slide in order to lock the hinge pin of the other side of the door inside the respective hinge seat.",
"According to the invention, the hinge arrangement further comprises a first stop means incorporated to each hinge seat, and a second stop means, which is fixed and slightly spaced in relation to each hinge pin, said first and second stop means being arranged so that, after the door has been opened by a certain degree from one side, the first stop means of the other side is angularly displaced around the respective hinge pin, from an inoperative position, in which it allows the liberation of said hinge pin from its hinge seat, to an operative position, in which it cooperates with the second stop means, avoiding said hinge pin from being liberated from its hinge seat, regardless of the locking slide being positioned in its median position.",
"With the construction cited above, after the door has been opened from one of its sides, by an angle sufficient to allow the user to access the positioning pin or the orifice that gives access to the V shaped cut of the locking slide, the stop means of the other side of the door are correspondingly angularly displaced relative to each other, from an inoperative position, in which they allow the adjacent hinge pins to be liberated from the fitting condition in the respective hinge seats, to an operative condition, in which they begin to mutually cooperate, locking the respective hinge pins in the respective hinge seats, even if the locking slide is unduly displaced to its median position, from which said non-opened side of the door may be detached from the cabinet if pulled by the user.",
"BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described below, with reference to the attached drawings, in which: FIG.",
"1 is a schematic upper plan view of the lower hinge arrangement of the present invention, with the door in the closed position; FIG.",
"2 illustrates an enlarged detail of part of FIG.",
"1, with the locking slide being partially cut at the end facing the side of the door to be kept articulated to the cabinet in a desired opening operation, in order to better illustrate the first and second stop means; FIG.",
"3 is a similar view to that of FIG.",
"1, but with the door in a partially open condition; FIG.",
"4 is a similar view to that of FIG.",
"2, but illustrating the same elements with the door in the already opened condition; FIG.",
"5 is a similar view to that of FIG.",
"4, but illustrating the opposite end of the partially opened door; FIG.",
"6 is a perspective view of an assembly comprising, in a single piece, a hinge pin and a door positioning means, said assembly being disposed to cooperate with the lower portion of a door when affixed to the cabinet.",
"FIG.",
"7 is a partially cut perspective view of a support plate, onto which is incorporated a pair of hinge pins, which are horizontally leveled at the lower end of the cabinet; FIG.",
"8 is a partially cut lower perspective view of a door of a refrigerator or freezer, to whose lower edge is mounted a seating plate, on which are formed two horizontally leveled hinge seats; and FIG.",
"9 is a partially sectional vertical view of an assembly comprising the cabinet and the almost closed door incorporating the seating plates and the support plates.",
"DESCRIPTION OF THE ILLUSTRATED EMBODIMENT As mentioned above, the hinge arrangement of the present invention is particularly adequate for providing the coupling of doors with automatical reversible opening to the cabinets of refrigerators or freezers.",
"The arrangement considered herein is of the type comprising four hinges in each door P, which are provided in pairs at the upper part and at the lower part of the door P, the hinges of each pair being provided close to the right and left end portions of said door P, respectively, and symmetrically in relation to the longitudinal axis of the door P or of the front face of the cabinet G. Each of said hinges comprises a hinge seat 10, which takes the form of a slot provided in a seating plate 1, horizontally affixed to the upper and lower ends of either the door P or the cabinet G, said slot having its axis usually provided transversal to the plane of the closing face of the part that carries said hinge seat 10, and having an open end portion 11 facing the front face of cabinet G, and a closed end portion 12 opposite to the open end portion 11 and in the form of a semicircle coinciding with the lateral edges of the slot, the radius of said closed end portion being slightly larger than that of a respective hinge pin 20, which defines the other element of each hinge and which is affixed to the other part consisting of the door P or the cabinet G, in order to be fitted into a respective hinge seat 10 upon closing the door, as illustrated in FIGS.",
"1 and 2.The fitting of each hinge pin 20 inside the respective hinge seat 10 is achieved by displacing the former along the slot that defines the hinge seat 10.On the same part consisting of either the door P or the cabinet G that carries the hinge seats 10 is mounted a locking slide 30, usually in the form of an elongated bar disposed parallel to the plane of the closing face of the part that carries it, so that it may be linearly displaced in opposite directions and from a median position corresponding to a closed door position (FIGS.",
"1 and 2).",
"Each end of the locking slide 30 is configured to define a cam portion 31 in the form of an inclined ramp, which remains seated against the respective hinge pin 20 when in the closed door condition, as illustrated in FIGS.",
"1 and 2.In this condition, in case the door P is pulled outwardly, simultaneously by both right and left sides, the two opposite cam portions 31 will avoid the liberation of the hinge pins 20 fitted in the respective hinge seats.",
"In order to open the door P, all the user has to do is to pull one of the sides thereof away from the cabinet G, making the force of both hinge pins 20 of this side of the door on the adjacent cam portions 31 of both locking slides 30 to provoke the linear displacement of the latter towards the other side of the door-cabinet assembly, so that a locking portion 32 of both locking slides 30 be positioned against the respective hinge pins 20, locking the latter, avoiding the liberation of the respective hinge seats 10 and consequently maintaining the door P articulated to the cabinet G during opening thereof.",
"As is known from the prior art, each pair of hinge seats 10 is usually provided in a frame, at least partially tubular, in which is guided a respective locking slide 30.Said frame is preferably mounted in the upper and lower end regions of door P. However, it should be understood that mounting the assembly formed by a pair of hinge seats 10 and by a locking slide 30 may be equally effected in the upper and lower ends of the cabinet G, in which case the hinge pins 20 are attached to the door P. In the figures of the appended drawings, the hinge pins 20 are attached to the cabinet G, while the hinge seats 10 and both locking slides 30 are mounted to the door P, with the description below being directed to this constructive option.",
"In order that the locking slide 30 be automatically and safely returned to the median position, after the door P has been closed, in which position the cam portions 31 are again positioned against the respective hinge pins 20, said locking slide 30 is medianly provided, on its longitudinal side turned to the front face of the cabinet G, with a V shaped cut 33, against which is pressed a positioning pin 40 which, in the illustrated embodiment, is mounted in the guiding frame of the locking slide 30, in an axially displaceable manner.",
"Thus, when a side of the door P is opened and the locking slide 30 is displaced to the opposite side, one of the sides of the V shaped cut 33 causes the displacement of the positioning pin 40 to the outermost region of the V shaped cut 33, whereby said positioning pin 40 becomes more salient in relation to the adjacent longitudinal side of the locking slide 30, as illustrated in FIG.",
"3.When the door P is returned to the closed position, the positioning pin 40 touches a surface of the cabinet in the final closing course, so that, as soon as the cam portion 31 of the open end of the locking slide 30 reaches, together with the door, a position that is posterior to that in which the adjacent hinge pin 20 is fitted in the respective hinge seat 10, said positioning pin 40 forces the locking slide 30 back to the median position, resetting the latter to a new door opening by any one of the right or left sides of said door.",
"While being preferred to mount the positioning pin 40 in an axially sliding way to the part carrying the hinge seats 10 and the locking slides 30, it should be understood that said positioning pin may be affixed to the other of said parts defined by the cabinet G and door P. In any of the constructions for the positioning pin 40 mentioned above, after the door P has been opened by a certain degree, such as that illustrated in FIG.",
"3, it is possible for any person to access the V shaped cut 33 of the locking slide 30, by pressing the movable positioning pin 40 or using any other similar instrument that exerts a transversal force on one of the sides of the V shaped cut 33, in order to provoke the displacement of the locking slide 30 to the median position, in which the application of the opening force onto the hinged side of the door will cause, by action of the hinge pin 20 against the adjacent cam portion 31 of the locking slide 30, the latter to be further displaced towards the open side of the door P, completely detaching said door from the cabinet G. For preventing said door P from being detached from the cabinet G, the present hinge arrangement is provided with a first stop means 15 in the form of a semi-circular projection incorporated to each hinge seat 10 and concentrically internally coinciding with the contour of the closed end portion 12 of the slot that defines each hinge seat 10, that is, having an inner radius of curvature that is slightly larger than the radius of the hinge pin 20.The present hinge arrangement further comprises a second stop means 25, which is affixed to the part of either the door P or to the cabinet G carrying the hinge pins 20, each second stop means 25 comprising a semi-circular wall, which has an inner radius of curvature that is larger than the outer radius of curvature of the first stop means 15, and which is concentric and radially spaced from a respective hinge pin 20 by a distance slightly larger than the radial thickness of the first stop means 15 in the form of a semi-circular projection.",
"The first and the second stop means 15, 25 are disposed substantially concentric and having the same axis of symmetry coinciding with the axis of the slot that defines the hinge seat 10, the chord of the second stop means 25 cutting the end portions of the first stop means 15 when the door P is in the closed position illustrated in FIGS.",
"1 and 2.In this condition, the first and second stop means remain in an inoperative condition.",
"When the door is opened by one side, the first stop means 15 of the other side and the associated hinge seat 10 are rotated around the respective hinge pin 20, soon reaching an operative position in which the axis of the slot of the hinge seat 10 no longer coincides with the axis of symmetry of the second stop means 25, as illustrated in FIG.",
"4.In this condition, the relative displacement of the hinge pin 20-second stop means 25 along the slot of the hinge seat 10 and outwardly from the latter is avoided by the interference between the first and the second stop means 15, 25.With the door P submitted to an opening of about 90° or slightly less, the rotation of the first stop means 15 around the hinge pin 20 reaches a condition in which it still cooperates with the hinge pin 20, defining an obstacle to the liberation path of the latter in relation to the respective hinge seat in which it is fitted and retained.",
"Thus, after the door has been opened by either of the sides thereof, the stop means of the side that remained articulated to the cabinet begin to interact, in order to guarantee the locking of the hinge pins 20 in the respective hinge seats 10, regardless of the position of the locking slide 30.In order to assure that the locking slide 30 is displaced to the hinged side of the door P upon closing thereof, each end of the locking slide 30 is further provided with a ramp 36, which is disposed on the longitudinal side provided with the V shaped cut 33 and which, by hitting a hinge pin 20 when the respective side of the door P is closed, forces the locking slide 30 to move to the other side, allowing it to pass by said hinge pin 20.Then, the positioning pin 40, by touching the other part of the door-cabinet assembly, forces the locking slide 30 to move back to the median position.",
"Due to the fact that the constructions of refrigerator doors, in which the opening may be achieved by either of the sides thereof, require a correct vertical positioning of said door during the final closing phase of the opened side, it is known the provision of support elements mounted to the door and to the cabinet and which mutually cooperate, in the condition in which the door is in the final closing phase, and in the complete closing phase, in order to assure that the door, even supporting the weight of its loaded inner shelves, has its opened side taken to its correct position, in the vertical direction, upon approaching the cabinet, so that the hinge pins of this same side may be correctly and smoothly engaged to the respective hinge seats in the final closing phase of this side of the door.",
"As shown in FIGS.",
"3, 4, 5 and 6 of the appended drawings, on each side of the door-cabinet assembly there is provided a support element 60, designed to support the weight of the door P in the final closing phase and which is preferably affixed to the cabinet G, in order to cooperate with a lower surface portion of the door P, from a certain closing condition of the latter.",
"In the embodiment cited above, the support element 60 comprises a rolling element 61, such as a ball or a roller, which is partially lodged and retained in a recess of a frame 62 and rotating freely when subject to tangential efforts in its spherical or cylindrical surface portion projecting from the frame 62.The frame 62 may be attached to any of the parts defined by the door P and the cabinet G, so that the ball 61 may operate against a surface (not illustrated), usually in the form of a ramp and provided in the other of said parts when the door P is closed, in order to force the door P to assume a project nominal leveling, adequate to the correct operation of the hinge arrangement with automatic reversible opening.",
"In FIG.",
"6 there is illustrated a construction in which the hinge pin 20 is mounted to a rod 21 projecting from a base 22 to be affixed, for example by non-illustrated screws, to the front face of cabinet G, which base 22 may incorporate, also in a single piece, the frame 62 carrying a ball shaped rolling element 61, of the support element 60, preferably mounted to the cabinet G to act under the lower end portion of the door P. In the embodiment shown in FIGS.",
"7, 8 and 9, a pair of horizontally leveled hinge seats 10, usually attached to the lower end of door P, is formed in a single seating plate 1 affixed along the width of the lower edge of the door P and which is provided in its lower edge with a plurality of free rotating rollers 18, which are adequately journalled on respective shaft extensions 19, which are usually coaxial to each other and parallel to said front edge of said seating plate 1, in order to allow the contour of the cylindrical surface of the rollers 18 to slightly project outwardly from the free external face of the seating plate 1.The constructive solution of the hinge seat mentioned above is designed to operate jointly with a respective pair of hinge pins 20 incorporated in a single support plate 2 which, in the illustrated construction, is affixed to the lower portion of the cabinet G, defining the lower threshold of the door P. As it may be observed from the drawings, both the hinge seats 10 incorporated to the respective lower seating plate 1 of the door P and the hinge pins 20 incorporated to the respective support plate 2 of the cabinet G are provided with the respective first stop means 15 and second stop means 25, as already described in relation to FIGS.",
"1-6.The support plate 2 is provided with a front edge 2a in the form of a smooth ramp, against which are seated the rollers 18 during the final closing phase of the door P, as schematically illustrated in FIG.",
"9.The rollers 18 follow the front edge 2a of the support plate 2, guaranteeing the correct leveling of the door immediately before the hinge means of the opened side initiate a new mutual operational contact.",
"While having been illustrated an arrangement in which the seating 1 is mounted to the lower edge of the door P, it should be understood that it is also possible to mount the seating plate 1 to the cabinet G and the support plate 2 to the door P. It is also possible to associate the rollers 18 to the support plate 2 affixed to the cabinet G, producing an inverted ramp on the seating plate 1."
]
] | Patent_10432540 |
[
[
"Method for identifying compounds or lead structures against rna target motifs and rna/protein interactions",
"Disclosed is a method for identifying compounds (lead structures) which specifically bind to (a) desired RNA target motif and can inhibit or eliminate the function thereof or (b) suppress a compound associated with a desired RNA target motif and can thereby inhibit or eliminate the function thereof.",
"The inventive method is based on the attachment of a ligand (=a compound to be identified) to a RNA target motif which is coupled to a modified ribozyme so that the ribozyme is transformed into an active or inactive conformation resulting in the cleaving of a signal-giving ribozyme substrate.",
"The identified compounds enabling modification of the cellular function of the RNA target motifs enable specific medicaments to be produced.",
"The invention also relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule.",
"The base pairing model of the polynucleotide, when the target molecule binds to the aptamer, is different from the base pairing model of the polynucleotide when the target molecule does not bind to the aptamer."
],
[
"1.A method for identifying compounds which bind specifically to a desired RNA-target motif and, as a result, can inhibit or eliminate the function of this and which has the following characteristics: (a) Generation of a construct (target reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA target motif (II), where (I) and (II) are linked to each other by an RNA linker and where the reporter ribozyme domain (I) changes its catalytic activity after specific binding of a compound to the RNA target motif (II); (b) Generation of a signaling ribozyme substrate which can specifically bind to the reporter ribozyme domain (I); (c) Bringing into contact of the TRK from step (a) and the ribozyme substrate from step (b) with the compound to be identified or a mixture which contains this compound; and (d) Determination of the binding of the compound to the RNA target motif.",
"2.A method for identifying compounds which can displace a compound that is associated with a desired RNA target motif and which, as a result, can inhibit or eliminate the function of this, being characterized by the following steps: (a) Production of a construct (target reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA target motif (II), where (I) and (II) are linked to each other by an RNA linker and where the reporter ribozyme domain (I) alters its catalytic activity after displacement of the compound associated with the desired RNA target motif from the RNA target motif (II); (b) Production of a signaling ribozyme substrate which specifically binds to the reporter ribozyme domain (I); (c) Bringing into contact of the TRK from step (a) with the compound which is naturally associated with the RNA target motif; (d) Bringing into contact of the complex from step (c) and the ribozyme substrate from step (b) with the compound which is to be identified or a mixture which contains this compound; and (e) Determination of the displacement of the compound associated with the RNA target motif.",
"3.The method according to claims 1 or 2, in which the ribozyme substrate can be cleaved by the reporter ribozyme domain (I) in step (b) and in which the binding is determined in the last step by the cleavage of the ribozyme substrate.",
"4.The method according to any of claims 1 to 3, in which the reporter ribozyme domain originates from a hammerhead ribozyme.",
"5.The method according to any of claims 1 to 4 in which the ribozyme substrate is a doubly labeled ribozyme substrate.",
"6.The method according to any of claims 1 to 5, in which the doubly labeled ribozyme substrate contains a fluorophore group and a fluorescence quenching group and in which the quenching of the fluorescence of the fluorophore by the fluorescence quenching group is prevented after cleavage of the reporter ribozyme domain.",
"7.The method according to claim 6, in which the fluorophore group is 6-carboxyfluorescein (FAM) and the fluorescence quenching group is 6-carboxytetramethylrhodamine (TAMRA).",
"8.A drug containing a compound which has been identified by a method according to any of claims 1 to 6.9.A kit for the performance of a method according to any of claims 1 to 9, where the kit comprises the following compounds: (a) A target reporter construct; and (b) A signaling ribozyme substrate.",
"10.A polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, where the binding site of the catalytic domain of the ribozyme for a ribozyme substrate is blocked to the binding of the substrate of the ribozyme in the absence of the target molecule of the aptamer.",
"11.The polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular according to claim 10, which also comprises the target molecule bound to the aptamer, where the binding site of the catalytic domain of the ribozyme for a ribozyme substrate is accessible for the binding of a substrate for the ribozyme in the presence of the target molecule of the aptamer.",
"12.The polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular a polynucleotide according to claims 10 or 11, in which the pattern of base pairing differs, depending on whether the target molecule of the aptamer is bound or absent.",
"13.The polynucleotide according to any of claims 10 to 12 which also comprises a substrate for the catalytic activity of the ribozyme.",
"14.The polynucleotide according to claim 13, characterized by the substrate being an FRET substrate.",
"15.The polynucleotide according to any of claims 10 to 12, comprising a nucleic acid sequence which is selected from the group comprising SEQ.",
"ID.",
"No.",
"51 and SEQ.",
"ID.",
"No 52.16.The polynucleotide according to any of claims 10 to 15, characterized by the polynucleotide being RNA, DNA or mixtures of these.",
"17.A biosensor, comprising a polynucleotide according to any of claims 10 to 16.18.The biosensor according to claim 17, characterized by the polynucleotide being bound to a solid carrier.",
"19.A method for identifying a compound which binds to a target molecule, including the following steps: a) Providing a polynucleotide according to any of claims 10 to 16, b) Optionally determining the catalytic activity of the ribozyme, c) Adding the target molecule, where the target molecule interacts with the nucleic acid which binds the target molecule, d) Optionally determining the catalytic activity of the ribozyme, e) Adding a candidate compound, f) Optionally determining the catalytic activity of the ribozyme, g) Providing a substrate for the catalytic activity of the polynucleotide and adding of the substrate to the reaction mixture, h) Determining the binding of the candidate compound to the target molecule.",
"20.The method according to claim 19, being characterized by that either before step f) or after step g), the candidate compound is submitted to a procedure, comprising the steps: a) Providing a polynucleotide, whereby the polynucleotide is an allosteric ribozyme and comprises a hammerhead ribozyme and an aptamer which is specific for the target molecule, preferably the aptamer of the polynucleotide according to claim 19, b) Optionally determining the catalytic activity of the ribozyme, c) Adding the target molecule, whereby the target molecule interacts with the nucleic acid which binds the target molecule, d) Optionally determining the catalytic activity of the ribozyme, e) Addition of the candidate compound, f) Optionally determining the catalytic activity of the ribozyme, g) Providing a substrate for the catalytic activity of the polynucleotide and adding the substrate to the polynucleotide, and h) Determining the binding of the candidate compound to the target molecule.",
"21.The method according to claim 19 or 20, characterized by the determination of the binding of the candidate molecule to the target molecule being performed by determining the enzymatic activity of the ribozyme.",
"22.The method according to claim 21, characterized by the substrate containing a fluorophore group and a fluorescence quenching group and the cleavage of the substrate by the catalytic activity of the ribozyme or the catalytic domain preventing or at least reducing the quenching of the fluorescence.",
"23.The method according to claim 22, characterized by the fluorophore group being 6-carboxyfluorescein and the fluorescence quenching group being 6-carboxytetramethyl rhodamine or Cy 3.24.A drug containing a compound which was identified according to any of claims 19 to 23.25.A kit for the performance of a procedure according to any of claims 19 to 23, comprising a) A polynucleotide in accordance with one of claims 10 to 16; and b) A signaling ribozyme substrate.",
"26.Use of coumermycin for the production of a drug for the treatment of HIV and/or FIV.",
"27.Use of nosiheptide for the production of a medicine for the treatment of HIV and/or FIV.",
"28.Use of patulin for the production of a medicine for the treatment of HIV and/or FIV."
],
[
"<SOH> SUMMARY <EOH>Described is a procedure for the identification of compounds (lead structures) which (a) bind specifically to a desired RNA target motif and which can, as a result, inhibit or eliminate the function of this or which (b) can displace a compound which is associated with a desired RNA target motif and which can as a result inhibit or eliminate the function of this or these.",
"The procedure in accordance with the invention is based on the binding of a ligand (=compound to be identified) to an RNA target motif which is coupled to a modified ribozyme, so that the ribozyme is converted to an active or to an inactive conformation, leading to the cleavage of a signaling ribozyme substrate.",
"The compounds identified in this way permit the modification of the cellular function of the RNA target motifs and permit the production of specific drugs.",
"The invention also relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for the target molecule, where there is a difference in the base pairing pattern of the polynucleotide when the target molecule is bound to the aptamer and when the target molecule is absent from the aptamer.",
"detailed-description description=\"Detailed Description\" end=\"tail\"?"
],
[
"The present invention relates to a procedure for the identification of compounds (lead structures) which either (a) specifically bind to a desired RNA-target motif and which can, as a result, inhibit or eliminate its function, or (b) displace a compound which is associated with a desired RNA-target motif and which can, as a result, inhibit or eliminate its function.",
"The procedure in accordance with the invention is based on the binding of a ligand (=compound to be identified) to an RNA-target motif which is coupled to a modified ribozyme, so that the ribozyme is transformed into an active or inactive (or more or less active) conformation, resulting in a measurable signal, for example, by cleavage of a signaling ribozyme substrate.",
"The compounds identified in this way serve as pharmaceutical lead substances for the production of drugs, as they can specifically influence the cellular function of an RNA-target motif or a compound which is associated with an RNA-target motif.",
"The present invention further relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer, a biosensor comprising this polynucleotide, a procedure for identifying a compound which binds to a target molecule and the use of coumermycin, nosiheptide and patulin.",
"Ribonucleic acids have essential functions in a multitude of cellular processes, for example, protein synthesis, the regulation of gene expression and the processing of mRNAs.",
"In addition, RNA molecules play a decisive role in the replication of retroviruses and therefore have an essential role in the manifestation of certain viral infections.",
"For this reason, functional RNAs are an important class of “drug targets” in pharmaceutical research [D. S. Eggleston, C. D. Prescott & N. D. Pearson (Eds.",
"), The Many Faces of RNA, Academic Press (1998) 83-96; Y. Tor et al., Chem.",
"Biol.",
"5 (1998), R277-R283; N. D. Pearson & C. D. Prescott, Chem.",
"Biol.",
"4 (1997), 409-414; T. Herrmann & E. Westhof, Curr.",
"Opin.",
"Biotech.",
"9 (1998), 66-73; M. Afshar et al., Curr.",
"Opin.",
"Biotech.",
"10 (1999), 59-63].",
"Small molecules which bind to RNA have been used for decades to treat bacterial infections [W. D. Wilson & K. Li, Curr.",
"Med.",
"Chem.",
"7 (2000), 73-98; Siegenthaler et al., Am.",
"J. Med.",
"80 (1986), 2-14].",
"For example, aminoglycosides, such as neomycin B, paromomycin or streptomycin, are capable of inhibiting bacterial translation by binding to ribosomal RNA, giving an antibiotic effect [J. Woodcock et al., EMBO J.",
"10 (1991), 3099-3103; D. Moazed & H. F. Noller, Nature 327 (1987), 389-394].",
"Moreover, a series of aminoglycosides of the 2-desoxystreptamine family are capable of binding specifically to the RNA structural motif “RRE” of the HIV genome, resulting in inhibition of the replication of the HI virus [M. L. Zapp et al., Cell 74 (1993), 969-978; W. K. C. Park, et al., JACS 118 (1996), 10150-10155].",
"Although these and other antibiotics have been of therapeutic value for a long time, they have forfeited much of their efficacy in the interim, as an ever increasing number of pathogenic organisms have acquired resistance genes to the established drugs, which has caused current massive problems in the health system [J. Davies, Science 264 (1994), 375-382; H. C. Neu, Science 257 (1992), 1064-1073; J. Davies & G. D. Wright, Trends Microbiol.",
"5 (1997), 234-240].",
"For various reasons, functional RNA structures are extraordinarily well suited as target molecules for drugs.",
"Just like proteins, RNAs and DNAs can form defined spatial structures and binding sites, where they can interact specifically with other molecules [O. C. Uhlenbeck et al., Cell 90 (1997), 833-840; S. M. Hecht, Bioorg.",
"Med.",
"Chem.",
"5 (1997), 1001-1248; C. S. Chow, Chem.",
"Rev.",
"97 (1997), 1489-1513].",
"There is also less danger that retroviruses will develop resistance to RNA drugs, as the RNA target molecules are highly conserved and are initially present in the host cell in the unduplicated form [F. Hamy et al., PNAS USA 94 (1997), 3548-3553; M. Afshar et al., Curr.",
"Opin.",
"Biotech.",
"10 (1999), 59-63].",
"In addition, cellular mechanisms for the repair of RNA damages are totally unknown, even today [T. Herrmann & E. Westhof, Curr.",
"Opin.",
"Biotech.",
"9 (1998)].",
"Although there are a wide variety of reasons to use RNAs as therapeutic target molecules, drugs with RNA structures as targets are much less established than drugs at the DNA or protein level.",
"There has therefore been great effort in modern biotechnology to develop antibiotics and antiviral drugs which are directed against defined RNA motifs [D. J. Ecker & R. H. Griffey, Drug Disc.",
"Today 4 (1999), 420-429].",
"There are in principle two distinct strategies: (a) rational drug design and (b) combinatorial drug research.",
"In rational drug design, one attempts to predict the conformation of an RNA-drug complex on the basis of experimental structural and functional data.",
"In particular, structural determination by NMR spectroscopy has contributed to the elucidation of the recognition of RNA by antibiotics at the molecular level [L. Jiang, et al., Chem.",
"Biol.",
"4 (1997), 35-50; D. Fourmy, et al., Science 274 (1996), 1367-1371].",
"A very wide variety of computer programs have been used to exploit these data for rational drug design by calculating RNA-drug structural models [e.g.",
"“Monte Carlo”: R. Rosenfeld et al., Annu.",
"Rev.",
"Biophys.",
"Biomol.",
"Struc.",
"24 (1995), 677-700; “DOCK”: Q. Chen et al., Biochemistry 36 (1997), 11402-11407; “Molecular Modeling”: T. Herrmann & E. Westhof, Curr.",
"Opin.",
"Biotech.",
"9 (1998), 66-73].",
"In comparison with this, there are relatively few approaches for determining the specificity of the RNA-drug interaction experimentally [M. Hendrix et al., J.",
"Am.",
"Chem.",
"Soc.",
"119 (1997), 3641-3648; Y. Wang et al., Biochemistry 35 (1996), 12338-12346].",
"Although the latest studies have led to interesting results, the current state of the art only offers very limited possibilities for the specific design of RNA-binding active substances.",
"To calculate RNA-active substance models, it is essential to understand the functional and structural principles of RNA-active substance recognition exactly, as these interactions occur at the levels of the primary, secondary and complex tertiary structure of the RNA [R. Schroeder et al., EMBO J.",
"19 (2000), 1-9; T. Herrmann & E. Westhof, Curr.",
"Opin.",
"Biotech.",
"9 (1998), 66-73].",
"In this context, the NMR structures of aminoglycoside-RNA complexes have provided valuable information on the principles of the interaction of RNA with small organic molecules [review: M. Afshar et al., Curr.",
"Opin.",
"Biotech.",
"10 (1999), 59-63].",
"In this way, it has, for example, been possible to employ the structure of the complex between ribosomal RNA and paromomycin as a basis to explain the binding properties of this antibiotic, its specificity for prokaryotic organisms and the development of resistance [D. Fourmy et al., Science 274 (1996), 1367-1371; S. C. Blanchard et al., Biochemistry 37 (1998), 7716-7724].",
"Aside from structural studies, specific functional analysis has contributed to the winning of insights into the molecular recognition of RNA-binding molecules.",
"However, only a few research groups are responding to this challenge, as many of the known RNA-binding molecules are complex natural substances which are difficult to modify [W. K. C. Park et al., JACS 118 (1996), 10150-10155; H. Wang & Y. Tor, JACS 119 (1997), 8734-8735; J. H.-H. Tok & R. R. Rando, JACS 120 (1998), 8279-8280].",
"The growing recognition that RNA molecules can play a decisive role in the development of the clinical picture of a disease is accompanied by increasing efforts to develop empirical procedures to identify RNA-binding active substances [M. Afshar et al., Curr.",
"Opin.",
"Biotech.",
"10 (1999), 59-63].",
"At the moment, the most promising approach appears to be to use the technologies of combinatorial chemistry, in which libraries of candidate molecules are screened for active substances, using high-throughput techniques.",
"There is increasing need today for high-throughput procedures, as these can be used to identify new lead structures to therapeutically relevant RNA structures—particularly those of known tertiary structure and regulatory significance.",
"Lead structures of this sort are very useful as they can, either directly or indirectly, lead to the development of new therapeutically active substances.",
"In addition, it would be extremely valuable for setting up data banks of RNA structure and RNA-function to identify as many low molecular weight substances as possible which bind specifically to defined RNA structures [P. Brion & E. Westhof, Annu.",
"Rev.",
"Biophys.",
"Biomol.",
"Struct.",
"26 (1997), 113-137].",
"Conventional standard methods for RNA analysis, such as gel retardation or filter binding experiments, assume kinetically stable RNA-ligand complexes for their detection and, for this reason alone, are unsuitable for the screening of large libraries of compounds on an industrial scale.",
"What would be much more desirable would be to have a robust, non-radioactive and widely applicable method which would allow the rapid and reliable identification of new RNA-binding molecules.",
"As will be seen in the following review of the methods currently used for this purpose, an assay of this sort is unknown according to the present state of the art.",
"H.-Y.",
"Mei et al.",
"describe a procedure for the identification of inhibitors of the group I self-splicing intron in Pneumocystis carinii [H.-Y.",
"Mei et al., NAR 24 (1996), 5051-5053].",
"Inhibitors of the group I intron splicing reaction are regarded as potential antibiotics for use in pulmonary infections which are caused by the fungus Pneumocystis carinii and which have a potentially fatal course for immune suppressed patients.",
"Mei et al.",
"carried out high-throughput screening and succeeded in identifying new inhibitory lead structures in a compound library of ca.",
"300,000 low molecular weight substances [H-Y Mei et al., Bioorg.",
"Med.",
"Chem.",
"5 (1997), 1185-1195].",
"However, the cleavage mechanism is specific for the group I intron, so that the procedure of Mei et al.",
"is not transferable to other ribozyme reactions or RNA target structures, with the result that it can only be used in a narrow context.",
"In addition, the procedure is based on radioactive detection and is also methodically demanding.",
"J. E. Arenas et al.",
"have developed a screening procedure (“SCAN”) which allows the rapid identification of low molecular weight ligands to various RNA target sequences [J. E. Arenas et al., Nucleic Acids Symp.",
"Series 41 (1999), 13-16].",
"With the help of this procedure, it was possible to isolate substances which bind to a specific regulatory RNA sequence, the so-called “epsilon-RNA” of the hepatitis B virus (HBV).",
"Some of the isolated substances exhibited very promising antiviral properties in a cell-based HBV replication model.",
"The procedure described by J. E. Arenas et al.",
"exploits the different hybridization properties of free and ligand-bound RNA to complementary nucleic acid probes.",
"Although this technique led to the identification of specific RNA-binding substances, the method is relatively demanding, as quantitative filtration steps must be performed to detect the RNA-ligand complexes.",
"In addition, Arenas et al.",
"used radioactively labeled RNAs for their high-throughput screening, evidently because other detection methods were not adequately sensitive.",
"In WO 98/18947A1, a procedure is described for the characterization and selection of RNA target molecules which bind to substances of therapeutic interest.",
"The method described is also suitable for the identification of new active substances with potential pharmacological activity.",
"RNA libraries are expressed for this purpose in living cells and brought into contact with the substance to be tested.",
"However, the identification of an RNA-target molecule pair is based on the phenotypic analysis of living cells, so that this procedure is unsuitable for the rapid high-throughput in vitro screening of large substance libraries.",
"In PCT/GB99/01761, a fluorescence-based procedure is described for the in vitro identification of RNA-binding substances.",
"It is necessary in this procedure to label both the target RNA structure being studied and an already known RNA ligand of this target structure with a fluorophore dye.",
"If RNA and ligand are spatially separated from each other, the fluorescence of the fluorophore groups can be measured.",
"In contrast, if a 1:1 complex between RNA and ligand is formed, the fluorescence is quenched.",
"The identification of a new RNA-binding substance is based on the elimination of fluorescence quenching in the presence of an RNA-binding competitor, leading to a measurable signal.",
"Although this procedure is suitable for the high-throughput screening of substance libraries, it is not generally applicable, as it always assumes that an RNA ligand has already been identified.",
"K. Hamasaki and R. Rando describe a fluorescence-based assay which can be used to examine specific interactions between RNA-binding substances and specific RNA structural motifs [Anal.",
"Biochem.",
"261 (1998), 183-190].",
"Its usefulness in principle was demonstrated with binding studies between pyrene-labeled aminoglycosides and 16S ribosomal RNA.",
"The procedure is however unsuitable for the high-throughput screening of large substance libraries, as every single substance to be tested must be provided with a fluorescent label.",
"The basic object of the present invention is therefore to provide a procedure which makes it possible to characterize interactions between RNA structures and RNA-binding molecules, rapidly, simply and reliably.",
"This procedure should not exhibit the disadvantages of the procedures according to the current state of the art, as shortly described above, and should make it possible to construct a robust, non-radioactive assay which can be automatized, for the identification of substances with potential pharmacological activity which either (a) bind directly to RNA structures or (b) inhibit the interaction between an RNA structure and an associated compound.",
"This object is solved by providing a procedure with the features characterized in the patent claims.",
"As shown in example 2, the procedure in accordance with the invention may be used particularly for searching for pharmaceutical lead substances to RNA-binding peptides or proteins.",
"With the procedure in accordance with the invention, substances with specific binding properties for short RNA structural motifs or RNA-binding molecules can be identified and characterized by using reporter ribozymes.",
"The procedure in accordance with the invention exploits the fact that (a) the binding of a molecule to a specific RNA-target motif or (b) the displacement of a molecule from a specific target motif can be directly measured, as the RNA-target motif is structurally linked to a reporter ribozyme domain.",
"An RNA construct of this sort as used in the assay is referred to below as a target reporter construct (TRK).",
"In accordance with the selected embodiment, (a) or (b), two cases can be distinguished: Case (a): If the RNA-target motif is present in the unbound form, a signal of a certain intensity can be detected.",
"As a consequence of a binding event (e.g.",
"binding of a low molecular weight substance to the target motif), the measured signal is changed.",
"As a result of this, it is possible to detect the binding of the substance to the RNA and to quantify the binding.",
"Case (b): If the RNA-target motif is present in the bound form (e.g.",
"to an RNA-binding protein) a signal of certain intensity can also be detected.",
"If the RNA-binding molecule is displaced from the binding site on the RNA, the measured signal is changed, making it possible to detect this displacement event.",
"In this way it is therefore possible to identify a substance with the desired binding properties for the original RNA-binding molecule.",
"The present invention therefore relates to a procedure for the identification of compounds which specifically bind to a desired RNA-target motif and which, as a result, can inhibit or eliminate the function of this motif.",
"This procedure is characterized by the following steps: (a) Preparing a construct (target-reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA-target motif (II), where (I) and (II) are connected to each other by an RNA-linker and where the reporter ribozyme domain (I) changes its catalytic activity after specific binding of a compound to the RNA-target motif (II); (b) Producing a signaling riboyzyme substrate which can bind specifically to the reporter ribozyme domain (I) and which can preferentially be cleaved by this; (c) Bringing into contact the TRK from step (a) and the ribozyme substrate from step (b) with the compound to be identified, for example a candidate from a substance library, or with a mixture containing this substance; and (d) Determining of the binding of the compound to the RNA-target motif, preferentially as a result of the cleavage of the ribozyme substrate.",
"On the other hand, the present invention also relates to a procedure for the identification of compounds which can displace a compound which is associated with the desired RNA-target motif (e.g.",
"a compound which is naturally associated with this RNA in the cell) and which can, as a result, inhibit or eliminate the function of this motif.",
"This procedure is characterized by the following steps: Production of a construct (target reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA target motif (II), where (I) and (II) are connected to each other through an RNA-linker and where the reporter ribozyme domain (I) changes its biological activity after displacement of the compound associated with the desired RNA target motif from the RNA target motif (II); Production of a signaling ribozyme substrate which can bind specifically to the reporter ribozyme domain (I) and which can, preferably be cleaved by this; Bringing into contact the TRK from step (a) and the compound associated with the RNA target motif; Bringing into contact the complex from step (c) and the ribozyme substrate from step (b) with the compound to be identified or with a mixture which contains this compound, for example, using a candidate from a substance library; and Detection of the displacement of a compound associated with the RNA target motif, preferentially by cleavage of the ribozyme substrate.",
"The step of the production of the signaling ribozyme substrate, as described above, can be eliminated if a substrate is already known for the reporter ribozyme domain used in the TRK.",
"An embodiment then comprises instead a step for the preparation of a signaling ribozyme substrate and, possibly, the addition of this to the procedure or reaction mixture.",
"The expression “reporter ribozyme domain”, as used herein, refers to a ribozyme which has been modified so that it is able to, for example, specifically cleave a suitable substrate RNA, giving a measurable signal.",
"Ribozymes, e.g.",
"hammerhead ribozymes (HHR), are catalytic RNA molecules which are capable of cleaving other RNA molecules at phosphodiester bonds in a sequence-specific fashion.",
"The hammerhead ribozyme structure includes three double stranded regions (helices I, II, and III), which flank the cleavable phosphodiester bond, and also two highly conserved single strand sequences [O. Uhlenbeck, Nature 328 (1987) 596-600].",
"For the purposes of the current invention, all ribozymes are in principle suitable which can cleave phosphodiester bonds in trans, i.e.",
"intermolecularly.",
"Apart from ribonuclease P [C. Guerrier-Takada et al., Cell 44 (1983), 849-857], the known ribozymes which occur in nature (hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme, Neurospora mitochondrial VS ribozyme, group I and group II introns) are however self-cleaving or self-splicing catalysts, which act in cis (intramolecularly) [reviewed in P. Turner (eds.",
"), Ribozyme protocols, Humana press (1997), 1-9].",
"By separating the catalytic unit from the sequence containing the site of cleavage, it was possible in all cases to produce variants of the ribozymes which cleave in trans: hammerhead ribozyme [J. Haselhoff and W. Gerlach.",
"Nature 334 (1988), 585-591]; hairpin ribozyme [A. Hampel and R. Tritz, Biochemistry 28 (1989), 4929-4933]; hepatitis delta ribozyme [M. Been, Trends Biochem.",
"Sci.",
"19 (1994) 251-256]; Neurospora mitochondrial VS ribozyme [H. Guo et al., J. Mol.",
"Biol.",
"232 (1993) 351-361]; group I intron from Tetrahymena [Zaug et al., Nature 324 (1986), 429-433]; group II intron [S. Augustin et al., Nature 34 (1990) 383-386].",
"Separation of the catalytic core sequence from a substrate sequence containing the site of cleavage made it possible to obtain ribozyme variants (corresponding to “reporter ribozyme domains”) which are capable of cleaving almost any target RNA intermolecularly under physiological conditions [J. Haselhoff, W. Gerlach, Nature 334 (1988) 585-591].",
"The hydrolysis of the target sequence to be cleaved is then always initiated by the formation of a catalytically active complex, consisting of ribozyme and substrate RNA.",
"After cleavage, the hydrolyzed substrate oligonucleotide dissociates from the ribozyme, which is then available for further conversions.",
"Trans-cleaving ribozymes can be developed on the basis of the ribozyme sequence.",
"For that purpose, the ribozyme is subdivided into two regions, where one contains the site of cleavage (substrate) and the other contains the catalytic site (in trans ribozyme).",
"Particularly active in trans ribozymes can be identified by experimental testing, i.e.",
"by the measurement of the cleavage activity of different ribozyme substrate constructs.",
"The synthetic and enzymatic production of ribozymes is known to the expert [Turner (eds.",
"), Ribozyme protocols, Humana press (1997) 51-111].",
"It has very recently been possible to optimize the kinetic properties (high turnover rates), the sequence length (minimal motifs) and substrate specificity of hammerhead ribozymes.",
"There are, for example, review articles on this subject in Birikh, Eur.",
"J. Biochem.",
"245 (1997), 1-16; Burke, Nature Biotech.",
"15 (1997), 414-415 and Eckstein, Lilley (Eds.",
"), Nucleic Acids and Molecular Biology 10, Springer Verlag (1996), 173-329.The catalytic activity of the ribozyme region then yields a measurable signal in response to the binding of a molecule of the RNA target domain or its displacement, where the term “ribozyme” includes both natural and modified ribozymes and DNA enzymes, the so-called desoxyribozymes (R. Breaker, Chem.",
"Rev.",
"97 (1997), 371-390; A. Jenne & M. Famulok, Top.",
"Curr.",
"Chem.",
"202 (1999), 102-131.As an alternative to the ribozymes which cleave nucleic acids, the present invention can also use other suitable signaling ribozymes, for example, ribozymes with RNA-ligase activity.",
"In the case of a ligase ribozyme, the binding event can be detected by PCR [M. P. Robertson & A. D. Ellington, Nat.",
"Biotechnol.",
"17 (1999), 62-66].",
"In this case, the detection can preferentially be performed by using the so-called “Taq-man” probes (K. J. Livak et al., PCR Methods Appl.",
"4 (1995), 357-362].",
"The term “RNA target motif”, as used herein, relates to RNA molecules or parts of these which fulfill a specific function within the cell on the basis of their sequence or structure.",
"The motifs can then either occur naturally in the cell or be of synthetic nature (e.g.",
"intracellularly expressed RNA aptamers, so-called “intramers”, see following; M. Blind, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 96 (1999), 3603-3610).",
"In the simplest case, the RNA target motif is identical with the reporter ribozyme, i.e.",
"the ribozyme itself is the target.",
"Examples of therapeutically relevant processes which are steered by ribozymes include self-splicing in pathogenic microorganisms, miss-splicing in human cells (e.g.",
"sickle cell anemia), tRNA processing by RNase P or RNA processing of the hepatitis delta virus genome [P. C. Turner (eds.",
"), Methods in Molecular Biology: Ribozyme Protocols, Vol.",
"74 (1997), Humana Press, Totowa, N. J., USA].",
"Structurally unique and highly conserved RNA structural elements with an important biological function are an important class of RNA target motifs [A. S. Brodsky & J. R. Williamson, J. Mol.",
"Biol.",
"267 (1997), 624-639; M. Afshar et al., Curr.",
"Opin.",
"Biotech.",
"10 (1999), 59-63].",
"Recognized examples of this include the structural elements “TAR” and “RRE” in the mRNA of the HI virus or the IRES sequence, which is responsible for the specific translation of certain proteins.",
"The significance and strategies for the selection of suitable RNA structural elements are discussed in detail in a recent review article by D. J. Ecker & R. H. Griffey [Drug Disc.",
"Today 4 (1999), 420-429].",
"The manner in which the structural element was found is nevertheless unimportant for the procedure in accordance with the invention.",
"However, for the signal detection to be as unambiguous as possible, the selected RNA target motif may not be too long, with a preferred maximum length of 60 nucleotides and a more preferred maximum length of 40 nucleotides.",
"RNA or DNA aptamers are particularly important with respect to the identification of substances directed against RNA-binding proteins [A. D. Ellington & J. Szostak, Nature 346 (1990), 818-822; C. Tuerk & L. Gold, Science 249 (1990), δ 05-510].",
"Aptamers are artificially selected nucleic acids with specific and sometimes high affinity binding properties to many different molecules [M. Famulok & A. Jenne, Curr.",
"Opin.",
"Chem.",
"Biol.",
"2 (1998), 320-327; M. Famulok, Curr.",
"Opin.",
"Struc.",
"Biol.",
"9 (1999), 324-329; S. E. Osborne & A. Ellington, Chem.",
"Rev.",
"97 (1997), 349-370].",
"As described in example 2, the claimed procedure can be used to identify lead substances against aptamer-binding molecules.",
"In this context, those aptamers are of particular interest which are directed against disease-relevant (intracellular) proteins (P. D. Good et al., Gene Ther.",
"4 (1997), 45-54; K. Konopka et al., Drug Target 5 (1998), 247-259; C. Tuerk & S. MacDougal-Waugh, Gene 137 (1993), 33-39; K. B. Jensen et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 19 (1995), 12220-12224; M. Blind, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 96 (1999), 3603-3610).",
"Aptamers and their intracellular equivalents, the intramers, are target-specific macromolecular lead substances which exhibit important pharmacological properties shared by the later therapeutically active product.",
"Aptamers are therefore extraordinarily well suited for the development of low molecular weight drugs, as the “therapeutic information” stored in the aptamer can be exploited by the procedure in this invention.",
"In principle, a measurable signal is produced in the procedure according to the invention when the macromolecular aptamer is displaced from its binding site on the target protein by another substance.",
"It can therefore be assumed in all probability that the identified substance possesses similar properties as the aptamer (e.g.",
"inhibition of the function of the target protein).",
"In contrast to many other screening assays, the native target protein can be used without potential interference from modifications, such as labeling with dyes or isotopes.",
"The term “target reporter construct”, as used herein, refers to the coupling of the reporter ribozyme domain and the RNA target motif through an RNA linker (see definition below), where the coupling occurs in such a way that the reporter ribozyme domain changes, keeps or loses its biologically active conformation after specific binding of a specific ligand (the compound to be identified) to the RNA target motif.",
"The expert can produce suitable target reporter constructs with the help of techniques which have now been established (Soukup and Breaker, Current Opinions in Structural Biology 10 (2000), 318-325).",
"The present definition also includes TRKs in which the reporter ribozyme domain and the RNA target motif are identical, and the presence of an RNA linker is eliminated, especially in this case.",
"In the context of the target reporter construct (TRK), the aptazyme (aptamer-ribozyme) will be discussed here shortly.",
"RNA molecules the function of which can be regulated by protein binding play an important role in many cellular processes [K. J. Addess et al., J. Mol.",
"Biol.",
"274 (1997), 72-83; M. J. Gait & J. Karn, Trends Biochem.",
"Sci.",
"18 (1993), 255-259].",
"It has recently been shown that controllable RNAs—in this case allosteric ribozymes—can be obtained by rational design or in vitro selection.",
"This was based on the fact that the spatial structure of a ribozyme can be stabilized or destabilized by structural changes and that major structural changes affect the catalytic activity of the ribozyme in most cases.",
"In addition, the fact was exploited that structural changes in RNA molecules can be coupled to the binding of a ligand.",
"Breaker et al.",
"succeeded in producing structural changes in hammerhead enzymes by evolutive methods or by rational design in such a way that the catalytic activity of the ribozymes could be structurally regulated by binding a low molecular weight ligand [J. Tang & R. R. Breaker, Chem.",
"Biol.",
"4 (1997), 453-459; J. Tang & R. R. Breaker, RNA 3 (1997), 914-925; J. Tang & R. R. Breaker, NAR 26 (1998), 4214-4221; G. A. Soukup & R. R. Breaker, PNAS 96 (1999), 3584-3589].",
"Ellington et al.",
"applied the principle of allosteric regulation to ligase ribozymes [M. P. Robertson & A. D. Ellington, Nat.",
"Biotechnol.",
"17 (1999), 62-66].",
"The term “aptazyme” has now become established in the literature for these ribozymes which can be allosterically regulated.",
"An aptazyme is characterized by two structural domains which are independent of each other—a catalytically active RNA motif (“ribozyme”) and a ligand-binding RNA-motif (“aptamer”), which serves as a receptor domain.",
"Binding of a ligand to the receptor domain causes structural changes, which result in increases or decreases in the catalytic activity.",
"Aptazymes are therefore suitable, for example, as molecular switches for the detection of low molecular weight substances or as control units for the conditional control of gene expression [see e.g.",
"WO 94/13791 and PCT 98/08974].",
"This also applies to the oligonucleotides in accordance with the invention.",
"In the target reporter construct in accordance with the invention, the RNA target motif must be linked to the ribozyme domain in a defined manner.",
"The target ribozyme construct is based on the previously described general principle of allosterically controllable ribozymes and must in principle exhibit the following properties.",
"The term “RNA linker”, as used herein, relates to an RNA sequence which connects the reporter ribozyme domain and the RNA target motif in such a way that signaling is communicated by conformational changes in the RNA structure.",
"This “RNA connecting link” is of particular importance in the procedure according to the invention [G. A. Soukup & R. R. Breaker, Structure 7 (1999), 783-791; G. A. Soukup & R. R. Breaker, Trends Biotech.",
"17 (1999), 469-476].",
"Suitable connecting links can be selected either by empirical testing of different known sequences or by in vitro selection [G. A. Soukup & R. R. Breaker, PNAS USA 96 (1999) 3584-3589].",
"The term “signaling ribozyme substrate”, as used herein, relates to any RNA molecule which can bind specifically to the reporter ribozyme domain, which is cleaved by it when it is in its biologically active conformation and which permits detection of this cleavage.",
"This assumes that the cleaved ribozyme substrate can be distinguished from the uncleaved ribozyme substrate and that a directly detectable signal is produced.",
"For example, the ribozyme substrate may have an anchor group at one end, which allows its immobilization to a suitable matrix, and has at the other end a reporter group, which serves to detect the immobilized (uncleaved) ribozyme substrate.",
"When the TRK is inactive (i.e.",
"when there is no suitable ligand for the RNA target motif), the ribozyme substrate remains intact and can be simply detected after immobilization on the matrix, as the anchor group is still bound to the reporter group.",
"In contrast, when the TRK is active (i.e.",
"after binding of the ligand to be identified to the RNA target motif), the reporter-specific signal is not detectable, as the reporter group is separated from the anchor group, as a result of the cleavage of the substrate.",
"As an alternative to an anchor group (such as biotin), the ribozyme substrate can also be immobilized by complementary sequence hybridization, if both, cleavage site and reporter group are distal to the hybridization site.",
"Reporter groups which are simple to detect and which are easy to couple to the ends of nucleic acids include 32P, dye molecules and molecules which are detectable with labeled antibodies.",
"However, it is also possible to detect the cleaved ribozyme substrate by a series of other procedures which are known to the expert, comprising gel electrophoresis and PCR.",
"The signaling ribozyme substrate is essentially complementary to the sequence or sequences of the reporter ribozyme domain which are responsible for substrate binding, i.e.",
"the substrate exhibits complementarity which allows binding to the ribozyme in such a manner that effective and specific cleavage of the ribozyme substrate is guaranteed.",
"Preferably, the ribozyme substrate is fully complementary to the sequences of the reporter ribozyme domain which are responsible for substrate binding.",
"The length of the binding region of the ribozyme substrate is preferably 8 to 14 nucleotides [P. Turner eds., Ribozyme protocols, Humana press (1997), 151-159, 253-264].",
"The ribozyme substrate may contain additional sequences at its 5′- and/or 3′-end which do not participate in the binding to the ribozyme.",
"The compounds to be identified or candidate compounds can in principle be any compounds of the widest variety of types.",
"The expert is also aware of a variety of sources which contain compounds which are suitable for the screening procedure in accordance with the invention.",
"For example, all conceivable substance libraries are suitable, including antisense nucleic acids; however, libraries of low molecular weight molecules are preferred, which fulfill certain conditions, for example, with respect to low toxicity [D. J. Ecker & R. H. Griffey, Drug Disc.",
"Today 4 (1999), 420-429].",
"The constructs which are necessary for the procedure in accordance with the invention (TRK, ribozyme substrate) and the oligonucleotides in accordance with the invention are preferably prepared in larger quantities by in vitro transcription of the corresponding DNA sequences.",
"For this purpose, DNA sequences are inserted into a vector which allows replication of the inserted DNA in a suitable host, under the control of a suitable promoter, preferably the T7 promotor.",
"Suitable vectors for the replication of prokaryotic or eukaryotic systems include, for example, pBR322, pNEB193, pUC18, pUC19 (Biolabs, USA.)",
"[J. Sampson and O. Uhlenbeck, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 85 (1988), 1033-1037].",
"The plasmids are subsequently isolated, purified and the in vitro translation is performed in accordance with standard procedures.",
"The constructs used for the procedure in accordance with the invention can however also be prepared by automated solid phase synthesis in accordance with standard procedures.",
"In a preferred embodiment of the procedure in accordance with the invention, the reporter ribozyme domain is derived from a hammerhead ribozyme.",
"Reference is made to the above discussion on hammerhead ribozymes and the production of variants which perform intermolecular cleavage.",
"In a preferred embodiment, the above or signaling ribozyme substrate is doubly labeled and the cleaved substrate is easy to distinguish from the intact substrate.",
"For example, a terminally biotinylated ribozyme substrate can be used, which is labeled with fluorescein at its other end.",
"After the reaction has been completed, the sample is incubated with a streptavidin-coated solid phase (e.g.",
"with a commercially available microtiter plate), to permit coupling of the biotinylated substrate end to the streptavidin matrix.",
"The matrix is then washed and tested.",
"If a ligand is present which binds specifically to the RNA target motif (reporter ribozyme domain is activated), there is no measurable fluorescein-specific fluorescence, or only weak and non-specific background fluorescence, as the fluorescein-labeled cleavage product could not be immobilized.",
"If however there is no activation of the reporter ribozyme construct, because of the absence of a ligand which binds specifically to the RNA target motif, the proportion of uncleaved and immobilized ribozyme substrate can be quantified by measurement of the fluorescein-specific fluorescence.",
"In a particularly preferred embodiment of the procedure in accordance with the invention, the doubly labeled ribozyme substrate contains a fluorophore group and a group which quenches fluorescence, whereby, after cleavage by the reporter ribozyme domain, the quenching of the fluorescence of the fluorophore by the quenching group is prevented.",
"Ribozyme substrates labeled in this way can, for example, be used in the FRET procedure [FRET=Fluorescence resonance energy transfer (J. R. Lakowicz, Principles of Fluorescent Spectroscopy; Plenum Press, New York (1983)].",
"FRET oligonucleotides are, for example, described in K. J. Livak, S. J.",
"A.",
"Flood, J. Marmaro, W. Giusti, K. Deetz, PCR Meth.",
"Appln.",
"4 (1995), 357-362.Particularly preferred FRET ribozyme substrates are RNA oligonucleotides or DNA-RNA hybrids in which a fluorescent group (e.g.",
"FAM=6-carboxyfluorescein, TET=tetrachloro-6-carboxyfluorescein or HEX=hexachloro-6-carboxyfluorescein) and a corresponding fluorescence quenching group, a so-called “quencher”, (e.g.",
"sulforhodamine 101, TAMRA=6-carboxytetramethylrhodamine or Cy 3), are incorporated close enough to each other for there to be effective quenching of the fluorescence of the fluorophore [Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York (1983), 303-339; V. Förster, Annals of Physics (Leipzig) 2 (1948), 55-75].",
"After cleavage of the ribozyme substrate by ribozyme-catalysed hydrolysis of a specific phosphodiester bond, the cleavage products can separate from each other in solution: the fluorescence of the fluorophore is now no longer subject to intramolecular quenching.",
"If therefore a suitable ligand binds to the RNA target motif, leading to a biologically active reporter ribozyme domain, this can be determined by production of a measurable fluorescence signal by cleavage of the substrate.",
"The procedure in accordance with the invention based on FRET technology is particularly suitable for the industrial high-throughput screening of substance libraries, as it is simple to perform and easy to adapt to different formats of microtiter plates [X. Chen et al., Genome Res.",
"8 (1998), 549-556; K. P. Bjornson et al., Biochemistry 33 (1994), 14306-14316; A. R. Gelsthorpe et al., Tissue Antigens 54 (1999), 603-614; J. E. Gonzalez et al., Drug Discov.",
"Today 4 (1999), 431-439].",
"In particular, radioactive waste is avoided, which otherwise must be expensively disposed of.",
"Moreover, this embodiment of the procedure in accordance with the invention has the advantage that it very sensitively detects the binding of the ligand, as the catalytic cleavage of the FRET substrate leads to clear signal amplification [for the very rapid determination of the cleavage activity of hammerhead enzymes by fluorescence measurement in the microtiter plate format, see also Jenne et al., Angew.",
"Chem.",
"111 (1999), 1383-1386.].",
"The embodiment of the procedure in accordance with the invention based on FRET technology is preferably designed so that initially, before the binding or displacement event which is to be measured, no or only a very small signal is measured and there is only a clear change in the fluorescence signal as a consequence of the binding or displacement.",
"This is mainly achieved as the result of selection of a suitable RNA linker, which connects the RNA target sequence to the ribozyme domain (see above).",
"Methods to label ribonucleic acids with fluorophores or with fluorescence-quenching groups and techniques to measure energy transfer (quenching) have already been described in detail [Turner (ed.",
"), Ribozyme protocols, Humana press (1997), 241-251].",
"5′-Fluorophore- and 3′-quencher-labeled RNA-oligonucleotides are commercially available (e.g.",
"5′-FAM- and 3′-TAMRA-labeled RNA from Eurogentec, Belgium).",
"It is favorable to perform the labeling at the RNA ends, so as not to influence the hybridization in the reporter ribozyme domain.",
"To avoid fluorescence emission from unwanted cleavage (e.g.",
"by nucleases) it is particularly advantageous to use nuclease-resistant ribozyme substrates (Eaton and Pieken, Annu.",
"Rev.",
"Biochem.",
"64 (1995), 837-863 and Shimayama et al., Nucleic Acids Res.",
"21 (1993), 2605-2611).",
"This is especially advantageous with respect to in vivo applications, in which the doubly labeled substrate is transfected exogenously into cells by suitable techniques (e.g.",
"microinjection, liposome transport, etc.)",
"(P. Turner (ed.",
"), Ribozyme protocols, Humana press (1997), 417-451).",
"In a particularly preferred embodiment, the double labeled substrates are then modified RNA oligonucleotides.",
"As long as the site of cleavage in the substrate is NUH ↓, (according to the IUB Code.",
": N=any base, H=A, U or C), the substrate can contain desoxyribonucleotides and/or modified bases and/or 2′-modified ribose units.",
"This increases the stability of the substrate in the cell extract (N. Taylor et al., Nucleic Acids Res.",
"20 (1992), 4559-4565).",
"The use of internally labeled rather than end labeled substrates can also contribute to an improvement in the signal to noise ratio, as fluorescence quenching may be enhanced when the distance between the two groups (fluorophore and quencher) is shorter.",
"The present invention also relates to a drug which contains a compound which has been identified by a procedure in accordance with the invention.",
"This also includes a compound derived from this, which can also bind to the RNA target motif, whereby this compound, for example, only includes a portion or a partial sequence of the originally identified compound or a portion or partial sequence which differs from this, with differing, but preferably enhanced affinity to the RNA target motif, in comparison to the original compound.",
"The drug is preferably combined with a suitable carrier.",
"Suitable carriers and the formulation of drugs of this sort are familiar to the expert.",
"For example, suitable carriers include phosphate-buffered saline, water, emulsions, for example, oil/water emulsions, wetting agents, sterile solutions, etc.",
"The administration of the drug can be either oral or parenteral.",
"The procedures for parenteral administration include topical, intra-arterial, intramuscular, subcutaneous, intramedullar, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration.",
"Finally, the present invention relates to a kit (or assay) for the performance of the procedures in accordance with the invention, where the kit includes the following compounds, as described above: (a) a target reporter construct (TRK) and (b) a signaling ribozyme substrate.",
"In a further aspect the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer which is specific for a target molecule, the aptamer portion of the polynucleotide preventing the formation of the catalytically active ribozyme by base pairing with sequences of the hammerhead ribozyme.",
"This is achieved in that the sequence which is essential for the catalytic activity of the hammerhead ribozyme, or a part of this, is, as a result of base pairing with the aptamer sequence, no longer available for the base pairing which is required for the catalytic activity.",
"A further aspect of the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer, the aptamer being specific for a target molecule, in particular a polynucleotide in accordance with the above aspect of the invention, which also includes the bound target molecule which is specific for the aptamer, and where the binding of the target molecule to the aptamer sequence leads to the development of enyzmic activity in the ribozyme.",
"Yet another aspect of the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, where the binding site of the catalytic domain of the ribozyme for a ribozyme substrate is blocked in the absence of the target molecule of the aptamer for binding of a substrate of the ribozyme.",
"A further aspect of the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular one in accordance with one of the other aspects of the present invention, where the binding site of the catalytic domain of the ribozyme is accessible for a ribozyme substrate in the presence of the target molecule of the aptamer for binding of a substrate of the ribozyme.",
"Finally, another aspect of the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular a polynucleotide in accordance with one of the other aspects of the present invention, where the pattern of base pairing of the polynucleotide on binding of the target molecule to the aptamer is different from that of the polynucleotide in the absence of the target molecule of the aptamer, in particular, when the target molecule does not bind to the aptamer.",
"In other words, the present invention also relates to a polynucleotide which comprises a hammerhead ribozyme and an aptamer which is specific for a target molecule, where both, the hammerhead ribozyme and the aptamer, are joined to each other and give different base pair hybridization patterns in the ribozyme, depending on the presence of the target molecule which is specific for the aptamer, which has the following effects: Binding of the target molecule to the aptamer leads to the formation of a catalytically active ribozyme in the polynucleotide in accordance with the invention.",
"In contrast, if the target molecule does not bind to the aptamer, the catalytically active ribozyme in the polynucleotide in accordance with the invention is not formed.",
"For example, the target molecule cannot bind to the aptamer sequence when a binding partner of the target molecule, for example, an inhibitor is present, which prevents a specific interaction between the aptamer nucleic acid part of the polynucleotide in accordance with the invention and the target molecule.",
"The polynucleotides described above are therefore both states of a polynucleotide, which are defined by the binding or non-binding of the target molecule of the aptamer.",
"The two states differ not only in their different secondary and tertiary structures, but additionally and particularly by the type and, in some cases, numbers of base pairs formed in the polynucleotide.",
"In other words, the two states differ in their patterns of base pairing.",
"This change in base pairing pattern constitutes a difference between the polynucleotides in accordance with the invention and the ribozymes known in the state of the art, including the allosteric ribozymes and the aptazymes, in which the allosteric effect of the binding of the allosteric effectors, such as the target molecule of the aptamer, is only evident as a change in the secondary and/or tertiary structure, but not in a change in the pattern of base pairing.",
"In the sense of the present disclosure, the oligonucleotides in accordance with the invention correspond to target receptor constructs, the hammerhead ribozymes to receptor ribozyme domains and the aptamers to RNA target motifs.",
"In the context of the present invention, the aptamer may also consist of deoxyribonucleic acid.",
"The herin described hammerhead ribozymes are typically those which consist of three helices, two of which form their “target RNA” by hybridization with the complementary sequence and the third of which is formed by a double strand within the ribozyme.",
"The catalytic domain of the ribozyme consists of nucleotides which are arranged between the double stranded structures.",
"The term “target RNA” means the RNA substrates which can be cleaved in cis or in trans by hammerhead ribozymes.",
"In the context of the present invention, the target RNA is typically RNA which is a substrate for the corresponding ribozyme, particularly a signaling substrate, in the sense of the present invention.",
"In one embodiment of the polynucleotides in accordance with the invention, it is planned that these include a substrate for the catalytic activity of the ribozyme.",
"Substrates of this type have already been described in the context of the above aspects of the invention, in particular the elaboration of the substrate as a FRET substrate.",
"Reference is made here to the corresponding disclosure.",
"In a further preferred embodiment, the polynucleotide in accordance with the invention exhibits a sequence which is selected from the group comprising SEQ ID No.",
"51 and SEQ ID No.",
"52.The polynucleotide in accordance with the invention can preferably embody RNA.",
"In the context of the present invention, at least regions of the polynucleotide in accordance with the invention may also consist of DNA, particularly those regions which participate in pairing with the substrate and also those which participate in intramolecular base pairing.",
"An example of substrates for the polynucleotides in accordance with the invention is the nucleic acid disclosed in SEQ ID No.",
"53.The configuration underlying the polynucleotides in accordance with the invention gives surprising advantages in comparison with the allosteric ribozymes known in the state of the art.",
"The most important of these advantages should be seen in the fact that the polynucleotides in accordance with the invention make it possible to produce an allosteric ribozyme for which the allosteric effector, i.e.",
"the target molecule which binds to the aptamer part of the polynucleotide, can be almost freely selected, without there being resulting impairment of the functionality of the allosteric ribozyme.",
"This is a difference between the polynucleotide in accordance with the invention and the aptazymes described according to the state of the art, in which a so-called communication module is interposed between the receptor part or domain, i.e.",
"the part of the allosteric ribozyme which binds the target molecule, and the catalytically active ribozyme domain, which is referred to here as the RNA linker.",
"These communication domains have a really considerable influence on both the effectiveness and the possibilities for designing the allosteric ribozyme, in particular concerning the choice of the aptamer to be used.",
"In some cases, this has the consequence that allosteric ribozyme systems of this sort cannot be used for certain target molecules and then, for example, cannot be used in the context of screening of compounds such as candidate compounds, which influence the interaction between the aptamer and the target molecule.",
"The procedures described here, using the polynucleotides in accordance with the invention, are practically free of this type of restriction.",
"The polynucleotides in accordance with the invention are namely standardized or easily standardizable allosteric ribozymes or target receptor constructs, of which the specificity can be surprisingly easily adjusted to each molecule to which an aptamer can be produced.",
"The reason for the flexibility of the specificity of the oligonucleotides in accordance with the invention, i.e.",
"the allosteric ribozymes in accordance with the invention, is based on the structure of the hammerhead ribozymes.",
"As three helices must be formed, it is possible to design at least one helix in such a way that it is complementary to the nucleic acid sequence of the aptamer part of the allosteric ribozyme.",
"As these double stranded regions of the hammerhead ribozymes have no catalytic function, any aptamer at all can be introduced into the allosteric ribozyme, with the result that, when the target molecule of the aptamer binds to the aptamer part of the allosteric ribozyme, the helices or non-helical nucleotides which are required for the catalytic activity of the ribozyme are present as such and are not paired with at least a part of the aptamer sequence.",
"The allosteric ribozyme is only catalytically active in the form that binds target molecule.",
"It follows from the above that binding of the target molecule to the aptamer part of the allosteric ribozyme results in another base pairing or base hybridization pattern in the oligonucleotide, than that in the absence of the target molecule.",
"It is this change in the pattern of base pairing and the resulting accessibility or non-accessibility of the catalytic part of the hammerhead ribozyme which differentiates the polynucleotide in accordance with the invention from the state of the art and which is the reason for the surprising advantage of the oligonucleotides in accordance with the invention.",
"A further aspect of the present invention relates to a biosensor comprising a polynucleotide in accordance with the invention, whereby it is planned in a preferred embodiment that the polynucleotide is bound to a solid carrier.",
"The basic possibilities for the use of biosensors also apply to the embodiments in accordance with the invention.",
"In yet another aspect, the invention relates to a procedure for the identification of a compound which binds to a target molecule and which includes the following steps: a) Providing a polynucleotide in accordance with one of the claims 10 to 16, b) Optionally determining the catalytic activity of the ribozyme, c) Adding the target molecule, the target molecule interacting with the nucleic acid which binds the target molecule, d) Optionally determining the catalytic activity of the ribozyme, e) Adding a candidate compound, f) Optionally determining the catalytic activity of the ribozyme, g) Providing a substrate for the catalytic activity of the polynucleotide and adding the substrate to the reaction mixture, h) Measuring the binding of the candidate compound to the target molecule.",
"The term candidate compound is used here to designate a compound which is used in these procedures and is investigated to establish whether it binds to the target molecule and, in particular, at a site which is recognized by the aptamer which is specific for the target molecule.",
"Candidate molecules of this sort are in principle suitable as pharmaceutical lead substances, provided that they influence the interaction between the aptamer and the target molecule.",
"In particular, a candidate compound is also a “compound to be identified” in the sense of the present disclosure.",
"It is planned in a preferred embodiment that the candidate compound should initially be tested for its ability to influence the interaction between the aptamer and the target molecule.",
"This can, for example, happen in that an allosteric ribozyme in accordance with the invention or an oligonucleotide in accordance with the invention are used which comprises an aptamer, which binds to the target molecule, but possibly at another site, and which then allows a limitation of the site at which there is an interaction between the aptamer and the target molecule.",
"The allosteric ribozymes used herein, more exactly aptazymes, differ in their specificity and/or affinity to the corresponding target molecules or parts of these.",
"It is also included in the context of the present invention that, after the candidate compound has been identified or validated by the procedure in accordance with the invention, it is subjected to a further identification or validation step, where the above applies and an appropriately planned procedure in accordance with the invention is used.",
"In the context of this additional, prior or subsequent, validation step, a procedure can also be used as shown, for example, in FIG.",
"8a.",
"An allosteric ribozyme is then provided which includes an aptamer which is specific for the same target molecule, as used in the context of the procedure in accordance with the invention, in which there is a change in the pattern of base pairing as a result of binding of the target molecule.",
"The catalytic activity of the ribozyme part of the allosteric ribozyme is optionally determined in advance and then the target molecule is added to the reaction mixture in accordance with the invention, whereby the target molecule interacts with the aptamer domain in the polynucleotide which binds the target molecule.",
"The catalytic activity of the allosteric enzyme in the presence of the target molecule can then, again, optionally be determined and then the candidate compound is added.",
"There is a further optional determination of the catalytic activity of the allosteric ribozyme in the presence of target molecule, before the candidate compound is added.",
"Followed by another optional determination of the catalytic activity of the allosteric ribozyme in presence of target molecule and candidate compound, the substrate for the catalytic activity of the polynucleotide is finally provided and added to the reaction mixture of the components named above.",
"Finally, the binding of the candidate molecule to the target molecule is determined with the help of the procedure in accordance with the invention.",
"In one embodiment, the allosteric ribozyme includes a hammerhead ribozyme part and an aptamer part, possibly joined together by a linker, for example an RNA linker, as described here, where the aptamer is directed against a molecule of molecular weight above 300 Da, preferably above 1000 Da, more preferably above 2 kDa and even more preferably of more than 5 kDa.",
"In preferred embodiments, the target molecule is a peptide or protein.",
"A protein-binding allosteric ribozyme of this type in accordance with the invention is described in the examples.",
"With the procedures in accordance with the invention, in particular with those which use allosteric ribozymes which bind to peptide or protein target molecules, it has been possible for the first time to use an inhibition assay to also identify those compounds which interact with peptides or proteins.",
"As peptides and proteins participate in many biological and pathological processes, the provision of allosteric ribozymes in accordance with the invention and their use in a procedure in accordance with the invention makes it possible for the first time to determine lead structures which bind to this highly relevant group of target molecules and which can therefore be used, either directly or indirectly, in the development of pharmaceutically active substances.",
"In a preferred embodiment, it is planned to couple the two procedures shown in FIG.",
"8 for the identification of compounds which specifically interact with a target molecule, i.e.",
"the compound which has already been identified or validated in the procedure shown in FIG.",
"8a will be additionally identified or validated in the procedures shown in FIG.",
"8b.",
"The advantage of coupling the two procedures is that the inherent disadvantages of the two procedures with respect to false positive or false negative results can compensate each other.",
"In particular, false positive results can arise if the candidate compound directly affects the catalytic activity of the ribozyme, for example by binding to the ribozyme part of the aptazyme, and then the effect observed in the experiment is not due to an interaction between the candidate compound and the target molecule.",
"Especially, since in one of the two procedures the polynucleotide in accordance with the invention is used in which binding of the target molecule causes a change in the base pair hybridization pattern and the resulting easy adaptability to different target molecules accompanying this kind of polynucleotides, coupling of the two procedures leads to particularly reliable identification of compounds which react specifically with the target molecule.",
"In particular, it is intended that, before step f) or after step g) of the procedure in accordance with the invention, the candidate compound should undergo a procedure which includes the steps: a) Providing a polynucleotide, whereby the polynucleotide is an allosteric ribozyme and comprises a hammerhead ribozyme and an aptamer which is specific for the target molecule, preferably the aptamer of the polynucleotide in accordance with claim 19, b) Optionally determining the catalytic activity of the ribozyme, c) Adding the target molecule, whereupon the target molecule interacts with the nucleic acid which binds the target molecule, d) Optionally determining the catalytic activity of the ribozyme, e) Adding a candidate compound, f) Optionally determining the catalytic activity of the ribozyme, g) Providing a substrate for the catalytic activity of the polynucleotide and adding the substrate to the polynucleotide, and h) Determining the binding of the candidate molecule to the target molecule.",
"In the procedure in accordance with the invention, it is intended that the binding of the candidate compounds to the target molecule should be determined by measuring the catalytic activity of the ribozyme.",
"The catalytic activity of the ribozyme is determined by using the FRET substrate, as herein initially described.",
"It is intended in a further embodiment that the substrate should contain a fluorophore and a fluorescence quencher group, where the quenching of the fluorescence is removed, or at least reduced, after cleavage of the substrate by the catalytic activity of the ribozyme or the catalytic domain of the polynucleotide in accordance with the invention.",
"In a particularly preferred embodiment, it is intended that the fluorophore group is 6-carboxyfluorescin and the fluorescence quencher group is 6-carboxytetramethylrhodamine or “Cy3”.",
"A further aspect of the present invention relates to a drug which includes a compound which has been identified by a procedure in accordance with the invention.",
"Yet another aspect of the invention relates to a kit for the performance of the procedure in accordance with the invention and which comprises: a) a polynucleotide in accordance with the invention and b) a signaling ribozyme substrate.",
"Yet another aspect of the present invention relates to the use of coumermycin for the production of a drug to treat HIV and/or FIV.",
"Another aspect of the invention relates to the use of nosiheptide for the production of a drug for the treatment of HIV and/or FIV.",
"Yet another aspect of the invention relates to the use of patulin for the production of a drug for the treatment of HIV and/or FIV.",
"Another aspect of the present invention relates to the use of coumermycin, nosiheptide or patulin for the treatment of HIV and/or FIV.",
"Another aspect of the invention relates to the use of the compounds which were identified or validated in accordance with the invention, e.g.",
"coumermycin, nosiheptide or patulin, for the development of pharmaceutically active compounds which can eventually be used as drugs.",
"The procedure can be as follows.",
"Firstly, it is possible that derivatives of the identified compounds can be prepared the pharmacological properties of which are optimized.",
"For example, such properties are efficacy, side-effects, toxicity, etc.",
"On the other hand, it is possible that, on the basis of the identified compounds, those elements, groupings or structures can be identified which a pharmaceutically active compound necessarily exhibits.",
"In particular, a corresponding profile can be developed on the basis of several compounds once these have been identified.",
"It lies in the context of the present invention that the ribozyme substrate for the catalytic activity of the ribozyme is covalently bound to the ribozyme or to the polynucleotide which comprises the aptamer, in accordance with one of the aspects of the present invention, so that the catalytic activity of the ribozyme is an intramolecular reaction.",
"The binding between the ribozyme and the ribozyme substrate is typically an internucleoside bond, which can also be present at both ends of the ribozyme or ribozyme substrate, or within the ribozyme or the nucleic acid sequence corresponding to the ribozyme substrate.",
"If the ribozyme is elaborated in this way, the steps in the procedure in accordance with the invention are simplified, so that separate provision of the ribozyme substrate can be dispensed with, although the other steps which characterize the procedure in accordance with the invention are retained.",
"The figures and examples below are used to further explain the invention in more detail, and give rise to further characteristics, embodiments and advantages.",
"FIG.",
"1: Secondary structures of construct TRK1 and substrate SK1.FIG.",
"2: Principle of the fluorescence measurement: A so-called FRET oligonucleotide (SK1) was selected as substrate for the reporter ribozyme TRK-1.This specific substrate includes a fluorophore group (FAM=6-carboxyfluorescein) which is spatially close to a molecule which quenches fluorescence (TAMRA=6-carboxytetramethyl rhodamine), so that the fluorescence emission of the fluorophore group is effectively quenched.",
"The binding of the doubly labeled FRET substrate leads to the formation of the catalytic complex.",
"After hydrolysis of the FRET substrate by the ribozyme, the cleavage fragments can be separated from each other in solution.",
"The fluorescence of the fluorophore is then no longer intramolecularly quenched and the ribozyme is available for the next catalytic cycle.",
"The removal of the FRET effect leads to a detectable increase in the FAM-specific fluorescence in the sample.",
"As the increase in fluorescence is directly proportional to the cleavage activity of the ribozyme, the rate of the reaction can be determined from the measured data.",
"FIG.",
"3: Principle of the identification of RNA-binding substances from combinatorial compound libraries.",
"The case is presented that the binding of a substance leads to a reduction in the fluorescence measured.",
"The results are evaluated by plotting the measured fluorescence against the initial reaction rate.",
"Comparison of the rates of reaction (=slope of the curve) is used to detect the interaction between RNA-binder and RNA.",
"In the illustrated example, the slope for (B) is significantly lower than for (A).",
"FIG.",
"4: Fluorescence measurements with TRK1/SL1 which were obtained by screening the substance library.",
"Time-dependent increase of the fluorescence signal for (a) the non-inhibited TRK1 reaction, (b) the TRK1 reaction in the presence of substance #332 and (c) the TRK1 reaction in the presence of substance #425.For each, the measured values, the regression lines for the uncorrected course of the reaction, the corresponding control reaction mixture without TRK1 and the corrected reaction are shown.",
"The values for the initial rates of the reaction (=slope of the corresponding regression lines) are also given.",
"FIG.",
"5: Principle of the screening assay.",
"Each microtiter plate contains (i) the target protein to be examined, (ii) a substance from the compound library (iii) and a suitable reporter ribozyme construct, for example, an intramer-ribozyme construct (see reporting text).",
"Fluorescence measurements can be used to identify substances which are capable of binding to the target protein being examined.",
"As this results in displacement of the RNA ribozyme construct from the protein binding domain, there is a detectable change in the fluorescence emission.",
"The fluorescence signal is enhanced in the example illustrated.",
"Microtiter wells A and C in the illustration contain no target protein-binding substance, so that only a weak signal is detected.",
"In contrast, well B gives a rapidly increasing signal, as this well contains a substance with the desired target-specific properties.",
"FIG.",
"6: Secondary structures of reporter ribozyme construct IRK1 and ribozyme substrate SK1.The selected Rev-binding sequence was wt RBE motiv, as isolated by D. P. Bartel et al.",
"[Cell 67 (1991), 529-536].",
"FIG.",
"7: Secondary structures of library (A) and of the isolated Rev-binding sequence (Seq 5) (B) [L. Giver et al., NAR 23 (1993), 5509-5516].",
"FIG.",
"8: High through-put systems based on RNA/protein interactions.",
"FIG.",
"8a) shows an allosteric hammerhead ribozyme, which comprises an aptamer component which binds specifically to Rev.",
"When Rev binds to the aptamer component of the ribozyme, the catalytic activity of the ribozyme is reduced.",
"If a suitable FRET substrate is used, which consists in this case of the two components FAM and TAMRA, and the sample is irradiated at 488 nm, there is then energy transfer between FAM and TAMRA, without fluorescence emission.",
"If various compounds, which could, for example, come from a compound library, are added to a reaction mixture containing these components and one of these compounds, small molecules in this case, interacts with Rev, this leads to release of Rev from the aptamer component, resulting in changes in the secondary or tertiary structure of the ribozyme, as a result of which the catalytic activity of the ribozyme is triggered or at least raised.",
"In this case, the FRET substrate is cleaved, and after irradiation at 488 nm, fluorescence emission at 520 nm is generated.",
"This fluorescence emission is the signal which shows that the respective compound, a small molecule in this example, herein also referred to as a candidate molecule, leads to a specific interaction with the target molecule and is accordingly a lead substance for the corresponding target molecule.",
"The Rev protein can be used at 1 μM in this system.",
"In FIG.",
"8a), the polynucleotide in accordance with the invention, an allosteric aptazyme, is shown in a procedure in accordance with the invention to identify a compound which interacts with a target molecule.",
"On binding the target molecule, in this case of Rev to the aptamer component of the polynucleotide in accordance with the invention, the ribozyme is present in a catalytically active form.",
"The FRET substrate with FAM and TAMRA, which is bound to the ribozyme and particularly to the catalytically active domain, is cleaved as a result of the catalytic activity, which abolishes the fluorescence quenching, leading to fluorescence emission at 520 nm on irradiation at 480 nm.",
"If one or several compounds to be identified are added to a reaction mixture of this type, and if the or one of these compounds interact with the target molecule, in the present case with the Rev protein, Rev dissociates from the aptamer component of the polynucleotide in accordance with the invention.",
"As a consequence of this, a different pattern of base pairing develops in comparison to the state when the Rev protein was bound to the aptamer component, leading to reduction or suppression of the catalytic activity of the ribozyme.",
"The substrate can no longer be cleaved and there is no longer fluorescence emission, where preferably the substrate can no longer bind to the area of catalytic activity as a result of a change in the pattern of base pairing.",
"FIG.",
"9 shows the sequences of the ribozymes in accordance with the invention used for the screening assays described in example 3, which are complexed with a 13mer substrate.",
"The aptamer-inhibited ribozyme AIR consists of a Rev-aptamer, which is bound through a penta-A linker to the 5′-end of the hammerhead ribozyme (HHR), corresponding to a recently discovered hammerhead ribozyme (1) and (2).",
"The aptamer domain forms a helix with the substrate binding site of the ribozyme domain.",
"In the absence of Rev, the aptamer component is not folded, but forms a helix with stem I of the ribozyme and prevents the binding of substrate in this way.",
"After the addition of Rev, the substrate binding site is released and the catalytic reaction (2) can occur.",
"The Rev response ribozyme (RRR) shown as (3) in FIG.",
"1 contains an HIV genome Rev binding element (RBE) in stem II.",
"RRR is active in the absence of Rev and is inhibited in its presence.",
"The three structures illustrated in FIG.",
"1 have been folded in accordance with the mfold server algorithm of M. Zucker and show the structures with minimal energy, namely 27.1 and 30.6 kcal/mol for AIR, and 25.4 kcal/mol for RRR.",
"The 3′-ends of the ribozyme and the 5′-ends of the substrate were connected by a tetraloop of GAAA for folding.",
"FIG.",
"10 shows the initial rates of reaction, expressed as measured fluorescence per time for the first five minutes, with the fluorescence of the negative control being subtracted.",
"Illustrated are the reactions with only ribozyme and FRET substrate, Rev reactions, containing 1 μM Rev-peptide in the case of HHR and RRR, 250 nM Rev in the case of AIR and reactions with Rev+compound 21 (Comp.",
"21), containing Rev and compound 21, coumermycin A1 at 100 μM.",
"The addition of Rev leads to inhibition of ribozyme RRR and activation of AIR.",
"In the case of RRR and AIR, the action of Rev is fully reversed by 100 μM coumermycin.",
"Wildtype HHR is hardly affected by Rev and compound 21 and serves as internal control.",
"FIG.",
"11 shows the concentration dependence of the activities of the identified compounds on Rev binding screening ribozymes, RRR and AIR.",
"The initial reaction rates were determined from the initial increase in fluorescence divided by the rate of the ribozyme reaction in the active state in which the compounds were absent.",
"FIG.",
"11a shows reactions containing 1 μM Rev peptide and various concentrations of coumermycin, novobiocin, nosiheptide and patulin.",
"FIG.",
"11b shows the AIR reactions which contain 250 nM Rev peptide and various concentrations of coumermycin, novobiocin and patulin.",
"FIG.",
"12 shows filter binding studies, where the filter binding was performed with 100 nM Rev protein, traces of 5′-32P-labeled reporter ribozymes, 200 nM FRET substrate and various concentrations of compounds in test buffer at room temperature.",
"The values shown are divided by a control reaction which contains no compound.",
"FIG.",
"12a shows binding studies with RRR and FIG.",
"12b binding studies with AIR.",
"FIG.",
"13 shows examples for the binding of identified antibiotics to Rev in the context of surface plasmon resonance studies.",
"20 μl 100 μM antibiotic in test buffer was injected at 25° C., rate 10 μl/s.",
"FIG.",
"13a shows the binding of coumermycin to surfaces which have been derivatized with Rev peptide and Rev protein.",
"There is no binding to RRR-derivatized surfaces detectable.",
"Coumermycin only binds to Rev, but not to ribozymes.",
"FIG.",
"13b illustrates the interaction between novobiocin, rosamicin, griseofulvin, streptolydigin and patulin with surfaces derivatized with Rev protein.",
"Only the antibiotics novobiocin and patulin, which were identified in the course of screening, exhibited binding to Rev.",
"FIG.",
"14 shows the cleavage activity which was measured as fluorescence per minute in the initial phase (5 min).",
"Negative control reactions without ribozymes were subtracted.",
"FIG.",
"14a illustrates how the initial cleavage activity for RRR and AIR depends on Rev peptide.",
"HHR is also slightly inhibited by more than 1 μM Rev peptide.",
"FIG.",
"14b shows how the initial cleavage activity for RRR and AIR depends on Rev protein.",
"HHR is also inhibited at more than 2 μM Rev protein.",
"There was no observed inhibition of AIR by high concentrations of Rev protein, as seen for Rev peptide in FIG.",
"14a.",
"FIG.",
"15 shows screening results, in which the ratios RRR/HHR and AIR/HHR were generated by subtraction of the fluorescence of the negative control reactions without ribozyme from the initial fluorescence, and division by the values obtained with active states of the ribozymes.",
"Then RRR/AIR values were divided by HHR values, to eliminate general effects on ribozyme function.",
"These reactions contained 10 nM ribozyme, 200 nM substrate, 1 μM (screen 1) or 250 nM (screen 2) Rev peptide and 100 μM antibiotics.",
"As a consequence of the total inhibition, the previously mentioned four most potent inhibitors of hammerhead ribozyme (#8, #31, #91, #92) were removed from the library of 96 antibiotics.",
"In screen 1, Rev effectors were identified by activation of RRR relative to HHR and in screen 2 by inhibition of AIR relative to HHR.",
"FIG.",
"16 shows Tables 1a and 1b.",
"Table 1a shows a comparison of the different values determined for selected antibiotics.",
"The column “Screening Results”: Identification with positive (RRR) and negative (AIR) measurements, where the illustrated show initial corrected fluorescence per time, relative to the active state of the ribozyme, and are divided by relative HHR reactions.",
"Novobiocin was not present in the library.",
"Kcomp: The antibiotic concentrations for half maximal recovery (RRR) or inhibition (AIR) of the cleavage activity obtained by adjusting the data shown in FIG.",
"11.Kfilter: The antibiotic concentration for the half maximal release of the ribozyme/Rev protein interaction, obtained by adjusting the data shown in FIG.",
"12.Table 1b: Kp values determined by surface plasmon resonance for the binding of antibiotics to the Rev protein.",
"FIG.",
"17 shows the procedure illustrated in FIG.",
"8, depicting the secondary structures.",
"The reporter system shown in FIG.",
"17(a) corresponds to that in FIG.",
"8(a) and the reporter system in FIG.",
"17(b) to that in FIG.",
"8(b).",
"EXAMPLE 1 Screening of Compounds which Bind to the A-Site Subdomain of 16S RNA With the help of the assay, a library of 500 uncharacterized low molecular weight substance mixtures (molecular weight under 3000 g/mol) was tested on microtiter plates for their binding properties to the target ribozyme construct TRK 1.The mixture of substances was obtained by filtration of bacterial extracts (Actinomycetes strains).",
"TRK1-RNA and the substrate SK1 were produced by automatic solid phase synthesis (for sequences see FIG.",
"1).",
"FIG.",
"2 shows the functional principle of the assay.",
"An inhibitory or activating effect on the cleavage activity of the ribozyme domain was measured as a reduced or increased fluorescence signal, respectively.",
"Table 1 shows a typical result of a screening experiment.",
"FIG.",
"3 shows a schematic depiction of the assay.",
"To increase the accuracy of the assay, control reaction mixtures without target ribozyme construct TRK1 were included on the same microtiter plate as the reaction mixture.",
"Subtraction of the fluorescence signals in the control mixtures made it possible to eliminate to a very large extent effects of the substance used on the FRET substrate which were non-specific for the ribozyme or target (e.g.",
"RNA aggregation, quenching effects or RNAse degradation).",
"As shown in FIGS.",
"4a-c, correction for these non-specific effects clearly reduced the errors in the measurement of the rates of reaction.",
"For example, incubation with substance #332 led to a significant decrease in the fluorescence signal from the control reaction (FIG.",
"4b).",
"In contrast, an increase in fluorescence was measured in the presence of substance #425, not only in the HHR-1 reaction, but also in the control sample (FIG.",
"4c).",
"Two substances from the tested library were identified on the basis of their clear inhibition on the rate of cleavage of the ribozyme domain: substance #122 and substance #387.To exclude the possibility that the observed inhibition or activation was a consequence of the fluorescence measurement procedure, a series of control experiments was performed, in which ribozyme cleavage was examined in a conventional manner with a 5′-32P labeled substrate (SKU1).",
"This analysis confirmed the results with substances #122 and #387.In a subsequent control experiment, the ribozyme domain (HHR1) of the TRK1 was examined separately, i.e.",
"without linker sequence and 16S RNA domain, in identically performed experiments.",
"The missing sequence was then replaced by the hairpin structure 5′-CCGGAUUGCCGG-3′.",
"It was shown that substance #122 inhibits ribozyme HHR1, while substance #387 has no measurable effect on the ribozyme.",
"A binding study with the 16S domain shown in FIG.",
"1 (Biacore analysis) confirmed that substance #122 binds specifically and with high affinity to the A-site subdomain.",
"Table 1: Typical results of a screening experiment (substances No 97-No192).",
"The table shows the relative activity of the reporter ribozyme in the presence of 100 μM of each substance.",
"The values were obtained by regression analysis, followed by normalization, where the initial rate for the non-inhibited reaction was set “1”.",
"Substances are highlighted where there was a clearly inhibitory effect.",
"All tests were performed with substrate excess, with 8 nM TRK1, 200 nM SK1 in 0.5×PBS at 32° C., with 8 mM MgCl2.The reactions were started by simultaneous addition of MgCl2 and substance.",
"Table 1: Typical results of the screening experiment (Substances 97-197).",
"EXAMPLE 2 Screening of Protein-Binding Substances by Using an Intramer-Ribozyme Construct Screening for a substance which binds to the viral Rev protein was performed on a library of 50 short RNA sequences, using the procedure in accordance with the invention.",
"The sequences are as follows: Seq 1: 5′-GGGAGUUGAUAACAGGCUCAAUGAGCCUGCUCGGUCAAC -3′ Seq 2: 5′-GGGAGUUGAUAGCAGGCUCAAUGAGCCUGAGUUCCCAAC -3′ Seq 3: 5′-GGGAGUUGAUAUCAGGCUCAAUGAGCCUGGUCGACCAAC -3′ Seq 4: 5′-GGGAGUUGAUACCAGGCUCAAUGAGCCUGCAAAGUCAAC -3′ Seq 5: 5′-GGGAGGUGGACUCCAGCUUCGGCUGUUGAGAUACACC-3′ Seq 6: 5′-GGGAGUUGGUACCAGGCUCAAUGAGCCUGAAAGCUCAAC -3′ Seq 7: 5′-GGGAGUUGCUACCAGGCUCAAUGAGCCUGGUUAAACAAC -3′ Seq 8: 5′-GGGAGUUGUUACCAGGCUCAAUGAGCCUGCGCGCGCAAC -3′ Seq 9: 5′-GGGAGUUGUAACCAGGCUCAAUGAGCCUGUAUAUACAAC -3′ Seq 10: 5′-GGGAGUUGUGACCAGGCUCAAUGAGCCUGAGAAUCCAAC -3′ Seq 11: 5′-GGGAGUUGUCACCAGGCUCAAUGAGCCUGCCUGGACAAC -3′ Seq 12: 5′-GGGAGUUGGCAUCAGGCUCAAUGAGCCUGUGGACACAAC -3′ Seq 13: 5′-GGGAGUUGGACUCAGGCUCAAUGAGCCUGGAAAAACAAC -3′ Seq 14: 5′-GGGAGUUGGAAUCAGGCUCAAUGAGCCUGAGGGGACAAC -3′ Seq 15: 5′-GGGAGUUGGCCUCAGGCUCAAUGAGCCUGCUUUUCCAAC -3′ Seq 16: 5′-GGGAGUUGGAAUCAGGCUCAAUGAGCCUGUCCCCUCAAC -3′ Seq 17: 5′-GGGAGUUGGCCUCAGGCUCAAUGAGCCUGACCCCACAAC -3′ Seq 18: 5′-GGGAGUUGGGGUCAGGCUCAAUGAGCCUGGUUUUGCAAC -3′ Seq 19: 5′-GGGAGUUGGUUUCAGGCUCAAUGAGCCUGCGGGGGCAAC -3′ Seq 20: 5′-GGGAGUUGAAUUCAGGCUCAAUGAGCCUGUAAAAUCAAC -3′ Seq 21: 5′-GGGAGUUGAACCCAGGCUCAAUGAGCCUGAUAUAUCAAC -3′ Seq 22: 5′-GGGAGUUGAACACAGGCUCAAUGAGCCUGUAUAUACAAC -3′ Seq 23: 5′-GGGAGUUGAAGGCAGGCUCAAUGAGCCUGAAAUUUCAAC -3′ Seq 24: 5′-GGGAGUUGAAGUCAGGCUCAAUGAGCCUGUUUAAACAAC -3′ Seq 25: 5′-GGGAGUUGAGUCCAGGCUCAAUGAGCCUGAAUUAACAAC -3′ Seq 26: 5′-GGGAGUUGUUUCCAGGCUCAAUGAGCCUGUUAAUUCAAC -3′ Seq 27: 5′-GGGAGUUGGGGGCAGGCUCAAUGAGCCUGCCCAAACAAC -3′ Seq 28: 5′-GGGAGUUGGCAACAGGCUCAAUGAGCCUGCACAGUCAAC -3′ Seq 29: 5′-GGGAGUUGACCCCAGGCUCAAUGAGCCUGGUGCAGCAAC -3′ Seq 30: 5′-GGGAGUUGCACACAGGCUCAAUGAGCCUGGUCAGCCAAC -3′ Seq 31: 5′-GGGAGUUGCAUACAGGCUCAAUGAGCCUGCAGUUACAAC -3′ Seq 32: 5′-GGGAGUUGUGAGCAGGCUCAAUGAGCCUGGGCAGUCAAC -3′ Seq 33: 5′-GGGAGUUGGGUCCAGGCUCAAUGAGCCUGUCAACUCAAC -3′ Seq 34: 5′-GGGAGUUGCGAUCAGGCUCAAUGAGCCUGACUAGGCAAC -3′ Seq 35: 5′-GGGAGUUGAAUCCAGGCUCAAUGAGCCUGUUGCACCAAC -3′ Seq 36: 5′-GGGAGUUGUCGGCAGGCUCAAUGAGCCUGACGUACCAAC -3′ Seq 37: 5′-GGGAGUUGUCCGCAGGCUCAAUGAGCCUGUGGUAACAAC -3′ Seq 38: 5′-GGGAGUUGAGAACAGGCUCAAUGAGCCUGCUCCGACAAC -3′ Seq 39: 5′-GGGAGUUGCCUCCAGGCUCAAUGAGCCUGACCUCGCAAC -3′ Seq 40: 5′-GGGAGUUGCCCGCAGGCUCAAUGAGCCUGGCAGCCCAAC -3′ Seq 41: 5′-GGGAGUUGGCGCCAGGCUCAAUGAGCCUGUCGAAGCAAC -3′ Seq 42: 5′-GGGAGUUGCGCGCAGGCUCAAUGAGCCUGUCACACCAAC -3′ Seq 43: 5′-GGGAGUUGCGAACAGGCUCAAUGAGCCUGAAAAAACAAC -3′ Seq 44: 5′-GGGAGUUGUCUUCAGGCUCAAUGAGCCUGUUUUUUCAAC -3′ Seq 45: 5′-GGGAGUUGGGAGCAGGCUCAAUGAGCCUGGGGGGGCAAC -3′ Seq 46: 5′-GGGAGUUGGCCUCAGGCUCAAUGAGCCUGCCCCCCCAAC -3′ Seq 47: 5′-GGGAGUUGGAUGCAGGCUCAAUGAGCCUGAAAUGGCAAC -3′ Seq 48: 5′-GGGAGUUGCCCUCAGGCUCAAUGAGCCUGAAAUGGCAAC -3′ Seq 49: 5′-GGGAGUUGCUCUCAGGCUCAAUGAGCCUGAUAUGGCAAC -3′ Seq 50: 5′-GGGAGUUGCUCGCAGGCUCAAUGAGCCUGAAAUGACAAC -3′ The DNA matrixes coding for the 50 RNA sequences were produced by in vitro transcription of oligonucleotides synthesized by solid phase synthesis.",
"After transcription, the RNAs were separated by length, using denaturing polyacrylamide gel electrophoresis in denaturing urea/polyacrylamide gels.",
"The corresponding bands were then visualized by fluorescence quenching.",
"For this purpose, the gels were packed in transparent foil, laid on DC aluminium foils (Kieselgel 60 F245, Merck) and irradiated with a pocket lamp at 254 nm.",
"Bands of the right length were cut out and the slices of gel were cut up into small pieces and covered with 300 mM sodium acetate (pH 5.2).",
"After incubation for 1 h at 65° C. and for 4 h at room temperature, the gel suspension was pressed through a syringe which was filled with glass wool.",
"After removing gel residues, the nucleic acids were precipitated and taken up in sterile water.",
"For the screening experiment, a binding RNA sequence (cf.",
"FIG.",
"7) for the Rev protein of HIV was isolated from the library of 50 different ribonucleic acids.",
"FIG.",
"5 shows how the assay functions in principle.",
"The experiment was performed with the sequences IRK1 and SK1 shown in FIG.",
"6, analogously to Example 1.The two sequences were produced by oligonucleotide solid phase synthesis and were purchased from Eurogentec (Belgium).",
"FIG.",
"7 shows a general formula for the sequences which do not bind to the Rev protein and for the identified Rev-binding RNA sequence.",
"The experiment was performed as follows: The sequences from the library and the IRK-1 were denatured separately in reaction buffer (10 mM HEPES (pH 7.4), 100 mM NaCl) for 1.5 min at 95° C. and then cooled at room temperature to renature.",
"The IRK-1 and SK-1 were mixed and incubated for 5 min at room temperature.",
"This incubation mixture was then divided equally between 50 individual reactions.",
"Rev protein was added to the reaction mixtures, which were then incubated for a further 15 min at room temperature.",
"The 50 RNA sequences from the library were then added to the 50 reaction mixtures.",
"After another 30 min, the cleavage reaction for substrate SK-2 was started by the addition of magnesium.",
"The final concentrations in the reaction mixtures were as follows: 10 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, IRK-18 nM, SK-1200 nM, RNA sequences from the library 2 μM, Rev protein 200 nM.",
"All components were prewarmed to 37° C. before mixing.",
"The evaluation was carried out as in Example 1 and led to the identification of the sequence in FIG.",
"7 as a Rev-binding sequence, which could compete with IRK-1 for binding to the protein.",
"The identification was made on the basis of the marked differences in fluorescence in comparison with the reaction mixtures from the other members of the library.",
"This experiment demonstrated that competitive inhibitors of an RNA/protein interaction can be easily isolated from combinatorial libraries using the procedure in accordance with the invention and fluorescence detection which is compatible with HTS procedures.",
"EXAMPLE 3 Assembly of a High-Throughput Screening System for the Identification of Compounds which Interact with the Target Protein Rev The data from this example are also the object of FIGS.",
"8 to 17.The interaction between the Rev protein of HIV and RRE facilitates the export of unspliced viral RNA, which is important for viral replication and which is therefore the object of many approaches to the development of antiviral therapies [Dayton, A. I.",
"& Zhang, M. J., Therapies directed against the Rev axis of HIV autoregulation, Adv Pharmacol 49, 199-228 (2000); Pollard, V. W. & Malim, M. H., The HIV-1 Rev protein, Annu Rev Microbiol 52, 491-532 (1998)].",
"In the context of the work described here, two different hammerhead ribozymes were constructed; their secondary structure is shown in FIG.",
"9 and their basic mode of action in FIG.",
"8.The ribozyme which can be activated by Rev is referred to as aptamer-inhibited ribozyme (AIR).",
"This was constructed by fusion of the Rev aptamer [Symensma, T. L., Giver, L., Zapp, M., Takle, G. B.",
"& Ellington, A. D., RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo, J Virol 70, 179-87 (1996)] at the 5′-end of the ribozyme, using a penta-A linker.",
"The second ribozyme was inhibited by Rev.",
"This was constructed by inserting the high affinity Rev-binding element (RBE) of the RRE HIV genomic sequence into stem II of the hammerhead ribozyme.",
"This yielded the R ev-responsive ribozyme (RRR) (cf.",
"FIGS.",
"8 and 9).",
"Ribozyme Reactions: Cleavage reactions with multiple turnover were performed at 32° C. in a test buffer containing 50 mM Tris (pH 7.9) and 25 mM NaCl.",
"The total volume of the reactions was 50 μl in plates with 96 flat bottom wells.",
"The fluorescence measurements were performed at an excitation wavelength of 489 nm, FAM emission at 520 nm and with the fluorescence detector Ascent Fluoroscan FL, as described by Jenne, A. et al., Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology, Nat Biotechnol 19, 56-61 (2001).",
"Ribozyme (10 nM) and FRET-labeled substrate (200 nM) were first incubated in test buffer at room temperature for 5 min, followed by optional addition of Rev and/or antibiotic.",
"The reactions were then incubated for 5 min at 32° C. and started by the addition of MgCl2 to a final concentration of 8 mM, using the dispensing function of the fluorometer.",
"Negative controls were always included for reactions without ribozyme and were subtracted after the measurements.",
"Filter Binding Studies: Reaction mixtures (20 μl) containing 100 nM Rev protein, traces of 5′-32P labeled RRR or AIR, 200 nM FRET substrate and various concentrations of antibiotic in test buffer were washed through a pre-rinsed 0.45 μm nitrocellulose membrane, followed by washing with 3 ml test buffer.",
"The sequence of additions and incubation times was performed as with the ribozyme reactions.",
"Surface Plasmon Resonance: Surface plasmon resonance studies were performed with a Biocore 3000 instrument in the automatic mode.",
"Rev protein and peptide surfaces were prepared by injection of 100 μM solutions in 60 mM NaOAc, pH 5.7 on EDC/NHS-activated CM5-Chips, which gave 1800 RU immobilized peptide and 5000 RU protein.",
"The RRR-RNA surface was produced by the injection of biotinylated RRR (75 nM) in Tris pH 7.5, 0.5 M NaCl on a streptavidin-derivatized chip and gave 1600 RU immobilized RRR.",
"Result: Both new apatazymes, AIR and RRR, are regulated by the RNA domain for the Rev protein, the intact Rev protein and by the Rev peptide, which includes aminoacids 34 to 50 of the Rev protein.",
"The surface plasmon resonance procedure gave Kp values as follows: 1.4 nM for the RRR/Rev protein, 9.3 nM for the RRR/Rev peptide, 1.3 nM for the AIR/Rev protein, and 9.0 nM for the AIR/Rev peptide.",
"The kinetic characterization of the new hammerhead ribozyme constructs under the conditions of multiple turnover showed that the initial reaction rate for the Rev-regulated RRR was 38% of the reaction rate of a well characterized hammerhead ribozyme, HHR (Jenne, A., Gmelin, W., Raffler, N. & Famulok, M., Real-Time Characterization of Ribozymes by Fluorescence Resonance Energy Transfer (FRET), Angew.",
"Chem.",
"Int.",
"Ed.",
"38, 1300-1303 (1999); Jenne, A. et al., Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology, Nat Biotechnol 19, 56-61 (2001).",
"In contrast to this, the initial rate with the aptamer-inhibited ribozyme AIR was only 1.4% of that with HHR (cf.",
"FIG.",
"11).",
"After adding 1 μM Rev peptide, RRR gave only 1.0% of the activity of HHR, corresponding to inhibition by a factor of 36.In contrast, the addition of 250 nM Rev peptide to ribozyme AIR gave 32-fold activation, to a level of 44% of the activity of HHR.",
"The inhibition of RRR at the half maximal concentration of Rev: K inh=464 for Rev peptide (cf.",
"FIG.",
"14a) and K inh=1.7 μM for Rev protein (cf.",
"FIG.",
"14b).",
"The activation of AIR: Kact=170 nM for Rev peptide (cf.",
"FIG.",
"14a) and K act=192 nM for the Rev protein (cf.",
"FIG.",
"14b).",
"At Rev peptide concentrations of above 800 nM, AIR is inhibited as well (cf.",
"FIG.",
"14a).",
"This effect can be explained as unspecific inhibition of the catalytic ribozyme function by the strongly positively charged peptide.",
"Even HHR is inhibited at Rev concentrations of above 1 μM.",
"As shown in FIG.",
"11, the initial activity is 93% at a concentration of 1 μM, although the cleavage is inhibited after some minutes.",
"When the Rev protein is used, AIR is still activated at concentrations of above 5 μM, whereas RRR is inhibited above 1 μM and HHR above 2 μM Rev protein (cf.",
"FIG.",
"14b).",
"Screening of 96 Antibiotics: Two different screening experiments were performed, one using the RRR reporter construct and the other using AIR.",
"The following standard reactions in duplicate were prepared for each plate with 96 wells: ribozyme (HHR and reporter construct) alone, ribozyme reactions containing Rev peptide (1 μM in the case of RRR screening, 0.25 μM in the case of AIR screening), two negative controls without ribozyme, the first only with substrate, and the second with Rev and substrate.",
"Each reaction which contained antibiotics also contained Rev peptide.",
"For each antibiotic, three different reactions were performed: HHR, reporter construct (RRR or AIR), negative controls, which only contained antibiotic and Rev and substrate.",
"The antibiotic concentrations were 100 μM in each case.",
"After subtraction of the negative control, the rise in fluorescence in the first five minutes, corresponding to the initial rate of reaction, was divided by the rise in fluorescence in the standard reaction without antibiotic, containing the active state of the ribozyme (see also FIG.",
"10).",
"In order to only describe specific effects on the Rev RNA interactions and not on the function of the hammerhead, the values obtained for the RRR and AIR were divided by the values for the HHR reactions.",
"In all, two screens were performed and the results of these are shown in FIG.",
"15.RRR inhibited by 1 μM Rev peptide was used for one, where active compounds were identified by the restoration of the cleavage catalytic activity of the ribozyme.",
"In the second screen, the ribozyme AIR was used; compounds which are capable of preventing the interaction between Rev and the Rev aptamer are identified on the basis of the inhibition of the Rev reporter construct, which is activated by 250 nM Rev peptide.",
"The most active inhibitors of the hammerhead ribozyme, antibiotics #8, #31, #91 and #92, could not be examined, as they inhibited the reaction completely.",
"Inactive compounds give readings of about zero in the RRR screen, because of the inhibition by Rev.",
"Active compounds give reactivation of the reporter construct RRR; total reactivation gives a reading of 1.Three compounds were identified which gave significant activation (above 0.75), namely coumermycin A1 (#21), nosiheptide (#58) and patulin (#63), as also shown in Table 1a.",
"In the AIR screen, inactive compounds give a reading of about 1, as they leave the reporter construct in its active Rev-binding state.",
"Active compounds gave low values near to zero and were identified on this basis (total inhibition).",
"It was striking that screening of the compound library of 96 antibiotics led to the identification of the same compounds (#21, #58 and #63), confirming that these are Rev inhibitors.",
"FIG.",
"11 shows the results of the measurement of the concentration dependence of the abolition of the interaction between Rev and the ribozyme.",
"Novobiocin, a natural analog of coumermycin A, was included in all further studies.",
"The results of the studies of relative initial rates are summarized in Table 1a.",
"Characterization of the Identified Antibiotics Conventional filter binding studies were performed as a further study of the interactions observed in fluorescence measurements.",
"Rev protein rather than Rev peptide was used for this purpose, as the peptide is too small to be retained by the filter.",
"50-60% of the 5′-32P-labeled reporter ribozymes RRR and AIR were retained on the filter in the presence of 100 nM Rev protein.",
"The addition of the identified antibiotics confirms once again that these antibiotics are capable of interfering with the interaction between RNA and the protein (cf.",
"FIG.",
"12).",
"It is very striking that these Rev protein binding experiments confirm the results obtained by fluorescence measurements with the Rev peptide.",
"The concentrations of the compounds at which 50% of the RNA protein complexes are dissociated are summarized in Table 1a.",
"Surface plasmon resonance was used for a more exact clarification of the interaction between the three components used in the test.",
"The binding of the antibiotics was tested on surfaces which had been derivatized with RRR-RNA, Rev peptide and with the Rev protein.",
"It was interesting that all four antibiotics demonstrated binding to both the Rev peptide and the Rev protein, whereas there was no detectable interaction for various other compounds in the library which had not been identified in the screening.",
"The four Rev-binding antibiotics exhibited no binding to RRR-RNA.",
"The KD values for the binding of the antibiotics are given in Table 1b.",
"With the exception of coumermycin, these values agree closely with the apparent binding constants determined by filter binding and fluorescence measurements.",
"As a control of the target protein reverse transcriptase, another nucleic acid binding protein from HIV-1 was tested, to check the specificity of the identified Rev-binding antibiotics.",
"No significant effects were observed at antibiotic concentrations of 100 μM in a standard transcriptase assay (while ddCTP gave total inhibition at 2.5 μM).",
"The conclusion from these results is therefore that the identified antibiotics and, in general, compounds identified by procedures in accordance with the invention, can be used as lead substances for future pharmaceutically active substances.",
"The features of the invention, as disclosed in the above description and in the claims and drawings may, either individually or in any combination, be essential for the implementation of the invention in its different embodiments.",
"SUMMARY Described is a procedure for the identification of compounds (lead structures) which (a) bind specifically to a desired RNA target motif and which can, as a result, inhibit or eliminate the function of this or which (b) can displace a compound which is associated with a desired RNA target motif and which can as a result inhibit or eliminate the function of this or these.",
"The procedure in accordance with the invention is based on the binding of a ligand (=compound to be identified) to an RNA target motif which is coupled to a modified ribozyme, so that the ribozyme is converted to an active or to an inactive conformation, leading to the cleavage of a signaling ribozyme substrate.",
"The compounds identified in this way permit the modification of the cellular function of the RNA target motifs and permit the production of specific drugs.",
"The invention also relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for the target molecule, where there is a difference in the base pairing pattern of the polynucleotide when the target molecule is bound to the aptamer and when the target molecule is absent from the aptamer."
]
] | Patent_10432560 |
[
[
"Electrochemical double-layer energy storage cells with high energy density and high power density",
"The invention concerns a method for preparing activated carbons based on wood, preferably softwood and in particular pine wood, for making electrodes for energy storage cells, particularly for super-capacitors.",
"Said activated carbons have a volume of mesopores less than 75% of the total pore volume and a volume of micropores less than 57% of the total pore volume.",
"The invention also concerns a method for making an electrode for energy storage cell, comprising the application of such an activated carbon on a support, preferably by coating derived from a slurry.",
"The energy storage cells using said activated carbons advantageously provide a better compromise between energy density and power density."
],
[
"1.A process for the preparation of a porous carbonaceous material comprising the following stages: a) carbonization of wood, preferably of softwood, and advantageously of pine wood, at a temperature of between 500 and 800° C.; b) thermal activation of the wood carbon obtained in a thin layer at a temperature of between 800 and 1 100° C. in the presence of steam and/or of carbon dioxide; the activated carbon obtained after stage b) exhibiting a volume of mesopores of less than 75% of the total pore volume and a volume of micropores of less than 75% of the total pore volume.",
"2.The process as claimed in claim 1, in which the activated carbon 15 obtained in stage b) exhibits a content of mesopores of between 40 and 60% of the total pore volume.",
"3.The process as claimed in claim 1, in which the activated carbon obtained in stage b) exhibits a content of micropores of between 20% and 20 40% of the total pore volume.",
"4.The process as claimed in claim 1, in which the activated carbon obtained in stage b) exhibits a pore volume of greater than 0.8 cm3/g, preferably of greater than 1 cm3/g.",
"5.The process as claimed in claim 1, in which the activated carbon obtained in stage b) exhibits a volume of micropores of between 0.2 and 0.6 cm3/g.",
"6.The process as claimed in claim 1, in which the activated carbon obtained in stage b) exhibits volume of mesopores is of between 0.4 and 0.8 cm3/g.",
"7.The process as claimed in claim 1, in which the activated carbon obtained after stage b) exhibits a specific surface of greater than 800 m2/g.",
"8.An electrode based on activated carbon comprising activated carbon capable of being obtained by the process as in claim 1.9.An electrode based on activated carbon comprising activated carbon based on wood exhibiting a volume of mesopores of less than 75% of the total pore volume and a volume of micropores of less than 75% of the total pore volume.",
"10.The electrode as claimed in claim 8, characterized in that the electrode comprises activated carbon binder in a ratio by weight of 10/90 to 90/10, 10 preferably of 30/70 to 70/30.11.The electrode as claimed in claim 8, characterized in that the binder is a polymer, preferably a thermoplastic and advantageously a polyether and/or polyalcohol.",
"12.A process for the manufacture of an electrode for an electrochemical double-layer energy storage cell comprising the stage of preparation of an activated carbon as claimed in claim 1; application of this activated carbon to a support.",
"13.The manufacturing process as claimed in claim 12, in which a slip is formed beforehand from the activated carbon derived from pine wood with a binder in a suitable solvent and that the solvent is evaporated after the application to a support.",
"14.The process as claimed in claim 12, in which the binder is a polymer, preferably a thermoplastic polymer and advantageously a polyether and/or a polyalcohol.",
"15.The process as claimed in claim 12, in which the activated carbon is mixed with the binder in a ratio by weight of 90/10 to 10/90, preferably of 30/70 to 70/30.16.The process as claimed in claim 12, in which the application 35 is carried out by coating.",
"17.An electrochemical double-layer energy storage cell comprising at least one electrode as claimed in claim 8.18.The cell as claimed in claim 16, exhibiting an energy density of greater than 3 Wh/kg, preferably of greater than 4 Wh/kg, and an energy power of greater than 4 kW/kg, preferably of greater than 5 kW/kg."
],
[
"The present invention relates to a process for the preparation of activated carbons based on wood, preferably on softwood, in particular on pine wood, having a specific pore structure for the manufacture of electrodes for electrochemical double-layer energy storage cells.",
"The invention also relates to the electrodes thus obtained and to the electrochemical double-layer energy storage cells comprising such electrodes and to a process for the manufacture of these electrodes.",
"The electrochemical storage of energy can be carried out via three different devices each having their own characteristics.",
"In a conventional electrochemical storage battery, the two nonpolarizable electrodes are separated by an ionic conductor.",
"Charge transfers take place via slow oxidation/reduction reactions.",
"The maximum power available is therefore low (<400 W/kg).",
"On the other hand, the energy stored is high (>30 Wh/kg).",
"In a conventional capacitor, the two polarizable electrodes are separated by a thin insulator.",
"In this type of system, the operating principle is based on the formation of an electrical double layer by accumulation of charges within the electrodes on either side of the insulator.",
"This phenomenon is very fast and allows charge-discharge periods of the order of a millisecond.",
"The pulse power provided by such systems is therefore extremely high (>104 W/kg).",
"On the other hand, the amount of energy stored is low (<10−2 Wh/kg).",
"In a supercapacitor, the two polarizable electrodes of high specific surface are separated by an ionic conductor.",
"As the amount of charge stored is proportional to the specific surface of these electrodes, there is great advantage in such a device in comparison with a conventional capacitor.",
"Thus, in terms of energy stored and of power available, the supercapacitor exists as a device intermediate between the storage battery and the capacitor.",
"The use of supercapacitors is well established in various applications.",
"Such capacitors can be described in terms of energy density (kilowatt-hour/kg) and power density (watt/kg) characteristics.",
"Capacitors with a high energy density store a relatively high capacitance, which is slowly discharged over a period of a few minutes.",
"On the other hand, capacitors with a high power density can deliver their energy rapidly (in a few milliseconds).",
"Various practical applications have different requirements in terms of energy and power.",
"For example, memory backup devices require a reasonably high energy density but do not require that the energy be delivered rapidly (low power, long discharge time).",
"Furthermore, an application such as starting an automobile engine requires a very high power and most of the energy has to be delivered in a few milliseconds.",
"Other applications require combinations of the energy and power densities which are intermediate between these two extremes.",
"Electrical devices for energy storage comprising electrodes based on activated carbons resulting from lignocellulose materials are known.",
"These devices, which are generally known as electrochemical carbon double-layer capacitor or CDLCs, are usually composed of a pair of electrodes (at least one which is a carbon paste electrode), a separator and a collector, impermeable to ions, which conducts current.",
"The activated carbons are characterized by a high total specific surface (generally in the range 500-2 500 m2/g).",
"They are differentiated by their origin or precursor (coal, wood, fruit shells, and the like) and by the type of activation, physical or chemical, which they have been subjected to.",
"The pores in the activated carbon are classified according to their size into micropores (diameter <2 nm), mesopores (diameter 2-50 nm) or macropores (diameter >50 nm).",
"High specific surfaces and a relatively low cost render activated carbons useful in many applications, including that of electrical energy storage devices.",
"It is known that some types of activated carbons have an influence on the energy and power densities of the CDLC.",
"This is because capacitors have been able to be improved either with regard to their power density or with regard to their energy density.",
"Carbons obtained by heat treatment of precursor which is activated in an alkaline bath at a high temperature are known, for example from U.S. Pat.",
"No.",
"5,430,606.The energy storage cells manufactured with these carbons exhibit a good energy density but prove to have a poor performance with regard to the power density.",
"Thus, their use is not made possible in applications requiring rapid delivery of the energy.",
"In addition, the preparation process is expensive.",
"CDLCs with a high energy density obtained from activated carbons having a specific pore structure composed essentially of micropores are also known from U.S. Pat.",
"No.",
"5,905,629.Furthermore, CDLCs with a high power density from activated carbons with an equivalent content of mesopores are also known from U.S. Pat.",
"No.",
"5,926,361.These carbons are obtained by an activation process followed by a heat treatment of the activated carbon precursor.",
"However, these CDLCs are not suitable for intermediate applications requiring both a high energy density and rapid delivery of the energy.",
"In addition, the process for manufacturing the carbons is expensive.",
"In addition, carbons having a pore volume of 0.3 to 2.0 cm3/g, including 10 to 60% of micropores, 20 to 70% of mesopores and not more than 20% of macropores, and exhibiting a specific surface of 1 000 to 2 500 m2/g are known from EP 1 049 116.The carbons disclosed are obtained exclusively from polymers.",
"It is therefore an object of the present invention to provide a process for the manufacture of activated wood carbon exhibiting a porosity profile suitable for the electrodes of electrochemical double-layer energy storage cells.",
"An object of the invention is thus to provide a process for the manufacture of a porous carbonaceous material.",
"Another object of the invention is to provide an electrode based on such materials and energy storage cells exhibiting a better compromise between the power density and the energy density in comparison with the preexisting cells of this type.",
"Another subject matter of the invention is a process for the manufacture of such improved energy storage cells.",
"In the context of this account, the term “energy storage cells” is understood to mean any device for the storage of electrochemical energy, supercapacitors and in particular CDLCs.",
"The cells according to the invention are obtained by virtue of activated carbons based on wood, preferably on softwood, in particular on pine wood, which exhibit a specific pore distribution and in particular have contents of mesopores and micropores of less than 75% of the total pore volume.",
"This specific pore distribution is partly due to the quality of the starting material, wood, preferably softwood, and in particular pine wood.",
"The carbons obtained from pine wood, which are particularly preferred, are characterized in addition by high purity.",
"The activated carbons exhibit a content of mesopores of less than 75%, preferably of between 40 and 60%, with respect to the total pore volume.",
"The volume of mesopores of the activated carbon used is preferably between 0.4 and 0.8 cm3/g.",
"Preferably, these carbons exhibit a pore volume of greater than 0.8 cm3/g, preferably of greater than 1 cm3/g, a median pore width of 15 to 50 nm and a specific surface of greater than 800 m2/g.",
"These activated carbons also preferably exhibit (as a function of the total pore volume) a content of macropores of less than 0.3 cm3/g.",
"The relative content of macropores is preferably less than the content of micropores and mesopores.",
"Thus, the activated carbon advantageously comprises less than 25%, preferably less than 10% and even more preferably less than 1% of macropores with respect to the total pore volume.",
"These carbons are subjected to an activation process so as to increase the surface area of the natural carbonaceous material.",
"Such an activation of the raw material is carried out either by a chemical process or by thermal process.",
"Activation process examples are indicated, for example, in patents U.S. Pat.",
"Nos.",
"4,107,084, 4,155,878, 5,212,144 and 5,270,017.An effective porosity of the activated carbons produced by thermal activation is the result of gasification of the carbon at high temperature (after an initial carbonization of the raw material), whereas the porosity of the products activated by chemical dehydration/condensation reaction are produced at low temperature.",
"The activated carbon precursor used according to the invention is wood, preferably softwood, and in particular pine wood.",
"The wood used can, for example, be in the form of wood chips, wood flour, wood dust, wood sawdust and combinations of these.",
"The activated carbon can be obtained by chemical activation or, preferably, by thermal or physical activation.",
"The chemical activation is generally carried out industrially in a simple furnace.",
"The precursor of the raw material is impregnated with a chemical activating agent and the mixture is heated at a temperature of 450° C.-700° C. The chemical activating agents reduce the formation of tars and of other derived products and thus increase the yield.",
"The appropriate chemical activating agents include hydroxides of alkali metal, carbonates, sulfides and sulfates; carbonates of alkaline earth metals, chlorides and phosphates; phosphoric acid; polyphosphoric acid; zinc chloride; sulfuric acid; fuming sulfuric acid; and combinations of these.",
"Phosphoric acid and zinc chloride are preferred among these agents.",
"The preferred among all is phosphoric acid.",
"The precursor is impregnated with activating agent and is then activated at approximately 550° C. As indicated above, the activated carbon is preferably obtained by thermal activation.",
"In this case, the precursor material is subjected to a carbonization heat treatment at a temperature of between 500 and 800° C. in order to obtain wood carbon, which is subsequently activated at a temperature of greater than 700° C., preferably of between 800 and 1 100° C., and more preferably still at a temperature of between 950 and 1 050° C. The thermal activation of the wood carbon takes place in a thin layer.",
"The term “thin” is understood to mean a layer with a thickness of approximately 2 to 5 cm.",
"The activation is preferably carried out in a furnace in which the precursor material moves by gravity from the top downward.",
"The activation is advantageously carried out in the presence of steam and/or of carbon dioxide.",
"The activated carbons capable of being obtained according to the process described above are particularly preferred in the manufacture of electrodes of electrochemical double-layer energy storage cells.",
"The process for the manufacture of these wood carbons is additionally advantageous in that it is economical.",
"A typical CDLC is composed of: (1) a pair of electrodes, at least one (preferably both) of which is a carbon paste electrode, (2) a porous separator which conducts ions and (3) a collector which is impermeable to ions, to provide electrical contact between the electrodes and an electrolyte.",
"The cell preferably exhibits an energy density of greater than 3 Wh/kg, in particular of greater than 4 Wh/kg, and an energy power of greater than 4 kW/kg, in particular of greater than 5 kW/kg.",
"The novel energy storage cells having a better power density/energy density compromise are derived from activated carbons based on wood.",
"These activated carbons are characterized in that they have a level of micropores with respect to the total pore volume of less than 75%, preferably of between 20 and 40%, with respect to the total pore volume.",
"Preferably, the volume of micropores of the activated carbon used is between 0.2 and 0.6 cm3/g.",
"The process for the manufacture of electrodes for CDLCs with a high power density and energy density comprises the application to a support of an activated carbon derived from wood having a volume of mesopores and of micropores as defined above.",
"For the manufacture of electrodes (1), the activated carbon is preferably ground to a size, expressed in d50, of approximately 30 micrometers and preferably to a d50 of approximately 10 micrometers.",
"Preferably, the application is carried out by preparing beforehand a slip comprising a powdered activated carbon, a binder and a solvent.",
"The slip is applied to the support and the solvent is subsequently evaporated to form a film.",
"According to the process of the invention, the activated carbons are mixed with a binder, such as a polymer binder, in an aqueous or organic solvent.",
"Thermoplastic or elastomeric polymers or their mixtures which are soluble in said solvent, for example, can be used as polymer binder.",
"Mention may in particular be made, among these polymers, of polyethers, such as polyoxyethylene (POE) or polyoxypropylene (POP), and/or of polyalcohols, such as polyvinyl alcohol (PVA), or of ethylene-vinyl acetate (EVA) copolymers.",
"The solvent can be any aqueous or organic solvent appropriate for dissolving the binder used.",
"Such a solvent is, for example, acetonitrile for polymer binders based on POE, POP, PVA and/or EVA.",
"The activated carbon is preferably mixed with the polymer in a ratio by weight of 10/90 to 60/40, preferably of 30/70 to 50/50.The paste obtained is subsequently applied to a support by coating.",
"It is advantageous for the coating to be carried out on a peelable support, for example using a template, generally flat in shape.",
"The solvent is subsequently evaporated, for example under a hood.",
"A film is obtained, the thickness of which depends in particular on the concentration of the carbon paste and on the deposition parameters but which is generally between a few micrometers and a millimeter.",
"The thickness is preferably between 100 and 500 micrometers and it is more preferably between 150 and 250 micrometers.",
"The appropriate electrolytes to be used to produce CDLCs with a high energy density and a high power density comprising at least one electrode based on activated carbon having the capacity to deliver improved energy densities and improved power densities consist of any medium highly conductive of ions, such as an aqueous solution of an acid, of a salt or of a base.",
"If desired, nonaqueous electrolytes (in which water is not used as solvent) can also be used, such as tetraethylammonium tetrafluoroborate (Et4NBF4) in acetonitrile or γ-butyrolactone or propylene carbonate.",
"In the structure of the cell, the electrolyte can have three general functions: as promoter of the conductivity of ions, as source of ions and, if appropriate, as binder for the carbon particles.",
"Sufficient electrolyte should be used to satisfy these functions (although a separate binder can be used to provide the binding function).",
"Preferably, the carbon paste comprises activated carbon, a binder and a solvent.",
"One of the electrodes can be composed of another material known in the art.",
"The current collector (3) which is impermeable to ions can be any electrically-conductive material which is nonconductive to ions.",
"Satisfactory materials to be used to produce these collectors comprise: carbon, copper, lead, aluminum, gold, silver, iron, nickel, tantalum, conductive polymers, nonconductive polymers filled with conductive material so as to render the polymer electrically conductive, and similar materials.",
"The collector (3) must be connected electrically to an electrode (1).",
"A separator (2), generally made of a highly porous material, is positioned between the electrodes, the functions of which separator are to provide electronic insulation between the electrodes (1) while allowing the ions of the electrolyte to pass.",
"The pores of the separator (2) have to be sufficiently small to prevent electrode-electrode contact between the opposite electrodes (contact would result in a short circuit and a rapid loss in the charges accumulated in the electrode).",
"Generally, any conventional battery separator can be used in a CDLC with a high power density and a high energy density.",
"The separator (2) can be a membrane which is permeable to ions which allows ions to pass through but which prevents electrons from passing.",
"The manufacturing process and the energy storage cell according to the invention are described in more detail in the following examples.",
"These examples are given by way of illustration and not by way of limitation of the invention.",
"EXAMPLES The activated carbons of the following examples 2S to 5S, sold by the Applicant Company, are obtained industrially according to the process of claim 1 by adjustment of the steam partial pressure and the increase in the residence time in the furnace, making it possible to change from grade 2S to 3S to 4S and to 5S by increasingly expanding the porosity.",
"Example 1 Thermally activated carbons derived from pine wood of 2S grade, available from Ceca, are used to produce carbon paste electrodes as described below.",
"This activated carbon is obtained by activation in a thin layer at a temperature of 1 000° C. in the presence of steam.",
"40 g of 2S activated carbon are first mixed with 60 g of polyoxyethylene (POE) 300 000 (available from Aldrich) in 500 ml of acetonitrile until a homogeneous slip is obtained.",
"This slip is subsequently applied by coating using a doctor blade in a PTFE template.",
"The solvent is allowed to evaporate under a hood at ambient temperature for approximately 12 hours.",
"A film is obtained, the dry thickness of which is approximately 200 micrometers.",
"Disks with a working surface area of 2 cm2 are cut out from this film using a hollow punch.",
"Example 2 Carbon paste electrodes are prepared in the same way as described in example 1 using the activated carbon derived from pine wood of 3S quality available from Ceca.",
"This activated carbon is activated in a thin layer at a temperature of 1 000° C. in the presence of steam.",
"Example 3 Carbon paste electrodes are prepared in the same way as described in example 1 using the activated carbon derived from pine wood of 4S grade available from Ceca.",
"This activated carbon is obtained by activation at a temperature of 1 000° C. in the presence of steam.",
"Example 4 Carbon paste electrodes are prepared in the same way as described in example 1 using the activated carbon derived from pine wood of 5S grade available from Ceca.",
"This activated carbon is obtained by activation at a temperature of 1 000° C. in the presence of steam.",
"Example 5 (Comparative Example) Carbon paste electrodes are prepared in the same way as described in example 1 using Osaka M15 activated carbon (available from Osaka Gas Co. Ltd) obtained from mesophase pitch.",
"Example 6 (Comparative Example) Carbon paste electrodes are prepared in the same way as described in example 1 using activated carbon of Osaka M20 grade (available from Osaka Gas Co. Ltd) obtained from mesophase pitch.",
"Example 7 (Comparative Example) Carbon paste electrodes are prepared in the same way as described in example 1 using activated carbon of Osaka M30 grade (available from Osaka Gas Co. Ltd) obtained from mesophase pitch.",
"Example 8 (Comparative Example) Carbon paste electrodes are prepared in the same way as described in example 1 using activated carbon of Puref-Low grade, available from (Norit Nederland) obtained from inorganic carbon.",
"Example 9 (Comparative Example) Carbon paste electrodes are prepared in the same way as described in example 1 using activated carbon of Norit SX+ grade, available from (Norit Nederland) obtained from peat.",
"Example 10 (Comparative Example) Carbon paste electrodes are prepared in the same way as described in example 1 using activated carbon of Norit SX Ultra grade, available from (Norit Nederland) obtained from peat.",
"The active surface of the samples is determined by nitrogen adsorption/desorption at 77K.",
"The mean size of the pores and the porosities characteristic of each of the samples are evaluated in the following way.",
"First, the surface of the pore volume having a diameter of less than 20 nm is determined by the method described in ASTM D4365.The concentration of mesopores is evaluated by the method according to ASTM 4641.Finally, the content of macropores is determined by means of the method according to ASTM D4284—intrusion of mercury.",
"The mean diameter of the pores is subsequently calculated from the total pore volume and the BET specific surface according to ASTM D4365 according to the formula D=4V/S.",
"The results are recorded in table 1 and 2.It is found from these results that the electrodes based on carbons obtained from pine wood have a pore structure differs fundamentally from that observed with regard to electrodes manufactured with other commercially available carbons.",
"Despite a total pore volume exhibiting a broad distribution, the electrodes according to the invention are clearly distinguished by their content of micropores and mesopores.",
"This is because, while the proportion of micropores and mesopores is in balance for the comparative examples, the electrodes according to the invention exhibit less than 32% by volume of micropores and more than 48% by volume of mesopores.",
"In conclusion, the samples obtained from carbons based on pine wood are clearly already distinguished from the comparative samples at the level of their pore structure.",
"TABLE 1 Specific surface and mean pore diameter BET specific surface Example (m2/g) BET Dmean (Å) 1 957 28.8 2 971 31.3 3 1196 29.7 4 1382 31.3 5 1508 18.6 6 2148 21.1 7 3284 23 8 885 29 9 1065 28.3 10 1165 29.9 The electrodes prepared according to examples 1 to 10 are subsequently used to assemble a measurement cell in order to evaluate their performance in a CDLC in terms of power density and energy density.",
"For this, the electrode is first impregnated with a liquid organic electrolyte, a 0.6M solution of tetraethylammonium tetrafluoroborate in γ-butyrolactone, for 1 h 30 at atmospheric pressure.",
"Subsequently, the impregnated electrodes are used to assemble a capacitor as follows.",
"Each of a pair of electrodes is positioned on a plate of treated aluminum and are then assembled face to face separated by a Puma 50/0.30 separating paper (available from Bolloré).",
"The two electrodes are connected to a potentiostat, one being connected first to a calibrated spring.",
"TABLE 2 Absolute and relative porosity V micro V meso V total Example (cm3/g) (cm3/g) (cm3/g) % micro % meso 1 0.236 0.481 0.84 28 57 2 0.268 0.576 1.03 26 56 3 0.306 0.645 1.11 27 58 4 0.455 0.704 1.46 31 48 5 0.647 0.107 0.84 77 13 6 0.719 0.43 1.30 55 33 7 1.608 1.332 3.28 49 41 8 0.294 0.38 0.89 33 43 9 0.384 0.42 1.06 36 40 10 0.431 0.5 1.23 35 40 When a potential difference is applied between the two electrodes of a CDLC, an electrochemical double layer is spontaneously formed at each of the electrode/electrolyte interfaces by accumulation of ionic entities on the side of the electrolyte and of electrical charges on the side of the electrode; the amount of charge thus accumulated is proportional to the voltage applied and to the surface capacity of the electrodes.",
"Each double layer is characterized by its capacity.",
"The overall system is thus defined by 2 capacities in series and the total capacity is expressed by: 1/C=1/C1+1/C2 The energy stored is directly proportional to the total capacity of the overall system.",
"The total resistance or alternatively the resistance in series of a capacitor is the second major parameter which characterizes the system.",
"The power of the CDLC is evaluated directly from its value.",
"The power density and energy density of the electrodes assembled as capacitors is evaluated by chronopotentiometry.",
"The current density used is 1.5 mA/cm2 and the limits of the galvanostatic cycling are 0 and 2.5 V. The series resistance and the capacity of the capacitor are deduced from the curve obtained.",
"The series resistance is calculated from the measurement of the ohmic drop at the beginning of the discharge.",
"The capacity of the capacitor is determined from the slope of the discharge curve: C=Idischarge(Δt/ΔU) The energy stored is directly proportional to this capacity, in agreement with E=1/2CV2 The resistance in series is measured from the ohmic drop at the beginning of discharge and after a relaxation phase: Rs=ΔU/Idischarge The power is subsequently determined from the resistance according to the following formula P=V2/4R The 2-cm2 electrodes are assembled in measurement cells in order to evaluate the energy density and power density.",
"The measurement results are presented in table 3 below.",
"TABLE 3 Energy density and power density Example E (Wh/kg) P (kW/kg) 1 4.051 4.200 2 4.340 5.157 3 5.008 5.669 4 7.750 7.247 5 4.886 0.657 6 9.177 1.276 7 12.478 1.878 8 1.680 3.818 9 2.480 4.895 10 3.673 3.980 It is seen, from the results, that the electrodes according to the invention exhibit a balanced power density and energy density and that the electrodes of this type are therefore suitable for CDLCs for intermediate applications requiring both a good energy density and a rapid delivery of the energy.",
"While the carbons which make it possible to deliver an improved power density and energy density are of use in producing the carbon paste used in CDLCs, these carbons can also be of use in other types of electrical devices in which the activated carbon is used as electrode material (such as batteries, “fuel cells”, and the like).",
"This listing of claims will replace all prior versions, and listings, of claims in the application:"
]
] | Patent_10432590 |
[
[
"Derivatives of 4-hydroxybutanoic acid and of its higher homologue as ligands of $g(g)-hydroxybutyrate (ghb) receptors, pharmaceutical compositions containing same and pharmaceutical uses",
"The invention concerns the field of synthesis organic chemistry applied to the pharmaceutical field and concerns novel derivatives of 4-hydroxybutanoic acid and its higher homologue, 5-hydroxypentanoic acid, their crotonic homologues, pharmaceutical compositions containing them and their pharmaceutical uses.",
"Said novel derivatives are capable of binding with γ-hydroxybutyrate (GHB)-specific receptors and hence capable of exhibiting agonist or antagonist properties, in particular for treating sleep disorders, anxiety and general diseases of the central nervous system.",
"The invention also concerns compounds of general formula (I) wherein the substituents are as defined in the description."
],
[
"1.Compounds with general formula I in which Ar represents one of the following mono-, bi- or tricyclics: in which: R1, R2, R3 and R independently represent a hydrogen atom, a halogen, an alkyl group, an aryl group, an aralkyl group, a hydroxyl group, a methoxy group, an acetyl group, a tosyl group, a COOEt group, an NHCOCH3 group, an NH2 group, a CON(CH3)2 group, an NO2 group or a COR5R6 group, in which R5 and R6 independently represent a hydrogen atom, a methyl group, a CH3H7 group or a benzyl group, each z independently represents a nitrogen or carbon atom, Y and Y′ independently represent a carbon, sulfur, oxygen or nitrogen atom, Y″ represents a methylene, ethylene or propylene group, each X′ independently represents a sulfur or oxygen atom, p has a value of 0, 1 or 2, in which n has a value of 0 or 1, in which X independently represents (CH2)2 or (CH2)3 or X═—CH═CH— (trans) and in which W represents COOH, COO−M+(M+ representing a counterion which is pharmaceutically acceptable), CH2OH, COOR (with R representing an alkyl group), SO3H or PO3H2 or a group chosen from the following: in which R7 and R8 independently represent a hydrogen atom, an alkyl group, an aryl group, an aralkyl group or a hydroxyl group, in which R9 independently represents a hydrogen atom or a methyl group and in which R10 independently represents an ethyl, C12H15 or adamantyl group.",
"2.Compounds according to claim 1 with general formula I′: in which W represents COOH or COO−M+ (M+ representing a counterion which is pharmaceutically acceptable), and in which Ar represents the groups defined in claim 1.3.A compound according to claim 1 or 2, characterized by the fact that it pertains to the salts with general formula I″ in which Ar, X and n are defined in claim 1 or 2.4.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-phenylbutanoic acid.",
"5.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(3,4-dichlorophenyl)-4-hydroxybutanoic acid.",
"6.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(3,4-dimethoxyphenyl)-4-hydroxybutanoic acid.",
"7.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(1-tosyl-1H-pyrrol-3-yl)butanoic acid.",
"8.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-pyridin-4-ylbutanoic acid.",
"9.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-pyridin-3-ylbutanoic acid.",
"10.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(2-thienyl)-4-hydroxybutanoic acid.",
"11.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-[4-(acetylamino)phenyl]-4-hydroxybutanoic acid.",
"12.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-[5-(ethoxycarbonyl)-1H-pyrrol-3-yl]-4-hydroxybutanoic acid.",
"13.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(2,3-dihydro-1-benzofuran-5-yl)-4-hydroxybutanoic acid.",
"14.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(2,3-dihydro-1,4-benzodioxin-6-yl)-4-hydroxybutanoic acid.",
"15.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-4-hydroxybutanoic acid.",
"16.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(1-naphthyl)butanoic acid.",
"17.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(2-naphthyl)butanoic acid.",
"18.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(2,3-dihydro-1H-inden-5-yl)-4-hydroxybutanoic acid.",
"19.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-[2-(ethoxycarbonyl)-1H-indol-5-yl]-4-hydroxybutanoic acid.",
"20.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(1H-indol-5-yl)butanoic acid.",
"21.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(1-benzothiphen-2-yl)-4-hydroxybutanoic acid.",
"22.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(4-oxo-1,4-dihydroquinolin-7-yl)butanoic acid.",
"23.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(4-oxo-1,4-dihydroquinolin-6-yl)butanoic acid.",
"24.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(7-methylimidazo[1,2-a]pyridin-3-yl)butanoic acid.",
"25.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(2-oxo-2,3-dihydro-1H-indol-5-yl)butanoic acid.",
"26.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-{2-[(dimethylamino)carbonyl]-1H-indol-5-yl}-4-hydroxybutanoic acid.",
"27.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(1,2-dihydroacenaphthylen-4-yl)-4-hydroxybutanoic acid.",
"28.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-dibenzo[b,d]thiophen-2-yl-4-hydroxybutanoic acid.",
"29.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-dibenzo[b,d]furan-2-yl-4-hydroxybutanoic acid.",
"30.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(9-acetyl-9H-carbazol-3-yl-4-hydroxybutanoic acid.",
"31.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(phenoxathiin-2-yl)butanoic acid.",
"32.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(phenoxathiin-3-yl)butanoic acid.",
"33.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)-4-hydroxybutanoic acid.",
"34.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(9H-xanthen-2-yl)butanoic acid.",
"35.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(9H-fluoren-3-yl)hydroxybutanoic acid.",
"36.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(4-oxo-4,5-dihydro-3H-pyridazo(4,5-b)indol-8-yl)butanoic acid.",
"37.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-4-(4-oxo-4,5-dihydro-3H-pyridazo(4,5-b)indol-6-yl)butanoic acid.",
"38.A compound according to claim 1, characterized by the fact that it pertains to N,4-dihydroxy-4-phenylbutanamide.",
"39.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of 4-hydroxy-5-phenylpentanoic acid.",
"40.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of 4(R)-hydroxy-5-phenylpentanoic acid.",
"41.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of 4(S)-hydroxy-5-phenylpentanoic acid.",
"42.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of 5-(3,4-dichlorophenyl)-4-hydroxypentanoic acid.",
"43.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of 5-hydroxy-5-phenylpentanoic acid.",
"44.A compound according to claim 3, characterized by the fact that it pertains to the sodium salt of 4-(3,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-4-hydroxybutanoic acid.",
"45.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of (E)-4-(4-chlorophenyl)-4-hydroxybut-2-enoic acid.",
"46.A compound according to claim 1, characterized by the fact that it pertains to the sodium salt of (E)-4-(4-acetylaminophenyl)-4-hydroxybut-2-enoic acid.",
"47.Use of a compound according to any one of claims 1 to 46 for obtaining a drug containing, as active ingredient, at least one compound with general formula I, I′ or I″ according to any one of claims 1 to 46 for the treatment of a disease which can be treated by administration of an agonist or antagonist of GHB receptors, in particular, neurological or mental disorders of the central nervous system and, in particular, regulation of sleep and of secretion of hormones, in particular, growth hormones, reduction of anxiety or increasing of alertness, antiepileptic activity, regulation of weight and food intake, regulation of mood or antidepressive activity, neuroleptic activity, regulation of circadian rhythm, hypnotic or anesthetic activity, neuroprotective or anti-ischemic activity, activity in the process of drug withdrawal and in addiction.",
"48.Use according to claim 47, characterized by the fact that the compound(s) used as active ingredient is(are) one or more sodium salts with general formula I″ obtained by neutralization of a compound with general formula I or I′ according to claim 1 or 2 and containing an acid function for the group W. 49.A pharmaceutical composition characterized by the fact that it contains, as active ingredient, at least one compound with general formula I, I′ or I″ according to any one of claims 1 to 46."
],
[
"The present invention relates to the field of synthesis organic chemistry applied to the pharmaceutical field and relates to new derivatives of 5-hydroxybutanoic acid and its higher homologue, 5-hydroxypentanoic acid, of their crotonoid homologues, to the pharmaceutical compositions containing them and to their pharmaceutical uses.",
"These new derivatives are capable of binding to the specific receptors of γ-hydroxybutyrate (GHB) and are consequently capable of having agonist and antagonist properties.",
"The present invention also relates to bioprecursors, isosteres and prodrugs of these compounds with improved oral therapeutic efficacy.",
"Finally, the present invention relates also to the specific uses of these GHB mimetics, particularly in the treatment of sleep disorders, anxiety and general disorders of the central nervous system (CNS).",
"γ-hydroxybutyrate was studied as early as the 1960s by Laborit, as an isosteric analogue of γ-aminobutyric acid (GABA) which is capable of passing through the blood-brain barrier.",
"In fact, GHB has been identified as being a natural component of the mammalian brain and is currently used in therapy as a well-tolerated general anesthetic.",
"Its catabolism is very rapid, the half-life being, as an indication, approximately one hour in cats.",
"GHB induces deep sedation with loss of postural reflexes and analgesia.",
"The electroencephalogram has been interpreted sometimes in certain animal species as being a sleep graph without paradoxical sleep disturbance, and sometimes as being a graph reminiscent of that of a “petit mal” epileptic episode.",
"The latter problem reduced the indications of GHB in anesthesia in elderly patients.",
"It was possible to demonstrate in the 1980s that GHB fulfilled the criteria generally used for qualification of a substance as neurotransmitter or neuromodulator, namely the presence of the synthetic enzyme of this compound in the presynaptic ends, calcium-dependent release by membrane depolarization, sodium-dependent high affinity active transport and, finally, high affinity saturable reversible binding on membrane preparations of synaptic origin.",
"All these properties make it possible to establish the existence of neuronal circuits in which a role of γ-hydroxybutyrate is directly implied.",
"The present invention aims to propose new derivatives of 5-hydroxybutanoic acid, 5-hydroxypentanoic acid and of their crotonoid homologues, which are capable of binding more effectively to the specific receptors of γ-hydroxybutyrate (GHB) and which have agonist and antagonist properties.",
"The inventors unexpectedly revealed that by functionalizing compounds of the family of 4-hydroxybutanoic acid or of 5-hydroxypentanoic acid (the acid, its esters, its sodium salts, its crotonoid derivatives, etc.",
"), in particular by aromatic substitution of the methyl carbon at the end of the chain, it was possible to obtain synthetic derivatives with increased affinity for GHB receptors compared to the affinity of natural GHB for these same receptors.",
"In a first aspect, the present invention relates to the compounds with general formula I: in which Ar represents one of the following mono-, bi- or tricyclics: in which: R1, R2, R3 and R4 independently represent a hydrogen atom, a halogen, an alkyl group, an aryl group, an aralkyl group, a hydroxyl group, a methoxy group, an acetyl group, a tosyl group, a COOEt group, an NHCOCH3 group, an NH2 group, a CON(CH3)2 group, an NO2 group or a COR5R6 group, in which R5 and R6 independently represent a hydrogen atom, a methyl group, a CH3H7 group or a benzyl group, each z independently represents a nitrogen or carbon atom, Y and Y′ independently represent a carbon, sulfur, oxygen or nitrogen atom, Y″ represents a methylene, ethylene or propylene group, each X′ independently represents a sulfur or oxygen atom, p has a value of 0, 1 or 2, in which n has a value of 0 or 1, in which X independently represents (CH2)2 or (CH2)3 or X═—CH═CH— (trans) and in which W represents COOH, COO−M+ (M+ representing a counterion which is pharmaceutically acceptable), CH2OH, COOR (with R representing an alkyl group), SO3H or PO3H2 or a group chosen from the following: in which R7 and R8 independently represent a hydrogen atom, an alkyl group, an aryl group, an aralkyl group or a hydroxyl group, in which R9 independently represents a hydrogen atom or a methyl group and in which R10 independently represents an ethyl, C12H15 or adamantyl group.",
"More preferably, it relates to the compounds with general formula I′: in which W represents COOH or COO−M+ (M+ representing a counterion which is pharmaceutically acceptable), and in which Ar represents the groups defined above.",
"In particular, it relates to the salts with general formula I″ in which Ar, X and n are defined above, which are obtained from compounds with general formula I and which can be prepared with techniques known to the expert in the field.",
"In a second aspect, the present invention provides pharmaceutical compositions which contain, as active ingredient, at least one compound with general formula I, I′ or I″.",
"It also relates to the use of the new synthesized derivatives for therapeutic purposes.",
"The present invention describes different routes of synthesis, relating to preferred embodiments, which are given as nonlimiting examples, and which are explained with reference to the appended drawings in which: FIG.",
"1 represents a simplified block diagram of the different processes of synthesis of the compounds according to the invention as well as of their main intermediate reaction products, FIG.",
"2 represents a simplified diagram of other processes of synthesis of compounds according to the invention, FIG.",
"3 represents another simplified diagram of other processes of synthesis of compounds according to the invention; and FIG.",
"4 represents a simplified block diagram of a process of synthesis of enantiomerically pure compounds according to the invention as well as of their main intermediate reaction products.",
"Except when indicated otherwise, the following terms used in the present description and the present set of claims have the following meanings: “alkyl” designates a straight saturated monovalent hydrocarbon radical with 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical with 3 to 6 carbon atoms, for example, methyl, ethyl, propyl, 2-propyl, butyl, etc.",
"“aryl” designates an aromatic hydrocarbon radical of the phenyl type, possibly substituted, that is, a phenyl group which is possibly substituted independently with one or two substituents chosen from the group formed by the halogen, —OR (in which R represents a hydrogen atom or an alkyl group) and —NRR′ (in which R and R′ independently represent a hydrogen atom or an alkyl group) groups.",
"“aralkyl” designates an aryl radical as defined above substituted by an alkylene group, that is, a straight saturated bivalent hydrocarbon radical with 1 to 6 carbon atoms or a branched saturated bivalent hydrocarbon radical with 3 to 6 carbon atoms containing at least one double bond, such as benzyl, phenylethyl, etc.",
"“halogen” designates a monovalent radical chosen from fluorine, chlorine, bromine and iodine.",
"The expressions “carbon atom” and “nitrogen atom” must be understood to designate said atoms, possibly provided with one or two hydrogen atoms and/or substituted by the aforementioned groups and/or having a chemical bond, in such a way as to satisfy the expected valences of carbon and nitrogen.",
"As examples, z can represent, depending on the case, N, NH, C, CH or CH2, Y can be, depending on the case, C, CH, CH2, S, O, etc.",
"The compounds according to the present invention can have one or more asymmetrical centers and can therefore be produced as individual stereoisomers (R) or (S), or as mixtures of them.",
"Except when specifically indicated, the present description and the present set of claims include both the individual enantiomers and their mixtures in any proportions, including the so-called racemic mixtures.",
"“Pharmaceutically acceptable excipient” designates a single excipient or a set of excipients which can be used in the preparation of a pharmaceutical composition which is generally safe, nontoxic and not undesirable biologically or otherwise, and includes one or more excipients which are acceptable for veterinary as well as for human pharmaceutical use.",
"“Pharmaceutically acceptable salt” designates a salt or a mixture of several salts which can be used in the preparation of a pharmaceutical composition which is generally safe, nontoxic and not undesirable biologically or otherwise and which has the desired pharmacological activity of the parent molecule.",
"Such salts include the salts formed when an acid proton of the parent molecule is replaced by a metal ion, for example, an alkali metal ion (preferably sodium or lithium) or an alkaline-earth ion, or is coordinated with an organic base such as ethanolamine, diethanolamine, the amino acids (lysine in particular), etc.",
"A “pharmaceutically acceptable counterion” designates an ion with a charge opposite that of the substance with which it is associated and which is pharmaceutically acceptable.",
"The term “prodrug” designates any compound which releases an active related drug according to general formula I, I′ or I″ in vivo when such a prodrug is administered to a mammalian subject.",
"The prodrugs are prepared by modifying the functional groups which are present in such a way that the modifications can be eliminated or neutralized in vivo in order to release the parent molecule, examples of prodrugs including, in a nonlimiting manner, esters (for example, the derivatives of acetate, formate, benzoate, etc.",
"), etc.",
"The terms “treating” or “treatment” of a disease include: preventing the disease, namely, causing the clinical symptoms of the disease not to develop in a mammal who may be exposed or predisposed to the disease but who is not experiencing or who does not yet have symptoms of the disease, inhibiting the disease, namely, stopping or reducing the development of the disease or of its clinical symptoms, or causing the disease to disappear, namely, bringing about the regression of the disease or of its clinical symptoms.",
"A “therapeutically effective quantity” designates the quantity of a compound which, when administered to a mammal for treating a disease, is sufficient to bring about such a treatment for the disease.",
"This quantity will vary as a function of the compound, the disease and its severity, and the age, weight, etc., of the mammal who is to be treated.",
"The starting compounds can be obtained commercially or can be synthesized according to the conventional processes.",
"It is understood that the present application is not limited to a particular route of synthesis and extends to other processes making production of the indicated compounds possible.",
"The compounds with general formula I″ are prepared as illustrated in the diagram of FIG.",
"1.The key intermediate products are the acids with general formula I and the corresponding keto acids or esters with general formula II: Ar—(CH2)n—(C*═O)—X—CO2R (II) in which R can be hydrogen or an alkyl group, preferably ethyl or methyl.",
"Depending on the nature of the aromatic (Ar), these derivatives are obtained by the Friedel-Crafts reaction (M. Kakushima et al.",
"JOC, 48(19), 1983, 3214; G. J. Quallich et al.",
"JOC, 55(16), 1990, 4971) or by condensation of a methylketone with glycoxylic acid (J. J. Bourguignon et al.",
"J. Med.",
"Chem., 1988, 31, 893-897) or by reaction of ArCO2Et with diethyl succinate in a basic medium (H. Michel et al.",
"Arch.",
"Pharmaz., 1974, 307, 689).",
"Application examples will be given in part A) hereafter (cf., in particular, Table 1 of this part).",
"These keto acids or esters II are subsequently reduced to corresponding alcohols with general formula III, using NaBH4 in the case in which R=Me or R=Et, and using KBH4 if R═H, and then salified using NaOH in order to obtain the corresponding sodium salts with general formula I″, as represented in FIG.",
"1.As indicated also in the diagram of FIG.",
"1, the reduced compounds III are lactonized into compounds with general formula IV by simple heating in an acid medium.",
"This reaction is well-known to the expert in the field and does not need to be developed in more detail here.",
"Of course, the expert in the field will be able to choose any acid suitable for obtaining the desired lactones.",
"As an example of a particularly suitable acid, it is possible to mention hydrochloric acid, as illustrated in a nonlimiting manner in the examples which follow.",
"The reaction of an amine with the lactones IV leads to the corresponding amides with general formula V. Furthermore, the keto acids for which R═H, X═(CH2)3 and n=0 can be transformed into γ-aroyl-γ-butyrolactones with general formula VI by reaction with bromine (K. Yamada et al., Tetrahedron, 1971, 27, 5445-5451).",
"These lactones VI can then be reduced to γ-benzyllactones with general formula IV by catalytic hydrogenation.",
"However, this last process cannot be applied if the aromatic Ar carries a halogen.",
"In this case, a reaction catalyzed by Pd(OAc)4 is called for.",
"Thus, by treating, for example, 1-chloro-4-iodobenzene, the methylidine-γ-butyrolactone with general formula VII is obtained (A. Arcadi et al.",
"JOC, 1992, 57, 976-982) which, by action of NaOH, leads to the corresponding keto acid II (cf.",
"FIG.",
"2).",
"The different steps mentioned in the preceding for synthesis of the compounds according to the invention will now be described in more detail by means of nonlimiting illustrative examples.",
"A) Access to the Intermediate Keto Acids or Keto Esters (II) The formation of the intermediate keto acids or keto esters with general formula II is described hereafter, the different processes for obtaining them being summarized in Table 1 which follows.",
"TABLE 1 Processes for obtaining intermediate keto acids or keto esters {circle over (2)}Procédé {circle over (1)}Réactifs de départ X n R n° ArH (CH2)2 0 H 1 ArH (CH2)2 0 Me 2 ArH (CH2)3 0 H 3 ArH CH═CH 1 H 4 ArH CH═CH 0 H 5 ArCOCH3 (CH2)2 0 H 6 ArCO2Et (CH2)2 0 H 7 (CH2)2 1 H 8 Key: 1Starting reagents 2Process No.",
"Eight processes and eleven examples of embodiments will be detailed in the following.",
"The keto esters which are obtained are reduced to lactones and salified as indicated in the diagram of FIG.",
"1.1) Process 1: Friedel-Crafts Reaction with Succinic Anhydride Four examples specifying the conditions of operation of solvents, temperature and reaction time are described hereafter.",
"Example 1 Synthesis of 4-[5-ethoxycarbonyl)-1H-pyrrol-3-yl]-4-oxobutanoic acid (keto acid derivative of Compound No.",
"9) Succinic anhydride (2.80 g, 27.9 mmol) is dissolved in dichloro-1,2-ethane (20 mL).",
"Aluminum chloride (11.20 g, 83.9 mmol) is added and then the pyrrolic derivative (2.00 g, 14.4 mmol) dissolved in the solvent (20 mL).",
"The reaction medium is stirred at room temperature for 3 h and then poured over ice.",
"The precipitate which forms is filtered and dried.",
"beige solid MM(C11H13NO5): 239.23 g mass: 2.62 g Yield: 76% {circle over (1)}RMN(1H, 300 MHz, DMSO): 12, 51{circle over (2)}(large s, 1H, COOH); 7, 75(s, 1H, CHpyrrol.",
"); 7, 15(s, 1H, CHpyrrol); 4, 27(q, 2H, —CH2—, J3 = 7, 0 Hz); 3, 04(t, 2H, —CH2—, J3 = 6, 2 Hz); 2, 52(t, 2H, —CH2—, J3 = 6, 2 Hz); 1, 30(t, 3H, —CH3, J3 = 7, 0 Hz).",
"Key: 1NMR* 2broad *[Editor's note: Within numbers in the NMR data and in the tables which follow, commas represent decimal points.]",
"Example 2 Synthesis of 4-(2,3-dihydro-1H-inden-5-yl)-4-oxobutanoic acid (keto acid derivative of Compound No.",
"15) A conventional Friedel-Crafts reaction is carried out in nitrobenzene (60 mL) at room temperature for 4 h: indane (4 mL, 32.66 mmol), AlCl3 (11.00 g, 82.5 mmol) and succinic anhydride (3.00 g, 30 mmol).",
"A slightly yellow solid is obtained, which is characterized as follows: slightly yellow solid MM (C13H14O3): 218.25 g mass: 5.03 g Yield: 71% {circle over (1)}RMN(1H, 200 MHz, DMSO): 12, 13(large s, 1H, —COOH); 7, 84(s, 1H, CHarom); 7, 78(d, 1H, CHarom., J3 = 7, 8 Hz); 7, 37(d, 1H, CHarom., J3 = 7, 8 Hz); 3, 23(t, 2H, —CH2—, J3 = 5, 9 Hz); 2, 93(m, 4H, 2- CH2—); 2, 57(t, 2H, —CH2—, J3 = 5, 9 Hz); 2, 05(m, 2H, —CH2—).",
"Key: 1NMR 2Broad Example 3 Synthesis of 4-(5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)-4-oxobutanoic acid (keto acid derivative of Compound No.30) The N-acetylated derivative of azepine (1.00 g, 4.21 mmol) is dissolved in CS2 (10 mL), and aluminum chloride (5.62 g, 42.1 mmol) is added.",
"The succinic anhydride (3.37 g, 33.8 mmol) is then added in small spatula loads.",
"The reaction medium is stirred (mechanical stirring) at reflux for 6 h. After cooling, this solution is poured over ice and extracted with ethyl acetate.",
"The organic phase is treated with 1N NaOH, and the aqueous phase is washed with ethyl acetate.",
"Then, it is then treated again in acid medium by addition of concentrated HCl.",
"The product is extracted with ethyl acetate, and dried using Na2SO4, and the solvents are evaporated.",
"The oil which is obtained is purified by silica gel column chromatography with a mixture of CH2Cl2/MeOH 9/1 as eluent.",
"yellow oil MM(C20H19NO4): 337.38 g mass: 900 mg Yield: 63% {circle over (1)}RMN(1H, 300 MHz, DMSO): 12, 17{circle over (2)}(large s, 1H, COOH); 8, 11-7, 22(m 7H, 7CHarom.",
"); 3, 47-3, 22(m, 4H, —CH2—CH2—); 2, 94-2, 84(m, 2H, —CH2—); 2, 59(t, 2H, —CH2—, J3 = 6, 2 Hz);1, 93 (s, 3H, —CH3).",
"Key: 1NMR 2Broad Example 4 Synthesis of 4-[4-(acetylamino)phenyl]-4-oxobutanoic acid (keto acid derivative of Compound No.",
"8) With mechanical stirring and argon flow, at room temperature, DMF (22 mL) is added dropwise to aluminum chloride (134 g, 1.00 mol).",
"After complete addition, the temperature is brought to 90° C., and the acetanilide (10 g, 74 mmol) and succinic acid (10 g, 100 mmol) mixture is added in small spatula loads.",
"The reaction medium is stirred at 80-90° C. for 2 h, and then poured over 500 g of crushed ice.",
"By addition of concentrated HCl, the pH is brought to 1.The precipitate which forms is filtered and recrystallized in DMF/H2O.",
"pale-yellow powder MM(C12H13NO4): 235.24 g mass: 12.10 g Yield: 70% {circle over (1)}RMN(1H, 300 MHz, DMSO): 12, 13{circle over (2)}(large s, 1H, COOH); 10, 31(s, 1H, NH); 7, 95(d, 2H, 2CHarom., J3 = 8, 7 Hz); 7, 74(d, 2H, 2CHarom., J3 = 8, 7 Hz); 3, 21(t, 2H, —CH2—, J3 = 6, 2 Hz); 2, 58(t, 2H, —CH2—, J3 = 6, 2 Hz); 2, 11(s, 3H, —CH3).",
"Key: 1NMR 2Broad 2) Process 2: Friedel-Crafts reaction with methyl-4-chloro-4-oxobutanoate EXAMPLE Synthesis of methyl 4-(7-methylimidazo[1,2-a]pyridin-3-yl)-4-oxobutanoate (methyl keto ester derivative of Compound No.",
"21) 7-methylimidazo[1,2-a]pyridine (0.50 g, 3.62 mmol) is dissolved in CS2 (8 mL).",
"The solution is cooled to 0° C., and aluminum chloride (1.40 g, 5.35 mmol) is added in small portions.",
"It is stirred for 30 min, and then the reaction medium is brought to reflux.",
"An acid chloride (1.50 g, 10 mmol) is added, and it is stirred at reflux for 2 h. The solution is cooled and poured over ice.",
"The pH is adjusted to 9 by addition of 3N NaOH.",
"It is extracted with ethyl acetate and then filtered using diatomaceous earth.",
"The ethyl acetate phase is dried using sodium sulfate and then evaporated to dryness.",
"Finally, it is triturated with ether, and the product obtained is filtered.",
"light-maroon powder MM(C13H14N2O3): 246.26 g mass: 100 g Yield: 12% {circle over (1)}RMN(1, 300 MHz, CDCl3): 9, 50(d, 1H, CHarom., J3 =6, 7 Hz); 8, 40(s, 1H, CHimidaz.",
"); 7, 50(s, 1H, CHarom.",
"); 6, 91(d, 1H, CHarom., J3 = 6, 7 Hz); 3, 75(s, 3H, —CH3); 3, 28(t, 2H, —CH2—, J3 =6, 7 Hz); 2, 81(t, 2H, —CH2—, J3 = 6, 7 Hz); 2, 50 (s, 3H, —CH3).",
"Key: 1 NMR 3) Process 3: Friedel-Crafts reaction with glutaric anhydride EXAMPLE Synthesis of 4-(4-methoxybenzoyl)butyric acid In a dry 250-mL two-necked flask in an argon atmosphere at 0° C., glutaric acid (5 g, 87.64 mmol) is added to a mixture of CH2Cl2 (125 mL) and anisole (14.2 g, 1.5 Eq., 0.13 mol).",
"AlCl3 (12.3 g, 1.05 Eq, 92 mmol) is added in portions, and the mixture is stirred at 0° C. for 4 h before being poured over ice (100 g).",
"The precipitate is collected by filtration, washed with cold water and dried under vacuum.",
"9 g of the desired product are obtained.",
"Yield: 50% White solid, MP = 133° C. {circle over (1)}RMN1H(200 MHz, CDCl3): δ 2, 08(p, 2H, 3J = 7, 2 Hz, H3); 2, 50(t, 2H, 3J = 7, 2 Hz, H2); 3, 04(t, 2H, 3J = 7, 2 Hz, H4); 3, 88(s, 3H, OCH3); 7, 47(syst{acute over (e+0 me AB, 4H arom, JAB +L =+0 8, 8 Hz, Δν+0 =+0 203 Hz))} Key: 1NMR 2System {circle over (1)}RMN 13C(50 MHz, CDCl3): δ 19, 18(C3); 33, 04 2(C2 ou C4); 36, 94(C2 ou C4); 55, 46(OCH3); 113, 72(C8); 129, 78(C6); 130, 30(C7); 163, 48(C9); 178, 80(Cl); 197, 99(C5).",
"Key: 1NMR 2Or Microanalysis: C % H % theoretical 64.85 6.35 actual 64.71 6.20 {circle over (1)}SM(IE, 70 eV) m/e(intensité relative %): 222(M+, 78), 135 [[(M-(CH2)3COOH)+, 100].",
"Key: 1Mass spectrometry 2Relative intensity % 4) Process 4: Friedel-Crafts Reaction with Glutaric Anhydride Followed by the Action of Bromine This process consists of a direct switch to 5-benzyllactone.",
"First of all, a Friedel-Crafts reaction is performed between the chosen aromatic nucleus and glutaric anhydride (cf.",
"Process 3), followed by a substitution with bromine and then lactonization in basic medium.",
"EXAMPLE Synthesis of 5-benzoyldihydrofuran-2-one In a 500-mL round-bottomed flask, 4-benzoylbutyric acid (20 g, 0.1 mol) is added to a mixture of dioxane (180 mL) and ether (60 mL).",
"Bromine (6.55 mL, 1.25 Eq, 0.125 mol) is added dropwise, and the solution is stirred for 3 h and then poured into an aqueous 30% NaHCO3 solution (150 mL) and again stirred for 6 h. The phases are separated, and the aqueous phase is extracted with CH2Cl2 (3×75 mL).",
"The organic phases are combined, washed with brine (150 mL), dried using anhydrous sodium sulfate, filtered and evaporated to dryness.",
"Silica column chromatography gives the desired compound (14.64 g).",
"Chromatography solvent: AcOEt/hexane, 30/70, Rf: 0.20 Yield: 74% White solid, MP = 91° C. {acute over (1)}RMN 1H(200 MHZ, CDCl3): δ 2, 44-2, 67(m, 4H, H2 et H3); 5, 79-5, 85(m, 1H, H4); 7, 48-7, 65(m, 3H arom.",
"meta et para); 7, 96-8, 00(m, 2H arom.",
"ortho).",
"{acute over (1)}RMN 13C(50 MHz, CDCl3): δ 24, 92(C3); 26, 76(C2); 78, 18(C4); 128, 75(CH arom.",
"); 128, 97(CH arom.",
"); 133, 49(C6); 134, 29 (CH arom.",
"); 176, 20(C1); 194, 24(C5).",
"Key: 1NMR 2Meta and para Microanalysis: C % H % theoretical 69.46 5.30 actual 69.36 5.29 {acute over (1)}SM(IE, 70 eV) m/e(intensité relative %): 190(M+, 38), 105[(M-PhCO)+, 100].",
"Key: 1Mass spectrometry 2Relative intensity % The keto lactones with general formula VI which are obtained are then reduced by H2 using Pd/C to give the compounds with general formula VI and the corresponding salts (cf.",
"FIG.",
"2).",
"EXAMPLE Synthesis of 5-benzyldihydrofuran-2-one To a solution of the keto lactone synthesized in the preceding (1 g, 5.25 mmol) in methanol (20 mL), Pd/C 10% (10 wt%, 0.1 g) and concentrated HCl (0.3 mL) are added.",
"The mixture is hydrogenated in a Paar apparatus at 70 psi (483 kPa) for 16 h and then filtered using Celite, washing with ethyl acetate.",
"Evaporation of the solvents and silica column chromatography give the desired compound (556 mg).",
"Chromatography solvent: AcOEt/hexane, 20/80, Rf: 0.23 Yield: 60% Colorless oil {circle over (1)}RMN 1H(200 MHz, CDCl3): δ 1, 85-1, 97 (m, 1H, Ha ou2Hb); 2, 13-2, 48(m, 3H, H2{acute over (3)}et Ha{acute over (2)}ou Hb); 2, 84-3, 08(m, 2H, H5); 4, 63-4, 76(m, 1H, H4); 7, 18-7, 33(m, 5H arom.)",
"{circle over (1)}RMN 13C(50 MHz, CDCl3): δ 26, 81(C3); 28, 34(C2); 40, 95(C5); 80, 56(C4); 126, 62(CH arom.",
"); 128, 31(CH arom.",
"); 129, 15(CH arom.",
"); 135, 73(C6); 176, 93(Cl).",
"Key: 1NMR 2Or 3And Microanalysis: C % H % theoretical 74.97 6.87 actual 74.85 7.00 {circle over (1)}SM(IE, 70 eV) m/e{acute over (2)}(intensité relative %): 176(M+, 45), 91 [(M-lactone)+, 31], 85(lactone+, 100).",
"Key: 1Mass spectrometry 2Relative intensity % 5) Process 5: Friedel-Crafts Reaction with Maleic Anhydride EXAMPLE Synthesis of p-(acetylamino)benzoyl-3 crotonic acid 2 g of acetanilide (0.015 mol) and 1.6 g of maleic anhydride (0.016 mol) are added to a suspension of AlCl3 (7.5 g, 0.055 mol) in 1,2-dichloroethane (35 mL).",
"The temperature climbs spontaneously to 60° C. The reaction medium is left to react for 20 h at room temperature and then is poured in ice and treated with concentrated HCl.",
"The solid which precipitates is filtered and dried and then recrystallized in ethane 70% (melting point—m.p.—: 244° C.).",
"MM(C12H11NO4): 233.23 g Yield: 45% {circle over (1)}RMN 1H(DMSO-d6): δ 7, 80(JAB = Hz, 4Haryl); 6, 55(d, HA, JAB = 15 Hz); 7, 77(d, HB, JAB = 15 Hz); 2, 10(s, 3H, CH3CONH).",
"Key: 1NMR 6) Process 6: Reaction of a Methyl Ketone with Glyoxalic Acid EXAMPLE Synthesis of (E)-4-(1H-indol-3-yl)-4-oxobut-2-enoic acid 0.09 mL of ketone and 0.1 mol of glyoxylic acid are mixed and then brought to 140° C. for 20 h. A Hieckmann apparatus (displacement of water) is then mounted on the flask and heated to 110° C. for 10 h and 145° C. for 3 h. The crude reaction product is taken up with ethyl acetate and extracted with potassium bicarbonate.",
"The aqueous phase is acidified with concentrated HCl at 0° C., and the precipitate obtained is filtered.",
"Beige solid MM(C12H9NO3): 215.21 g Yield: 62% TM > 200° C. {circle over (1)}RMN(1H 200 MHz,, CDCl3): 8, 10-8, 30(m, 2H, CH indol.",
"+NH); 6, 9-7, 7(m, 4H); 7, 80(d, 1H, CHA = C, J3 = 16, 0 Hz); 6, 70(d, 1H, CHB = C, J3 = 160 Hz).",
"Key: 1NMR 7) Process 7: Reaction of Diethyl Succinate with Aryl Esters EXAMPLE a) Synthesis of diethyl 2-(pyridin-3-ylcarbonyl)succinate.",
"Ethyl nicotinate (10 mL, 73 mmol) is dissolved in ether (50 mL).",
"Diethyl succinate (24 mL, 144 mmol) is added, followed by NaH (7.00 g, 60% in oil, 175 mmol) in small spatula loads.",
"The reaction medium is stirred at room temperature in an argon atmosphere for 2 days.",
"It is then poured over crushed ice.",
"The aqueous phase is collected and treated with 6N HCl in order to bring the pH to 4.5.The product is extracted with chloroform and dried using Na2SO4.After evaporation of the solvents, the crude oil obtained is purified by silica gel column chromatography, with AcOEt/Hexane 1/2 and then 1/1 and 7/3 as eluent.",
"thick orange oil MM(C14H17NO5): 279.29 g mass: 8.87 g Yield: 43% {circle over (1)}RMN(1H, 300 MHz, CDCl3): 9, 25(d, 1H, CHarom., J4 = 2, 0 Hz); 8, 81(dd, 1H, CHarom., J 3 = 4, 8 Hz et J4 = 2, 0 Hz); 8, 33-8, 29(M, 1H, CHarom.",
"); 7, 47-7, 43(m, 1H, CHarom.",
"); 4, 82(m, 1H, —CH—); 4, 18-4, 09(m, 4H, 2CH2); 3, 24-2, 99(m, 2H, CH2-COO-); 1, 28-1, 13(m, 6H, 2-CH3).",
"Key: 1NMR b) Synthesis of 4-oxo-4-pyridin-3-ylbutanoic acid The keto ester synthesized in the preceding (8.87 g, 31.76 mmol) is dissolved in 1N sulfuric acid (80 mL).",
"The reaction medium is stirred at reflux for 3 h and then cooled.",
"The pH is brought to 4.5 by dropwise addition of a saturated solution of NaHCO3.The precipitate which forms is filtered and dried.",
"beige solid MM(C9H9NO3): 179.18 g mass: 5.56 g Yield: 98% {circle over (1)}RMN(1H, 300 MHz, DMSO): 9, 17(s, 1H, CHarom.",
"); 8, 80(d, 1H, CHarom., J3 = 4, 3 H; 8, 32(d, 1H, CHarom., J3 = 7, 6 Hz); 7, 58(dd, 1H, CHarom., J3 = 4, 3 Hz et J3 = 7, 6 Hz); 3, 31(t, 2H, —CH2—, J3 = 6, 2 Hz); 2, 62(t, 2H, —CH2—, J3 = 6, 2 Hz).",
"Key: 1NMR Process 8: Synthesis of a Compound of the Compound Type with General Formula VII: EXAMPLE Synthesis of 5(E)-[(4-chlorophenyl)methylidene]tetrahydrofuran-2-one (case in which R′═H) In a dry 250-mL two-necked flask provided with a cooling device, in an argon atmosphere, one places tetrabutylammonium chloride (7.5 g, 1.5 Eq., 0.025 mol) which is dried under vacuum with a vane pump at 60° C overnight.",
"1-chloro-4-iodobenzene (4.07 g, 0.017 mol), 4-pentynoic acid (2.5 g, 1.5 Eq., 0.025 mol) and acetonitrile (50 mL) are added.",
"Then with stirring, Pd(OAc)2(PPh3)2 (5 mol %, 639 mg, 0.85 mmol) is added, followed by triethylamine (50 mL).",
"The solution is heated to 60° C. for 1 h, cooled to room temperature and then poured quickly into 3N HCl.",
"The aqueous phase is extracted with AcOEt (4×75 mL), and the organic phases are combined, washed with water (3×100 mL) then with brine (100 mL), dried using anhydrous sodium sulfate in the presence of activated charcoal, filtered using Celite and evaporated to dryness.",
"Silica column chromatography gives the desired compound (2.18 g).",
"Chromatograph solvent: AcOEt/hexane, 20/80, Rf: 0.36 Yield: 61% Beige solid, MP = 140-141° C. {circle over (1)}RMN 1H(200 MHz, CDCl3): δ 2, 69-2, 81(m, 2H, H2); 3, 07-3, 19(m, 2H, H3); 6, 24-6, 29(m, 1H, H5); 7, 23{circle over (2)}(systéme AB, 4H arom., JAB = 8, 5 Hz, Δν 33 Hz).",
"{circle over (1)}RMN 13C(50 MHz, CDCl3): δ 25, 05(C3); 27, 54(C2); 105, 96(C5); 128, 78(C7 ou C8); 128, 89(C7 ou C8); 132, 24(C6 ou C9); 132, 79(C6 3ou C9); 151, 50(C4); 173, 92(C1).",
"Key: 1 NMR 2 System 3 Or Microanalysis: C % H % theoretical 63.32 4.35 actual 63.26 4.37 {circle over (1)}SM(IE, 70 eV) m/e{circle over (2)}(intensité relative %): 208 (M+, 94), 152[(M-CH2CH2CO)+, 72], 124[(H—C—Ph-pCl)+, 57], 89(100).",
"Key: 1Mass spectrometry 2Relative intensity % B) Reduction of the Intermediate Keto Acids or Keto Esters with General Formula II Into Corresponding Alcohols III 1) Reduction of Keto Acids EXAMPLE Synthesis of 4-hydroxy-4-(1-tosyl-1H-pyrrol-3-yl)butanoic acid (Compound No.",
"4) The keto acid (2.00 g, 62 mmol) is dissolved in a 1M aqueous solution of KHCO3 (20 mL).",
"KBH4 (540 mg, 10 mmol) is added in small spatula loads.",
"The reaction medium is stirred at room temperature for 12 h and then cooled to 0° C. Concentrated HCl is added dropwise to bring the pH to 1.It is left to be stirred cold for 1 h, and the precipitate which forms is then filtered and left to dry in the oven.",
"white solid MM(C15H17NO5S): 323.28 g mass: 1.80 g Yield: 89.5% {circle over (1)}RMN(1H, 300 MHz, DMSO): 7, 85(d, 2H, 2CHarom., J3 = 8, 1 Hz); 7, 44(d, 2H, 2CHarom.",
"J3 = 8, 1 Hz); 7, 26(s, 1H, CHpyrrol.",
"); 7, 12(s, 1H, CHpyrrol.",
"); 6, 32(s, 1H, CHpyrrol.",
"); 4, 42(t, 1H, CH—O—, J3 = 6, 2 Hz); 2, 39(s, 3H, —CH3); 2, 20 (m, 2H, —CH2—); 1, 77(m, 2H, —CH2—).",
"Key: 1NMR 2) Reduction of Keto Esters with General Formula II by Switching to Lactones with General Formula VI EXAMPLE Synthesis of 5-(1H-indol-5-yl)dihydrofuran-2(3H)-one The keto ester (300 mg, 1.10 mmol) is dissolved in dry MeOH (10 mL).",
"NaBH4 (46 mg, 1.22 mmol) is added.",
"The reaction medium is stirred at room temperature for 24 h, with addition every 6 h of a spatula tip of NaBH4.The methanol is evaporated, and the residue is taken up in a water/AcOEt mixture.",
"The organic phase is collected, dried using Na2SO4 and evaporated.",
"The crude product is purified by silica gel column chromatography with CH2Cl2/AcOEt 9/1 as eluent.",
"white powder MM(C12H11NO2): 201.23 g mass: 110 g Yield: 47% {circle over (1)}RMN(1H, 300 MHZ, CDCl3): 8, 58{acute over (2)}(large s, 1H, NH); 7, 61(s, 1H, CHarom.",
"); 7, 38(d, 1H, CHarom., J3 = 9, 3 Hz); 7, 23(m, 1H, CHindol.",
"); 7, 13(d, 1H, CHarom., J3 = 9, 3 Hz); 6, 55-6, 54(m, 1H, CHindol.",
"); 5, 58(m, 1H, CHlacton); 2, 76-2, 57(m, 3H, CH2—CH); 2, 38-2, 22(m, 1H, CH).",
"Key: 1NMR 2Broad The acid alcohols can be lactonized by hydrolysis with heat (60° C., concentrated HCl in THF) for one night.",
"C) Access to the Sodium Salts (Final Products) The acid alcohols III obtained by reduction followed by hydrolysis or opening of the lactones IV are salified by an alkaline solution, for example, by a 1M solution of sodium carbonate in water (0.9 Eq.).",
"The aqueous phase is washed with ethyl acetate, then lyophilized or evaporated, followed by trituration with ether.",
"This salification reaction is well-known to the expert in the field and does not need to be developed in more detail here.",
"Of course, the expert in the field will be able to choose, instead of the aforementioned sodium hydroxide, any other base suitable for obtaining the desired corresponding salts.",
"As examples of suitable bases, it is possible to mention, in a nonlimiting manner, mineral bases such as LiOH, Ca(OH)2, etc., as well as organic bases conventionally used in synthesis organic chemistry, in particular those used for the synthesis of compositions intended for pharmaceutical use.",
"D) Particular Cases: 1) Formation of Amides with General Formula V (cf.",
"FIG.",
"2) As indicated in the diagram of FIG.",
"2, the opening of reduced lactones IV can be done by addition of a primary or secondary amine or by addition of a hydroxamic acid, in order to end up with the amides or with the corresponding hydroxamino-substituted V. Three embodiments given as nonlimiting examples relating to this operation will hereafter be given in more detail.",
"Example 1 Synthesis of a primary amide, 4-(1-benzothiophen-2-yl)-4-hydroxybutanamide (amide derivative of Compound No.",
"18) The lactone (103 mg, 0.47 mmol) is dissolved in THF (5 mL), and NH40H (2 mL, 25% in water) is added.",
"It is heated to 50° C. for 1 h 30 min and left to cool and evaporate the THF.",
"The residue is taken up in AcOEt and dried using Na2SO4.The solvents are evaporated, and the crude oil which is obtained is purified by silica gel column chromatography, with CH2Cl2/MeOH 9/1 as eluent.",
"Thick oil MM(C12H13NO2S): 235.31 g mass: 61 g Yield: 55% {circle over (1)}RMN(1H, 300 MHz, CDCl3): 7, 89-7, 85(m, 2H, 2CHarom.",
"); 7, 44(s, 1H, CHthienyl); 7, 41-7, 33(m, 2H, 2CHarom.)",
"5, 59-et 5, 45(2s, 2H, —NH2); 5, 24(m, 1H, CH—O); 3, 50(s, 1H, —OH); 2, 50-2, 21(m, 4H, —CH2—CH2).",
"Key: 1NMR Example 2 Synthesis of a secondary amide, 4(1-benzothiophen-2-yl)-N-benzyl-4-hydroxybutanamide The lactone (0.102 g, 0.47 mmol) is dissolved in ether (5 mL).",
"Benzylamine (51 μL, 0.47 mmol) is added, and the reaction medium left with stirring for 48 h. The ether is evaporated, and the crude product is purified by silica gel column chromatography, with CH2Cl2/MeOH 95/5 as eluent.",
"very thick translucent oil MM(C19H20NO2S): 326.45 g mass: 107 g Yield: 70% {circle over (1)}RMN(1H, 300 MHz, CDCl3): 7, 84(m, 2H, 2CHarom.",
"); 7, 43-7, 28(m, 8H, 8CHarom.",
"); 5, 89(large s, 1H, —OH); 5, 24(m, 1H, CH—O); 4, 46(d, 2H, CH2, J3 = 5, 0 Hz); 3, 82(d, 1H, —NH, J3 = 5, 0 Hz); 2, 47-2, 19(m, 4H, —CH2—CH2—).",
"Key: 1NMR 2Broad Example 3 Treatment with Hydroxamic Acid in Order to Obtain N,4-dihydroxy-4-phenylbutanamide (Compound No.",
"34) Metallic sodium (100 mg, 4.34 mmol) is dissolved in methanol (4 mL).",
"Hydroxylamine (0.30 g, 4.32 mmol) is added, and then lactone (0.50 g, 3.09 mmol) dissolved in methanol (5 mL).",
"The reaction medium is stirred at room temperature for 12 h. The methanol is evaporated, and the residue is taken up in ethyl acetate.",
"The insoluble material is filtered, and the filtrate is dried using Na2SO4.The solvents are evaporated, and the oil which is obtained is triturated with ether.",
"very hygroscopic white solid MM(C10H13NO3): 195.22 g mass: 220 mg Yield: 37% {circle over (1)}RMN(1H, 300 MHz, DMSO): 7, 41-7, 21(m, 5H, 5CHarom.",
"); 4, 55(m, 1H, —CH—O); 2, 04-1, 73(2m, 4H, —CH2CH2—).",
"Key: 1NMR 2) Syntheses of Compounds of Types VIII, IX and X As shown in the diagram of FIG.",
"3, the presence in position 3 or 4 of an amine on ethyl β-benzoyl propionate (compound with general formula II with Ar=PhNH2, n=0, X═(CH2)2 and R=Et) made possible the synthesis of dihydroquinolones (compounds VIII) and phenylquinoxalines (compounds X).",
"In effect, condensation of the amine with Meldrum acid leads, as intermediate, to the amine methylene-dioxane-dione (compound IX) which is cyclized thermally (cf.",
"left diagram of FIG.",
"3).",
"The reaction with phenylglyoxal leads to the expected phenylquinoxaline with formula X.",
"The keto esters which are obtained are then reduced to lactones and salified as described in the diagram of FIG.",
"1.The following indicative examples describe in more detail the conditions of operation used for these types of reactions.",
"a) Synthesis of Compounds of Type VIII Example 1 Synthesis of ethyl 4-(4-{[2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]amino}phenyl)-4-oxobutanoate The mixture, Meldrum acid (0.70 g, 0.46 mmol) and methyl orthoformate (15 mL), is heated to reflux (140° C.) for 2 h. The amine (1.00 g, 4.52 mmol) is added all at once.",
"The reaction mixture is stirred at reflux for 12 h. After cooling, the solvent is evaporated, and the residue is purified by recrystallization in methanol.",
"orange-gold powder MM(C19H21NO7): 375.39 g mass: 1.26 g Yield: 75% TM: 154° C. {circle over (1)}RMN(1H, 300 MHz, CDCl3): 11, 32(large d, 1H, —NH, J3 = 14, 0 Hz); 8, 70(d, 1H, CH, J3 = 14, 0 Hz); 8, 07(m, 2H, 2CHarom.",
"); 7, 34(m, 2H, 2CHarom.",
"); 4, 15(q, 2H, —CH2—, J3 7, 2 Hz); 3, 29(t, 2H, −CH2—, J3 = 6, 5 Hz); 2, 77(t, 2H, —CH2—, J3 6, 5 Hz); 176 (s, 6H, 2-CH3); 1, 27(t, 3H, —CH3, J3 = 7, 2 Hz).",
"Key: 1NMR 2Broad Example 2 Synthesis of ethyl 4-oxo-4-(4-oxo-1,4-dihydroquinolin-6-yl) butanoate Diphenyl ether (10 mL) is heated to 240° C. (slight reflux); the Meldrum derivative (1.00 g, 2.66 mmol) is added all at once.",
"The reaction mixture is then stirred at 240° C. for 10 min and then poured very slowly over petroleum ether (150 mL).",
"The precipitate which forms is filtered and purified by silica gel column chromatography (eluent: CH2Cl2/MeOH 9/1).",
"beige solid MM (C15H15NO4): 273.33 g mass: 0.52 g Yield: 71% {circle over (1)}RMN (1H, 200 MHz, CDCl3 + MeOD): 8.90(d, 1H, CHarom., J4=2.0 Hz); 8.13(dd, 1H, CHarom., J3=8.8 Hz, J4=2.0 Hz); 7.62(d, 1H, CHvinyl., J3=7.3 Hz); 7.37(d, 1H, CHarom., J3=8.8 Hz); 6.30(d, 1H, CHvinyl., J3=7.3 Hz); 4.16(q, 2H, —CH2—, J3=7.1 Hz); 3.41(t, 2H, —CH2—, J3=6.6 Hz); 2.77(t, 2H, —CH2—, J3=6.6 Hz); 1.26(t, 3H, —CH3, #J3=7.1 Hz).",
"Key: 1 NMR b) Syntheses of Compounds of Type X Example 1 4-[4-(acetylamino)-3-nitrophenyl]-4-oxobutanoic acid Fuming HNO3 (12 mL) is cooled to 0° C., and the derivative which is to be nitrated (4.00 g, 17.0 mmol) is then quickly added.",
"It is left to be stirred at 0° C. for 10 min.",
"Then, the medium is poured over ice, and the precipitate which forms is filtered.",
"yellow powder MM (C12H12N2O6): 280.23 g mass: 2.97 g Yield: 62% TM: 145° C. {circle over (1)}RMN (1H, 200 MHz, DMSO): 10.6(s, 1H, NH); 8.44(d, 1H, CHarom., J4=2.0 Hz); 8.29(dd, 1H, CHarom., J3=8.5 Hz, J4=2.0 Hz); 7.86(d, 1H, CHarom., J3=8.5 Hz), 3.30(t, 2H, —CH2—, J3=6.2 Hz); 2.61 (t, 2H, —HC2—, J3=6.2 Hz).",
"Key: 1 NMR Example 2 Ethyl 4-(4-amino-3-nitrophenyl)-4-oxobutanoate The N-acetylated derivative (2.00 g, 7.51 mmol) is mixed with concentrated HCl (10 mL) and heated to reflux for 15 min.",
"Then, EtOH (50 mL) is added, and it is stirred at reflux for 12 h. It is then allowed to cool, and the solvents are evaporated to the maximum extent.",
"The residue is extracted with ethyl acetate, and the pH is brought to 8-9 by addition of KHCO3.The organic phase is dried, and the solvents are evaporated.",
"Finally, the crude product is purified by silica gel column chromatography (eluent: AcOEt/Hexane 1/3).",
"orange oil MM (C12H14N2O5): 266.25 g mass: 1.36 g Yield: 68% {circle over (1)}RMN (1H, 300 MHz, CDCl3): 8.81(d, 1H, CHarom., J4=2.0 Hz); 8.02(dd, 1H, CHarom., J3=8.8 Hz, J4=2.0 Hz); 6.86(d, 1H, CHarom., J3=8.8 Hz); 4.17(q, 2H, —CH2-, J3=7.1 Hz); 3.26(t, 2H, —CH2—, J3=6.5 Hz); 2.77(t, 2H, —CH2—, J3=6.5 Hz); 1.27(t, 3H, —CH3, J3=7.1 Hz).",
"Key: 1 NMR Example 3 Ethyl 4-(3,4-diaminophenyl)-4-oxobutanoate The nitrated derivative (1.31 g, 4.9 mmol) is dissolved in 95% ethanol (30 mL).",
"SnCl2, 2H2O (4.4 g, 19.6 mmol) is added and heated to reflux for 2 h. The solvents are evaporated, and the residue is extracted with ethyl acetate; then, the pH is brought to 7 by addition of KHCO3.The precipitate of tin salts which forms is filtered, and the organic phase is dried using Na2SO4 and evaporated to dryness.",
"Finally, the purification of the crude product is done by silica gel column chromatography (eluent: AcOEt/Hexane 1/1).",
"Thick oil MM (C12H16N2O3): 236.01 g mass: 700 mg Yield: 60% {circle over (1)}RMN (1H, 300 MHz, CDCl3): 7.46-7.38(m, 2H, 2CHarom.,); 6.68(d, 1H, CHarom., J3=8.1 Hz); 4.16(q, 2H, —OCH2—); 3.89(large{circle over (2)}s, 2H, —NH2); 33.37(large{circle over (2)}s, 2H, —NH2); 3.22(t, 2H, —CH2—, J3=6.8 Hz); 2.72(t, 2H, —HC2—, J3=6.8 Hz); 1.27(t, 3H, —CH3).",
"Key: 1 NMR 2 Broad Example 4 Ethyl-4-oxo-4-(2-phenylquinoxalin-6-yl) butanoate In a round-bottomed flask, the diamino ester is dissolved in EtOH (10 mL).",
"Phenylglyoxal, hydrate (130 mg, 0.85 mmol) is added and heated to reflux for 5 h. It is allowed to cool, and the precipitate which forms is filtered.",
"yellow powder MM (C20H18N2O3): 334.37 g mass: 233 mg Yield: 82% {circle over (1)}RMN (1H, 200 MHz, CDCl3): 9.43(s, 2H, CHquinox.",
"); 8.74(d, 1H, CHarom., J4=1.9 Hz); 8.36(dd, 1H, CHarom., J3=8.9 Hz, J4=1.9 Hz); 8.26-8.19(m, 4H, 4CHarom.,; 7.60-7.58(m, 3H, 3CHarom.",
"); 4.19(q, 2H, —CH2—, J3=7.1 Hz); 3.50(t, 2H, —CH2—, J3=6.6 Hz); 2.86(t, 2H, —CH2—, J3=6.6 Hz); 1.30(t, 3H, —CH3, J3=7.1 Hz).",
"Key: 1 NMR E) Syntheses of Enantiomerically Pure Compounds The present invention also describes the enantioselective synthesis of the (R) and (S) acid sodium salts of γ-benzyl-γ-hydroxybutanoic acid.",
"These syntheses involve the L or D glutamic acid as illustrated in the diagram of FIG.",
"4.For this, optically pure (R) and (S) acid chlorides were prepared according to processes described in the literature (ref.",
"1: M. Larcheveque et al., Bull.",
"Soc.",
"Chim.",
"Fr.",
"1987,116-122; G. Eguchi et al., Bull.",
"Chim.",
"Soc.",
"Jpn., 1974, 47, 1704-1708).",
"These compounds were then treated with tetraphenyltin in the presence of a catalyst in HMPT (ref.",
"2: J. W. Labadie et al., J.A.C.S., 1983, 105, 6129).",
"The (R) and (S) γ-benzoyl-γ-butyrolactones are then obtained, which are reduced by catalytic hydrogenation (process 4), giving the two optically pure enantiomers (compounds VI′ (R) and VI″ (S).",
"These are salified as described in the preceding, leading to the corresponding (R) and (S) sodium salts.",
"F) Characterization of the Compounds Which are Obtained The following table gives the main physicochemical characteristics of specific compounds belonging to the families with general formula I, I′ or I″ as synthesized in practice and which are explicitly claimed in the present invention.",
"Of course, the corresponding acid compounds with general formula I or I′ in which W is COOH, which enable one to obtain the sodium salts, are also considered to be part of this table and of the explicitly claimed compounds.",
"TABLE 2 Main physicochemical characteristics of the compounds which are obtained E A C D Masse F N° du B N° du formule molaire RMN “1H”, 200 ou 300MHz, solvant composé Nom Procédé brute g/mol ou microanalyses 1 Sel de sodium de l'acide 1 C10H10O3Na 201.18 Microanalyses 4-hydroxy-4- C % H % phénylbutanoïque Th.",
"59.70 5.01 G Pr.",
"59.41 5.14 2 Sel de sodium de l'acide 1 C10H9Cl2O3Na 271.08 1H, 200MHz, D2O: 7.35-7.30(m, 2H, 4-(3,4 dichlorophényl)- 2CHarom.",
"); 7.11-7.06(m, 1H, CHarom.",
"); 4-hydroxybutanoïque 4.50(t, 1H, CH—O—, J3=6.6Hz); 2.08-2.00(m, 2H, —CH2—); 1.88-1.75(m, 2H, —CH2—) 3 Sel de sodium de l'acide 1 C12H15O5Na 262.23 1H, 200MHz, D2O: 6.97(m, 3H, 4-(3,4- CHarom.",
"); 4.59(t, 1H, CH—O—, diméthoxylphényl)-4- J3=8.3Hz); 3.82(s, 3H, —O—CH3—); 3.80(s, hydroxybutanoïque 3H, —O—CH3—); 2.16-1.89(m, 4H, —CH2—CH2—) 4 Sel de sodium de l'acide 1 C15H16NO5SNa 345.37 1H, 300MHz, D2O: 7.63(d, 2H, 2CHarom., 4-hydroxy-4-(1-tosyl- J3=7.7Hz); 7.22(d, 2H, 2CHarom., 1H-pyrrol-3-yl)- J3=7.7Hz); 7.13(s, 1H, CHpyrrol.",
"); butanoïque 6.29(s, 2H, 2 CHpyrrol.",
"); 4.47(t, 1H, CH—O—, J3=6.4Hz); 2.22(s, 3H, —CH3—); 2.03-1.82(2m, 4H, —CH2—CH2) 5 Sel de sodium de l'acide 7 C9H10O3Na 189.17 1H, 200MHz, D2O: 8.36 J (large s, 2H, 4-hydroxy-4-pyridin-4- 2CHarom.",
"); 7.33 J (large s, 2H, 2CHarom.",
"); yl-butanoïque 4.75(m, 1H CH—O); 2.13-1.89(m, 4H, —CH2—CH2) 6 Sel de sodium de l'acide 7 C9H10O3Na 189.17 1H, 200MHz, D2O: 8.34(m, 2H, 2CHarom.",
"); 4-hydroxy-4-pyridin-3- 7.74(d, 1H, CHarom., J3=8.0Hz); 7.33(dd, yl-butanoïque 1H, CHarom., J3=4.9 M et 8.0Hz); 4.65(t, 1H, CH—O—, J3=6.3Hz); 2.15-1.89(m, 4H, —CH2—CH2) 7 Sel de sodium de l'acide 1 C8H9NaO3S 208.22 1H, 300MHz, D2O: 7.34-7.32(m, 1H, 4-(2-thiényl)-4- CHthinyl.",
"); 7.01-6.69(m, 2H, hydroxybutanoïque 2CHthinyl.",
"); 4.91(t, 1H, CH—O—, J3=6.8Hz); 2.26-1.99(m, 4H, —CH2—CH2) 8 Sel de sodium de l'acide 1 C12H14NO4Na 259.24 1H, 200MHz, D2O: 7.24(m, 4H, 4-[4- 4CHarom.",
"); 4.51(t, 1H, J3=6.8Hz, CH—O); acétylamino)phényl]-4- 1.84-2.07(m, 4H —CH2—CH2); 1.93(s, 3H, —CH3) hydroxybutanoïque 9 Sel de sodium de l'acide 1 C11H14NO5Na 263.23 1H, 300MHz, D2O: 7.00(s, 1H, 4-[5-(éthoxycarbonyl)-1H-pyrrol-3- CHpyrrol.",
"); 6.90(s, 1H, CHpyrrol.",
"); 4.58(t, yl]-4-hydroxybutanoïque 1H, CH—O—, J3=6.2Hz); 4.20(q, 2H, —CH2—, J3=7.2Hz); 2.14-1.92(2m, 4H, —CH2—CH2); 1.25(t, 3H, —CH3, J3=7.2Hz) 10 Sel de sodium de l'acide 1 C12H13O4Na 244.22 1H, 300MHz, D2O: 7.17(s, 1H, CHarom.",
"); 4-(2,3-dihydro-1- 7.01(d, 1H, CHarom., J3=8.1Hz); 6.69(d, benzofuran-5-yl)-4- 1H, CHarom., J3=8.1Hz); 4.47(m, 3H, —CH2 hydroxybutanoïque et CH—O); 3.09(t, 2H, —CH2—, J3=8.7Hz); 2.08-1.78(m, 4H, —CH2—CH2) 11 Sel de sodium de l'acide 1 C12H13O5Na 260.92 1H, 300MHz, D2O: 6.82 J (large s, 3H, 4-(2,3-dihydro-1,4- 3CHarom.",
"); 4.48(t, 1H, —CH—0—, benzodioxin-6-yl)-4- J3=8.2Hz); 4.18(s, 4H, O—CH2—CH2—O); hydroxybutanoïque 2.15-1.78(m, 4H, —CH2—CH2—) 12 Sel de sodium de l'acide 1 C14H16NO4Na 285.28 1H, 200MHz, D2O): 7.82(d, 1H, CHarom., 4-(1-acétyl-2,3-dihydro- J3=8.6Hz); 7.16(s, 1H, CHarom.",
"); 7.08(d, 1H-indol-5-yl)-4- 1H, CHarom., J3=8.6Hz); 4.52(t, 1H, —CH—O, hydroxybutanoïque J3=6.4Hz); 3.93(t, 2H, —CH2, J3=8.2Hz); 3.02(t, 2H, —CH2, J3=8.2Hz); 2.07(s, 3H, —CH3); 2.04-1.88(2m, 4H, —CH2—CH2—) 13 Sel de sodium de l'acide 1 C14H13O3Na 252.14 1H, 300MHz, D2O: 8.10(d, 1H, 4-hydroxy-4-(1- CHarom.",
"); 7.89-7.78(m, 2H, 2CHarom.",
"); naphthyl)-butanoïque 7.58-7.44(m, 4H, 4CHarom.",
"); 5.44(t, 1H, —CH—O, J3=5.8Hz); 2.24-1.98(m, 4H, —CH2—CH2—) 14 Sel de sodium de l'acide 1 C14H13O3Na 252.14 1H, 300MHz, D2O: 7.84-7.82(m, 3H, 4-hydroxy-4-(2- 3CHarom.",
"); 7.75(s, 1H, CHarom.",
"); 7.48-7.42(m, naphthyl)-butanoïque 3H, 3CHarom.",
"); 2.15-1.94(m, 4H, —CH2—CH2—) 15 Sel de sodium de l'acide 1 C13H15O3Na 242.25 1H, 300MHz, D2O: 7.17(s, 1H, CHarom.",
"); 4-(2,3-dihydro-1H- 7.15(s, 1H, CHarom.",
"); 7.15(d, 1H, inden-5-yl)-4-hydroxy- CHarom., J3=7.6Hz); 7.05(d, 1H, butanoïque CHarom., J3=7.6Hz); 4.54(t, 1H, CH—O, J3=6.5Hz); 2.80-2.73(m, 4H, 2—CH2); 2.14-1.82(m, 3H, 3—CH2) 16 Sel de sodium de l'acide 1 C15H16NO5Na 313.28 1H, 200MHz, D2O: 7.56(s, 1H, CHarom.",
"); 4-[2-(éthoxycarbonyl)- 7.32-7.36(m, 2H, 2CHarom.",
"); 7.05(s, 1H, 1H-indol-5-yl]-4- CHindol.",
"); 4.71(m, 1H, —CH—O); 4.20(q, hydroxybutanoïque 2H, —CH2—); 2.20-2.10(m, 4H, —CH2—CH2—); 1.3(t, 3H, —CH3—) 17 Sel de sodium de l'acide 1 C12H12NO3Na 241.22 1H, 200MHz, D2O: 7.55(s, 1H, CHarom.",
"); 4-hydroxy-4-(1H-indol- 7.43(d, 1H, CHarom., J3=8.4Hz); 7.30(d, 5-yl)-butanoïque 1H, CHindol., J3=2.9Hz); 7.13(d, 1H, CHarom., J3=8.4Hz); 6.48(d, 1H, CHindol., J3=2.9Hz); 4.70 K (en dessous du pic de l'eau, 1H, CH—O); 2.17-1.91(m, 4H, —CH2—CH2—) 18 Sel de sodium de l'acide 1 C12H11SONa 258.28 1H, 300MHz, D2O: 7.89-7.84(m, 2H, 4-(1-benzothiophène-2- 2CHarom.",
"); 7.44(s, 1H, CHthio.",
"); 7.40-7.30(m, yl)-4-hydroxybutanoïque 2H, 2CHarom.",
"); 5.01(t, 1H, —CH—O, J3=4.0Hz); 2.15(m, 4H, —CH2—CH2—) 19 Sel de sodium de l'acide H C13H12NO4Na 269.23 1H, 300MHz, D2O: 8.08(d, 1H, CHarom., 4-hydroxy-4-(4-oxo- cas J3=8.1Hz); 8.00(d, 1H, CHarom., J3=7.2Hz); 1,4-dihydroquinolin-7- parti- 7.54(s, 1H, CHarom.",
"); 7.41(d, yl)-butanoïque culier 1H, CHarom., J3=8.1Hz); 6.37(d, 1H, CHarom., J3=7.2Hz); 4.80(t, —CH—O, J3=6.4Hz); 2.15(t, 2H, —CH2, J3=7.2Hz); 1.99(t, 2H, —CH2, J3=7.2Hz) 20 Sel de sodium de l'acide H C13H12NO4Na 269.23 1H, 300MHz, D2O: 7.93(d, 1H, CHarom., 4-hydroxy-4-(4-oxo- cas J4=1.9Hz); 7.86(d, 1H, CHvinyl., J3=7.2Hz); 1,4-dihydroquinolin-6- parti- 7.61(dd, 1H, CHarom., J3=8.8Hz, yl)-butanoïque culier J4=1.9Hz); 7.45(d, 1H, CHarom., J3=8.8Hz); 6.27(d, 1H, CHvinyl., J3=7.2Hz); 4.75(t, 1H, —CH—O, J3=3.7Hz); 2.15(t, 2H, —CH2, J3=7.2Hz); 2.17-1.94(2m, 4H, —CH2—CH2—) 21 Sel de sodium de l'acide 2 C12H13N2O3Na 256.23 1H, 200MHz, D2O: 8.08(d, 1H, CHarom., 4-hydroxy-4-(7-méthyl- J3=6.6Hz); 7.37(s, 1H, CHimidaz.",
"); 7.19(s, imidazo[1,2-a]pyridin- 1H, CHarom.",
"); 6.72(d, 1H, CHarom., 3-yl)-butanoïque J3=6.6Hz); 4.93(m, 1H, —CH—O); 2.21(s, 3H, —CH3); 2.16-2.27(m, 4H, —CH2—CH2—) 22 Sel de sodium de l'acide 1 C12H12NO4Na 257.28 1H, 200MHz, D2O: 12.31(s, 1H, NH); 4-hydroxy-4-(2-oxo- 7.16(s, 1H, CHarom.",
"); 7.08(d, 1H, 2,3-dihydro-1H-indol-5- CHarom., J3=8.8Hz); 6.74(d, 1H, yl)-butanoïque CHarom., J3=8.8Hz); 4.47(s, 1H, CHO); 3.44(s, 2H, CH2); 2.21 et 1.79(2s, 4H, 2CH2) 23 Sel de sodium de l'acide 1 C15H17N2O4Na 312.30 1H, 300MHz, D2O): 7.60(s, 1H, CHarom.",
"); 4-{2- 7.28-,7.42(m, 2H, 2CHarom.",
"); 7.05(s, [(diméthylamino)carbon 1H, CHindol.",
"); 3.35(m, 4H, —CH2—CH2—); yl]-1H-indol-5-yl}-4- 2.50(s, 6H, 2—CH3) hydroxybutanoïque 24 Sel de sodium de l'acide 1 C16H15O3Na 278.29 1H, 200MHz, D2O: 7.68(d, 1H, CHarom., 4-(1,2- J3=8.6Hz); 7.46-7.35(m, 2H, 2CHarom.",
"); dihydroacénaphthylen-4-yl)-4- 7.20(m, 2H, 2CHarom.",
"); 5.23(t, 1H, hydroxybutanoïque —CH—O, J3=6.1Hz); 3.17(t, 4H, —CH2—CH2—, J3=7.6Hz); 2.16-2.00(m, 4H, —CH2—CH2—) 25 Sel de sodium de l'acide 1 C16H13O3SNa 308.34 1H, 200MHz, D2O: 8.10-8.00(m, 2H, 4-dibenzo[b,d]thiophène- 2CHarom.",
"); 7.98-7.75(m, 2H, 2-yl-4- 2CHarom.",
"); 7.40-7.30(m, 3H, hydroxybutanoïque 3CHarom.",
"); 4.74(m, 1H, —CH—O); 2.15-2.02(m, 4H, —CH2—CH2—) 26 Sel de sodium de l'acide 1 C16H13O4Na 292.27 1H, 300MHz, DMSO: 8.04(m, 2H, 4-dibenzo[b,d]furan-2- 2CHarom.",
"); 7.70-7.35(m, 5H, 5CHarom.",
"); yl-4-hydroxybutanoïque 4.78(m, 1H, —CH—O); 2.13(2H, —CH2); 1.84(m, 2H, —CH2—) 27 Sel de sodium de l'acide 1 C18H16O4NNa 333.33 1H, 300MHz, D2O: 7.57-7.01(m, 7H, 4-(9-acétyl-9H- 7CHarom.",
"); 4.64(m, 1H, —CH—O); 2.30(s, carbazol-3-yl-4- 3H, —CH3); 2.20-1.82(2m, 4H, —CH2—CH2—) hydroxybutanoïque 28 Sel de sodium de l'acide 1 C16H13O4SNa 324.34 1H, 200MHz, D2O: 7.06-6.83(m, 7H, 4-hydroxy-4- 7CHarom.",
"); 4.43(m, 1H, —CH—O); (phénoxathiin-2 ou 3- 2.01-1.77(2m, 4H, —CH2—CH2—) yl)butanoïque isomère A 29 Sel de sodium de l'acide 1 C16H13O4SNa 324.34 1H, 300MHz, D2O: 7.77-7.63(m, 4H, 4-hydroxy-4- 4CHarom.",
"); 738-7.32(m, 3H, 3CHarom.",
"); (phénoxathiin-2 ou 3- 4.62(m, 1H, —CHO); 2.23-1.96(2m, 4H, yl)butanoïque —CH2—CH2—) isomère B 30 Sel de sodium de C20H20NO4Na 361.38 1H, 200MHz, D2O: 7.32-7.16(m, 7H, l'acide 4-(5-acétyl- 7CHarom.",
"); 7.51(m, 1H, —CH—O—); 10,11-dihydro-5H- 3.27-3.07(m, 2H, —CH2—); 2.78-2.64(m, dibenzo[b,f]azépin-3- 2H, —CH2—); 1.97-1.76(m, 7H, yl)-4-hydroxybutanoïque —CH2—CH2— et —CH3) 31 Sel de sodium de l'acide 1 C17H15O4Na 306.30 1H, 200MHz, D2O: 7.16-6.91(m, 7H, 4-hydroxy-4-(9H- 7CHarom.",
"); 4.58(m, 1H, —CH—O); 3.86(s, xanthen-2- 2H, —CH2); 2.11-1.97(2m, 4H, —CH2—CH2) yl)butanoïque 32 Sel de sodium de l'acide 1 C17H15O3Na 290.30 1H, 300MHz, D2O: 7.65(m, 2H, 4-(9H-fluoren-3- 2CHarom.",
"); 7.45(m, 2H, 2CHarom.",
"); yl)hydroxybutanoïque 7.29-7.23(m, 3H, 3CHarom.",
"); 4.62(m, 1H, —CH—O); 3.37(s, 2H, —CH2—); 2.17-1.91(2m, 4H, —CH2—CH2—) 33 Sels de sodium des 1 C14H12N3O3Na 293.25 1H, 300MHz, D2O: 8.06(s, 1H, CHarom.",
"acides 4-hydroxy-4-(4- L des 2 isomères); 7.50(s, 1H, CHarom.",
"L des 2 oxo-4,5-dihydro-3H- isomères); 7.39(d, 1H, CHarom.",
"iso.",
"6, pyridazo(4,5-b)indol-8 J3=7.6Hz); j 7.28(2d, 1H, CHarom.",
"iso.",
"8 et 6-yl)butanoïque M et CHarom.",
"iso.",
"6, J3=8.7Hz); 7.16(d, 1H, mélange isomère 8/6 CHarom.",
"iso.",
"8, J3=8.7Hz); 7.06(t, 1H, CHarom.",
"iso.",
"6, J3=7.6Hz); 4.90(m, 1H, CH—O— iso.",
"6); 4.66(m, 1H, CH—O— iso.",
"8); 2.29-1.95(2m, 4H, —CH2—CH2— L des 2 isomères) 34 N,4-dihydroxy-4- H C10H13NO3 195.22 1H, 300MHz, DMSO: 7.41-7.21(m, 5H, phénylbutanamide cas 5CHarom.",
"); 4.55(m, 1H, —CH—O); parti- 2.04-1.73(2m, 4H, —CH2—CH2—) culier 35 Sel de sodium de l'acide 4 C11H13NaO3 216.21 Microanalyses: 4-hydroxy-5- C % H % phénylpentanoïque.",
"Th.",
"56.40 6.45 G Pr.",
"56.94 6.15 36 Sel de sodium de l'acide I C11H13NaO3 216.21 Microanalyses: 4(R)-hydroxy-5- énantio- C % H % phénylpentanoïque.",
"mère Th.",
"56.40 6.45 pur G Pr.",
"56.50 6.39 37 Sel de sodium de l'acide I C11H13NaO3 216.21 Microanalyses: 4(S)-hydroxy-5- énantio- C % H % phénylpentanoïque.",
"mère Th.",
"56.40 6.45 pur G Pr.",
"56.47 6.44 38 Sel de sodium de l'acide 8 C11H11Cl2NaO3 285.10 Microanalyses: 5-(3,4-dichlorophényl)- C % H % 4-hydroxypentanoïque.",
"Th.",
"46.34 3.89 G Pr.",
"46.12 3.90 39 Sel de sodium de l'acide 3 C11H13O3Na 216.21 1H, 200MHz, D2O: 7.20(m, 5H, 5-hydroxy-5- 5CHarom.",
"); 4.49(t, 1H, CH—O); 2.04(t, phénylpentanoïque 2H, CH2COO, J3=6.5Hz); 1.36-1.63(m, 4H, CH2—CH2) 40 Sel de sodium de l'acide 5 C15H16NO4Na 297.26 1H, 200MHz, D2O: 7.30(s, 1H, CHarom.",
"); 4-(3,3-diméthyl-2-oxo- 7.13(d, 1H, CHarom., J3=7.9Hz); 6.84(d, 1,2,3,4- 1H, CHarom., J3=7.9Hz); 6.55(m, 1H, tétrahydroquinolin-6- CH═C; 5.95(d, 1H, CH═C, J3=10.8Hz); yl)-4-hydroxybutanoïque 5.24(d, 1H, CH—O, J3=3.3Hz); 2.35(s, 2H, CH2); 1.17(s, 6H, 2CH3) 41 Sel de sodium de l'acide 6 C10H8O3CINa 234.60 Microanalyses: (E)-4-(4-chlorophényl)- C % H % 4-hydroxybut-2-ènoïque Th.",
"56.48 4.27 Pr.",
"56.37 4.02 42 Sel de sodium de l'acide 6 C12H12NO4Na 257.23 1H, 200MHz, D2O: 720(m, 4H, (E)-4-(4- CHarom.",
"); 6.56(dd, 1H, JAB=15.6Hz; acétylaminophényl)-4- JAX=5.6Hz); 5.94(d, 1H, JAB=15.6Hz); hydroxybut-2-ènoïque 5.16(d, JAX=5.6Hz, CH—O); 1.95(s, 3H, CH3CO) Key: A Compound No.",
"B Name C Process No.",
"D Total formula E Molar mass g/mol F “1H” NMR, 200 or 300 MHz, solvent or microanalyses G Actual H Particular case I Pure enantiomer J Broad K Above the peak of water L Of the 2 isomers M And 1 Sodium salt of 4-hydroxy-4-phenylbutanoic acid 2 Sodium salt of 4-(3,4 dichlorophenyl)-4-hydroxybutanoic acid 3 Sodium salt of 4-(3,4-dimethoxyphenyl)-4-hydroxybutanoic acid 4 Sodium salt of 4-hydroxy-4-(1-tosyl-1H-pyrrol-3-yl)butanoic acid 5 Sodium salt of 4-hydroxy-4-pyridin-4-ylbutanoic acid 6 Sodium salt of 4-hydroxy-4-pyridin-3-ylbutanoic acid 7 Sodium salt of 4-(2-thienyl)-4-hydroxybutanoic acid 8 Sodium salt of 4-[4-(acetylamino)phenyl]-4-hydroxybutanoic acid 9 Sodium salt of 4-[5-(ethoxycarbonyl)-1H-pyrrol-3-yl]-4-hydroxybutanoic acid 10 Sodium salt of 4-(2,3-dihydro-1-benzofuran-5-yl)-4-hydroxybutanoic acid 11 Sodium salt of 4-(2,3-dihydro-1,4-benzodioxin-6-yl)-4-hydroxybutanoic acid 12 Sodium salt of 4-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-4-hydroxybutanoic acid 13 Sodium salt of 4-hydroxy-4-(1-naphthyl)butanoic acid 14 Sodium salt of 4-hydroxy-4-(2-naphthyl)butanoic acid 15 Sodium salt of 4-(2,3-dihydro-1H-inden-5-yl)-4-hydroxybutanoic acid 16 Sodium salt of 4-[2-(ethoxycarbonyl)-1H-indol-5-yl]-4-hydroxybutanoic acid 17 Sodium salt of 4-hydroxy-4-(1H-indol-5-yl)butanoic acid 18 Sodium salt of 4-(1-benzothiphen-2-yl)-4-hydroxybutanoic acid 19 Sodium salt of 4-hydroxy-4-(4-oxo-1,4-dihydroquinolin-7-yl)butanoic acid 20 Sodium salt of 4-hydroxy-4-(4-oxo-1,4-dihydroquinolin-6-yl)butanoic acid 21 Sodium salt of 4-hydroxy-4-(7-methylimidazo[1,2-a]pyridin-3-yl)butanoic acid 22 Sodium salt of 4-hydroxy-4-(2-oxo-2,3-dihydro-1H-indol-5-yl)butanoic acid 23 Sodium salt of 4-{2-[(dimethylamino)carbonyl]-1H-indol-5-yl}-4-hydroxybutanoic acid 24 Sodium salt of 4-(1,2-dihydroacenaphthylen-4-yl)-4-hydroxybutanoic acid 25 Sodium salt of 4-dibenzo[b,d]thiophen-2-yl-4-hydroxybutanoic acid 26 Sodium salt of 4-dibenzo[b,d]furan-2-yl-4-hydroxybutanoic acid 27 Sodium salt of 4-(9-acetyl-9H-carbazol-3-yl-4-hydroxybutanoic acid 28 Sodium salt of 4-hydroxy-4-(phenoxathiin-2 or 3-yl)butanoic acid isomer A 29 Sodium salt of 4-hydroxy-4-(phenoxathiin-2 or 3-yl) butanoic acid isomer B 30 Sodium salt of 4-(5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)-4-hydroxybutanoic acid 31 Sodium salt of 4-hydroxy-4-(9H-xanthen-2-yl)butanoic acid 32 Sodium salt of 4-(9H-fluoren-3-yl)hydroxybutanoic acid 33 Sodium salts of 4-hydroxy-4-(4-oxo-4,5-dihydro-3H-pyridazo(4,5-b)indol-8 and 6-yl)butanoic acids 8/6 isomer mixture 34 N,4-dihydroxy-4-phenylbutanamide 35 Sodium salt of 4-hydroxy-5-phenylpentanoic acid 36 Sodium salt of 4(R)-hydroxy-5-phenylpentanoic acid 37 Sodium salt of 4(S)-hydroxy-5-phenylpentanoic acid 38 Sodium salt of 5-(3,4-dichlorophenyl)-4-hydroxypentanoic acid 39 Sodium salt of 5-hydroxy-5-phenylpentanoic acid 40 Sodium salt of 4-(3,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-4-hydroxybutanoic acid 41 Sodium salt of (E)-4-(4-chlorophenyl)-4-hydroxybut-2-enoic acid 42 Sodium salt of (E)-4-(4-acetylaminophenyl)-4-hydroxybut-2-enoic acid G) Tests of Therapeutic Activity 1) In Vitro Tests Approximately forty compounds according to the invention were synthesized and tested in experiments of binding on the brains of rats using 3H-GHB as radioligand and according to the experimental protocol described hereafter.",
"For the displacement studies, the reference ligand used is tritiated GHB (100 Ci/mmol, CEA, Saclay).",
"The receptors studied come from membranes of brains of Wistar rats raised in the laboratory.",
"The animals are sacrificed quickly by decapitation, and the brains are removed excluding the cerebellum and the brain stem.",
"The brains are then homogenized in 10 volumes (weight/volume) of 0.32M sucrose containing 5 mM EDTA (pH 6.0).",
"After a first centrifugation at 800 G intended for eliminating the cellular debris and the nuclei, the supernatant is centrifuged at 16,000 G so as to eliminate the P2 residue (synaptosomes+mitochondria).",
"This residue is then homogenized in a suitable apparatus, for example, of the type known by the term “polytron,” in 70 volumes of distilled water at 0° C. containing 5 mM EDTA.",
"After centrifugation at 20,000 G (4° C., 20 min), the residue obtained is washed with the same medium supplemented with 0.5% CHAPS (3-[3-cholamidopropyldimethylammonio]-1-propane sulfonate).",
"After another centrifugation at 20,000 G, the membranes obtained are resuspended in a potassium phosphate buffer with pH=6.0.After recentrifugation, the residue is stored at −80° C. for use the next day.",
"The incubation of the membranes with the radioactive GHB and the various compounds at variable concentrations (between 10−9 and 10−4 M) is done in a potassium phosphate buffer also with pH=6.0 for a duration of 30 min in ice.",
"After this incubation, the separation of the bound 3H-GHB from the free 3H-GHB is done by filtration under suction with filters made of glass fiber (Whatmann GF/B).",
"The membranes retained by the filter are quickly washed under suction three times with 1 mL of incubation buffer maintained at 0° C. The filters are then “counted” by liquid scintillation in a counter, in the presence of a scintillation liquid.",
"The results are expressed in percentage of total reversible binding determined in the presence of an excess of nonradioactive GHB (500 μM, 60-80% total binding).",
"The statistical analyses of the IC50 values (concentration of synthetic analogues capable of displacing 50% of the reversible binding of the 3H-GHB) are summarized in the table hereafter.",
"The lower the IC50 value, the greater the affinity of the ligand for the receptor.",
"The statistical analyses of the displacement curves are carried out using the GraphPad Prism software (San Diego, Calif.).",
"The results obtained are gathered in the following table: TABLE 3 In vitro results {circle over (1)} N° du {circle over (2)} composé CI50 μM GHB 5.60 1 6.80 2 0.30 3 0.30 4 1.60 5 3.90 6 2.50 7 1.80 8 0.80 9 2.30 10 0.60 11 0.90 12 0.60 13 0.10 14 0.30 15 0.70 16 0.20 17 1.40 18 0.10 19 1.10 20 24.7 21 16.2 22 1.20 23 0.20 24 0.08 25 0.1 26 0.30 27 0.20 28 0.10 29 0.90 30 0.50 31 0.10 32 0.30 33 0.20 34 23.5 35 2.30 36 1.80 37 25.0 38 0.80 39 34.2 40 1.10 41 3.80 42 0.60 Key: {circle over (1)} Compound No.",
"{circle over (2)} IC50 μM As can be observed, certain compounds among those synthesized are approximately ten times more active than GHB and therefore have improved sedative properties.",
"The compounds of the present invention are therefore of particular value with regard to their use for obtaining a drug intended for the treatment of neurological or mental disorders in which the central nervous system plays a part.",
"This pertains in particular to disorders in which the GHB receptors are involved and which can benefit from the effects of an agonist or an antagonist of the GHB receptors: regulation of sleep and secretion of hormones, in particular of growth hormones, reduction of anxiety or increased alertness, antiepileptic activity, regulation of weight and food intake, regulation of mood or antidepressive activity, neuroleptic activity, regulation of circadian rhythm, hypnotic or anesthetic activity, neuroprotective or anti-ischemic activity, activity in the process of drug withdrawal and in addiction.",
"These drugs are characterized by the fact that they contain, as active ingredient, at least one compound with general formula I, I′ or I″.",
"Preferabley, the aforementioned compound(s) used as active ingredient is(are) one or more sodium salts with general formula I″ obtained by neutralization of a compound with general formula I or I′ containing an acid function for the group W. 2) In Vivo Trials: Electroencephalographic Study (EEG) of Rats Who Received 2-4 mmol/kg of GHB Synthesis Analogues Male Wistar rats, weighing 200 g at the beginning of the experiment, coming from the Centre d'Elevage Janvier (Route des Chenes-Secs, Le Genest St-Isle, 53940 France) were used for this study.",
"After eight days of adaptation to the breeding conditions at the Animalerie Centrale de la Faculte de Medecine (11, rue Humann 67084 Strasbourg), these animals were placed in individual cages (Makrolon type cages 3H, 425×266×150) in a standard (7 a.m./7 p.m.) day/night rhythm, with water and food (UAR ref.",
"A04) continually available to them.",
"The animals were then transferred to an experimentation facility on supports capable of receiving 24 cages.",
"a) Implantation of Frontoparietal Cortical Electrodes The surgical procedure is the following: after one month of becoming accustomed to the (10 a.m./10 p.m.) day/night cycle, the rats are weighed and then anesthetized with ketamine (Imalgene 500 Merial) at a dose of 150 mg/kg i.p.",
"After having placed the animal in a stereotactic frame (Narishige), a rostrocaudal incision is made using a sterilized scalpel (No.",
"3 blade, Swann-Morton England).",
"The parietal bone sutures, Lambda and Bregma, are exposed serve as stereotactic reference (point 0).",
"After having perforated the cranial casing using a dental drill without infringing on the meninges (Minitor Narishige), two stainless steel screws 500 μm in diameter (Magister, 4 rue du Lac 25130 Villers Le Lac) are implanted at the following coordinates: Bregma AP: ±4 mm and ML: ±3 mm.",
"Two copper wires are soldered with tin to the screws, on one hand, and to the female connector, on the other hand (VP Electronic, Square de la Poteme, 91302 Massy Cedex).",
"The assembly which is realized is then covered with a polymerizing resin (Paladur, Kulzer, Germany).",
"The implanted rats are then placed in their respective cages.",
"A post-operative period greater than 48 h is complied with before any recording.",
"b) EEG Recording All the EEG recordings are made during the first hours of the dark [sic; light] phase (that is, between 10 a.m. and 1 p.m.), which represents the period of awakening and intense activity of the animals.",
"The rats are placed in a cage made of Plexiglas (170×170×300), and after a 30 min period of becoming accustomed to their new environment, they are recorded continuously for a duration of 3 h after i.p.",
"injection (2 mL/kg) of 0.9% NaCl or the ligand to be studied.",
"The EEG graph is made using an 8-track recorder (Alvar Electronic, 6 rue du Progres, 93511-Montreuil) with a running speed of 0.5 cm/s.",
"c) Calculation of the Duration of Slow Wave Sleep (SWS) The total durations of slow wave sleep for each animal are evaluated in 20 min sections for the total duration of the 3 h recording (1 cm=2 seconds).",
"d) Statistical Analysis and Graphic Representation The statistical comparison is made using an analysis of variance test (Anova) followed by a multiple comparison test.",
"The animals (6 to 8/group/dose) are recorded both on 0.9% NaCl (reference value) and after administration of the product to be studied in the amount of 2 mmol/kg.",
"The results are represented by expressing the means ±SEM of the total durations in min of slow wave sleep in 20 min time sections with respect to the affinity of the ligand for the GHB receptor (IC50) in the table hereafter.",
"The numbers in parentheses correspond to the percentages of slow wave sleep with respect to the total duration of sleep.",
"TABLE 4 In vivo results Augmentation N° du Dosage Durée de de la durée du Composé CI50 utilisé sommeil lent sommeil lent testé μM mmole/kg i.p.",
"profond (mn) profond (mn) {circle over (1)} {circle over (2)} {circle over (3)} {circle over (4)} {circle over (5)} NaCl — — 11 ± 4 (7%) — 2 0.30 0.28 53 ± 7 42 (23%) 24 0.08 0.28 61 ± 8 50 (28%) 24 0.08 0.15 53 ± 7 42 (23%) Key: {circle over (1)} No.",
"of the tested compound {circle over (2)} IC50 μM {circle over (3)} Dosage used mmol/kg i.p.",
"{circle over (4)} Duration of slow wave sleep (min) {circle over (5)} Increase of the duration of slow wave sleep (min) As can be observed, the compounds according to the invention make possible a significant increase of the duration of slow wave sleep.",
"Consequently, the present invention also relates to a pharmaceutical composition containing, as active ingredient, at least one compound with general formula I, I′ or a salt with general formula I″.",
"The claimed pharmaceutical compositions moreover contain other pharmaceutically acceptable excipients or vehicles.",
"The present invention makes possible the use of a compound according to said invention for obtaining a drug containing, as active ingredient, at least one compound with general formula I, I′ or I″ for the treatment of a disease which can be treated by administration of an agonist or antagonist of GHB receptors, in particular, neurological or mental disorders of the central nervous system and, in particular, regulation of sleep and of secretion of hormones, in particular, growth hormones, reduction of anxiety or increased alertness, antiepileptic activity, regulation of weight and food intake, regulation of mood or antidepressive activity, neuroleptic activity, regulation of circadian rhythm, hypnotic or anesthetic activity, neuroprotective or anti-ischemic activity, activity in the process of drug withdrawal and in addiction.",
"Thanks to the compounds of the present invention, it also becomes possible to propose a process for treatment of a disease in a mammal which can be treated by administration of a GHB agonist, in particular, diseases of the central nervous system, and particularly, diseases relating to sleep and to anxiety, said treatment including the administration to the mammal of a therapeutically effective quantity of at least one compound with general formula I, I′ or I″ according to the present invention, preferably at least one sodium salt chosen from the compounds with general formula I″, in particular those mentioned in Table 2.Of course, the invention is not limited to the embodiments which have been described.",
"Modifications remain possible, particularly from the standpoint of the constitution of the various elements or by substitution of equivalent techniques, without consequently leaving the field of protection of the invention."
]
] | Patent_10432692 |
[
[
"Process for preparing siloxane-filler compositions using an extruder mixer",
"Methods describing a process for the production of polysiloxane containing masses, containing surface treated filler material, by continuously feeding materials to a screw extruder (12) in which they are mixed and conveyed to an outlet (14) with removal of gaseous materials therefrom.",
"The screw extruder (12) has at least two screws (22) located in communicating chambers distributed radially about a common axis (26) and extending with the axis of each screw parallel to the common axis (26).",
"The materials being mixed in the extruder typically comprise (a) a polysiloxane, (b a reinforcing filler and (c) a hydrophobing agent.",
"Each material is fed into the extruder (12) as the material itself or in admixture with any one or more of the others of (a), (b) and (c)."
],
[
"1.A process for production of polysiloxane containing masses that contain surface treated filler material the process comprising (I) continuously feeding materials to a screw extruder in which they are mixed and (II) thereafter conveying the masses to an outlet while removing gaseous materials therefrom, wherein the screw extruder comprises more than two screws located in communicating chambers that are distributed radially about a common axis and that extend with the axis of each screw parallel to the common axis, said materials being mixed in the extruder comprising; a) a polysiloxane having more than 40 siloxane units selected from the group consisting of (i) trialkylsilyl end blocked polysiloxanes and (ii) polysiloxanes having at least one silicon bonded group selected from the group consisting of alkenyl groups, hydroxyl groups and hydrolysable groups; b) a reinforcing filler material selected from the group consisting of finely divided silica, surface treated finely divided silica, finely divided calcium carbonate, surface treated finely divided calcium carbonate, quartz powder, aluminium hydroxide, zirconium silicate, diatomaceous earth and titanium dioxide, and c) a hydrophobing agent selected from the group consisting of disilazanes and water and polydiorganosiloxanes of up to 40 siloxane units having silicon bonded groups selected from the group consisting of hydroxyl groups and amino groups, each of the materials having been fed into the extruder individually, or in admixture with any one or more of (a), (b) and (c).",
"2.A process according to claim 1 further characterized in that the finely divided filler is selected from the group consisting of fumed and precipitated silica.",
"3.A process according to claim 2 further characterized in that a mixture (d) comprising polysiloxane (a) and silica (b) in a ratio from 0.7 to 1.8 parts a) to 1 part silica, is fed to the extruder.",
"4.A process according to claim 3 further characterized in that mixture (d) is selected from the group consisting of a paste and a powder.",
"5.A process according to claim 3 further characterized in that the mixture (d) is stored in a reservoir after having been mixed and before introduction to the extruder.",
"6.A process according to claim 5 further characterized in that mixture (d) has been aged for a period of not less than ten minutes after mixing and before delivery to the extruder.",
"7.A process according to claim 3 further characterized in that mixture (d) is fed to the extruder from a separate continuous mixing unit where the component materials have a residence time between thirty seconds and five minutes.",
"8.A process according to claim 1 further characterized in that the extruder has twelve screws arranged to co-operate in mixing and conveying materials through the extruder to its outlet.",
"9.A process according to claim 8 further characterized in that the length of each extruder screw is from 25 to 60 times the diameter of the screw.",
"10.A process according to claim 8 further characterized in that the diameter of each extruder screw is from 20 to 160 mm.",
"11.A process according to claim 8, further characterized in that the screws are rotated in the same sense and at a speed from 50 to 1200 rpm.",
"12.A process according to claim 1 further characterized in that the interior of the extruder provides zones which in a first zone, a first portion of each screw meshes with a first portion of each of those screws adjacent to it so that the regime in this first zone is predominantly feeding of mixture towards a subsequent zone, and in a which there is a second zone in which portions of the screws are arranged to promote intensive kneading and dispersing of the mixture as well as feeding it to subsequent zones, and a third zone in which portions of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it towards the outlet.",
"13.A process according to claim 12 further characterized in that the first zone has a length of 5 to 30 times the diameter of a screw.",
"14.A process according to claim 12 further characterized in that the second zone has a length of 5 to 15 times the diameter of a screw.",
"15.A process according to claim 12, further characterized in that the third zone has a length of 5 to 30 times the length of a screw.",
"16.A process according to claim 12 further characterized in that the material in the first three zones of the extruder comprises between 70 and 180 parts of (a) per 100 parts of (b).",
"17.A process according to claim 12 further characterized in that material in the third zone is subject to reduced pressure.",
"18.A process according to claim 12 further characterized in that it comprises a fourth zone in which portions of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it, this zone having a length of 5 to 15 times the diameter of a screw.",
"19.A process according to claim 2 further characterized in that the materials fed to the screw extruder comprise a polysiloxane (a) which is a polysiloxane having more than 40 siloxane units which has at least one silicon bonded alkenyl group and a product (e) formed by mixing finely divided silica, a polysiloxane having more than 40 siloxane units which has at least one silicon bonded alkenyl group, water and an hydrophobing agent comprising a disilazane or polydiorganosiloxanes of up to 40 siloxane units having silicon bonded hydroxyl groups.",
"20.A process for production of polysiloxane containing masses containing surface treated filler material that comprises (I) continuously feeding materials to a screw extruder in which the materials are mixed and thereafter (II) conveying the mixed materials to an outlet, with removal of gaseous materials therefrom, characterized, in that, the screw extruder comprises more than two screws located in communicating chambers distributed radially about a common axis and extending with the axis of each screw parallel to the common axis and, in that, the materials fed to the screw extruder consist essentially of A. a polysiloxane having more than 40 siloxane units selected from the group consisting of trialkylsilyl end blocked polysiloxanes, polysiloxanes having at least one silicon bonded group selected from the group consisting of alkenyl groups, hydroxyl groups, and hydrolysable group; B. a reinforcing filler material selected from the group consisting of finely divided silica, calcium carbonate, quartz powder, aluminium hydroxide, zirconium silicate, diatomaceous earth and titanium dioxide, C. an hydrophobing agent selected from the group consisting of (i) disilazanes and water (ii) polydiorganosiloxanes of up to 40 siloxane units having silicon bonded groups selected from the groups consisting of a. hydroxyl groups, and b. amino groups and, (iii) the product formed by mixing any of A, B, C(i), C(ii) and C(iii).",
"21.",
"(canceled) 22.A process according to claim 20 further characterized in that, the materials delivered from the outlet of the extruder are further compounded with at least one material, selected from curatives, catalysts, inhibitors, plasticizers, extenders and non-reinforcing fillers, to provide a curable product.",
"23.A process according to claim 12 further characterized in that the first zone has a a temperature of the material controlled at less than 100° C. 24.A process according to claim 12 further characterized in that the first zone has a length of 5 to 30 times the diameter of a screw and the temperature of material in the first zone is controlled at less than 100° C. 25.A process according to claim 12 further characterized in that the temperature of the materials in the second zone is controlled at less than 150° C. 26.A process according to claim 12 further characterized in that the second zone has a length of 5 to 15 times the diameter of a screw and the temperature of material in the second zone is controlled at less than 150° C. 27.A process according to claim 12 further characterized in that the temperature of material in the third zone is controlled at 100° C. to 350° C. 28.A process according to claim 12 further characterized in that it comprises a fourth zone in which portions of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it, the temperature of material in the fourth zone being controlled at less than 250° C. 29.A process according to claim 12 further characterized in that it comprises a fourth zone in which portions of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it, the fourth zone having a length of 5 to 15 times the diameter of a screw and the temperature of the material in the fourth zone being controlled at less than 250° C. 30.A process according to claim 1 further characterized in that, the materials delivered from the outlet of the extruder are further compounded with other materials, including materials selected from the group consisting of curatives, catalysts, inhibitors, plasticizers, extenders and non-reinforcing fillers, to provide a curable product."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Silicone compositions that can be applied or shaped in fluid condition and then cured are well known.",
"Among such compositions are those that are intended to have a low viscosity during storage and application and yet provide products of high mechanical strength.",
"Such compositions may be used for a variety of purposes and are especially favoured for use in coating or moulding operations in which they become cured, with or without a separate heating operation.",
"Typically such compositions contain reinforcing filler, the most commonly used one being silica, to enhance mechanical properties of the cured product.",
"Whilst assisting in reinforcing the final product, silica also tends to associate progressively with the polysiloxanes present in the fluid composition with corresponding increase in viscosity of the composition.",
"It has become a practice to render the silica hydrophobic by suitable treatment of its surface.",
"Surface treatment of silica can be done with the silica in dry powder form and before introduction to the silicone composition, but is commonly done in-situ in the composition.",
"Surface treating materials, for example hexamethyldisilazane or divinyltetramethyldisilazane, are therefore usually incorporated with water into the composition of polysiloxane and silica.",
"Commonly, this mixing is done batchwise, but this is a slow process and the product may suffer from significant variations in quality between batches.",
"Continuous processes for in-situ treatment of silica have been proposed which employ twin screw extruders.",
"The twin screw extruders may be extended in length to achieve mixing of materials but those employed are generally limited to a maximum length of 60 times the diameter of the screw, for engineering reasons.",
"The proposed processes can be inefficient or uneconomical due to lack of residence time of the mixture in the extruder and low throughput of mixture through the extruder.",
"Also, low density fillers contain a high proportion of air which leads to problems when continuously incorporating them into silicone polymers in a twin screw extruder.",
"The rate of incorporation of filler into a composition has been one of the main throughput limitations in a continuous process."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a plan of a multiple screw extruder used in the illustrative process, FIG.",
"2 is a side elevation of a portion of the extruder shown in FIG.",
"1 , FIG.",
"3 is sectional view taken substantially on the line A-A of FIG.",
"2 , FIG.",
"4 is a diagram of a screw located in the extruder.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?",
"The illustrative process is for production of polysiloxane containing masses incorporating surface treated filler material, which comprises continuously feeding materials to an inlet ( 10 ) of a screw extruder ( 12 ) in which they are mixed and conveyed to an outlet ( 14 ) with removal of gaseous materials therefrom via vents ( 16 , 18 , 20 ).",
"The vent ( 16 ) may be arranged to vent air from the extruder as mixing is carried out or to feed material into the extruder for mixing with other ingredients.",
"Means are provided for heating and/or cooling various sectors of the extruder as required during operation of the extruder.",
"The screw extruder ( 12 ) comprises twelve screws ( 22 ) located in communicating chambers ( 24 ) distributed radially about a common axis ( 26 ) and extending with the axis of each screw parallel to the common axis ( 26 ).",
"The extruder comprises a housing having a generally cylindrical outer casing ( 28 ) disposed with its axis substantially horizontal and providing the common axis ( 26 ).",
"A cavity ( 30 ) extends through it about the substantially horizontal common axis ( 26 ).",
"The interior of the outer casing ( 28 ) is profiled to define a plurality of part cylindrical surfaces ( 32 ) equidistant from the common axis and spaced around equal angular sectors around the perimeter of the cavity ( 30 ).",
"An inner casing portion ( 34 ) of the housing is secured to the outer casing within the cavity ( 30 ).",
"Its outer surface is profiled to define part cylindrical surfaces ( 36 ) complementary to the surfaces ( 32 ) of the outer casing ( 28 ).",
"The surfaces ( 32 ) and ( 36 ) together define the chambers ( 24 ) which communicate with adjacent chambers and each receive a screw ( 22 ) arranged for rotation about an axis parallel to that of the housing.",
"The diameter of each extruder screw is 30 mm.",
"The length of each extruder screw was 32 times the diameter of the screw.",
"In processes according to the invention, the screws are rotated in the same sense and at a speed from 50 to 1200 rpm.",
"Each screw 22 has portions profiled to promote desired kneading or feeding of mixture within the extruder.",
"These portions of the screws are positioned on the screws and within the extruder so as to provide a first ( 40 ), second ( 42 ), third ( 44 ) and fourth ( 46 ) mixing zones disposed axially in the extruder.",
"In the first zone ( 40 ) a first portion ( 41 ) of each screw meshes with a first portion ( 41 ) of each of those screws adjacent to it so that the regime in this first zone is predominantly feeding of mixture towards subsequent zones.",
"In the second zone ( 42 ), portions ( 43 ) of the screws are arranged to promote intensive kneading and dispersing of the mixture as well as feeding it to subsequent zones.",
"In the third zone ( 44 ), portions ( 45 ) of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it towards the outlet.",
"In the fourth zone ( 46 ) portions ( 47 ) of the screws are arranged to promote further kneading and dispersing of the mixture as well as feeding it, In a preferred process the first zone has a length of 5 to 30 times the diameter of a screw and the temperature of material in the first zone is controlled at less than 100° C. The second zone has a length of 5 to 15 times the diameter of a screw and the temperature of material in the second zone is controlled at less than 150° C. The third zone has a length of 5 to 30 times the length of a screw and the temperature of material in the third zone is controlled at 100° C. to 350° C. The fourth zone has a length of 5 to 15 times the diameter of a screw and the temperature of material in the fourth zone is controlled at less than 250° C. Preferably, material in the third zone is subject to reduced pressure of between −100 and −1000 mbar (i.e.",
"between −10 4 and −10 5 Pa), pulled via the vents ( 18 , 20 )."
],
[
"FIELD OF INVENTION This invention is concerned with improvements in or relating to mixing compositions and to silicone compositions produced by such mixing.",
"BACKGROUND OF THE INVENTION Silicone compositions that can be applied or shaped in fluid condition and then cured are well known.",
"Among such compositions are those that are intended to have a low viscosity during storage and application and yet provide products of high mechanical strength.",
"Such compositions may be used for a variety of purposes and are especially favoured for use in coating or moulding operations in which they become cured, with or without a separate heating operation.",
"Typically such compositions contain reinforcing filler, the most commonly used one being silica, to enhance mechanical properties of the cured product.",
"Whilst assisting in reinforcing the final product, silica also tends to associate progressively with the polysiloxanes present in the fluid composition with corresponding increase in viscosity of the composition.",
"It has become a practice to render the silica hydrophobic by suitable treatment of its surface.",
"Surface treatment of silica can be done with the silica in dry powder form and before introduction to the silicone composition, but is commonly done in-situ in the composition.",
"Surface treating materials, for example hexamethyldisilazane or divinyltetramethyldisilazane, are therefore usually incorporated with water into the composition of polysiloxane and silica.",
"Commonly, this mixing is done batchwise, but this is a slow process and the product may suffer from significant variations in quality between batches.",
"Continuous processes for in-situ treatment of silica have been proposed which employ twin screw extruders.",
"The twin screw extruders may be extended in length to achieve mixing of materials but those employed are generally limited to a maximum length of 60 times the diameter of the screw, for engineering reasons.",
"The proposed processes can be inefficient or uneconomical due to lack of residence time of the mixture in the extruder and low throughput of mixture through the extruder.",
"Also, low density fillers contain a high proportion of air which leads to problems when continuously incorporating them into silicone polymers in a twin screw extruder.",
"The rate of incorporation of filler into a composition has been one of the main throughput limitations in a continuous process.",
"DETAILED DESCRIPTION OF THE INVENTION We have now found that mixtures of polysiloxane and reinforcing filler can be prepared with improved efficiency by feeding selected materials to a screw extruder comprising more than two screws located in communicating chambers distributed radially about a common axis and extending with the axis of each screw parallel to the common axis.",
"The present invention provides in accordance with one of its aspects a process for production of polysiloxane containing masses containing surface treated filler material which comprises continuously feeding materials to a screw extruder in which they are mixed and conveyed to an outlet with removal of gaseous materials therefrom, the screw extruder comprising more than two screws located in communicating chambers distributed radially about a common axis and extending with the axis of each screw parallel to the common axis and the materials being mixed in the extruder comprising; a) a polysiloxane having more than 40 siloxane units selected from the group consisting of trialklysilyl end blocked polysiloxanes or polysiloxanes having at least one silicon bonded alkenyl, hydroxyl or hydrolysable group, b) a reinforcing filler material selected from the group consisting of finely divided silica, surface treated finely divided silica, calcium carbonate, surface treated finely divided calcium carbonate, quartz powder, aluminium hydroxide, zirconium silicate, diatomaceous earth and titanium dioxide; c) an hydrophobing agent selected from the group consisting of disilazanes with water and polydiorganosiloxanes of up to 40 siloxane units having silicon bonded hydroxyl or amino groups, each of the materials having been fed into the extruder as the material itself or in admixture with any one or more of the others of (a), (b), and (c).",
"In a process according to the invention, we prefer to employ an extruder in which three or more screws are spaced around equal angular sectors such for example as more fully described in U.S. Pat.",
"No.",
"5,836,682.Preferably the screws are confined in a housing having inner and outer casing elements that together define the chambers in which the screws rotate.",
"Preferably the inner and outer casing elements are secured together and disposed with the common axis at least substantially horizontal.",
"Preferably each screw has a plurality of processing elements that closely interengage on adjacent shafts.",
"Preferably the extruder has ten or more, for example twelve, interengaging screws arranged with their axes disposed radially equidistant from the common axis so as to co-operate in mixing and conveying materials through the extruder to its outlet.",
"In a preferred extruder, the twelve screws intermesh closely and are arranged in a circle around a core.",
"Material within the extruder is mixed whilst being caused to move axially of the extruder and in the ring provided by the chambers, as in a spiral.",
"Preferably the length of each extruder screw is from 25 to 60 times the diameter of the screw.",
"Preferably the diameter of each extruder screw is from 20 to 160 mm.",
"In a preferred process according to the invention, the screws are rotated in the same sense and at a speed from 50 to 1200 rpm.",
"In a process according to the present invention, we prefer to arrange that the interior of the extruder provides at least first, second and third mixing zones spaced along the common axis.",
"Preferably, in a first of the zones a first portion of each screw meshes with a first portion of each of those screws adjacent to it so that the regime in this first zone is predominantly feeding of mixture towards subsequent zones.",
"Preferably in a second of the zones, portions of the screws are arranged to promote intensive kneading and dispersing of the mixture as well as feeding it to subsequent zones.",
"Preferably, in a third zone, portions of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it towards the outlet.",
"Preferably, there is also a fourth zone in which portions of the screws are arranged to promote further kneading and dispersing of the mixture as well as feeding it, In a preferred process the first zone has a length of 5 to 30 times the diameter of a screw.",
"Preferably the temperature of material in the first zone is controlled at less than 100° C. Preferably the second zone has a length of 5 to 30 times the diameter of a screw.",
"Preferably the temperature of material in the second zone is controlled at less than 150° C. Preferably, the third zone has a length of 5 to 15 times the length of a screw.",
"Preferably the temperature of material in the third zone is controlled at 100° C. to 350° C. Preferably, material in the third zone is subject to reduced pressure.",
"When present, the fourth zone preferably has a length of 5 to 15 times the diameter of a screw.",
"Preferably the temperature of material in the fourth zone when present is controlled at less than 250° C. The polysiloxanes (a) used in a process according to the invention may be linear or branched and have a viscosity from a fluid to a gum.",
"These materials comprise siloxane chain units XpRqSiO(4−(p+q))/2 and chain terminating units preferably of the general formula XR2SiO1/2, in which each R represents a monovalent hydrocarbon group containing up to 12 carbon atoms, for example methyl or phenyl which may be substituted or not, X represents a group R or an alkenyl, hydroxyl or hydrolysable group, p has a value of 0, 1 or 2, q has a value of 0, 1 or 2 and the sum of p+q has a value of 0, 1 or 2.Preferred polysiloxanes (a) are at least substantially linear materials consisting predominantly of chain units of the general formula R2SiO where R represents the methyl group.",
"Suitable materials include trimethylsilyl terminated polydimethylsiloxanes having a viscosity of 350 mPas or more, methylvinylpolysiloxanes of viscosities from about 2000 to 60000 mPas, α,ω-dihydroxypolydiorganosiloxanes having viscosities from about 5000 to 11000 mPas and fluorinated polydiorganosiloxanes.",
"In a process according to the invention, the reinforcing filler (b) may be any of those normally employed in silicone compositions, for example high surface area silicas or calcium carbonates, quartz powder, aluminium hydroxide, zirconium silicate, diatomaceous earth and titanium dioxide.",
"If desired, the reinforcing filler fed to the extruder may consist in whole or in part of surface treated silica or calcium carbonate for example silica which has been rendered hydrophobic by treatment of its surface with silane or silazane.",
"When a reinforcing filler is used which has not been surface treated to render it hydrophobic, it is preferred to additionally feed an hydrophobing agent (c) to the extruder.",
"Preferably, the hydrophobing agent is selected from the group consisting of disilazanes with water and polydiorganosiloxanes of up to 40 siloxane units having silicon bonded hydroxyl or amino groups.",
"Suitable disilazanes include hexamethyldisilazane and tetramethyldivinyldisilazane.",
"The polydiorganosiloxanes referred to are preferably linear polydiorganosiloxanes having chain units as referred to above.",
"In a process according to the invention we prefer that one of the materials fed to the extruder is a mixture (d) which is a product formed by mixing polysiloxane (a) and filler (b) (which is preferably fumed or precipitated silica) and optionally other materials.",
"Conveniently such a mixture (d) may be mixed in a separate continuous mixing apparatus for example one which operates by way of a rotor stator mixer with forced axial transport or a twin screw mixer with a very high outer diameter to inner diameter ratio in order to allow a high free volume and is therefore able to mix larger amounts of light fillers, such a twin screw will typically be co-rotating, with overlapping screw agitators which rotate at a consistent speed as the ingredients are fed at various points along its length.",
"Depending on the formulation and the output of this mixer, it may feed directly to the extruder or alternatively the mixing apparatus may feed to a reservoir in which the mixture is stored and aged before delivery to the extruder.",
"In a process according to the invention, the mixture (d) is preferably a powder or a paste which has been formed by mixing materials (a), (b) and (c) in proportions by weight such that for 100 parts silica there are employed 50 to 180 parts polysiloxane (a).",
"When the material is a paste it preferably contains about 110 to 280, parts by weight of polysiloxane (a) per 100 parts by weight of the filler, and when the mixture (d) is a powder, it preferably contains about 70 to 110 parts by weight of polysiloxane (a) per 100 parts by weight filler.",
"Preferably (c) is present in about 10 to 30, most preferably 15 to 25 parts by weight and d is present in the range of 2 to 10 parts by weight most preferably 3 to 6 by weight per 100 parts by weight filler.",
"In a process according to the invention, the materials are preferably fed to the screw extruder in proportions to provide in the first second and third zones a ratio of 70 to 180 parts by weight of polysiloxane (a) to 100 parts by weight of filler (b).",
"A process according to the invention is useful for processing filler/polysiloxane polymer masses generally and is especially suitable for preparation of masterbatches intended for further processing to provide formulations in one or more parts for curing to provide a finished product.",
"The invention provides in another of its aspects a process for production of a masterbatch for processing into a curable product which comprises continuously feeding materials to a screw extruder in which they are mixed and conveyed to an outlet with removal of gaseous materials therefrom characterised in that the screw extruder comprises more than two screws located in communicating chambers distributed radially about a common axis and extending with the axis of each screw parallel to the common axis and in that the materials fed to the screw extruder consist essentially of a) a polysiloxane having more than 40 siloxane units selected from the group consisting of trialklysilyl end blocked polysiloxanes and polysiloxanes having at least one silicon bonded alkenyl, hydroxyl or hydrolysable group, b) a reinforcing filler material selected from the group consisting of finely divided silica, calcium carbonate, quartz powder, aluminium hydroxide, zirconium silicate, diatomaceous earth and titanium dioxide, c) an hydrophobing agent selected from the group consisting of disilazanes and polydiorganosiloxanes of up to 40 siloxane units having silicon bonded hydroxyl or amino groups and d) water and/or e) the product formed by mixing any two or more of a), b), c) and d).",
"In accordance with another aspect of the invention, the materials from the outlet of the extruder may be further compounded with other materials, including for example curatives, catalysts, inhibitors plasticisers, extenders and non-reinforcing fillers.",
"Curatives that may be incorporated include for example polysiloxanes incorporating silicon-bonded hydrogen atoms for reaction with alkenylpolysiloxanes of the masterbatch, and hydrolysable silanes and siloxanes for reaction with polysiloxanes of the masterbatch having hydroxyl or hydrolysable groups.",
"Catalysts that may be incorporated include, for example, platinum catalysts for the hydrosilylation reaction, organometal salts or complexes for promoting condensation cure, and peroxides.",
"Inhibitors may be incorporated to vary the rate of reaction in the cured product.",
"Plasticisers and extenders that may be incorporated include for example trimethylsilyl-terminated polydimethylsiloxane.",
"Non reinforcing fillers which may be incorporated may be selected from, for example iron oxide, zinc oxide, carbon black, glass microballoons, and carbonates of calcium, magnesium, barium or zinc and barium sulphate.",
"Processes according to the invention contribute to improved efficiency of production facilities and yield products of good consistency comparatively quickly.",
"In order that the invention may become clearer there now follows a description to be read with the accompanying drawings of a process selected for description to illustrate the invention by way of example.",
"In the examples, all parts are by weight and all viscosities were measured at 25° C. and it should be understood that Premixes A, B, C and D discussed in the following examples are alternative mixtures referred to above as mixture (e).",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a plan of a multiple screw extruder used in the illustrative process, FIG.",
"2 is a side elevation of a portion of the extruder shown in FIG.",
"1, FIG.",
"3 is sectional view taken substantially on the line A-A of FIG.",
"2, FIG.",
"4 is a diagram of a screw located in the extruder.",
"The illustrative process is for production of polysiloxane containing masses incorporating surface treated filler material, which comprises continuously feeding materials to an inlet (10) of a screw extruder (12) in which they are mixed and conveyed to an outlet (14) with removal of gaseous materials therefrom via vents (16, 18, 20).",
"The vent (16) may be arranged to vent air from the extruder as mixing is carried out or to feed material into the extruder for mixing with other ingredients.",
"Means are provided for heating and/or cooling various sectors of the extruder as required during operation of the extruder.",
"The screw extruder (12) comprises twelve screws (22) located in communicating chambers (24) distributed radially about a common axis (26) and extending with the axis of each screw parallel to the common axis (26).",
"The extruder comprises a housing having a generally cylindrical outer casing (28) disposed with its axis substantially horizontal and providing the common axis (26).",
"A cavity (30) extends through it about the substantially horizontal common axis (26).",
"The interior of the outer casing (28) is profiled to define a plurality of part cylindrical surfaces (32) equidistant from the common axis and spaced around equal angular sectors around the perimeter of the cavity (30).",
"An inner casing portion (34) of the housing is secured to the outer casing within the cavity (30).",
"Its outer surface is profiled to define part cylindrical surfaces (36) complementary to the surfaces (32) of the outer casing (28).",
"The surfaces (32) and (36) together define the chambers (24) which communicate with adjacent chambers and each receive a screw (22) arranged for rotation about an axis parallel to that of the housing.",
"The diameter of each extruder screw is 30 mm.",
"The length of each extruder screw was 32 times the diameter of the screw.",
"In processes according to the invention, the screws are rotated in the same sense and at a speed from 50 to 1200 rpm.",
"Each screw 22 has portions profiled to promote desired kneading or feeding of mixture within the extruder.",
"These portions of the screws are positioned on the screws and within the extruder so as to provide a first (40), second (42), third (44) and fourth (46) mixing zones disposed axially in the extruder.",
"In the first zone (40) a first portion (41) of each screw meshes with a first portion (41) of each of those screws adjacent to it so that the regime in this first zone is predominantly feeding of mixture towards subsequent zones.",
"In the second zone (42), portions (43) of the screws are arranged to promote intensive kneading and dispersing of the mixture as well as feeding it to subsequent zones.",
"In the third zone (44), portions (45) of the screws are arranged to promote kneading and dispersing of the mixture as well as feeding it towards the outlet.",
"In the fourth zone (46) portions (47) of the screws are arranged to promote further kneading and dispersing of the mixture as well as feeding it, In a preferred process the first zone has a length of 5 to 30 times the diameter of a screw and the temperature of material in the first zone is controlled at less than 100° C. The second zone has a length of 5 to 15 times the diameter of a screw and the temperature of material in the second zone is controlled at less than 150° C. The third zone has a length of 5 to 30 times the length of a screw and the temperature of material in the third zone is controlled at 100° C. to 350° C. The fourth zone has a length of 5 to 15 times the diameter of a screw and the temperature of material in the fourth zone is controlled at less than 250° C. Preferably, material in the third zone is subject to reduced pressure of between −100 and −1000 mbar (i.e.",
"between −104 and −105 Pa), pulled via the vents (18, 20).",
"EXAMPLE 1 100 parts of a vinyl functional polydiorganosiloxane having a viscosity of 55,000 mPas (a) were mixed with 100 parts of a fumed silica having surface area of 255 m2/g (b), 22.5 parts of hexamethyldisilazane (c) and 6 parts of water in a twin shaft extruder having an outer diameter: inner diameter ratio of 2.7 to form a powder (Premix A).",
"Premix A was transferred into the feed section of the barrel of a twin screw extruder in which the screws were each of 30 mm diameter and of a length of 45 times their diameter and rotating at 500 rpm.",
"60 parts of the vinyl functional polydiorganosiloxane (a) were added in the feed section of the twin screw extruder and the mixture was compounded to a uniform paste (in an extruder feed and intensive mixing section of the extruder) having a temperature of less than 100° C. In a third extruder section, the paste was heated to 275° C. and exposed to reduced pressure of −900 mbar (−9×104 Pa) to remove hexamethyldisilazane and water as well as by-products.",
"In a fourth section, a further 71 parts of the polydiorganosiloxane (a) were added to form a homogeneous paste suitable for formulation to provide a curable composition.",
"The obtained paste was dispensed from the extruder and cooled.",
"The maximum output of the extruder was 6.6 kg/h.",
"Its viscosity was measured 24 hours after dispensing using a cone and plate viscometer at 10 s−1 shear rate and found to be 1490 mPas.",
"EXAMPLE 2 94 parts of the vinyl functional polydiorganosiloxane having a viscosity of 55,000 mPas (a) used in Example 1 were mixed with 100 parts of the silica (b) used in Example 1, 21.4 parts of hexamethyldisilazane (c) and 6.6 parts of water in a rotor stator mixer with forced axial transport to form a powder (Premix B).",
"Premix B was transferred into the inlet (10) of the twelve screw extruder in which the screws were rotating at 230 rpm.",
"64 parts of the vinyl functional polydiorganosiloxane (a) were added in the feed section of the screw extruder and the mixture was compounded to a uniform paste (in the extruder feed and intensive mixing section of the extruder) having a temperature of less than 100° C. In the third extruder section, the paste was heated to 210° C. and exposed to reduced pressure of −900 mbar (−9×104 Pa) to remove hexamethyldisilazane and water as well as by-products.",
"In the fourth section, a further 73 parts of the polydiorganosiloxane (a) were added and mixing continued to form a homogeneous paste suitable for formulation to provide a curable composition.",
"The obtained paste was dispensed from the extruder and cooled.",
"The maximum output of the extruder was 103 kg/hr.",
"Its viscosity was measured, 24 hours after dispensing using a cone and plate viscometer at 10 s−1 shear rate and found to be 860 mPas.",
"As can be seen from the data, the process used in Example 2 provided a composition of lower viscosity than that of Example 1 with the same overall formulation, indicating better treatment of the silica and better stripping of volatiles.",
"Additionally the throughput achieved in Example 2 is more than 15 times higher compared to Example 1, making the process of Example 2 economically much more attractive than that of Example 1.EXAMPLE 3 91 parts of the vinyl functional polydiorganosiloxane having a viscosity of 55,000 mPas (a) used in Example 1 were mixed with 100 parts of the silica (b) used in Example 1, 20 parts of hexamethyldisilazane (c) and 6 parts of water in a continuous rotor stator mixer with forced axial transport to form a powder (Premix C).",
"Premix C was transferred into the inlet (10) of the twelve screw extruder in which the screws were rotating at 230 rpm.",
"61 parts of the vinyl functional polydiorganosiloxane (a) were added in the feed section of the screw extruder and the mixture was compounded to a uniform paste (in the extruder feed and intensive mixing section of the extruder) having a temperature of less than 100° C. In the third extruder section, the paste was heated to 210° C. and exposed to reduced pressure of −900 mbar (−9×104 Pa) to remove hexamethyldisilazane and water as well as by-products.",
"In the fourth section, a further 79 parts of the polydiorganosiloxane (a) were added and mixing continued to form a homogeneous paste suitable for formulation to provide a curable composition.",
"The obtained paste was dispensed from the extruder and cooled.",
"The maximum output of the extruder was 110 kg/hr.",
"Its viscosity was measured, 24 hours after dispensing using a cone and plate viscometer at 10 s−1 shear rate and found to be 822 mPas.",
"A 2 mm thick layer of the obtained paste coated onto a plastic foil was observed to have a smooth appearance with no visible particles.",
"EXAMPLE 4 91 parts of the vinyl functional polydiorganosiloxane having a viscosity of 55,000 mPas (a) used in Example 1 were mixed with 100 parts of the silica (b) used in Example 1, 20 parts of hexamethyldisilazane (c) and 6 parts of water in a rotor stator mixer with forced axial transport to form a powder (Premix D).",
"Premix D was transferred into the inlet (10) of the twelve screw extruder in which the screws were rotating at 230 rpm.",
"175 parts of the vinyl functional polydiorganosiloxane (a) were added in the feed section of the screw extruder and the mixture was compounded to a uniform paste (in the extruder feed and intensive mixing section of the extruder) having a temperature of less than 100° C. In the second and third extruder sections, the paste was heated to 210° C. and exposed to reduced pressure of −900 mbar (−9×104 Pa) to remove hexamethyldisilazane and water as well as by-products.",
"The obtained paste was dispensed from the extruder and cooled.",
"The maximum output of the extruder was 150 kg/hr.",
"Its viscosity was measured, 24 hours after dispensing using a cone and plate viscometer at 10 s−1 shear rate and found to be 1729 mPas.",
"A 2 mm thick layer of the obtained paste coated onto a plastic foil was observed to have a grainy appearance with many silica particles visible.",
"The results show that the paste obtained in Example 4 had a higher viscosity than the one produced in Example 3, despite having a higher content of polysiloxane.",
"Additionally, the appearance of the paste obtained in Example 4 had a poorer appearance due to inadequately dispersed filler particles."
]
] | Patent_10432749 |
[
[
"Self-closing packaging for moisture-sensitive material",
"Packaging for moisture-sensitive material, in particular detergent material, comprising an outer container comprising a container body and a lid cover hingedly fixed to the container body, and an inner bag made of water-impermeable flexible sheet material for receiving the moisture-sensitive material, the mouth of the inner bag being fixed both at the lid cover and at the sidewalls of the container body in such a way that, when the lid cover is lifted, the lower part of the inner bag is significantly lifted from the bottom of the container body."
],
[
"1.Packaging for moisture-sensitive material, in particular detergent material, comprising an outer container (10) comprising a container body (11, 12) and a lid cover (13) hingedly fixed to the container body, and an inner bag (20) made of water-impermeable flexible sheet material for receiving the moisture-sensitive material (30), the mouth of the inner bag being fixed both at the lid cover (13) and at the sidewalls (11) of the container body (11, 12) in such a way that, when the lid cover (13) is lifted, the lower part of the inner bag (20) is significantly lifted from the bottom (12) of the container body.",
"2.Packaging according to claim 1 wherein the mouth of the inner bag (20) is fixed at the edge portion (14) of the lid cover (13) being opposite to the hinged portion (15) thereof and at the edge portion (16) of the container body being adjacent to the said edge portion (14) of the lid cover (13) in the closed state of the outer container (10).",
"3.Packaging according to claim 1 wherein the mouth of the inner bag (20) is fixed at the edge portion (14) of the lid cover (13) being opposite to the hinged portion (15) thereof and at the sidewall (11) of the container body being opposite to the sidewall of the container where the hinge (15) of the lid is formed.",
"4.Packaging according to claim 1 wherein the inner bag (20) is fixed at no other portion of the outer container (10).",
"5.Use of a packaging according to claim 1 for packaging material containing volatile ingredients.",
"6.Use of a packaging according to claim 1 for packaging water-soluble sachets containing detergent material.",
"7.Use of a packaging material according to claim 2 for packaging water-soluble sachets containing detergent material.",
"8.Use of a packaging material according to claim 3 for packaging water-soluble sachets containing detergent material."
],
[
"The invention is directed to a packaging for moisture-sensitive material, in particular detergent material.",
"Commonly, moisture-sensitive material, such as detergent material, e.g.",
"detergent powder, granules, tablets or detergents contained in water-soluble sachets, are packaged in containers made of cardboard material with a lining of water-impermeable sheet material to protect the contents from absorption of water from the atmosphere.",
"However, the user has to make sure that the packaging is closed again after usage.",
"If not, the moisture-sensitive material can chemically degrade potentially affecting the activity of ingredients or suffer a negative impact on its physical properties (i.e.",
"resistance, solubility, .",
".",
".",
").",
"Additionally, there may be a substantial loss of volatile material such as perfume from the material, when the container is not tightly closed.",
"Although this negative impact on moisture is undesirable in general, it is particularly problematic in the case of tablets not individually wrapped in a moisture isolating film or even worse in the case of detergents packaged in water-soluble monodose sachets, for instance in PVOH sachets.",
"Such sachets are getting sticky when absorbing water from the atmosphere, resulting in adhering to each other.",
"When trying to separate such adhered sachets, destroying of the water-soluble film material is highly likely.",
"Moreover, undesirable loss of water from the contents of such sachets is another problem if they are not contained in a tight packaging.",
"The object of the invention therefore is to provide for a tight packaging ensuring reliable protection of the contents thereof from moisture in the atmosphere and/or avoiding escape of volatile material from the packaging.",
"This object is solved by a packaging comprising an outer container comprising a container body and a lid cover hingedly fixed to the container body, and an inner bag made of water-impermeable flexible sheet material for receiving the moisture-sensitive material, the mouth of the inner bag being fixed both at the lid cover and at the sidewalls of the container body in such a way that, when the lid cover is lifted, the lower part of the inner bag is significantly lifted from the bottom of the container body.",
"In a preferred embodiment of the invention, the mouth of the inner bag is fixed at the edge portion of the lid cover being opposite to the hinged portion thereof and at least at the edge portion of the container body being adjacent to the said edge portion of the lid cover in the closed state of the outer container.",
"In the most preferred embodiment, the inner bag is fixed at no other portion of the outer container.",
"The invention is also directed to the use of a packaging according to the invention for packaging material containing volatile ingredients or water-soluble sachets containing detergent material.",
"The packaging of the present invention provides for a system ensuring automatic self-closing of the packaging after having been opened by the user to take out a portion of the powder or granules or one or more tablets or sachets in order to protect the moisture-sensitive contents thereof.",
"The invention is based on providing an inner bag which is relatively loose within the outer container, i.e.",
"basically only the mouth of the inner bag being fixed thereto.",
"However, the inventive packaging does not only provide for a reliable protection of the contents thereof from moisture in the atmosphere, but is also a tight packaging in another sense, namely by avoiding escape of volatile matter from the packaging.",
"Such volatile material may, for example, be perfume commonly used in detergent material.",
"In case of the preferred use of the inventive packaging, namely for water-soluble sachets containing detergent material, the tight packaging of the present invention also avoids “drying out” of the sachets, i.e.",
"loss of water or other liquids from the material contained therein by migration across the film material of the sachets.",
"It is essential to at least fix one portion of the mouth of the inner bag to the lid cover and another portion to a sidewall of the container to allow opening of the inner bag by lifting the lid cover.",
"For stability of the package, the inner bag filled with the moisture-sensitive material will preferably be close to or even at least partly supported on the bottom of the container in its closed state.",
"It is, however, essential for the invention that the lower part of the inner bag will be substantially lifted from the bottom when lifting the lid cover for opening the package.",
"Only then, it can be guaranteed that the weight of the contents of the inner bag, by gravitational effect, will draw back the lid cover to its closed state when released by the user.",
"There are no specific restrictions for the materials used for the outer container and the inner bag of the package of the present invention with the exception that the inner bag should be made of water-impermeable flexible sheet material to allow protection of the contents and, by the form of a loose bag, to function according to the invention.",
"Such water-impermeable sheet material may be polyethylene or any other polymeric sheet material with comparable characteristics.",
"The outer container may be made out of cardboard material although not restricted thereto.",
"In an alternative embodiment to the present invention there is provided packaging for moisture sensitive material comprising an outer container including a container body and a lid movable between an open and a closed position relative to the container body, and an inner container made of water-impermeable material for receiving the moisture sensitive material, the inner container being fixed to both the container body and the lid of the outer container such that when the lid is moved from the closed position to the open position, a lower part of the inner container is lifted relative to its position when the lid is in the closed position such that in use, the lid is biased to the closed position.",
"The lid may be pivotable between the open and the closed position, or it may be slidable between the two positions.",
"The inner container may be comprised of foldable sheet water-impermeable material or from flexible water impermeable material.",
"The invention is explained in more detail by the following examples represented by the drawings wherein FIG.",
"1 shows a cross-sectional view of a packaging according to a first embodiment of the present invention in its closed state; and FIG.",
"2 shows a cross-sectional view of the same packaging in its opened state.",
"FIG.",
"3 shows a perspective view of the same packaging in its opened state.",
"FIG.",
"4 shows a cross-sectional view of a packaging according to a second embodiment of the present invention in its opened state.",
"The packaging of the present invention comprises an outer container 10 comprising a container body consisting of upright sidewalls 11 and a bottom 12.At position 15 at the upper end of one of the sidewalls 11 a lid cover 13 is hingedly fixed thereto.",
"The portion of the lid cover 13 being opposite to the hinged portion 15 is designated in the drawings by reference numeral 14 and"
]
] | Patent_10432765 |
[
[
"System and process for validating, aligning and reordering one or more genetic sequence maps using at least one ordered restriction map",
"A method and system are provided for comparing ordered segments of a first DNA restriction map with ordered segments of a second DNA restriction map to determine a level of accuracy the first DNA map and/or the second DNA map.",
"In particular, the first and second DNA maps can be received (the first DNA map corresponding to a sequence DNA map, and the second DNA map corresponding to a genomic consensus DNA map as provided in an optical DNA map).",
"Then, the accuracy of the first DNA map and/or the second DNA map is validated based on information associated with the first and second DNA maps.",
"In addition, a method and system are provided for aligning a plurality of DNA sequences with a ordered DNA restriction map.",
"The DNA sequences and the DNA map are received (the DNA sequences being fragments of a genome and the DNA map corresponding to a genomic consensus DNA map which relates to an optical ordered DNA map).",
"Then, a level of accuracy of the DNA sequences and the DNA map is obtained based on information associated with the DNA sequences and the DNA map by means of the method and system described above.",
"The locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map are located.",
"Furthermore, it is possible to obtain locations of the DNA map (without the validation) by locating an optimal one of the locations for each of the DNA sequences for each of the locations."
],
[
"1.A process for comparing ordered segments of a first DNA map with ordered segments of a second DNA map to determine a level of accuracy of at least one of the first DNA map and the second DNA map, comprising the steps of: a) receiving the first and second DNA maps, the first DNA map corresponding to a sequence DNA map, the second DNA map corresponding to a genomic consensus DNA map as provided in an ordered restriction DNA map; and b) validating the accuracy of at least one of the first DNA map and the second DNA map based on information associated with the first and second DNA maps.",
"2.The process according to claim 1, wherein the validating step comprises determining whether one or more matches exist between ordered segments of the first DNA map and the ordered segments of the second DNA map.",
"3.The process according to claim 2, wherein the validating step further comprises obtaining a number of the matches which exist between the segments of the first DNA map and the segments of the second DNA map after the determining substep.",
"4.The process according to claim 1, wherein the validating step comprises determining whether the first DNA map includes one or more cuts which are missing from the second DNA map.",
"5.The process according to claim 4, wherein the validating step further comprises obtaining a number and locations of the missing cuts, after the determining substep, based on the first and second DNA maps.",
"6.The process according to claim 1, wherein the validating step comprises determining whether the second DNA map includes one or more cuts which are absent from the first DNA map.",
"7.The process according to claim 6, wherein the validating step further comprises obtaining a number and locations of the absent cuts, after the determining substep, based on the first and second DNA maps.",
"8.The process according to claim 3, wherein the validating step further comprises the substeps of: i. determining whether the first DNA map includes one or more cuts which are missing from the second DNA map, ii.",
"after substep i, obtaining a first number and locations of the missing cuts based on the first and second DNA maps, iii.",
"determining whether the second DNA map includes one or more cuts which are absent from the first DNA map, and iv.",
"after substep iii, obtaining a second number and locations of the absent cuts based on the first and second DNA maps.",
"9.The process according to claim 8, further comprising the step of: c) generating an error indication if at least one of: i. the number of the matches is less than a match threshold, ii.",
"the first number of the missing cuts is greater than a first predetermined threshold, and iii.",
"the second number of the absent cuts is greater than a second predetermined threshold.",
"10.The process according to claim 1, wherein the first DNA map is an in-silico ordered restriction map obtained from a DNA sequence.",
"11.The process according to claim 10, wherein the first DNA map includes identification data and at least one vector of the segments of the first DNA map.",
"12.The process according to claim 11, wherein the at least one vector of the first segments encodes a size of base-pairs of the DNA sequence.",
"13.The process according to claim 12, wherein the second DNA map includes identification data and at least one variable-length vector representing its ordered segments.",
"14.The process according to claim 1, wherein the second DNA map is a subsequence of a genome-wide ordered restriction map of an optical DNA map.",
"15.The process according to claim 1, wherein the validation step comprises determining the accuracy of at least one of the first DNA map and the second DNA map using the following probability density function: Pr(D|Ĥ(σ,pc,pƒ)) where: D is the second DNA map, Ĥ is the first DNA map, σ is a standard deviation summarizing map-wide standard deviation data, pc is a probability of a positive cut of a DNA sequence, and pƒ is a probability of a false-positive cut of the DNA sequence.",
"16.The process according to claim 1, wherein the accuracy is validated as a function of an orientation of the first DNA map with respect to an orientation of the second DNA map.",
"17.The process according to claim 1, wherein the validation step comprises the substeps of: i. executing a dynamic programming procedure (“DPP”) on the first and second DNA maps to generate a first table of partial and complete alignment scores, and first auxiliary tables and first data structures to keep track of number and locations of cuts and segment matches, ii.",
"receiving a third DNA map which is a reverse map of the first DNA map, iii.",
"executing the DPP on the second and third DNA maps to generate a second table of partial and complete alignment scores, and second auxiliary tables and second data structures to keep track of number and locations of the cuts and the segment matches, and iv.",
"analyzing the last row of the first table and a last row of the second table to obtain at least one optimum alignment of the first and second DNA maps, and v. reconstructing at least one of an optimum alignment and sub-optimal alignments using the first and second auxiliary tables and data structures.",
"18.The process according to claim 1, wherein the accuracy is validated by matching an extension of a first left end segment of the segments of the first DNA map to at least one of the segments of the second DNA map.",
"19.The process according to claim 1, wherein the accuracy is validated by matching an extension of a first right end segment of the segments of the first DNA map to at least one of the segments of the second DNA map.",
"20.The process according to claim 1, further comprising the step of: d) detecting an alignment of the first DNA map with respect to the second DNA map, the alignment being indicative of sequence positions of the segments of the first DNA map along the second DNA map.",
"21.A software system which, when executed on a processing device, configures the processing device to compare segments of a first DNA map with segments of a second DNA map to determine a level of accuracy of at least one or both of the first DNA map and the second DNA map, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) receives the first and second DNA maps, the first DNA map corresponding to a sequence DNA map, the second DNA map corresponding to a genomic consensus DNA map as provided in an ordered restriction DNA map, and b) validates the accuracy of at least one of the first DNA map and the second DNA map based on information associated with the first and second DNA maps.",
"22.The software system according to claim 21, wherein, when validating the accuracy, the processing subsystem determines whether one or more matches exists between at least one of the segments of the first DNA map and at least one of the segments of the second DNA map.",
"23.The software system according to claim 22, wherein, when validating the accuracy, the processing subsystem obtains a number of the matches which exist between the segments of the first DNA map and the segments of the second DNA map.",
"24.The software system according to claim 21, wherein, when validating the accuracy, the processing subsystem determines whether the first DNA map includes one or more cuts which are missing from the second DNA map.",
"25.The software system according to claim 4, wherein, when validating the accuracy, the processing subsystem obtains number and location of the missing cuts based on the first and second DNA maps.",
"26.The software system according to claim 21, wherein, when validating the accuracy, the processing subsystem determines whether the second DNA map includes one or more cuts which are absent from the first DNA map.",
"27.The software system according to claim 24, wherein, when validating the accuracy, the processing subsystem obtains number and location of the absent cuts based on the first and second DNA maps.",
"28.The software system according to claim 23, wherein, when validating the accuracy, the processing subsystem: i. determines whether the first DNA map includes one or more cuts which are missing from the second DNA map, ii.",
"obtains number and location of the missing cuts based on the first and second DNA maps, iii.",
"determines whether the second DNA map includes one or cuts which are absent from the first DNA map, and iv.",
"obtains a second number of the absent cuts based on the first and second DNA maps.",
"29.The software system according to claim 28, wherein, when executed on the processing device, the processing subsystem further configures the processing device to generate an error indication if at least one of: i. the number of the matches is less than a match threshold, ii.",
"the first number of the missing cuts is greater than a first predetermined threshold, and iii.",
"the second number of the absent cuts is greater than a second predetermined threshold.",
"30.The software system according to claim 21, wherein the first DNA map is an in-silico ordered restriction map obtained from a DNA sequence.",
"31.The software system according to claim 30, wherein the first DNA map includes identification data and a variable-length vector of the segments of the first DNA map.",
"32.The software system according to claim 31, wherein the vector of the segments of the first DNA map encodes a size of base-pairs of the DNA sequence.",
"33.The software system according to claim 32, wherein the second DNA map includes identification data and a variable-length vector of the segments of the second DNA map.",
"34.The software system according to claim 21, wherein the second DNA map is a genome-wide ordered restriction map of an optical DNA map.",
"35.The software system according to claim 21, wherein, when validating the accuracy, the processing subsystem determines the accuracy of the at least one of the first DNA map and the second DNA map using the following probability density function: Pr(D|Ĥ(σ,pc,pƒ)) where: D is the second DNA map, Ĥ is the first DNA map, σ is a standard deviation summarizing map-wide standard deviation data, pc is a probability of a positive cut of a DNA sequence, and pƒ is a probability of a false-positive cut of the DNA sequence.",
"36.The software system according to claim 21, wherein the accuracy is validated as a function of an orientation of the first DNA map with respect to an orientation of the second DNA map.",
"37.The software system according to claim 21, wherein, when validating the accuracy, the processing subsystem: i. executes a dynamic programming procedure (“DPP”) on the first and second DNA maps to generate a first table of partial and complete alignment scores, and first auxiliary tables and data structures to keep track of the number and locations of cuts and segment matches, ii.",
"receives a third DNA map which is a reverse map of the first DNA map, iii.",
"executes the DPP on the second and third DNA maps to generate a second table of partial and complete alignment scores, and second auxiliary tables and data structures to keep track of the number and locations of cuts and segment matches, iv.",
"analyzes a last row of the first table and a last row of the second table to obtain at least one optimum alignment of the first and second DNA maps, and v. reconstructing at least one of an optimum alignment and sub-optimal alignments using the first and second auxiliary tables and data structures.",
"38.The software system according to claim 21, wherein the accuracy is validated by matching an extension of a first left end segment of the segments of the first DNA map to at least one of the segments of the second DNA map.",
"39.The software system according to claim 21, wherein the accuracy is validated by matching an extension of a first right end segment of the first DNA map to at least one of the segments of the second DNA map.",
"40.The software system according to claim 21, wherein, when executed on the processing device, the processing subsystem further configures the processing device to determine an alignment of the first DNA map with respect to the second DNA map, the alignment being indicative of sequence positions of the first segments along the second DNA map.",
"41.A process for aligning a plurality of DNA sequences with a DNA map, comprising the steps of: a) receiving the DNA sequences and the DNA map, the DNA sequences being fragments of a genome, the DNA map corresponding to a genomic consensus DNA map which relates to an ordered restriction DNA map; b) validating a level of accuracy of at least one of the DNA sequences and the DNA map based on information associated with the DNA sequences and the DNA map; and c) determining locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map.",
"42.The process according to claim 41, wherein the locations are determined for each of the DNA sequences.",
"43.The process according to claim 41, wherein the locations are positions on the DNA map at which the corresponding DNA sequences are anchorable.",
"44.The process according to claim 41, wherein the locations define at least one alignment of the DNA sequences with respect to the DNA map.",
"45.The process according to claim 44, wherein the at least one alignment includes multiple alignments of the DNA sequences with respect to the DNA map.",
"46.The process according to claim 45, further comprising the step of: d) ranking the multiple alignments based on a predetermined criteria to obtain a score set which includes a particular score for each of the multiple alignments.",
"47.The process according to claim 46, wherein the determining step comprises: i. providing the DNA sequences in a first order of the multiple alignments with respect to the DNA map, and ii.",
"determining a position for each of the DNA sequences, with respect to the DNA map, by selecting the DNA sequences to be in a second order corresponding to the score set.",
"48.The process according to claim 41, wherein the determining step comprises restricting each of the DNA sequences to be associated with only one of the locations on the DNA map.",
"49.The process according to claim 48, wherein the determining step produces a single alignment of the DNA sequences with respect to the DNA map.",
"50.The process according to claim 41, wherein the determining step includes: i. locating an optimal one of the locations for each of the DNA sequences to obtain an alignment solution, and ii.",
"repeating substep ii for each of the locations.",
"51.The process according to claim 50, wherein the locating substep is repeated for each subsequent one of the locations and excluding the alignment solution from a preceding locating substep.",
"52.The process according to claim 51, wherein each subsequent locating substep is performed by relaxing at least one particular constraint to determine the respective locations.",
"53.The process according to claim 52, wherein the at least one particular constraint includes a first requirement that two of the DNA sequences are prevented from overlapping when associated with the respective locations on the DNA map.",
"54.The process according to claim 52, wherein the particular constraint includes at least one of: i. a second requirement that a maximum number of the DNA sequences are associated with the respective locations on the DNA map, and ii.",
"a third requirement that an overall score of the alignment of the DNA sequences with respect to the locations on the DNA map is minimized or maximized.",
"55.The process according to claim 54, further comprising the step of: e) assigning respective weighs to the second requirement and the third requirement.",
"56.The process according to claim 41, wherein the ordered restriction DNA map is an optical DNA map.",
"57.A software system which, when executed on a processing device, configures the processing device to align a plurality of DNA sequences with a DNA map, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) receives the DNA sequences and the DNA map, the DNA sequences being fragments of a genome, the DNA map corresponding to a genomic consensus DNA map which relates to an ordered restriction DNA map, b) validates a level of accuracy of at least one of the DNA sequences and the DNA map based on information associated with the DNA sequences and the DNA map, and c) determines locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map.",
"58.The software system according to claim 57, wherein the locations are determined for each of the DNA sequences.",
"59.The software system according to claim 57, wherein the locations are positions on the DNA map at which the corresponding DNA sequences are anchorable.",
"60.The software system according to claim 57, wherein the locations define at least one alignment of the DNA sequences with respect to the DNA map.",
"61.The software system according to claim 60, wherein the at least one alignment includes multiple alignments of the DNA sequences with respect to the DNA map.",
"62.The software system according to claim 61, wherein the processing subsystem, when executed on the processing device, configures the processing device to rank the multiple alignments based on a predetermined criteria to obtain a score set which includes a particular score for each of the multiple alignments.",
"63.The software system according to claim 62, wherein the processing device is configured by the processing subsystem to determine the locations of the DNA sequences by: i. providing the DNA sequences in a first order of the multiple alignments with respect to the DNA map, and ii.",
"determining a position for each of the DNA sequences, with respect to the DNA map, by selecting the DNA sequences to be in a second order corresponding to the score set.",
"64.The software system according to claim 57, wherein the processing device is configured by the processing subsystem to determine the locations on the DNA sequences by restricting each of the DNA sequences to be associated with only one of the locations of the DNA map.",
"65.The software system according to claim 64, wherein the processing device is configured by the processing subsystem to determine the locations of the DNA sequences for producing a single alignment of the DNA sequences with respect to the DNA map.",
"66.The software system according to claim 57, wherein the processing device is configured by the processing subsystem to determine the locations of the DNA sequences by: i. locating an optimal one of the locations for each of the DNA sequences to obtain an alignment solution, and ii.",
"repeating substep ii for each of the locations.",
"67.The software system according to claim 66, wherein the locating of the optimal one of the locations is repeated for each subsequent one of the locations and the alignment solution is excluded from a preceding locating iteration.",
"68.The software system according to claim 67, wherein the processing subsystem configures the processing device to perform each subsequent locating iteration by relaxing at least one particular constraint to determine the respective locations.",
"69.The software system according to claim 68, wherein the at least one particular constraint includes a first requirement that two of the DNA sequences are prevented from overlapping when associated with the respective locations on the DNA map.",
"70.The software system according to claim 68, wherein the particular constraint includes at least one of: i. a second requirement that a maximum number of the DNA sequences are associated with the respective locations on the DNA map, and ii.",
"a third requirement that an overall score of the alignment of the DNA sequences with respect to the locations on the DNA map is minimized or maximized.",
"71.The software system according to claim 70, wherein the processing subsystem, when executed on the processing device, configures the processing device to assign respective weighs to the second requirement and the third requirement.",
"72.The software system according to claim 70, wherein the ordered restriction DNA map is an optical DNA map.",
"73.A process for aligning a plurality of DNA sequences with a DNA map, comprising the steps of: a) receiving the DNA sequences and the DNA map, the DNA sequences being fragments of a genome, the DNA map corresponding to a genomic consensus DNA map which relates to an ordered restriction DNA map; and b) determining locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map, the locations of the DNA sequences being determined by: i. locating an optimal one of the locations for each of the DNA sequences, and ii.",
"repeating substep i for each of the locations.",
"74.A software system which, when executed on a processing device, configures the processing device to align a plurality of DNA sequences with a DNA map, the software system comprising: a processing subsystem which, when executed on the processing device, configures the processing device to perform the following steps: a) receives the DNA sequences and the DNA map, the DNA sequences being fragments of a genome, the DNA map corresponding to a genomic consensus DNA map which relates to an ordered restriction DNA map, and b) determines locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map by: i. locating an optimal one of the locations for each of the DNA sequences, and ii.",
"repeating the locating of the optimal one of the locations for each of the locations."
],
[
"<SOH> BACKGROUND INFORMATION <EOH>The sequence of nucleotide bases present in strands of nucleotides, such as DNA and RNA, carries the genetic information encoding proteins and RNAs.",
"The ability to accurately determine a nucleotide sequence is crucial to many areas in molecular biology.",
"For example, the study of genetics relies on complete nucleotide sequences of the organism.",
"Many efforts have been made to generate complete nucleotide sequences for various organisms, including humans, mice, worms, flies and microbes.",
"There are a variety of well-known methods to sequence nucleotides, including the Sanger dideoxy chain termination sequencing technique and the Maxam-Gilbert chemical sequencing technique.",
"However, the current technology limits the length of a nucleotide sequence that may be sequenced.",
"Techniques have been developed to sequence larger nucleotide sequences.",
"In general, these methods involve fragmenting the large sequence into fragments, cloning the fragments, and sequencing the cloned fragments.",
"The sequences can be fragmented through the use of restriction enzymes or mechanical shearing.",
"Cloning techniques include the use of cloning vectors such as cosmids, bacteriophage, and yeast or bacterial artificial chromosomes (YAC or BAC).",
"The nucleotide sequence of the fragments can then be compared, overlapping regions identified, and the sequences assembled to form “contigs,” which are sets of overlapping clones.",
"By assembling the overlapping clones, it is possible to determine the sequence of nucleotide bases of the full length sequence.",
"These methods are well known to those having ordinary skill in the art.",
"The accuracy of nucleotide sequence data is limited by numerous factors.",
"For example, there may be missing sections due to incomplete representation of the genomic DNA.",
"There may also be spurious DNA sequences intermixed with the desired genomic DNA.",
"Common sources of contamination are vector-derived DNA and host cell DNA.",
"Also, the accuracy of the identification of bases tends to degrade toward the end of long sequence reads.",
"Additionally, repeated sequences can create errors in the re-assembly and/or the mismatching of contigs.",
"In order to reduce the sequence data errors, sequencing of the fragments is generally performed multiple times.",
"To help reduce errors such as mismatching or misassembly resulting from repeated sequences, the “hierarchical shotgun sequencing” approach (also referred to as “map-based,” “BAC-based” or “clone by clone”) can be used.",
"This approach involves generating and organizing a set of large insert clones covering the genome and separately performing shotgun sequencing on appropriately selected clones.",
"Because the sequence information is local, the issue of long-range misassembly is eliminated and the risk of short-range misassembly is reduced.",
"Other known sequencing and characterization techniques involve generating restriction fragment fingerprints to determine whether close overlaps are present, thereby assembling the BACs into fingerprint clone contigs.",
"Fingerprint clone contigs can be positioned along the chromosome by anchoring them with sequence-tagged sites (STS) markers from existing genetic and physical maps.",
"These fingerprint clone contigs can be associated with specific STSs by probe hybridization or direct search of the sequenced clones.",
"Clones can also be positioned by fluorescence in situ hybridization.",
"Each of these known techniques are costly and time consuming.",
"Another approach for characterizing nucleotide sequences involves the use of ordered restriction maps of single molecules.",
"One specific technique used to produce single molecule ordered restriction maps is “Optical Mapping”.",
"Optical mapping is a single molecule methodology for the rapid production of ordered restriction maps from individual DNA molecules.",
"Ordered restriction maps are preferably constructed using fluorescence microscopy to visualize restriction endonuclease cutting events on individual fluorochrome-stained DNA molecules.",
"Restriction enzyme cleavage sites are visible as gaps that appear flanking the relaxed DNA fragments (pieces of molecules between two consecutive cleavages).",
"Relative fluorescence intensity (measuring the amount of fluorochrome binding to the restriction fragment) or apparent length measurements (along a well-defined “backbone” spanning the restriction fragment) have proven to provide accurate size-estimates of the restriction fragment and have been used to construct the final restriction map.",
"Such restriction map created from one individual DNA molecule is limited in its accuracy by the resolution of the microscopy, the imaging system (CCD camera, quantization level, etc.",
"), illumination and surface conditions.",
"Furthermore, depending on the digestion rate and the noise inherent to the intensity distribution along the DNA molecule, with some probability, one is likely to miss a small fraction of the restriction sites or introduce spurious sites.",
"Additionally, investigators may sometimes (rather infrequently) lack the exact orientation information (whether the left-most restriction site is the first or the last).",
"Thus, given two arbitrary single molecule restriction maps for the same DNA clone obtained this way, the maps are expected to be roughly the same in the following sense—if the maps are “aligned” by first choosing the orientation and then identifying the restrictions sites that differ by small amount, then most of the restrictions sites will appear roughly at the same place in both the maps.",
"For instance, in the original method, fluorescently-labeled DNA molecules were elongated in a flow of molten agarose containing restriction endonucleases, generated between a cover-slip and a microscope slide, and the resulting cleavage events were recorded by fluorescence microscopy as time-lapse digitized images.",
"The second generation optical mapping approach, which dispensed with agarose and time-lapsed imaging, involves fixing elongated DNA molecules onto positively-charged glass surfaces, thus improving sizing precision as well as throughput for a wide range of cloning vectors (cosmid, bacteriophage, and yeast or bacterial artificial chromosomes (YAC or BAC)).",
"A DNA sequence map is an “in silico” order restriction map that is obtained for a nucleotide sequence by simulating a restriction enzyme digestion process.",
"The sequence data is analyzed and restriction sites are identified in a predetermined manner.",
"The resulting sequence map has some piece of identification data plus a vector of fragments, whose elements encode the size in base-pairs.",
"Sequenced clones can be associated with fingerprint clone contigs in the physical map by using the sequence data to calculate a partial list of restriction fragments in silico and comparing that list with the experimental database of BAC fingerprints.",
"Genomic consensus maps are generated from optical maps using, e.g., “Gentig” software which is a conventional software that generates optical ordered restriction maps.",
"It was previously unknown how to determine the accuracy of the DNA sequence maps.",
"Indeed such determination was either impossible or provided a small level of surety.",
"It is one of the objects of the present invention to enable a validation of the DNA ordered sequence maps against the optical maps.",
"Another object of the present invention is to enable an alignment and reordering of the DNA sequence maps based on the optical mapping.",
"Approaches to aligning or reconstructing restriction maps have been described in E. W. Myers et al., “An O(N2 lg N) Restriction Map Comparison and Search Algorithm”, Bulletin of Mathematical Biology, 54(4):599-618, 1992; R. M. Karp et al., “Algorithms for Optical Mapping”, RECOMB 98, 1998; Parida, L., A Uniform Framework for Ordered Restriction Map Problems, Journal of Computational Biology, Vol 5, No 4, Mary Ann Liebert Inc. Publishers, pp 725-739, 1998; Gusfield, D., Algorithms on Strings, Trees, and Sequences, Cambridge University Press, 1997; and Lee, J. K., Dancik, V., and M. S. Waterman, “Estimation for restriction sites observed by optical mapping using reversible-jump Markov Chain Monte Carlo”, J. Comp.",
"Biol., 5, 505-516, 1997.However, none of these publications disclose the novel processes and systems described herein below."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In general, an exemplary embodiment of the system and process for validating and aligning the simulated ordered restriction map against the optical ordered restriction map according to the present invention can be implemented as follows.",
"First, each molecule may be cut in several places using a digestion process by one or more restriction enzymes as is known to those having ordinary skill in the art.",
"Each of these “cut” molecules can represent a partial DNA (optical) ordered restriction map.",
"Then, it is possible to reconstruct a complete Genome Wide (optical) ordered restriction map.",
"Such reconstruction process can be carried out by an iterative process which maximizes the likelihood of a plausible hypothesis given the partial map and the model of the error sources (e.g., a Bayesian-based process).",
"It should be understood that the inputs to the Validation/Alignment system and process are preferably restriction maps (which include DNA sequences therein) and Genome wide (e.g.",
"optical) ordered restriction maps (which can be represented as variable length vectors of segment/fragment information fields).",
"Each segment information has two pieces of information associated therewith: size and standard deviation.",
"The size may be a measure of the segment, which is proportional to the number of nucleotides present in the segment.",
"The standard deviation preferably represents the error associated with the segment size measurement.",
"Each map has associated therewith, e.g., two measures of how reliable the detection of cuts by the procedure is, i.e., the false positive probability and the digestion probability.",
"The first measure relates to the event that the cut is detected incorrectly.",
"The second measure relates to the event that the cut actually appears where it is reported.",
"According to the present invention, the optical and simulated ordered restriction maps are compared to one another to determine whether and to what extent they match.",
"The accuracy of a match is computed by minimizing the error committed by matching one map against the other at a given position.",
"An exemplary mathematical model and procedure underlying this computation is preferably a Bayesian-based procedure/algorithm.",
"The computation is based on a Dynamic Programming Procedure (“DPP”).",
"However, it should be understood that other procedures and algorithms can be utilized to compare to one another these maps to validate and align at least one such map, according to the present invention.",
"Using the Bayesian-based exemplary procedure with the system and method of the present invention, hypothesis can be obtained and the probability of a given event (based on the hypothesis) may be formulated.",
"This probability is preferably a mathematical formula, which is then computed using a conventional model of various error sources.",
"An exemplary optimization process which uses such formula may maximize or minimize the formula.",
"In order to find the extreme value of the overall probability formula over all possible combinations of matches, a conventional DPP can be used on the problem which was defined by the Bayesian-based exemplary procedure as described above.",
"For example, the DPP may preferably compute a set of extreme values for a mathematical formula defined above by extending a partial solution in a predetermined manner while keeping track of a particular number of alternatives.",
"All of the alternatives may be maintained in a table, and thus do not have to be recomputed every time the associated likelihood or score function needs to be evaluated.",
"Accordingly, a method and system according to the present invention are provided for comparing ordered segments of a first DNA map with ordered segments of a second DNA map to determine a level of accuracy the first DNA map and/or the second DNA map.",
"In particular, the first and second DNA maps can be received (the first DNA map corresponding to a sequence DNA map, and the second DNA map corresponding to a genomic consensus DNA map as provided in an optical DNA map).",
"Then, the accuracy of the first DNA map and/or the second DNA map is validated based on information associated with the first and second DNA maps.",
"In another embodiment of the present invention, the first DNA map and/or the second DNA map are validated by determining whether one or more matches exist between ordered segments of the first DNA map and the ordered segments of the second DNA map.",
"In addition, a number of the matches which exist between the segments of the first DNA map and the segments of the second DNA map can be obtained.",
"In yet another embodiment of the present invention, the first DNA map and/or the second DNA map are validated by determining whether the first DNA map includes one or more cuts which are missing from the second DNA map.",
"Also, a number and locations of the missing cuts based on the first and second DNA maps can be obtained thereafter.",
"According to a further embodiment of the present invention, the first DNA map and/or the second DNA map are validated by determining whether the second DNA map includes one or more cuts which are absent from the first DNA map.",
"The validation can also be performed by determining whether the first DNA map includes one or more cuts which are missing from the second DNA map, obtaining a first number and locations of the missing cuts based on the first and second DNA maps, determining whether the second DNA map includes one or cuts which are absent from the first DNA map, and obtaining a second number and locations of the absent cuts based on the first and second DNA maps.",
"Furthermore, it is possible to generate an error indication if the number of the matches is less than a match threshold, the first number of the missing cuts is greater than a first predetermined threshold, and/or the second number of the absent cuts is greater than a second predetermined threshold.",
"In another embodiment of the present invention, the first DNA map is an in-silico ordered restriction map obtained from a DNA sequence, which may include identification data and at least one vector of the segments of the first DNA map.",
"At least one vector of the first segments can encode a size of base-pairs of the DNA sequence.",
"Further, the second DNA map can include identification data and at least one variable-length vector representing its ordered segments.",
"In still another embodiment of the present invention, the second DNA map is defined as a subsequence of a genome-wide ordered restriction map.",
"Also, the validation is performed by determining the accuracy of at least one of the first DNA map and the second DNA map using the following probability density function: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"Pr ( D|Ĥ (σ, p c ,p ƒ )) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"where D is the second DNA map, Ĥ is the first DNA map, σ is a standard deviation summarizing map-wide standard deviation data, p c is a probability of a positive cut of a DNA sequence, and p ƒ is a probability of a false-positive cut of the DNA sequence.",
"In another embodiment of the present invention, the accuracy can be validated as a function of an orientation of the first DNA map with respect to an orientation of the second DNA map.",
"Also, the validation can be performed by executing a dynamic programming procedure (“DPP”) on the first and second DNA maps to generate a first table of partial and complete alignment scores, and first auxiliary tables and first data structures to keep track of number and locations of cuts and segment matches, receiving a third DNA map which is a reverse map of the first DNA map, executing the DPP on the second and third DNA maps to generate a second table of partial and complete alignment scores, and second auxiliary tables and second data structures to keep track of number and locations of the cuts and the segment matches, analyzing a last row of the first table and a last row of the second table to obtain at least one optimum alignment of the first and second DNA maps, and reconstructing an optimum alignment and/or sub-optimal alignments using the first and second auxiliary tables and data structures.",
"According to still another embodiment of the present invention, the accuracy can be validated by matching an extension of one or more left end segment of the segments of the first DNA map to at least one segment of the second DNA map and/or by matching an extension of one or more right end segment of the segments of the first DNA map to at least one segment of the second DNA map.",
"Furthermore, it is possible to detect an alignment of the first DNA map with respect to the second DNA map, the alignment being indicative of sequence positions of the segments of the first DNA map along the second DNA map.",
"In addition, other embodiments of the process and system according to the present invention are provided for aligning a plurality of DNA sequences with a DNA map.",
"First, the DNA sequences and the DNA map can be received (the DNA sequences being fragments of a genome and the DNA map corresponding to a genomic consensus DNA map which relates to an ordered restriction—e.g.",
"optical—DNA map).",
"Then, a level of accuracy of the DNA sequences and the DNA map is validated based on information associated with the DNA sequences and the DNA map.",
"The locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map are located.",
"Furthermore, it is possible to obtain locations of the DNA map (without the validation) by locating an optimal one of the locations for each of the DNA sequences for each of the locations.",
"In another embodiment of the present invention, the locations are determined for each of the DNA sequences, they may be positions on the DNA map at which the corresponding DNA sequences are anchorable, and these locations can define at least one alignment of the DNA sequences with respect to the DNA map.",
"The alignment may include multiple alignments of the DNA sequences with respect to the DNA map, and the multiple alignments may be ranked based on a predetermined criteria to obtain a score set which includes a particular score for each of the multiple alignments.",
"The determination may be performed by providing the DNA sequences in a first order of the multiple alignments with respect to the DNA map and determining a position for each of the DNA sequences, with respect to the DNA map, by selecting the DNA sequences to be in a second order corresponding to the score set.",
"In still another embodiment of the present invention, the determination of the locations can be performed by restricting each of the DNA sequences to be associated with only one of the locations on the DNA map.",
"Also, such determination may produce a single alignment of the DNA sequences with respect to the DNA map.",
"In yet another embodiment of the present invention, the determination can be performed by locating an optimal one of the locations for each of the DNA sequences to obtain an alignment solution for each of the locations.",
"Also, the locating of the optimal location may be repeated for each subsequent one of the locations and excluding the alignment solution from a preceding locating procedure.",
"Furthermore, each subsequent locating procedure can be made by relaxing at least one particular constraint to determine the respective locations.",
"The particular constraint preferably includes a first requirement that two of the DNA sequences are prevented from overlapping when associated with the respective locations on the DNA map.",
"The particular constraint can include a second requirement that a maximum number of the DNA sequences are associated with the respective locations on the DNA map, and a third requirement that an overall score of the alignment of the DNA sequences with respect to the locations on the DNA map is minimized or maximized.",
"It is also possible to assign respective weighs to the second requirement and the third requirement."
],
[
"FIELD OF THE INVENTION The present invention relates to a system and process for a sequence validation based on at least one ordered restriction map, and more particularly to validating, aligning and/or reordering one or more genetic sequence maps (e.g., ordered restriction enzyme DNA maps) using such ordered restriction map via map matching and comparison.",
"BACKGROUND INFORMATION The sequence of nucleotide bases present in strands of nucleotides, such as DNA and RNA, carries the genetic information encoding proteins and RNAs.",
"The ability to accurately determine a nucleotide sequence is crucial to many areas in molecular biology.",
"For example, the study of genetics relies on complete nucleotide sequences of the organism.",
"Many efforts have been made to generate complete nucleotide sequences for various organisms, including humans, mice, worms, flies and microbes.",
"There are a variety of well-known methods to sequence nucleotides, including the Sanger dideoxy chain termination sequencing technique and the Maxam-Gilbert chemical sequencing technique.",
"However, the current technology limits the length of a nucleotide sequence that may be sequenced.",
"Techniques have been developed to sequence larger nucleotide sequences.",
"In general, these methods involve fragmenting the large sequence into fragments, cloning the fragments, and sequencing the cloned fragments.",
"The sequences can be fragmented through the use of restriction enzymes or mechanical shearing.",
"Cloning techniques include the use of cloning vectors such as cosmids, bacteriophage, and yeast or bacterial artificial chromosomes (YAC or BAC).",
"The nucleotide sequence of the fragments can then be compared, overlapping regions identified, and the sequences assembled to form “contigs,” which are sets of overlapping clones.",
"By assembling the overlapping clones, it is possible to determine the sequence of nucleotide bases of the full length sequence.",
"These methods are well known to those having ordinary skill in the art.",
"The accuracy of nucleotide sequence data is limited by numerous factors.",
"For example, there may be missing sections due to incomplete representation of the genomic DNA.",
"There may also be spurious DNA sequences intermixed with the desired genomic DNA.",
"Common sources of contamination are vector-derived DNA and host cell DNA.",
"Also, the accuracy of the identification of bases tends to degrade toward the end of long sequence reads.",
"Additionally, repeated sequences can create errors in the re-assembly and/or the mismatching of contigs.",
"In order to reduce the sequence data errors, sequencing of the fragments is generally performed multiple times.",
"To help reduce errors such as mismatching or misassembly resulting from repeated sequences, the “hierarchical shotgun sequencing” approach (also referred to as “map-based,” “BAC-based” or “clone by clone”) can be used.",
"This approach involves generating and organizing a set of large insert clones covering the genome and separately performing shotgun sequencing on appropriately selected clones.",
"Because the sequence information is local, the issue of long-range misassembly is eliminated and the risk of short-range misassembly is reduced.",
"Other known sequencing and characterization techniques involve generating restriction fragment fingerprints to determine whether close overlaps are present, thereby assembling the BACs into fingerprint clone contigs.",
"Fingerprint clone contigs can be positioned along the chromosome by anchoring them with sequence-tagged sites (STS) markers from existing genetic and physical maps.",
"These fingerprint clone contigs can be associated with specific STSs by probe hybridization or direct search of the sequenced clones.",
"Clones can also be positioned by fluorescence in situ hybridization.",
"Each of these known techniques are costly and time consuming.",
"Another approach for characterizing nucleotide sequences involves the use of ordered restriction maps of single molecules.",
"One specific technique used to produce single molecule ordered restriction maps is “Optical Mapping”.",
"Optical mapping is a single molecule methodology for the rapid production of ordered restriction maps from individual DNA molecules.",
"Ordered restriction maps are preferably constructed using fluorescence microscopy to visualize restriction endonuclease cutting events on individual fluorochrome-stained DNA molecules.",
"Restriction enzyme cleavage sites are visible as gaps that appear flanking the relaxed DNA fragments (pieces of molecules between two consecutive cleavages).",
"Relative fluorescence intensity (measuring the amount of fluorochrome binding to the restriction fragment) or apparent length measurements (along a well-defined “backbone” spanning the restriction fragment) have proven to provide accurate size-estimates of the restriction fragment and have been used to construct the final restriction map.",
"Such restriction map created from one individual DNA molecule is limited in its accuracy by the resolution of the microscopy, the imaging system (CCD camera, quantization level, etc.",
"), illumination and surface conditions.",
"Furthermore, depending on the digestion rate and the noise inherent to the intensity distribution along the DNA molecule, with some probability, one is likely to miss a small fraction of the restriction sites or introduce spurious sites.",
"Additionally, investigators may sometimes (rather infrequently) lack the exact orientation information (whether the left-most restriction site is the first or the last).",
"Thus, given two arbitrary single molecule restriction maps for the same DNA clone obtained this way, the maps are expected to be roughly the same in the following sense—if the maps are “aligned” by first choosing the orientation and then identifying the restrictions sites that differ by small amount, then most of the restrictions sites will appear roughly at the same place in both the maps.",
"For instance, in the original method, fluorescently-labeled DNA molecules were elongated in a flow of molten agarose containing restriction endonucleases, generated between a cover-slip and a microscope slide, and the resulting cleavage events were recorded by fluorescence microscopy as time-lapse digitized images.",
"The second generation optical mapping approach, which dispensed with agarose and time-lapsed imaging, involves fixing elongated DNA molecules onto positively-charged glass surfaces, thus improving sizing precision as well as throughput for a wide range of cloning vectors (cosmid, bacteriophage, and yeast or bacterial artificial chromosomes (YAC or BAC)).",
"A DNA sequence map is an “in silico” order restriction map that is obtained for a nucleotide sequence by simulating a restriction enzyme digestion process.",
"The sequence data is analyzed and restriction sites are identified in a predetermined manner.",
"The resulting sequence map has some piece of identification data plus a vector of fragments, whose elements encode the size in base-pairs.",
"Sequenced clones can be associated with fingerprint clone contigs in the physical map by using the sequence data to calculate a partial list of restriction fragments in silico and comparing that list with the experimental database of BAC fingerprints.",
"Genomic consensus maps are generated from optical maps using, e.g., “Gentig” software which is a conventional software that generates optical ordered restriction maps.",
"It was previously unknown how to determine the accuracy of the DNA sequence maps.",
"Indeed such determination was either impossible or provided a small level of surety.",
"It is one of the objects of the present invention to enable a validation of the DNA ordered sequence maps against the optical maps.",
"Another object of the present invention is to enable an alignment and reordering of the DNA sequence maps based on the optical mapping.",
"Approaches to aligning or reconstructing restriction maps have been described in E. W. Myers et al., “An O(N2 lg N) Restriction Map Comparison and Search Algorithm”, Bulletin of Mathematical Biology, 54(4):599-618, 1992; R. M. Karp et al., “Algorithms for Optical Mapping”, RECOMB 98, 1998; Parida, L., A Uniform Framework for Ordered Restriction Map Problems, Journal of Computational Biology, Vol 5, No 4, Mary Ann Liebert Inc. Publishers, pp 725-739, 1998; Gusfield, D., Algorithms on Strings, Trees, and Sequences, Cambridge University Press, 1997; and Lee, J. K., Dancik, V., and M. S. Waterman, “Estimation for restriction sites observed by optical mapping using reversible-jump Markov Chain Monte Carlo”, J. Comp.",
"Biol., 5, 505-516, 1997.However, none of these publications disclose the novel processes and systems described herein below.",
"SUMMARY OF THE INVENTION In general, an exemplary embodiment of the system and process for validating and aligning the simulated ordered restriction map against the optical ordered restriction map according to the present invention can be implemented as follows.",
"First, each molecule may be cut in several places using a digestion process by one or more restriction enzymes as is known to those having ordinary skill in the art.",
"Each of these “cut” molecules can represent a partial DNA (optical) ordered restriction map.",
"Then, it is possible to reconstruct a complete Genome Wide (optical) ordered restriction map.",
"Such reconstruction process can be carried out by an iterative process which maximizes the likelihood of a plausible hypothesis given the partial map and the model of the error sources (e.g., a Bayesian-based process).",
"It should be understood that the inputs to the Validation/Alignment system and process are preferably restriction maps (which include DNA sequences therein) and Genome wide (e.g.",
"optical) ordered restriction maps (which can be represented as variable length vectors of segment/fragment information fields).",
"Each segment information has two pieces of information associated therewith: size and standard deviation.",
"The size may be a measure of the segment, which is proportional to the number of nucleotides present in the segment.",
"The standard deviation preferably represents the error associated with the segment size measurement.",
"Each map has associated therewith, e.g., two measures of how reliable the detection of cuts by the procedure is, i.e., the false positive probability and the digestion probability.",
"The first measure relates to the event that the cut is detected incorrectly.",
"The second measure relates to the event that the cut actually appears where it is reported.",
"According to the present invention, the optical and simulated ordered restriction maps are compared to one another to determine whether and to what extent they match.",
"The accuracy of a match is computed by minimizing the error committed by matching one map against the other at a given position.",
"An exemplary mathematical model and procedure underlying this computation is preferably a Bayesian-based procedure/algorithm.",
"The computation is based on a Dynamic Programming Procedure (“DPP”).",
"However, it should be understood that other procedures and algorithms can be utilized to compare to one another these maps to validate and align at least one such map, according to the present invention.",
"Using the Bayesian-based exemplary procedure with the system and method of the present invention, hypothesis can be obtained and the probability of a given event (based on the hypothesis) may be formulated.",
"This probability is preferably a mathematical formula, which is then computed using a conventional model of various error sources.",
"An exemplary optimization process which uses such formula may maximize or minimize the formula.",
"In order to find the extreme value of the overall probability formula over all possible combinations of matches, a conventional DPP can be used on the problem which was defined by the Bayesian-based exemplary procedure as described above.",
"For example, the DPP may preferably compute a set of extreme values for a mathematical formula defined above by extending a partial solution in a predetermined manner while keeping track of a particular number of alternatives.",
"All of the alternatives may be maintained in a table, and thus do not have to be recomputed every time the associated likelihood or score function needs to be evaluated.",
"Accordingly, a method and system according to the present invention are provided for comparing ordered segments of a first DNA map with ordered segments of a second DNA map to determine a level of accuracy the first DNA map and/or the second DNA map.",
"In particular, the first and second DNA maps can be received (the first DNA map corresponding to a sequence DNA map, and the second DNA map corresponding to a genomic consensus DNA map as provided in an optical DNA map).",
"Then, the accuracy of the first DNA map and/or the second DNA map is validated based on information associated with the first and second DNA maps.",
"In another embodiment of the present invention, the first DNA map and/or the second DNA map are validated by determining whether one or more matches exist between ordered segments of the first DNA map and the ordered segments of the second DNA map.",
"In addition, a number of the matches which exist between the segments of the first DNA map and the segments of the second DNA map can be obtained.",
"In yet another embodiment of the present invention, the first DNA map and/or the second DNA map are validated by determining whether the first DNA map includes one or more cuts which are missing from the second DNA map.",
"Also, a number and locations of the missing cuts based on the first and second DNA maps can be obtained thereafter.",
"According to a further embodiment of the present invention, the first DNA map and/or the second DNA map are validated by determining whether the second DNA map includes one or more cuts which are absent from the first DNA map.",
"The validation can also be performed by determining whether the first DNA map includes one or more cuts which are missing from the second DNA map, obtaining a first number and locations of the missing cuts based on the first and second DNA maps, determining whether the second DNA map includes one or cuts which are absent from the first DNA map, and obtaining a second number and locations of the absent cuts based on the first and second DNA maps.",
"Furthermore, it is possible to generate an error indication if the number of the matches is less than a match threshold, the first number of the missing cuts is greater than a first predetermined threshold, and/or the second number of the absent cuts is greater than a second predetermined threshold.",
"In another embodiment of the present invention, the first DNA map is an in-silico ordered restriction map obtained from a DNA sequence, which may include identification data and at least one vector of the segments of the first DNA map.",
"At least one vector of the first segments can encode a size of base-pairs of the DNA sequence.",
"Further, the second DNA map can include identification data and at least one variable-length vector representing its ordered segments.",
"In still another embodiment of the present invention, the second DNA map is defined as a subsequence of a genome-wide ordered restriction map.",
"Also, the validation is performed by determining the accuracy of at least one of the first DNA map and the second DNA map using the following probability density function: Pr(D|Ĥ(σ,pc,pƒ)) where D is the second DNA map, Ĥ is the first DNA map, σ is a standard deviation summarizing map-wide standard deviation data, pc is a probability of a positive cut of a DNA sequence, and pƒ is a probability of a false-positive cut of the DNA sequence.",
"In another embodiment of the present invention, the accuracy can be validated as a function of an orientation of the first DNA map with respect to an orientation of the second DNA map.",
"Also, the validation can be performed by executing a dynamic programming procedure (“DPP”) on the first and second DNA maps to generate a first table of partial and complete alignment scores, and first auxiliary tables and first data structures to keep track of number and locations of cuts and segment matches, receiving a third DNA map which is a reverse map of the first DNA map, executing the DPP on the second and third DNA maps to generate a second table of partial and complete alignment scores, and second auxiliary tables and second data structures to keep track of number and locations of the cuts and the segment matches, analyzing a last row of the first table and a last row of the second table to obtain at least one optimum alignment of the first and second DNA maps, and reconstructing an optimum alignment and/or sub-optimal alignments using the first and second auxiliary tables and data structures.",
"According to still another embodiment of the present invention, the accuracy can be validated by matching an extension of one or more left end segment of the segments of the first DNA map to at least one segment of the second DNA map and/or by matching an extension of one or more right end segment of the segments of the first DNA map to at least one segment of the second DNA map.",
"Furthermore, it is possible to detect an alignment of the first DNA map with respect to the second DNA map, the alignment being indicative of sequence positions of the segments of the first DNA map along the second DNA map.",
"In addition, other embodiments of the process and system according to the present invention are provided for aligning a plurality of DNA sequences with a DNA map.",
"First, the DNA sequences and the DNA map can be received (the DNA sequences being fragments of a genome and the DNA map corresponding to a genomic consensus DNA map which relates to an ordered restriction—e.g.",
"optical—DNA map).",
"Then, a level of accuracy of the DNA sequences and the DNA map is validated based on information associated with the DNA sequences and the DNA map.",
"The locations of the DNA map at which the DNA sequences are capable of being associated with particular segments of the DNA map are located.",
"Furthermore, it is possible to obtain locations of the DNA map (without the validation) by locating an optimal one of the locations for each of the DNA sequences for each of the locations.",
"In another embodiment of the present invention, the locations are determined for each of the DNA sequences, they may be positions on the DNA map at which the corresponding DNA sequences are anchorable, and these locations can define at least one alignment of the DNA sequences with respect to the DNA map.",
"The alignment may include multiple alignments of the DNA sequences with respect to the DNA map, and the multiple alignments may be ranked based on a predetermined criteria to obtain a score set which includes a particular score for each of the multiple alignments.",
"The determination may be performed by providing the DNA sequences in a first order of the multiple alignments with respect to the DNA map and determining a position for each of the DNA sequences, with respect to the DNA map, by selecting the DNA sequences to be in a second order corresponding to the score set.",
"In still another embodiment of the present invention, the determination of the locations can be performed by restricting each of the DNA sequences to be associated with only one of the locations on the DNA map.",
"Also, such determination may produce a single alignment of the DNA sequences with respect to the DNA map.",
"In yet another embodiment of the present invention, the determination can be performed by locating an optimal one of the locations for each of the DNA sequences to obtain an alignment solution for each of the locations.",
"Also, the locating of the optimal location may be repeated for each subsequent one of the locations and excluding the alignment solution from a preceding locating procedure.",
"Furthermore, each subsequent locating procedure can be made by relaxing at least one particular constraint to determine the respective locations.",
"The particular constraint preferably includes a first requirement that two of the DNA sequences are prevented from overlapping when associated with the respective locations on the DNA map.",
"The particular constraint can include a second requirement that a maximum number of the DNA sequences are associated with the respective locations on the DNA map, and a third requirement that an overall score of the alignment of the DNA sequences with respect to the locations on the DNA map is minimized or maximized.",
"It is also possible to assign respective weighs to the second requirement and the third requirement.",
"BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the present invention and its advantages, reference is now made to the following description, taken in conjunction with the accompanying drawings, in which: FIG.",
"1 is a first exemplary embodiment of a system for validating, aligning and/or reordering a genetic sequence using an optical map via map matching and comparison according the present invention; FIG.",
"2 is a second exemplary embodiment of a system for validating, aligning and/or reordering a genetic sequence using the optical map; FIG.",
"3 is an exemplary embodiment of a validation procedure of a process according to the present invention; FIG.",
"4 is an exemplary embodiment of the process according to the present invention for simulating a restriction digestion of the sequence map, and then validating the accuracy of the consensus optical order restriction map and/or the simulated map; FIG.",
"5A is a detailed flow chart of an exemplary validation technique utilized in the process shown in FIG.",
"4; FIG.",
"5B is a detailed illustration of an exemplary flow diagram of particular steps of FIG.",
"5A in which fragments of the optical ordered restriction map are compared to fragment of the simulated ordered restriction map to obtain one or more set(s) of most likely matches; FIG.",
"6A is a first exemplary illustration of a technique for matching a sequence map against a consensus optical map; FIG.",
"6B is a second exemplary illustration of the technique for matching the sequence map against the consensus optical map in which the consensus optical map does not possess any false enzyme cuts and the sequence map does not have any missing enzyme cut(s); FIG.",
"6C is a third exemplary illustration of the technique for matching the sequence map against the consensus optical map in which the consensus optical map does not possess any false enzyme cuts while the sequence map is missing the enzyme cut(s); FIG.",
"6D is a fourth exemplary illustration of the technique for matching the sequence map against the consensus optical map in which the consensus optical map has a false enzyme cut and the sequence map does not have any missing enzyme cuts; FIG.",
"6E is a fifth exemplary illustration of the technique for matching the sequence map against the consensus optical map in which the consensus optical map has a false enzyme cut and the sequence map is missing the enzyme cut; FIG.",
"6F is a sixth exemplary illustration of the technique for matching the sequence map against the consensus optical map in which left fragments of each of the consensus optical and sequence maps are mismatched; FIG.",
"6G is a sixth exemplary illustration of the technique for matching the sequence map against the consensus optical map in which right fragments of each of the consensus optical and sequence maps are mismatched; FIG.",
"7 is a detailed illustration of the exemplary flow diagram of the validation procedure according to the present invention which utilizes dynamic programming principles and the sequence and consensus maps illustrated in FIGS.",
"6F and 6G; FIG.",
"8 is an exemplary embodiment of the process according to the present invention in which an alignment of a simulated order restricted map takes place after (or during) the validation technique has been implemented to determine the accuracy of the simulated order restricted map(s) and/or the consensus optical map(s); FIG.",
"9 is a detailed illustration of the flow diagram of the process show in FIG.",
"8; FIG.",
"10 is a flow diagram of a particular set of steps in the process illustrated in FIG.",
"9 in which best matches are selected for each sequence map and an overall alignment thereof is constructed; and FIG.",
"11 is an illustration of an example of a possible alignment of a chromosome arrangement using the system and process of the present invention.",
"DETAILED DESCRIPTION FIG.",
"1 illustrates a first exemplary embodiment of a system for validating, aligning and/or reordering a genetic sequence using an optical (consensus) map via map matching and comparison according to the present invention.",
"In this embodiment, the system includes a processing device 10 which is connected to a communications network 100 (e.g., the Internet) so that it can receive optical sequence mapping data and DNA sequence data.",
"The processing device 10 can be a mini-computer (e.g., Hewlett Packard mini computer), a personal computer (e.g., a Pentium chip-based computer), a mainframe (e.g., IBM 3090 system), and the like.",
"The DNA sequence data can be provided from a number of sources.",
"For example, this data can be GenBank Data 110 obtained from GenBank database (NIH genetic sequence database), Sanger Data 120 obtained from Sanger Center database, and/or Celera Data 130 obtained from the Celera Genomics database.",
"These are publicly available genetic databases, or—in the last case—private commercial genetic databases.",
"The optical sequence mapping data correspond to optical mapping data 140 that can obtained from external systems.",
"For example, such optical map data, i.e., optical mapping ordered restriction data, can be generated using the methods described in U.S. Pat.",
"No.",
"6,174,671, the entire disclosure of which is incorporated herein by reference.",
"In particular, the methods described in this U.S. patent produce high-resolution, high accuracy ordered restriction maps based on data created from images of populations of individual DNA molecules digested by restriction enzymes.",
"As shown in FIG.",
"1, after the processing device 10 receives the optical mapping data and the DNA sequence data via the communications network 100, it can then generate one or more results 20 which can be a validation/determination of the accuracy of the DNA sequence data and/or of the optical mapping data, an alignment of the DNA sequence data based on the results of the validation procedure, and reordering thereof FIG.",
"2 illustrates another embodiment of the system 10 according to the present invention in which the optical mapping data 140 is transmitted to the system 10 directly from an external source, without the use of the communications network 100 for such transfer of the data.",
"In this second embodiment of the system as shown in FIG.",
"2, the DNA sequence data 110, 120, 130 is also transmitted directly from the one or more of the DNA sequence databases (e.g., the Sanger Center database, the Celera Genomics database and/or the GenBank database), without the need to use the communications network 100 shown in the first embodiment of FIG.",
"1.It is also possible for the optical mapping data 140 to be obtained from a storage device provided in or connected to the processing device 10.Such storage device can be a hard drive, a CD-ROM, etc.",
"which are known to those having ordinary skill in the art.",
"A. Validation Process and System General Flow Diagram FIG.",
"3 is an exemplary embodiment of the process according to the present invention which is preferably executed by the processing device 10 of FIGS.",
"1 and 2.In this exemplary embodiment, the optical mapping data 140 is forwarded to a technique 250 which constructs one or more consensus maps 260, based on this data 140 by considering the local variations among aligned single molecule maps.",
"One example of such technique 250 is a “gentig” computer program as described in T. Anantharaman et al., “Genomics via Optical Mapping II: Ordered Restriction Maps”, Journal of Computational Biology, 4(2), 1997, pp.",
"91-118, and T. Anantharaman et al., “Genomics via Optical Mapping III: Contiging Genomic DNA and Variations”, AAAI Press, 7th International Conference on Intelligent Systems for Molecular Biology, ISMB 99, Vol.",
"7, 1999, pp.",
"18-27, the entire disclosure of which are incorporated herein by reference.",
"In particular, “gentig” software uses a Bayesian-based (probabilistic) approach to automatically generate “contigs” from optical mapping data.",
"For example, “contigs” can be assembled over whole microbial genomes.",
"The “gentig” software repeatedly combines two islands that produce the greatest increase in probability density, excluding any “contigs” whose false positive overlap probability are unacceptable.",
"For example, four parameters in the program can be altered to change the number of molecules that the program “contigs” together, thus forming the consensus maps.",
"The details of the consensus maps shall be described herein below in further details.",
"According to the exemplary embodiment of the present invention, the DNA sequence data (e.g., the GenBank data 110, the Sanger data 120 and the Celera data 13) can be collected at a database collection junction 200, which can be a computer program executed by the processing device 10.This collection can be initiated and/or controlled either manually (e.g., by a user of the processing device 10 to obtain particular DNA sequences) and/or automatically using the processing device 10 or another external device.",
"Upon the collection of the DNA sequence data from one or more of the DNA sequence databases 110, 120, 130, the database collection junction 200 outputs a particular DNA sequence 210 or a portion of such DNA sequence.",
"Thereafter, the data for this DNA sequence 210 (or a portion thereof) is forwarded to a technique 220 which simulates a restriction enzyme digestion process to generate an “in silico” ordered restriction sequence map 230.Thereafter, the system and process of the present invention executes a validation algorithm 270 which determines the accuracy of the ordered restriction sequence map 230 based on the data provided in the optical consensus map(s) 260.This result can be output as or more results 280 in the form of a response a score (e.g., a rank for each ordered restriction map), a binary output (e.g., the accuracy validated vs. unvalidated), etc.",
"Provided herein below is a detailed information regarding the consensus maps and the sequence maps.",
"Consensus (Optical) Map The consensus optical map can be defined as a genome-wide, ordered restriction map which is represented as a structured item consisting of particular identification data and a variable length vector composed of fragments.",
"For example, the consensus map can be represented by a vector of fragments, where each fragment is a triple of positive real numbers.",
"<ci,li,σi>εR3 and where ci is defined as the cut probability associated with a Bernoulli Trial, li is the fragment size, related to the mean of a random variable with Gaussian distribution having an estimated standard deviation equal to σi.",
"For example, the total length of the fragment vector as can be defined as N. Also, it is possible to define an index the vector of fragments from 0 to N−1.The consensus maps can be created from several long genomic single molecule maps, where each molecule map thereof may be obtained from the images of the molecules stretched on a surface and further combined by a Bayesian algorithm implemented in the “gentig” program.",
"As described above, the “gentig” program is capable of constructing consensus maps by considering local variations among the aligned single molecule maps.",
"Sequence Map As is generally known, a sequence is a string of letters obtained from a set {A, C, G, T, N, X}.",
"These letter have a standard meaning in the art if bio-informatics.",
"In particular, the letters A, C, G, T are DNA bases, N is “unknown”, and X is a “gap”.",
"A sequence map is an “in silico” ordered restriction map obtained from the sequence by simulating a restriction enzyme digestion process.",
"Hence, each sequence map has some piece of identification data plus the vector of fragments, whose elements encode exactly the size in base-pairs.",
"The sequence map fragment vector j-th element is defined as a number aj which is the size of the fragment.",
"The total length of the sequence map fragment vector is defined as M. The fragment vector is indexed from 0 to M−1.Thus, each sequence map has at least a portion of identification data of the DNA sequence data 110, 120, 130, in addition to the vector of fragments whose elements encode exactly the size in base-pairs.",
"The sequence map fragment vector j-th element is indicative of a number aj which corresponds to the size of the fragment.",
"As an example, the total length of the ordered restriction sequence map fragment vector can be M. Thus, the fragment vector can be indexed from 0 to M−1.Overall Process Description FIG.",
"4 shows an exemplary flow chart of the embodiment of the process according to the present invention for simulating a restriction digestion of the sequence map, and then validating the accuracy of the consensus optical order restriction map and/or the simulated ordered map.",
"This process can be performed by the processing device 10 which is shown in FIGS.",
"1 and 2.As shown in this flow chart, the processing device 10 receives the optical ordered restriction data in step 310, which can be the consensus optical map(s) 260 shown in FIG.",
"3.Then, in step 320, the processing device 10 receives the DNA sequence data, which is preferably the DNA sequence 210 which is also shown in FIG.",
"3.In step 330, the restriction digestion of the sequence data is simulated to obtain the simulated (in silico) ordered restriction map which is also shown in FIG.",
"3 as the sequence map(s) 230.Thereafter, in step 340, the accuracy of the optical ordered restriction map and/or of the simulated ordered restriction map is validated, preferably to locate likely matches within one another.",
"Finally, the results of the validation are generated in step 350.Exemplary Embodiment of Validation Procedure of the Exemplary Process FIG.",
"5A shows a detailed flow chart of an embodiment of the exemplary validation procedure utilized in step 340 of the process shown in FIG.",
"4.In particular, a current fragment of the optical ordered restriction map is compared to a respective fragment of the simulated ordered restriction map to obtain one or more set(s) of most likely matches (step 3410).",
"Then, the processing device 10 determines if all fragments of the simulated ordered restriction map were checked in step 3420.If not, the process takes the next fragment of the simulated ordered restriction map to be the current fragment for checking performed in step 3430, and the comparison of step 3410 is repeated again for the current fragment of the simulated ordered restriction map.",
"Otherwise, because it is determined that all fragments of the simulated ordered restriction map were checked, all of the matches are ranked in step 3440, and the processing device 10 determines the best match(s) in step 3450.If the processing device 10 determines that the rank of the best match(s) is greater than a predetermined threshold (step 3460), the processing device 10 validates the accuracy of the optical ordered restriction map and/or of the simulated ordered restriction map (step 3470).",
"Otherwise such accuracy is not validated in step 3480.It should be understood that the exemplary validation procedure shown in FIG.",
"5A can be performed for one or multiple iterations over the fragments.",
"FIG.",
"5B shows a detailed illustration of an exemplary flow diagram of steps 3410-3430 of FIG.",
"5A in which the fragments of the optical ordered restriction map are compared to the fragment of the simulated ordered restriction map to obtain one or more set(s) of most likely matches.",
"Particularly, in step 4010, Probability Pr(D|H(pc,pƒ)), as shall be described in further detail below, is calculated for each possible alignment of the fragments of the optical ordered restriction map (i.e., the consensus map) against fragments of simulated ordered restriction map (i.e., the sequence map).",
"Then, in step 4020, an overall match probability as a maximum likelihood estimate (“MLE”) is calculated by extending the computation over all fragments of the consensus map and all fragments of the sequence map.",
"The exemplary applications of the exemplary embodiment of the process according to the present invention on the sequence and consensus maps are provided in further detail below with reference to FIGS.",
"6A-6G.",
"Statistical Description of the Problem FIG.",
"6A shows an exemplary setup of the matching procedure involving a sequence map (corresponding to the simulated ordered restriction map) and a consensus map (corresponding to the optical ordered restriction map).",
"The sequence map is preferably considered to be an ideal map, i.e., viewed as the hypothesis H of a Bayesian problem to be analyzed, while the consensus map is preferably considered to be of data D to be validated against hypothesis H. In this manner the following probability density function is formed Pr(D|H(,pc,pƒ)), where is a standard deviation which summarizes maps wide standards deviation data (e.g., =ƒ(i) for some function ‘ƒ’), pc is the cut probability, and pƒ is the false positive cut probability.",
"This calculation is shown in FIG.",
"5b and discussed above.",
"Ideal Scenario In an ideal scenario, the orientations of the sequence maps are known, there are no false cuts, and no missing cuts, i.e., pc=1, and pƒ=0, thus the terms associated with these parameters vanish, as it shall be described in further detail below.",
"For example, if a position h in the consensus map is taken, the consensus map fragment sub-vector is provided from the position h to N−1.Also, the full fragment vector of the sequence map can be, e.g., from 0 to M−1.For the sake of simplicity of the explanation of the present invention, it is possible to remove the h position term of the consensus map fragment sub-vector, and count the consensus map fragments from the position term 0 so that expressions such as li, instead of lh+i, can be utilized.",
"To obtain a “match” between the i-th fragments of the consensus map and the corresponding fragments of the sequence map, it is preferable to evaluate to what extent the consensus map and the sequence map deviate from one another.",
"A Gaussian distribution should preferably be utilized for the i-th fragment of each of the maps, and the following expression may be evaluated: 1 2 πσ i 2 ⅇ - ( l i - a j ) 2 2 σ i 2 Given the above expression, and with the assumption that the sequence map is correct (i.e., Pr(H)=1), the overall Pr(D|H(σ, .",
".",
". ))",
"function can be provided as: Pr ( D ❘ H ( , … ) ) = ∏ i = 0 n ( 1 2 π σ i ⅇ - ( l i - a j ) 2 2 σ i 2 ) .",
"To maximize the likelihood of the validation, it is preferable to utilize the logarithm of the simplified expression and obtain the following expression: ln ( Pr ( D ❘ H ( , … ) ) ) = ∑ i = 0 n ln ( 1 2 π σ i 2 ) - ∑ i = 0 n ( ( l i - a j ) 2 2 σ i 2 ) This express maximizes logarithmic likelihood, therefore it provides a Maximum Likelihood Estimate (“MLE”).",
"Since it is possible to assume that the first term of the MLE does not vary extensively from one location to another, it is preferable to simplify the problem by minimizing a “weighted sum-of-error-square” cost function.",
"F ( D ) = ∑ i = 0 n ( ( l i - a i ) 2 2 σ i 2 ) Minimizing function F(D, .",
".",
". )",
"may yield the “best match” of the sequence map (represented as H) against the consensus map (represented as D).",
"According to the present invention, it is preferable to take into account the two possible orientations of the sequence map with respect to the consensus map.",
"Below, false cuts and missing cuts in the consensus map are considered.",
"Orientation Since the sequence map can be evaluated against the consensus map by “reversing” its orientation, the expression for Pr(D, , .",
".",
".",
"|H) can be rewritten as: Pr(D,|H( .",
".",
".",
"))=max[Pr1(D,|H( .",
".",
".",
")),Pr2(D|HR( .",
".",
".",
")], where HR represents the reversed sequence map.",
"As provided previously, it is possible to construct the function F as: F(D,H)=max[F1(D,H),F2(D,HR)].",
"Thus, the expression for F2(D, HR) will be as follows: F 2 ( D , H R ) = ∑ i = 0 n ( ( l i - a ( n - i ) ) 2 2 σ i 2 ) False Cuts and Missing Cuts In order to correctly model errors in the matching process, it is preferable to take into account false cuts and missing cuts.",
"For example, the matching process can be modeled with two parameters: Missing restriction sites in the sequence map are preferably modeled by a probability pc (i.e., a “cut” probability).",
"In particular, pc=1 means that the restriction sites are actually present in the map, 0≦pc<1 means that there are some missing cuts, etc.",
"False restriction sites in the consensus map are preferably modeled by a rate parameter pƒ (i.e., a “false” cut probability).",
"In an exemplary case, 0<pƒ≦1 means that the consensus map may have some false cuts.",
"These parameters should preferably be included in the expression describing Pr( .",
".",
". )",
"and, therefore in the function F( .",
".",
". )",
"described above.",
"EXAMPLE 1 No missing cuts and no false cuts.",
"In this example as shown in FIG.",
"6B, the term for the matching of the i-th fragment of the sequence map 610 against the i-th fragment of the consensus map 620 should preferably take into account the cut probability pc.",
"Thus, the expression is as follows: p c ⨯ 1 2 πσ i 2 ⅇ - ( l i - a j ) 2 2 σ i 2 .",
"which yields the cost function, after taking the negative log likelihood.",
"ln ( 2 π σ i 2 p c ) + ( l i - a j ) 2 2 σ i 2 .",
"EXAMPLE 2 Missing cuts and no false cuts.",
"In this example and as shown in FIG.",
"6C, the exemplary embodiment of the system and method of the present invention considers a cut in the sequence map 630 that has no corresponding cut in the consensus map 610.A match is attempted of the i-th consensus map fragment against the aggregation of the j and j−1 fragments in the sequence map 630.For example, the computation of the Gaussian expression should be “penalized” by taking into account the missing cut.",
"The main term is preferably modeled as: p c ⨯ 1 2 π σ i 2 ⅇ - ( l i - ( a j + a ( j - 1 ) ) ) 2 2 σ i 2 ⨯ ( 1 - p c ) .",
"yielding a cost function: ln ( 2 π σ i p c ) + ( l i - ( a j + a ( j - 1 ) ) ) 2 2 σ i 2 + ln ( 1 1 - p c ) .",
"EXAMPLE 3 No missing cuts and some false cuts.",
"In this case and as shown in FIG.",
"6D, the converse case of Example 2 is being considered.",
"A false cut event of the consensus map 640 can be modeled as a Bernoulli trial with probability pƒ.",
"For example, the full term for such matching would likely aggregate fragments i and i−1 of the consensus map 640 against the j-th fragment of the sequence map 620.The full term would likely be: p c ⨯ 1 2 π ( σ i 2 + σ ( i - 1 ) 2 ) ⅇ - ( ( l i + l ( i - 1 ) ) - a j ) 2 2 ( σ i 2 + σ ( i - 1 ) 2 ) ⨯ p f .",
"Taking the negative log likelihood again, the following expression is obtained: ln ( 2 π ( σ i 2 + σ ( i - 1 ) 2 ) p c ) + ( ( l i + l ( i - 1 ) ) - a j ) 2 2 ( σ i 2 + σ ( i - 1 ) 2 ) + ln ( 1 p f ) .",
"It should be noted that for the current data obtained from the optical mapping process, pƒ 10−5.This current data often dominate the complete expression.",
"EXAMPLE 4 Some missing cuts and some false cuts.",
"Of course, it is conceivable that there may be missing cuts and false cuts together as shown in FIG.",
"6E.",
"It is possible to accurately match or align the i−u cut in the sequence map 660 against the j−v cut in the consensus map 650.It is also possible to properly match the (i+1)-th cut (the cut immediately following the i-th fragment in both the consensus map 650 and the sequence map 660) in the two maps by appropriately treating all the intervening missing cuts in sequence map 660 and all the intervening false cuts in the consensus map 650.In this case, the “matching term” has the following general form: p c ⨯ 1 2 π ( σ i 2 + σ ( i - 1 ) 2 + … + σ ( i - v ) 2 ) ⨯ ⅇ - ( ( ( l i + l ( i - 1 ) + … + l ( i - v ) ) - ( a j + a ( j - 1 ) + … + a ( j - u ) ) ) 2 2 ( σ i 2 + σ ( i - 1 ) 2 + … + σ ( i - v ) 2 ) ) ⨯ ( 1 - p c ) ( u - 1 ) ⨯ p f ( v - 1 ) .",
"Taking the negative log likelihood, the following expression is obtained: - ln p c + ln ( 2 π ( σ i 2 + σ ( i - 1 ) 2 + … + σ ( i - v ) 2 ) ) + ( ( l i + l ( i - 1 ) + … + l ( i - v ) ) - ( a j + a ( j - 1 ) + … + a ( j - u ) ) ) 2 2 ( σ i 2 + σ ( i - 1 ) 2 + … + σ ( i - v ) 2 ) + ( u - 1 ) ln 1 1 - p c + ( v - 1 ) ln 1 p f .",
"B.",
"Dynamic Programming Procedure The validation of a sequence map against the optical map can be implemented as a dynamic programming procedure (“DPP”).",
"Detailed descriptions of the DPP are provided in T. H. Cormen et al., “Introduction to Algorithms”, The MIT Press and McGraw-Hill, 1990, and D. Gusfield, “Algorithms on Strings, Trees, and Sequences”, Cambridge University Press, 1997, the entire disclosures of which is incorporated herein by reference.",
"An exemplary DPP for the process according to the present invention is as follows: Procedure sequence-map-validate (sequence-map, consensus-map)/*Other parameters will be specified .",
".",
".",
"e.g., pƒ, pc, k, etc.",
"*/begin run DPP on consensus-map and sequence map; run DPP on consensus-map and reversed sequence map; collect the k “best” alignments by examining the last row of both DPP tables and “return” them; end This DPP procedure can be executed two or more times.",
"It is improbable for two alignments for the sequence map and for its reversed version to have equivalent scores.",
"It is preferable to start from the DPP's main recurrence to obtain a formulation of the sequence map vs. consensus map matching expression.",
"Dynamic Programming “Main” Recurrence For the description provided below, index i shall be used to indicate a fragment in the consensus map, and the index j to indicate a fragment in the sequence map.",
"Assuming that the consensus map has M fragments and that the sequence map has N fragments, the DPP may preferably utilize a N×M matching table T. Considering the entry T[i, j], this entry will likely contain the partially computed value of the matching function F( .",
".",
".",
").",
"For example, F( .",
".",
". )",
"would be incrementally computed from “left” to “right” by taking into consideration all possible fragment by fragment matches.",
"The main recurrence for entry T[i, j] is provided as follows: T [ i , j ] := min 0 < u ≤ i 0 < v ≤ j ( T [ i - u , j - v ] + ln ( 2 π ( σ i 2 + σ ( i - 1 ) 2 + … + σ ( i - v ) 2 ) p c ) + ( ( l i + l ( i - 1 ) + … + l ( i - v ) ) - ( a j + a ( j - 1 ) + … + a ( j - u ) ) ) 2 2 ( σ i 2 + σ ( i - 1 ) 2 + … + σ ( i - v ) 2 ) + ( u - 1 ) ln 1 1 - p c + ( v - 1 ) ln 1 p f ) .",
"The determination of the respective sizes of u and v should be performed.",
"In one exemplary embodiment of the present invention, the sizes of u and v should preferably depend on i's.",
"In another exemplary embodiment of the present invention, u and v may also depend also on the digestion rate of the “in vivo” experiment that breaks up the DNA molecule.",
"However, a pragmatic bound may be equal to, e.g., 3 times the overall standard deviation (which in practice can be approximated by the value 3).",
"This bound may preferably become a parameter of the DPP.",
"In this way, the computation for each entry T[·,·] should consider approximately 9 neighboring or adjacent entries.",
"A simple model for the initial conditions should preferably be as follows: T[i,0]:=∞, for iε[1,N].",
"T[i,0]:=0, for jε[1,M] In this model, it is preferably to never match or strongly penalize a match of the first fragments of the consensus map against an “inner” fragment of the sequence map (cf.",
"first column having a ∞ value).",
"Also, the match of any fragment of the consensus map can be made against the first fragment of the sequence map rather neutral (with the first two zero values).",
"A more complex model initializes the first row of the dynamic programming table by taking into account, e.g., only the size of the i-th fragment.",
"Provided below is an exemplary description of a complete model for the above-referenced boundary conditions.",
"Left and Right End Fragment Computations.",
"It is possible to provide a more sophisticated and accurate model for the left fragments and right fragments calculations (i.e.",
"for the initial and final conditions).",
"Such models take into consideration the case in which certain fragments on either the left or the right of the sequence map do not “properly match” any fragment in the consensus map.",
"I.",
"Left End Penalty Computation As shown in FIG.",
"6F, the first “matching fragments” are a2 from the sequence map 680, and lj from the consensus map 670, identified by their size.",
"The general case is for fragment i of the sequence map 680 to match fragment j of the consensus map 670.An analysis of the fragment α0 of the sequence map 680 is as follows.",
"Most of the time, the left end of this fragment α0 (which can assume not to be corresponding to an actual restriction site) will fall within the boundaries of fragment i−n of the consensus map 670 (for 0≦n≦i).",
"Within this framework, the minimum value that can be assigned to a “match” of the left end fragments of the sequence map 680 corresponds to one of three cases: Match by extension of the first left end fragment of the sequence map 680.Bad matches until fragment i of the sequence map matches fragments j of the consensus map 670.Match without extension to some fragment in the consensus map 670.EXAMPLE 1 Extending α0 by x leads to a match.",
"If α0 is “extended” by an extra size x (as shown in FIG.",
"6F), x is extended as far to the left as possible to match the cut on the left of fragments i−n (e.g., fragment of size li−2 illustrated in FIG.",
"6F).",
"The value of this match (which is built on top of the derivation performed for the “regular case”) is provided by the following expression: ln ( 2 π ( σ ( i - n ) 2 + σ ( i - ( n - 1 ) ) 2 + … + σ i 2 ) p c ) + ( ( l ( i - n ) + l ( i - ( n - 1 ) ) + … + l i ) - ( x + a 0 + a 1 + … + a j ) ) 2 2 ( σ ( i - n ) 2 + σ ( i - ( n - 1 ) ) 2 + … + σ i 2 ) + x L + ( n - 1 ) ln 1 p f + j ln 1 1 - p c .",
"This case express depends on two parameters which did not appear in the regular case: x being the size extension (please note it in the second and the third term), and L being the molecule map average fragment size.",
"The second sub-term is preferably the regular “sizing error” penalty which takes into account the extension x.",
"The third sub-term may add an extra penalty based on the amount of the end fragment being stretched with respect to the overall structure of the expression.",
"To utilize the expression, it is beneficial to find where its minimum with respect to the position of x.",
"By differentiating in this manner, the expression can be minimized by setting x as follows: x = ( ( l ( i - n ) + l ( i - ( n - 1 ) ) + … + l i ) - ( a 0 + a 1 + … + a j ) ) - ( σ ( i - n ) 2 + σ ( i - ( n - 1 ) ) 2 + … + σ i 2 ) L By substituting this value for x in the original expression, the following expression is obtained: ln ( 2 π ( σ ( i - n ) 2 + σ ( i - ( n - 1 ) ) 2 + … + σ i 2 ) p c ) + ( ( l ( i - n ) + l ( i - ( n - 1 ) ) + … + l i ) - ( a 0 + a 1 + … + a j ) ) L + ( - 1 2 L 2 ) ( σ ( i - n ) 2 + σ ( i - ( n - 1 ) ) 2 + … + σ i 2 ) + n ln 1 p f + j ln 1 1 - p c .",
"Again, the last two sub-terms may account for the false cuts and the missing cuts, respectively.",
"It is possible to assume that there is at least one “good” cut in the sequence map.",
"EXAMPLE 2 No extension and bad matches until i and j.",
"In this case, the first “good match” is located when fragment i of the sequence map matches fragments j of the consensus map.",
"The expression corresponding to this case is n ln ( 1 p f ) + ( j + 1 ) ln ( 1 1 - p c ) This expression takes into consideration (and possibly corrects) all missing matches and the false matches in both maps (e.g., the j+1 term takes into account the 0-th cut as a missing cut).",
"Case 3: Match without extension to some fragment in the consensus map.",
"It shall be assumed that a “good match” exists between fragment i of the consensus map and fragments j of the sequence map, and, as with Example 1 of this subsection, the fragment from the consensus map (which is within which the end of fragment 0— size α0—of the sequence map lies) is indexed i−n.",
"A match of the fragment 0 of the sequence map to any of the n fragments up to fragment i of the consensus map as then attempted.",
"All possible missing cuts and false cuts along the way are taken into consideration.",
"The attempt of minimizing the following expression (dependent on k) will likely compete against the expressions in Examples 1 and 2 for the best end match.",
"min 0 ≤ k ≤ i ( ( ( l ( i - k ) + l ( i - ( k - 1 ) ) + … + l i ) - ( x + a 0 + a 1 + … + a j ) ) 2 2 ( σ ( i - k ) 2 + σ ( i - ( k - 1 ) ) 2 + … + σ i 2 ) + ( k - 1 ) ln 1 p f + j ln 1 1 - p c ) .",
"II.",
"Right End Penalty Computation FIG.",
"6G shows an exemplary illustration of the maps which are utilized for the right end penalty computation, i.e., for fragments trailing the end of the sequence map 690 and/or of the consensus map 680.This computation is almost symmetric to the left end penalty computation described above.",
"However, there is a difference to be taken into account for the right end computation which makes the computation asymmetrical with respect to the left end penalty computation described above.",
"When the “last good match” between fragment i of the consensus map 670 and fragment j of the sequence map 690 is considered, a consideration of what is the score of the match up to that point should also be undertaken.",
"In particular, the value T[, i] should be considered (thus assumed to be available at that point).",
"Thus, as per the left end computation, three terms should be considered.",
"They are analogous to the three terms for the left end computation, but they should be augmented with T[j, i] to be meaningful.",
"III.",
"Description of the Exemplary Validation Procedure FIG.",
"7 shows a detailed illustration of the exemplary flow diagram and architecture of the validation procedure according to the present invention which utilizes dynamic programming principles and the sequence and consensus maps illustrated in FIGS.",
"6F and 6G.",
"Each box represents the solution of a “dynamic programming”-like problem.",
"In particular, the map data is provided to a left end table 360 which then passes at least a portion of such data to a middle table 365.The output of both the left end table 360 and the middle table 365 are combined in block 370, and the combined results are forwarded to a results table I 375.Then, at least a portion of the data from the results table I 375 is passed to a right end table 380, and the combined results are forwarded to a results table II 385.The data in the results table I 375 and the results table II 385 are computed using the scores contained in the other tables (e.g., the left end table 360, the middle table 365 and the right end table 380).",
"The overall computation uses these three tables 360, 365, 380 as follows: * the T[.,.]",
"for the middle table computation; the TL[.,.]",
"for the left end penalty computation; and the TR[.,.]",
"for the right end penalty computation.",
"It is also possible to re-use certain tables to save memory and system resources of the processing device 10.The flow of control produces the content of each table 360, 365, 380, in turn, and the final resulting table (e.g., the results table II 385) can be examined to reconstruct the alignment trace-back.",
"IV.",
"Possible Optimization Filling the entire T[.,.]",
"table, i.e., the middle table 365, may take on the order of 4 times O(N2M min(N,M)) to complete, where N is the size of the sequence map and M is the size of the consensus map.",
"However, it is possible to optimize the filling of the middle table 365 down to O(NM min(N,M)) by utilizing the limiting argument on the computation performed for each entry T[i, j].",
"Because of the limit on u and v, the computation time for each entry can be considered “constant”.",
"In a simple setup, the middle table 365 may take up O(NM) space, hence it too may be quadratic even when extra “backtrace recording” is considered, as described in Gusfield, D., “Algorithms on Strings, Trees, and Sequences”, Cambridge University Press, 1997.It is also possible to optimize the execution time via a hashing scheme similarly to the scheme used in the “gentig” program.",
"In such case, the time complexity can be reduced by a further order of magnitude.",
"Experimental Results The first experiments using software based on the system and method described above checked “in silico” maps obtained from Plasmodium falciparum sequence data against optical ordered restriction maps for the same organism.",
"I. Plasmodium falciparum Sequence Data The sequence for the Pasmodium falciparum 's 14 chromosomes was obtained from the Sanger Institute database (www.sanger.ac.uk) and from the TIGR database (www.tigr.org).",
"The experiment cut the sequences “in silico” using the BamHI restriction enzyme.",
"The resulting maps were fed to the software (implementing the process according to the present invention) along with appropriate optical ordered restriction maps.",
"The results of the experiments on chromosome 2 and chromosome 3 (showing a number pf fragments) are provided below, as well as the experiment on all chromosomes using a particular enzyme (e.g., NheI).",
"Number of Fragments Chromosome from DB reversed chr 2 30 23 chr 3 36 28 Two “in silico” maps were provided for the chromosome 2 and chromosome 3 sequences with the fragment numbers obtained being provided in the table above.",
"The molecule maps thus produced were then sent to the validation checker alongside various consensus maps.",
"II.",
"Plasmodium falciparum Optical Ordered Restriction An optical ordered restriction map published in J. Jing et al., “Optical Mapping of Plasmodium Falciparum Chromosome 2”, Genome Research, 9:175-181, 1999 and Z. Lai et al., “A shotgun optical map of the entire Plasmodium Falciparum genome”, Nature Genetics, 23:309-313, 1999, and the maps generated by the “gentig” program were utilized for this experiment.",
"The “gentig” program provided the use of the indication of the overall standard deviation to be used for each fragment of the consensus map.",
"The parameter used was: {circumflex over (σ)}=4.4754 Kbps, and each fragment was assigned a standard deviation of: σ ^ l L , Kbps where l is the fragment size and L is the average consensus map fragment size.",
"III.",
"Validation Procedure Results The validation DPP according to the present invention was executed on chromosome 2 and chromosome 3.The DPP ran with the following limitations: The u and v parameters for the main recurrence formula were set to 3.The procedure for matching the left and right ends of the sequence maps using the special computations described above was not utilized.",
"The summary of the results are provided below in Tables 1-3.Table 1 and 3 show the match of the sequence maps for chromosomes 2 and 3 against the consensus maps generated by the “gentig”.",
"Table 2 shows the match of the sequence maps against the consensus map which as published in M. J. Gardner et al., “Chromosome 2 sequence of the human malaria parasite Plasmodium Falciparum”, Science, 282:1126-1132, 1998.The position of the matches of the sequence against the consensus maps are also shown in Tables 1-3.TABLE 1 Chromosome 2 Validation Summary A # missing # false rank matches score map id cuts cuts 1 29 80.869 1302 0 1 2 28 105.861 1302 2 1 3 18 126.956 1326 12 4 4 22 127.488 1305 8 4 5 18 132.890 1414 12 2 In particular, Table 1 shows the data for the best “matches” found by the validation procedure of the present invention for the case of Plasmodium falciparum chromosome 2.The “in silico” sequence map was obtained from the TIGR database sequence.",
"The sequence map (as well as its reversed) was checked against 75 (optical) consensus maps produced by the gentig program.",
"The 75 optical maps cover the entire Plasmodium falciparum genome.",
"The validation procedure located its best matches against the map tagged 1302.TABLE 2 Chromosome 2 Validation Summary B # missing # false rank matches score map id cuts cuts 1 29 77.308 NYU-WISC 1 0 2 22 125.088 NYU-WISC 8 2 3 22 130.866 NYU-WISC 8 4 4 24 131.475 NYU-WISC 6 1 5 24 132.838 NYU-WISC 6 4 Table 2 shows the data for the best “matches” found by the validation procedure of the present invention for the case of Plasmodium falciparum chromosome 2.The “in silico” sequence map was obtained from the TIGR database sequence.",
"The sequence map (as well as its reverse) was checked against the map published in M. J. Gardner et al.",
"publication.",
"TABLE 3 Chromosome 3 Validation Summary # missing # false rank matches score map id cuts cuts 1 35 108.360 1365 1 0 2 32 117.571 1365 4 1 3 32 119.956 1365 4 2 4 35 121.786 1296 1 3 5 31 125.265 1365 5 1 Table 3 shows the data for the “best” matches found by the validation procedure of the present invention for the case of Plasmodium falciparum chromosome 3.The “in silico” sequence map was obtained from the Sanger Institute database sequence.",
"The sequence map (as well as its reversed) was checked against 75 (optical) consensus maps produced by gentig.",
"The 75 optical maps cover the entire Plasmodium falciparum genome.",
"The validation procedure located its best matches against the map tagged 1365.The processing device 10 of the present invention was executed at approximately 75×4=300 DPP instances in about 5 minutes during the experiment.",
"Also, during this experiment, the processing device 10 kept track of all the intermediate results and made them available for interactive inspection after the actual execution.",
"Also, the sequence, the sequence map, and the consensus maps, were always available for inspection and manipulation IV.",
"Conclusion The statistical model of an exemplary embodiment of the present invention is essentially a formulation of a maximum likelihood problem which is solved by minimizing a weighted sum-of-square-error score.",
"The solution is computed by constructing a “matching table” using a dynamic programming approach whose overall complexity is of the order O(M min(N, M)) (for our non-optimized solution), where N is the length of the consensus map and M is the length of the consensus map.",
"The preliminary results of the experiment described above illustrate how the process and system of the present invention can be used in assessing the accuracy of various sequence and map data currently being published in a variety of formats from a many different sources.",
"B. Alignment and Reordering Process and System Overall Alignment Process Flow Diagram FIG.",
"8 shows an exemplary embodiment of the process for aligning sequences using optical maps according to the present invention which can also be executed by the processing device 10 of FIGS.",
"1 and 2.In this exemplary embodiment and similarly to the validation process illustrated in FIG.",
"3, the optical mapping data 140 is forwarded to a technique 250 (e.g., the “gentig” program) which constructs one or more consensus maps 260 based on the optical mapping data 140 by considering the local variations among aligned single molecule maps.",
"According to this exemplary embodiment of the alignment process of the present invention, the particular DNA sequence 210 or a portion of such DNA sequence is provided.",
"Thereafter, the data for this DNA sequence (or a portion thereof) is forwarded to a technique 220 which simulates a restriction enzyme digestion process to generate an “in silico” ordered restriction sequence map 230.The system and process of the present invention may then executes the validation algorithm 270 which determines the accuracy of the ordered restriction sequence map 230 based on the data provided in the optical consensus map(s) 260.As with the validation procedure of FIG.",
"3, this result can be output 280 in the form of a response a score (e.g., a rank for each ordered restriction map), a binary output (e.g., the accuracy validated vs. unvalidated), etc.",
"The exemplary embodiments of the validation process and system of the present invention have been described in great detail herein above.",
"Finally, the simulated ordered restriction sequence map(s) can be aligned against the optical ordered restriction map in block 400.In one exemplary embodiment of the alignment process of the present invention, for each simulated ordered restriction map, the best anchoring position of such map is located on the ordered restriction consensus map (e.g.",
"an optical consensus map).",
"The result of such location procedure is the generation of the entire set of anchoring positions of the simulated ordered restriction maps.",
"In one preferred embodiment, the best anchoring positions are provided first to effectuate the best possible alignment.",
"This can be done using a one-dimensional Dynamic Programming Procedure.",
"Those having ordinary skill in the art would clearly understand that it is possible to produce multiple alignments for the simulated ordered restriction maps due to many anchoring positions than may be available.",
"Provided below are further details of the alignment process and system according to the present invention.",
"Detailed Flow Diagram of Alignment Process FIG.",
"9 shows an exemplary flow chart of the embodiment of the process according to the present invention for simulating a restriction digestion of the sequence map, validating the accuracy of the consensus optical order restriction map and/or the simulated map, and constructing an alignment therefore.",
"This process can be performed by the processing device 10 which is shown in FIGS.",
"1 and 2.Similarly to the validation process shown in FIG.",
"4, the processing device 10 receives the optical ordered restriction data in step 410, which can be the consensus optical map(s) 260 shown in FIG.",
"8.Then, in step 420, the processing device 10 receives the sequence data, which is preferably the DNA sequence data 210 also shown in FIG.",
"8.In step 430, the restriction digestion of the sequence data is simulated to obtain the simulated (in silico) ordered restriction map which is also shown in FIG.",
"8 as the sequence map(s) 230.Thereafter, the optical ordered restriction map is compared to the simulated ordered restriction map to obtain one or more sets of most likely matches (step 440).",
"The processing device 10 then determines if all the simulated ordered restriction maps were checked in step 445.If not, the process takes the next simulated ordered restriction map to be the current simulated ordered restriction map to be checked in step 450, and the comparison of step 440 is repeated again for the current simulated ordered restriction map.",
"Otherwise, since it is determined that all the simulated ordered restriction maps were checked, all of the matches are ranked in step 460, and the processing device 10 determines the best match(s) for each simulated ordered restriction map based on the respective ranks in step 470.Then, in step 480, the alignment of the simulated ordered restriction map is constructed with respect to the optical ordered restriction maps based on the score of the matches.",
"Global Alignment To reiterate, the validation process and system of the present invention described above can match an ordered restriction sequence map against an ordered restriction consensus map.",
"This validation process and system can be possibly described as a positioning process of the sequence map against the consensus map.",
"When many sequences positioning are taken into consideration, it may be possible to describe the validation process as a “global” collective alignment against a particular consensus map.",
"Thus, for the sake of clarity, the output of the procedure that produces this final result shall be referred to herein below as an alignment.",
"For example, the result of n “validation experiments” can be identified as n sets of possible sequence positions along the consensus map.",
"Each of these results can be denoted as set Si (with 0<i≦n), with |Si|=k.",
"Each of the k items in each Si is a triple [si, x(i,j), v(i,j)]—where Si is a sequence map identifier, x(i,j) is the j-th alignment of si against the consensus map, and v(i,j) is the sequence alignment score (with 0<j≦k) obtained from the single sequence (map) positioning process.",
"The set containing every Si (with 0<i≦n) is called S. An exemplary embodiment of the procedure to perform the matching, ranking and alignment steps 440-480 using the sequence maps and costs described above is provided below with reference to FIG.",
"10.The end result will preferably be an alignment whose overall cost C can be computed by summing all the costs v(i,j) eventually selected.",
"Initially, in step 510, the global cost C is set to infinity.",
"Then, in step 520, the best matches out of each set Si of simulated ordered restriction maps (i.e., sequence maps) against the optical ordered restriction map (i.e., the consensus map) are selected.",
"The best matches are grouped into a set of triples called TS, and the cost v(i,j) and the position x(i,j) of each respective sequence Si are analyzed.",
"The cost V of this set of triples TS is then computed using, e.g., a specialized ID Dynamic Programming Procedure (step 540), and compared to C. If V is equal to C plus or minus a tolerance value (step 550), then the set of triples Ts is determined to be the result of the alignment procedure (step 580).",
"If V is not equal to C plus or minus a tolerance value, then first C is equated to V, and the triple [si, x(i,j), v(i,j)] corresponding to the best of the “second best” among the Si's is selected (step 570).",
"The triple [si, x(i,j), v(i,j)] is then removed from the set of triples TS, and the triple [si, x(i,j), v(i,j)] (with j different from j′) is inserted into the set of triples TS (step 575).",
"A new V is then computed from the updated set of triples TS (step 540).",
"Provided below is an exemplary map-based alignment algorithm/problem which can be utilized with the alignment process and system of the present invention.",
"Let S=∪iSi.",
"For example, at most one triple from each Si, can be selected while satisfying the following global conditions/objectives which can possibly be relaxed: 1.When anchoring two or more selected triples within the alignment TS, two selected sequences sp and sq anchored at their respective x(p,b) and x(q,a), preferably do not overlap (for suitable p, q, a, and b and p not equal to q); 2.Σ(Ii×v(i,j)) is minimized over each j in the sequences set Si so that as many as possible sequence maps Si's are included in the alignment; and 3.the number of non-selected sequences, n−ΣiIi is minimized.",
"where Ii is an indicator variable assuming a value 1 if the triplet from the sequence Si is included in the chosen set TS, and 0 otherwise.",
"It should be understood that the objectives (2) and (3) provided above may conflict.",
"In particular, the minimum of the objective (2) is achieved when no sequence is selected, while with the objective (3), it is preferable to choose as many sequences as possible, irrespective of the score values.",
"This conflict may be resolved by, e.g., a weighting scheme involving a Lagrangian-like term which linearly combines the two contradictory objectives.",
"It is possible to solve this problem by using various approximation algorithms.",
"For example, the following two algorithms/procedures: 1.a “Greedy” algorithm/procedure, and 2.a “Dynamic Programming” algorithm/procedure.",
"During the experimentation of the alignment system and process of the present invention, the Greedy algorithm/procedure and the Dynamic Programming algorithm/procedure were utilized with successful results.",
"Provided below are the detailed description of these algorithms/procedures (1)-(2) of the present invention.",
"Greedy Algorithm/Procedure A solution P can be constructed such that each Si is ordered by value v(i,j).",
"Then, the best item from each sequence Si is placed in the partial solution P by selecting the sequences in the order imposed by each x(i,j).",
"It should be understood that the final solution P is not guaranteed to be optimal; however, this solution may provide the results which may be acceptable to the implementers of the alignment procedures.",
"Dynamic Programming/Procedure This algorithm/procedure is based on the traditional dynamic programming approach.",
"Indeed, the implementation of this algorithm/procedure is straight forward and space-efficient as provided below.",
"The problem can first be considered for one exemplary case when k=1, and an appropriate algorithm can be selected.",
"Next, the general case when k>1 can be considered, and good approximation heuristics may be devised.",
"(a) Alignment procedure for Sequence number k being 1.If the number of sequences k present in each set Si of triples is restricted to be 1 (e.g., being the best score), then the problem yields to a feasible and efficient algorithm.",
"In general, if the sequence matches uniquely to one map location, then this case should apply.",
"An exemplary embodiment of the alignment algorithm for the dynamic programming solution, constructing the solution P, is described below.",
"In particular, 1.Sort all the triples of sequence, cost and position, <si, x(i,j), v(i,l)> in ascending x(i,l) order, and store the result in a list L. Thereafter, the indices i and j can be assumed to range over the list L. 2.Construct two vectors C[i] and B[i] (0<i≦n), where each entry in global cost C is defined to be the cost of including si in an alignment that already contains sequences, or a subset thereof, up to Sj; and the index j is stored in B[i].",
"The update rules for C[i] and B[i] preferably search backward in the C vector for values which minimize the cost function, and set B to “point back” to the chosen point.",
"For example, C[i]=max (C[j]+W(λ;i)) such that Si does not overlap with Sj, 0<j<i B[i]=j.",
"W(λ; i) function takes into consideration the conflicting nature of the objectives described above.",
"Since it is most likely not possible to optimize both objectives simultaneously, a weight function can be generated (where a user may supply the parameter λ) which would preferably account for both objectives.",
"Two exemplary W functions are provided below: Wi(λ;i)=|Si|−λ·vi, W2(λ;i)=1−λ·vi.",
"Wi takes into account the “span” covered by the selected sequences (where |Si| is the size of the sequence).",
"W2 takes into account the number of sequences which were selected.",
"The parameter λ is controlled by the user.",
"(b) Alignment Procedure for Sequence Number k>1.If sequence number k>1, then the procedure may be more complex.",
"Since for each set Si, there may be k number of alignments to select from, the complexity involved in a straightforward generalization of the preceding procedure is conjectured to grow exponentially.",
"It is possible to use a heuristic procedure/algorithm to produce an acceptable solution in the case when the sequence number k>1.The concept of this procedure is to iterate or repeat the dynamic programming procedure (i.e., k=1 case) on an input set that takes the best possible solutions from each sequence Si while ignoring the non-overlapping constraint.",
"This solution can be further improved in the subsequent iteration by constructing a new input to the DPP procedure (i.e., where k=1) that consists of the preceding solution augmented with an element from each sequence Si excluded in the preceding solution.",
"Because the preceding solution is also a solution of the new expression, the new solution is at least as effective as the solution previously provided.",
"In each iteration, the basic solution can also be a general (and possibly suboptimal) solution.",
"Because when an item is removed from consideration, it is never again reconsidered; thus, according to a preferred embodiment of the present invention, there can be only O(kn) iterations, and each iteration involves O(n2) work.",
"Hence a naive analysis yields an O(kn3) time algorithm.",
"Experimental Results FIG.",
"11 shows an illustration of a possible alignment of an exemplary chromosome arrangement using the system and method of the present invention.",
"In particular, a region of the alignment of P. falciparum's Chromosome 12 is shown therein which was generated using the software implementing an exemplary embodiment of the validation, alignment and reordering system and method of the present invention.",
"The two underlined maps in position 39 and 50 of the figure illustrate an acceptable anchoring of “contigs” 11 and 13 to the optical ordered restricted map.",
"Also, the alignment was obtained without any overlap filter.",
"One having ordinary skill in the art would clearly recognize that many other applications of the embodiments of the system and process for validating and aligning of the simulated ordered restriction maps according to the present invention.",
"Indeed, the present invention is in no way limited to the exemplary applications and embodiments thereof described above."
]
] | Patent_10432766 |
[
[
"Thinners for invert emulsions",
"A method of reducing the viscosity of oil-based drilling fluids and well service fluids at low temperatures and a thinner compound for use in such drilling fluids and well service fluids is disclosed.",
"The method comprises adding to said drilling fluids or well service fluids a thinner having the formula: R—(C2H4O)n(C3H6O)m(C4H8O)k-H where R is a saturated or unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging form about 0 to about 10, and k is a number ranging from about 0 to about 10."
],
[
"1.A method of influencing the rheology of a drilling fluid or well service fluid comprising an invert emulsion, said method comprising adding to said drilling fluid or well service fluid a compound having the formula: R—(C2H4O)n(C3H6O)m(C4H8O)k-H where R is a saturated or unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging from about 0 to about 10, and k is a number ranging from about 0 to about 10.2.The method of claim 1 wherein, in said formula, k is zero and m is a number ranging from about 1 to about 10, or m is zero and k is a number ranging from about 1 to about 10.3.The method of claim 1 wherein in said formula, n is a number ranging from about 1 to about 6, m is a number ranging from about 1 to about 6, and k is zero.",
"4.The method of claim 1 wherein said invert emulsion comprises a continuous oil phase comprising compounds or compositions flowable and pumpable at temperatures at least as low as about 40 degrees Fahrenheit.",
"5.The method of claim 1 wherein said invert emulsion comprises a continuous oil phase comprising compounds or compositions flowable and pumpable at temperatures above about 32 degrees Fahrenheit.",
"6.The method of claim 5 wherein said oil phase comprises compounds or compositions selected from the group comprising: (f) carboxylic esters of the formula: R′—COO—R″ where R′ is a saturated or unsaturated, linear or branched, alkyl radical having about 1 to about 23 carbon atoms and R″ is an alkyl radical, branched or unbranched, saturated or unsaturated, having about 1 to about 23 carbon atoms; (g) linear or branched olefins having about 8 to about 30 carbon atoms; (h) water-insoluble symmetric or asymmetric ethers of monohydric alcohols of natural or synthetic origin, said alcohols containing about 1 to about 24 carbon atoms; (i) water-insoluble alcohols of the formula: R′″—OH where R′″ is a saturated, unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms; and (j) carbonic diesters.",
"7.The method of claim 1 wherein said compound is added to said drilling fluid or well service fluid in an amount sufficient to effect a reduction in the viscosity of said drilling fluid or well service fluid.",
"8.The method of claim 1 wherein said compound is added to said drilling fluid or well service fluid in an amount sufficient to maintain the flowability and pumpability of said drilling fluid or well service fluid at temperatures less than about 50 degrees Fahrenheit.",
"9.The method of claim 1 wherein said compound is added to said drilling fluid or well service fluid in quantities ranging from about 0.5 pounds to about 15.0 pounds of said compound per barrel of said drilling fluid or well service fluid.",
"10.The method of claim 1 wherein said compound reduces the viscosity of said drilling fluid or well service fluid at low temperatures.",
"11.The method of claim 10 wherein said compound does not significantly affect the viscosity of said fluid at high temperatures.",
"12.The method of claim 1 wherein said compound is added to said fluid when said fluid is prepared.",
"13.The method of claim 1 wherein said compound is added to said fluid while said fluid is circulating in a wellbore.",
"14.A drilling fluid or well service fluid comprising a continuous oil phase, water dispersed in said oil phase, solids insoluble in said oil phase, and a compound having the formula: R—(C2H4O)n(C3H6O)m(C4H7O)k-H where R is a saturated or unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging from about 0 to about 10, and k is a number ranging from about 0 to about 10.15.The drilling fluid or well service fluid of claim 14 wherein said compound is added in sufficient amounts to reduce the viscosity of said fluid at low temperatures.",
"16.The drilling fluid or well service fluid of claim 14 having a density of from about 8 to about 18 lbs/gal.",
"17.The drilling fluid or well service fluid of claim 14 having a yield point of not more than about 75 lbs/100 ft2 at about 40° F. 18.A method of reducing the viscosity of an invert emulsion drilling fluid or well service fluid at low temperatures comprising adding to said fluid an effective amount of compound having the formula: R—(C2H4O)n(C3H6O)m(C4H8O)k-H where R is a saturated or unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging from about 0 to about 10, and k is a number ranging from about 0 to about 10.19.The method of claim 18 wherein said compound does not significantly affect the viscosity of the drilling fluid at high temperatures.",
"20.The method of claim 18 further comprising circulating said fluid in a wellbore and adding said compound to said fluid during said circulation.",
"21.The method of claim 18 further comprising preparing said fluid and adding said compound to said fluid during said preparation."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention is generally related to methods and compositions for drilling and servicing wellbores in hydrocarbon bearing subterranean formations.",
"Particularly, this invention is related to oil-based drilling fluid systems comprising water-in-oil invert emulsions, and to thinners that enhance or enable use of such fluids, at temperatures at or below about 50 degrees Fahrenheit (about 10 degrees Centigrade).",
"2.Description of Relevant Art A drilling fluid, or “mud” which a drilling fluid is also often called, is a specially designed fluid that is circulated in a wellbore as the wellbore is being drilled to facilitate the drilling operation.",
"The various functions of a drilling fluid include removing drill cuttings from the wellbore, cooling and lubricating the drill bit, aiding in support of the drill pipe and drill bit, and providing a hydrostatic head to maintain the integrity of the wellbore walls and prevent well blowouts.",
"Specific drilling fluid systems are selected to optimize a drilling operation in accordance with the characteristics of a particular geological formation.",
"A drilling fluid typically comprises water and/or oil or synthetic oil or other synthetic material or synthetic fluid (“synthetic”) as a base fluid, with solids in suspension.",
"A non-aqueous based drilling fluid typically contains oil or synthetic as a continuous phase and may also contain water dispersed in the continuous phase by emulsification so that there is no distinct layer of water in the fluid.",
"Such dispersed water in oil is generally referred to as an invert emulsion or water-in-oil emulsion.",
"A number of additives may be included in such oil based drilling fluids and invert emulsions to enhance certain properties of the fluid.",
"Such additives may include, for example, emulsifiers, weighting agents, fluid-loss additives or fluid-loss control agents, viscosifiers or viscosity control agents, and alkali.",
"Further general discussion and description of oil-based drilling fluids is provided in P. A. Boyd, et al., New Base Oil Used In Low Toxicity Oil Muds, Journal of Petroleum Technology, pages 137-142 (1985), which is incorporated herein by reference.",
"An essential criterion for assessing the utility of a fluid as a drilling fluid or as a well service fluid is the fluid's rheological parameters, particularly under drilling and wellbore conditions.",
"For use as a drilling fluid, or as a fluid for servicing a well, the fluid must be capable of maintaining certain viscosities suitable for drilling and circulation in the wellbore.",
"Preferably, a drilling fluid will be sufficiently viscous to be capable of supporting and carrying to the surface of the well drill cuttings without being so viscous as to interfere with the drilling operation.",
"Moreover, a drilling fluid must be sufficiently viscous to be able to suspend barite and other weighting agents.",
"However, increased viscosity can result in problematic sticking of the drill string, and increased circulating pressures can contribute to lost circulation problems.",
"Thinners may be added to the drilling fluid or drilling mud systems before and in the course of drilling.",
"Anionic surfactants particularly from the group of the fatty alcohol sulfates, the fatty, alcohol ether sulfates and the alkylbenzenesulfonates are examples of such thinners known in the prior art.",
"Although such compounds have been shown to effect thinning of drilling fluids, problems with such prior art thinners may occur when using the drilling muds at low temperatures (temperatures at or below about 50° F. (10° C.)).",
"At such low temperatures, despite the use of known prior art thinners, oil based drilling fluids typically have high or increased viscosity, which may render the fluids unusable for drilling.",
"After pumping into the wellbore, drilling fluids may undergo heating from the formation, depending on the depth of the wellbore and the temperature of the formation.",
"For example, heating in the range of about 150° to about 250° F. (about 66° to about 121° C.) is not uncommon and subterranean temperatures as high as about 350° F. (about 178° C.), particularly in very deep wellbores, are known.",
"The Arctic region, for example, is known to have very low surface temperatures but very high subterranean temperatures.",
"Even more problematic are deepwater wells (i.e., typically wells below at least about 1500 feet), which subject drilling fluids to chilling from cold waters surrounding the riser as the fluid returns to the surface from the high temperature subterranean formation.",
"Such chilling of oil based drilling fluids typically increases their viscosity while such subterranean heating of oil based drilling fluids typically reduces their viscosity.",
"Preferably, thinners which reduce the viscosity of drilling fluids at low temperatures will not affect the viscosity of the fluids at high temperatures.",
"That is, in many cases, a thinner is desired that is capable of “selectively” influencing the rheology or particularly reducing the viscosity of oil-based drilling fluids only at lower temperatures, such as may be encountered at the ground surface of the wellbore, or in the riser surrounded by waters above a deepwater offshore well, for example.",
"Thinners and other additives to drilling fluids, as well as drilling fluids employed in onshore and offshore wells, must commonly meet stringent environmental regulations related to biodegradability and toxicity.",
"Further, drilling fluids and additives to drilling fluids must be able to withstand subterranean conditions that the fluids will typically encounter in a wellbore, such as high temperatures, high pressures, and pH changes.",
"A need exists for improved rheology-modifying or viscosity reducing additives to oil-based drilling fluids, and particularly to drilling fluids comprising invert (water-in-oil) emulsions, which are expected to be used in or to encounter low temperatures in drilling operations.",
"As used herein, unless indicated otherwise, “low temperatures” shall be understood to mean temperatures at or below about 50° F. (about 10° C.)."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>According to the method of the present invention, a compound is added to a water-in-oil or invert emulsion drilling fluid or well service fluid which reduces the viscosity of the drilling fluid or well service fluid at low temperatures or which enables or enhances the ability of the drilling fluid or well service fluid to maintain its viscosity at low temperatures.",
"The compound, which may be generally called a “thinner,” continues to have this effect on a drilling fluid or well service fluid in drilling or servicing wellbores in subterranean formations, particularly hydrocarbon bearing subterranean formations.",
"Further, this compound does not significantly affect the viscosity of the emulsion at high temperatures.",
"The compound has the following formula: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"R—(C 2 H 4 O) n (C 3 H 6 O) m (C 4 H 8 O) k -H in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"where R is a saturated or unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging from about 0 to about 10, and k is a number ranging from about 0 to about 10.The invention also comprises the composition of a water-in-oil or invert emulsion drilling fluid or well service fluid containing this thinner compound."
],
[
"BACKGROUND OF THE INVENTION 1.Field of the Invention This invention is generally related to methods and compositions for drilling and servicing wellbores in hydrocarbon bearing subterranean formations.",
"Particularly, this invention is related to oil-based drilling fluid systems comprising water-in-oil invert emulsions, and to thinners that enhance or enable use of such fluids, at temperatures at or below about 50 degrees Fahrenheit (about 10 degrees Centigrade).",
"2.Description of Relevant Art A drilling fluid, or “mud” which a drilling fluid is also often called, is a specially designed fluid that is circulated in a wellbore as the wellbore is being drilled to facilitate the drilling operation.",
"The various functions of a drilling fluid include removing drill cuttings from the wellbore, cooling and lubricating the drill bit, aiding in support of the drill pipe and drill bit, and providing a hydrostatic head to maintain the integrity of the wellbore walls and prevent well blowouts.",
"Specific drilling fluid systems are selected to optimize a drilling operation in accordance with the characteristics of a particular geological formation.",
"A drilling fluid typically comprises water and/or oil or synthetic oil or other synthetic material or synthetic fluid (“synthetic”) as a base fluid, with solids in suspension.",
"A non-aqueous based drilling fluid typically contains oil or synthetic as a continuous phase and may also contain water dispersed in the continuous phase by emulsification so that there is no distinct layer of water in the fluid.",
"Such dispersed water in oil is generally referred to as an invert emulsion or water-in-oil emulsion.",
"A number of additives may be included in such oil based drilling fluids and invert emulsions to enhance certain properties of the fluid.",
"Such additives may include, for example, emulsifiers, weighting agents, fluid-loss additives or fluid-loss control agents, viscosifiers or viscosity control agents, and alkali.",
"Further general discussion and description of oil-based drilling fluids is provided in P. A. Boyd, et al., New Base Oil Used In Low Toxicity Oil Muds, Journal of Petroleum Technology, pages 137-142 (1985), which is incorporated herein by reference.",
"An essential criterion for assessing the utility of a fluid as a drilling fluid or as a well service fluid is the fluid's rheological parameters, particularly under drilling and wellbore conditions.",
"For use as a drilling fluid, or as a fluid for servicing a well, the fluid must be capable of maintaining certain viscosities suitable for drilling and circulation in the wellbore.",
"Preferably, a drilling fluid will be sufficiently viscous to be capable of supporting and carrying to the surface of the well drill cuttings without being so viscous as to interfere with the drilling operation.",
"Moreover, a drilling fluid must be sufficiently viscous to be able to suspend barite and other weighting agents.",
"However, increased viscosity can result in problematic sticking of the drill string, and increased circulating pressures can contribute to lost circulation problems.",
"Thinners may be added to the drilling fluid or drilling mud systems before and in the course of drilling.",
"Anionic surfactants particularly from the group of the fatty alcohol sulfates, the fatty, alcohol ether sulfates and the alkylbenzenesulfonates are examples of such thinners known in the prior art.",
"Although such compounds have been shown to effect thinning of drilling fluids, problems with such prior art thinners may occur when using the drilling muds at low temperatures (temperatures at or below about 50° F. (10° C.)).",
"At such low temperatures, despite the use of known prior art thinners, oil based drilling fluids typically have high or increased viscosity, which may render the fluids unusable for drilling.",
"After pumping into the wellbore, drilling fluids may undergo heating from the formation, depending on the depth of the wellbore and the temperature of the formation.",
"For example, heating in the range of about 150° to about 250° F. (about 66° to about 121° C.) is not uncommon and subterranean temperatures as high as about 350° F. (about 178° C.), particularly in very deep wellbores, are known.",
"The Arctic region, for example, is known to have very low surface temperatures but very high subterranean temperatures.",
"Even more problematic are deepwater wells (i.e., typically wells below at least about 1500 feet), which subject drilling fluids to chilling from cold waters surrounding the riser as the fluid returns to the surface from the high temperature subterranean formation.",
"Such chilling of oil based drilling fluids typically increases their viscosity while such subterranean heating of oil based drilling fluids typically reduces their viscosity.",
"Preferably, thinners which reduce the viscosity of drilling fluids at low temperatures will not affect the viscosity of the fluids at high temperatures.",
"That is, in many cases, a thinner is desired that is capable of “selectively” influencing the rheology or particularly reducing the viscosity of oil-based drilling fluids only at lower temperatures, such as may be encountered at the ground surface of the wellbore, or in the riser surrounded by waters above a deepwater offshore well, for example.",
"Thinners and other additives to drilling fluids, as well as drilling fluids employed in onshore and offshore wells, must commonly meet stringent environmental regulations related to biodegradability and toxicity.",
"Further, drilling fluids and additives to drilling fluids must be able to withstand subterranean conditions that the fluids will typically encounter in a wellbore, such as high temperatures, high pressures, and pH changes.",
"A need exists for improved rheology-modifying or viscosity reducing additives to oil-based drilling fluids, and particularly to drilling fluids comprising invert (water-in-oil) emulsions, which are expected to be used in or to encounter low temperatures in drilling operations.",
"As used herein, unless indicated otherwise, “low temperatures” shall be understood to mean temperatures at or below about 50° F. (about 10° C.).",
"SUMMARY OF THE INVENTION According to the method of the present invention, a compound is added to a water-in-oil or invert emulsion drilling fluid or well service fluid which reduces the viscosity of the drilling fluid or well service fluid at low temperatures or which enables or enhances the ability of the drilling fluid or well service fluid to maintain its viscosity at low temperatures.",
"The compound, which may be generally called a “thinner,” continues to have this effect on a drilling fluid or well service fluid in drilling or servicing wellbores in subterranean formations, particularly hydrocarbon bearing subterranean formations.",
"Further, this compound does not significantly affect the viscosity of the emulsion at high temperatures.",
"The compound has the following formula: R—(C2H4O)n(C3H6O)m(C4H8O)k-H where R is a saturated or unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging from about 0 to about 10, and k is a number ranging from about 0 to about 10.The invention also comprises the composition of a water-in-oil or invert emulsion drilling fluid or well service fluid containing this thinner compound.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 2 at different temperatures.",
"FIG.",
"2 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 3 at different temperatures.",
"FIG.",
"3 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 4 at different temperatures.",
"FIG.",
"4 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 5 at different temperatures.",
"FIG.",
"5 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 6 at different temperatures.",
"FIG.",
"6 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 7 at different temperatures.",
"FIG.",
"7 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 8 at different temperatures.",
"FIG.",
"8 is a graph comparing yield point of mud systems with and without thinners of the invention tested as reported in Table 9 at different temperatures.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention provides a method of influencing the rheology, and particularly reducing the viscosity, of drilling fluids or well service fluids comprising invert (water-in-oil) emulsions.",
"The method is particularly applicable to fluids for use in wellbores penetrating hydrocarbon bearing subterranean formations and has particular advantage in applications where the fluids are subjected to low temperatures, as in drilling or in servicing deepwater offshore wells.",
"Such drilling fluids and well service fluids typically comprise a continuous oil phase, water dispersed in the oil phase, solids insoluble in the drilling fluid or well service fluid suspended in the fluid, and various additives.",
"As the term is used herein, “invert emulsion” or “oil-in-water emulsion” is understood to mean the liquid portion of the drilling fluid comprising an emulsion (excluding solids).",
"The term “invert emulsion drilling fluid” means the total sum of what is circulated as a drilling fluid.",
"In the method of this invention, a composition or compound having the following formula (I) is added to the invert emulsion or oil-based drilling fluid (or well service fluid) to reduce the viscosity of the fluid or to enhance the ability of the fluid to maintain its viscosity or to resist increasing viscosity at low temperatures.",
"The compound may be added to the fluid during initial preparation of the fluid or later as the fluid is being used for drilling or well service purposes in the formation.",
"The quantity added is an effective amount to maintain or effect the desired viscosity of the drilling fluid.",
"For purposes of this invention, an “effective amount” of thinner of formula (I) is preferably from about 0.5 to about 15 pounds per barrel of drilling fluid or mud.",
"A more preferred amount of thinner ranges from about 1 to about 5 pounds per barrel of drilling fluid and a most preferred amount is about 1.5 to about 3 pounds thinner per barrel of drilling fluid.",
"Formula (I) is: R—(C2H4O)n(C3H6O)m(C4H8O)k-H (I) where R is a saturated or unsaturated, linear or branched, alkyl radical having about 8 to about 24 carbon atoms, n is a number ranging from about 1 to about 10, m is a number ranging from about 0 to about 10, and k is a number ranging from about 0 to about 10.Preferably, R has about 8 to about 18 carbon atoms; more preferably, R has about 12 to about 18 carbon atoms; and most preferably, R has about 12 to about 14 carbon atoms.",
"Also, most preferably, R is saturated and linear.",
"The compositions or compounds of formula (1) may be prepared by customary techniques of alkoxylation, such as alkoxylating the corresponding fatty alcohols with ethylene oxide and/or propylene oxide or butylene oxide under pressure and in the presence of acidic or alkaline catalysts as is known in the art.",
"Such alkoxylation may take place blockwise, i.e., the fatty alcohol may be reacted first with ethylene oxide, propylene oxide or butylene oxide and subsequently, if desired, with one or more of the other alkylene oxides.",
"Alternatively, such alkoxylation may be conducted randomly, in which any desired mixture of ethylene oxide, propylene oxide and/or butylene oxide is reacted with the fatty alcohol.",
"In formula (I), the subscripts n and m respectively represent the number of ethylene oxide (EO) and propylene oxide (PO) molecules or groups in one molecule of the alkoxylated fatty alcohol.",
"The subscript k indicates the number of butylene oxide (BO) molecules or groups.",
"The subscripts n, m, and k need not be integers, since they indicate in each case statistical averages of the alkoxylation.",
"Included without limitation are those compounds of the formula (I) whose ethoxy, propoxy, and/or butoxy group distribution is very narrow, such as for example, “narrow range ethoxylates” also called “NREs” by those skilled in the art.",
"To accomplish the purposes of this invention, the compound of formula (I) must contain at least one ethoxy group.",
"Preferably, the compound of formula I will also contain at least one propoxy group (C3H6O—) or butoxy group (C4H80—).",
"Mixed alkoxides containing all three alkoxide groups—ethylene oxide, propylene oxide, and butylene oxide—are possible for the invention but are not preferred.",
"Preferably, for use according to this invention, the compound of formula (I) will have a value for m ranging from about 1 to about 10 with k zero or a value for k ranging from about 1 to about 10 with m zero.",
"Most preferably, m will be about 1 to about 10 and k will be zero.",
"Other preferred compounds for use in the invention having the formula (I) above will have n ranging from about 1 to about 6, m ranging from about 1 to about 6, and k zero.",
"Still other preferred compounds for use in the invention having the formula (I) above will have n ranging from about 2 to about 5, and m being about 3 or about 4 with k zero.",
"It is particularly advantageous to establish the distribution of ethylene oxide and propylene oxide groups in the compounds of formula (I) in an ethylene oxide to propylene oxide ratio of about 1:1 to about 2:1, or even more preferably, about 2:1.5.Additional preferred compounds for use in the invention having formula (I) above will have alkyl radicals containing about 12 to about 18 carbon atoms, or more preferably about 12 to about 14 carbon atoms, with subscripts n and m each having values of about 4 or about 5.Used as thinners according to the method of the invention, the compounds of formula (I) reduce the viscosity or lower the yield point of the drilling fluid to which they are added.",
"These thinners are particularly effective at low temperatures, i.e., temperatures at or below about 50° F. (about 10° C.) and most particularly effective at temperatures at or below about 40° F. (about 4° C.).",
"The lower limit of effectiveness for these thinners is about 14° F. (about −10° C.).",
"The thinners do not significantly influence or affect the rheology of drilling fluids at high temperatures, particularly temperatures ranging from about 100 to about 250° F. or more.",
"The compounds of formula (I) are biodegradable and are of little or no toxicity.",
"They are expected to be capable of meeting increasingly stringent environmental regulations affecting the oil and gas industry worldwide.",
"Example drilling fluids comprising invert (water-in-oil) emulsions of particular use in the method of the invention generally have an oil phase comprising diesel oil, paraffin oil and/or mineral oil, or a synthetic oil.",
"Alternatively, other carrier fluids may be used such as carboxylic esters, alcohols, ethers, internal olefins, alphaolefins (IO and/or AO), and polyalphaolefins (PAO), which may be branched or unbranched but are preferably linear and preferably ecologically acceptable (non-polluting oils).",
"Preferably, the oils or carrier fluids used for the oil phase of the drilling fluid will be comprised of compounds which are flowable and pumpable at temperatures above about 32° F. (about 0° C.) or at least as low as about 40° F. (about 5° C.) as well as at higher temperatures.",
"For example, compounds selected from one or more of the following groups or classes below are believed particularly suitable to comprise the oil phase of drilling fluids used in the present invention: (a) most preferably, carboxylic esters of the formula: R′—COO—R″ (II) where R′ is a saturated or unsaturated, linear or branched, alkyl radical having about 5 to about 23 carbon atoms and R″ is an alkyl radical, branched or unbranched, saturated or unsaturated, having about 1 to about 22 carbon atoms; (b) also preferably, linear or branched olefins having about 8 to about 30 carbon atoms; (c) water-insoluble symmetric or asymmetric ethers of monohydric alcohols of natural or synthetic origin, said alcohols containing about 1 to about 24 carbon atoms; (d) water-insoluble alcohols of the formula: R′″—OH (III) where R′″ is a saturated, unsaturated, linear or branched alkyl radical having about 8 to about 24 carbon atoms; and (e) carbonic diesters.",
"Such suitable oils are taught further, for example, in: European Patent Applications 0 374 671, 0 374,672,0 382 070, and 0 386 638 of Cognis; European Laid-Open Specification 0 765 368 of Cognis (linear olefins); European Application 0 472 557 (water insoluble symmetric or asymmetric ethers of monohydric alcohols of natural or synthetic origin containing about 1 to about 24 carbon atoms); European Application 0 532 570 (carbonic diesters).",
"Carboxylic esters of formula (II) above are preferred for the oil phase of drilling fluids used in this invention and particularly preferred are the esters described in European Laid-Open Specification EP 0 374 672 and EP 0 386 636.In a preferred embodiment of this invention, compounds of formula (I) are added to drilling fluids comprising invert emulsions having an oil phase comprising esters of formula (II) where the radical R′ in formula (II) is an alkyl radical having about 5 to about 21 carbon atoms (or more preferably about 5 to about 17 carbon atoms or most preferably about 11 to about 17 carbon atoms).",
"Particularly suitable alcohols for making such esters are branched or unbranched alcohols with about 1 to about 8 carbon atoms, for example, methanol, isopropanol, isobutanol, and 2-ethylhexanol.",
"Alcohols having about 12 to about 18 carbon atoms may alternatively be preferred for making other esters suitable for the invention.",
"For example, additional preferred esters for the oil phase of drilling fluids used in the invention include, without limitation: saturated C12-C14 fatty acid esters and unsaturated C16-C18 fatty acids (with isopropyl-, isobutyl- or 2-ethylhexanol as the alcohol component); 2-ethylhexyl octanoate; acetic acid esters, especially acetates of C16-C18 fatty alcohols; branched carboxylic esters disclosed in WO 99/33932 of Chevron or EP 0 642 561 of Exxon; alpha olefin mixtures disclosed in EP 0 765 368 A1 of Cognis and Halliburton; and blends of these various esters.",
"The oil phase of the emulsions of the drilling fluids used in the invention is preferably comprised of at least about 50% by volume of one or more preferred compounds (a)-(e) above.",
"More preferably, such preferred compounds comprise about 60% to about 80% by volume of said oil phase, and most preferably, such preferred compounds comprise about 100% of the oil phase.",
"Water is preferably present in the liquid phase of the drilling fluids used in the invention, and preferably in amounts not less than about 0.5% by volume (excluding solids in the liquid phase).",
"In a preferred embodiment of this invention, thinners of formula (I) are added to drilling fluids comprising invert emulsions containing about 15 to about 35% by volume water and more preferably 20% by volume water and about 80% by volume oil phase.",
"To compensate for the osmotic gradient between the drilling mud and the formation or connate water, water in drilling fluids used in the present invention typically includes fractions of electrolytes, such as calcium salts and/or sodium salts.",
"CaCl2 in particular is frequently used, although other salts from the group of alkali metals and/or alkaline earth metals are also suitable, with potassium acetates and formates being common examples.",
"Preferred drilling fluids used in this invention have the following rheology: plastic viscosity (PV) in the range of about 10 to about 60 cP, and preferably in the range of about 15 to about 40 cP, and yield point (YP) in the range of about 5 to about 40 lb/100 ft2, and preferably in the range of about 10 to about 25 lb/100 ft2, at about 122° F. (about 50° C.).",
"At lower temperatures, i.e., at or below about 40° F. (about 4° C.), the YP should not exceed about 75 lb/100 ft2, and should preferably be in the range of about 10 to about 65 lb/100 ft2, more preferably about 15 to about 45 lb/100 ft2, and most preferably less than about 35 lb/100 ft2.A preferred practicable lower limit for YP for drilling fluids used in this invention is about 5 lb/100 ft2.Methods for determining these parameters of PV and YP are well known to those skilled in the art.",
"An example reference is “Manual of Drilling Fluids Technology”, particularly the chapter on Mud Testing, available from Baroid Drilling Fluids, Inc., in Houston, Tex.",
"(USA), incorporated herein by reference.",
"The solids content (not including low gravity solids), or the amount of weighting agents, in drilling fluids used in this invention is preferably about 0 to about 500 lb/bbl, and most preferably about 150 to about 350 lb/bbl.",
"The mud weight, i.e., the density of the drilling fluids, is preferably in the range of about 8 to about 18 lb/gal.",
"and more preferably about 9 to about 15 lb/gal.",
"Such solids, or weighting agents, which serve to increase the density of the drilling fluids, may be any solids known to those skilled in the art as useful for such purpose, but will preferably be inert or environmentally friendly.",
"Drilling fluids used in this invention may optionally also contain other additives known to those skilled in the art, such as fluid-loss control additives and emulsifiers.",
"Alkali may also be used, preferably lime (calcium hydroxide or calcium oxide), to bind or react with acidic gases (such as CO2 and H2S) encountered during drilling in the formation.",
"Such alkali, or an alkali reserve, is known to prevent hydrolysis by acidic gases of generally acid-labile esters of the drilling fluid.",
"Preferred quantities of free lime in the drilling fluids range from about 1 to about 10 lbs/bbl, and more preferably about 1 to about 4 lbs/bbl, although lower ranges such as less than about 2 lbs/bbl are preferred for certain esters that tend to hydrolyze in the presence of alkaline compounds as will be known to those skilled in the art.",
"Other suitable agents as an alternative to lime may also be used to adjust and/or stabilize invert emulsions of the drilling fluids with respect to acids.",
"An example of such alternative agents is a protonated amine, as described in U.S. Pat.",
"No.",
"5,977,031.Further optional additives that may be present in the drilling fluids used in this invention include electrolytes, such as calcium chloride, organophilic bentonite and organophilic lignite.",
"Glycols and/or glycerol may also be added.",
"Still further, dispersion aids, corrosion inhibitors and/or defoamers may be used.",
"These and other suitable auxiliaries and additives are used in amounts known to those skilled in the art depending on the conditions of the particular wellbore and subterranean formation.",
"Although the invention has primarily been described in the context of a method of using compounds of formula (I) as thinners for drilling fluids at low temperatures, the compounds of formula (I) may also be effective as thinners for well service fluids such as spotting fluids or workover fluids at low temperatures.",
"Further description and use of the invention is shown by the following examples: EXAMPLES To show the effect of the invention, the following experiments were conducted.",
"In each case an invert emulsion drilling mud system of the following general composition was prepared: Ester bbl 0.496 Water bbl 0.233 Emulsifier lb 6.0 Organophilic bentonite lb 1.0 Organophilic lignite lb 5.0 Alkali reserve (lime) lb 1.5 CaCl2 × 2 H2O lb 27.2 Barite lb 314.0 Dispersing auxiliary lb 0.5 Thinner lb/bbl 2.0 The oil phase (A) used was a 2-ethylhexyl octanoate as disclosed in EP 0 386 636.The emulsifier used was the product EZ MUL NTE (Baroid Drilling Fluids Inc., Houston, Tex.).",
"The oil/water ratio was 70/30 in each case.",
"Measurements were carried out on a system without thinner (C1), and with a C12/14 fatty alcohol sulfate +2 EO, sodium salt (C2), with a C12 ether sulfate, sodium salt (C3) and with an oleic acid sulfonate disodium salt (C4), respectively, as prior art thinners, and comparison was made with these thinners and with compounds of formula (I) in accordance with the invention.",
"The formula (I) compounds used for this purpose were as follows: E1 C12/C14 fatty alcohol containing 2 EO and 4 PO E2 C12/C14 fatty alcohol containing 5 EO and 4 PO E3 C12/C18 fatty alcohol containing 5 EO and 4 PO E4 C12/C14 fatty alcohol containing 6 EO and 4 PO The invert muds were prepared in a conventional manner and subsequently, at 40° F. and 122° F., the rheological characteristics of plastic viscosity (PV) and yield point (YP) and the gel strength after 10 seconds and 10 minutes using a Fann SR12 rheometer (from Fann) were determined.",
"Measurements E5, E6 and E7 were carried out using the thinners E1, E2 and E4, but in contrast to the details above, 45 lb of solids (rev dust, i.e., filter ash) were also added to each of the muds, in order to demonstrate the advantageous action of the compounds of formula (I) used in accordance with the invention in the case of high solids loading of the emulsions.",
"In these cases, the measurements were taken only after 16 hours of aging at 150° F. The thinner was not added to the muds E5 to E7 until after aging.",
"The results of the measurements are given in Tables 1a and 1b below.",
"TABLE 1a C1 C1 C2 C2 C3 C3 C4 C4 E1 E1 E2 E2 E3 E3 E4 E4 Temp.",
"40 122 40 122 40 122 40 122 40 122 40 122 40 122 40 122 ° F. PV 94 28 105 30 n.m. 33 91 24 93 31 87 28 94 28 83 29 (cP) YP 68 29 71 35 n.m. 62 69 20 70 41 34 33 62 41 30 30 lb/100 ft2 Gels 27/29 12/13 24/29 15/15 n.m. 26/31 25/25 6/7 25/28 17/19 11/13 14/16 20/24 17/18 8/11 13/14 10″/10′ n.m: not measurable TABLE 1b Measurements with addition of 45 lb rev dust C1 C1 E5 E5 E6 E6 E7 E7 Temperature 40 122 40 122 40 122 40 122 ° F. PV 94 28 107 37 108 40 106 37 (cP) YP 68 29 37 23 72 42 46 30 lb/100 ft2 Gels 27/29 12/13 12/14 7/9 26/30 14/18 17/19 12/14 10″/10′ The data, especially for the yield point (YP), clearly indicate the advantageous thinning effect of the compounds of formula (I) used in the method and in the emulsions of the invention, especially at low temperatures, in comparison to the prior art.",
"The higher plastic viscosity for E5 to E7 is attributable to the higher proportion of solids in the mud systems.",
"Further experiments may be seen in Tables 2 to 9.In these cases, the yield point (YP) of the systems tested was investigated at different temperatures and depicted as a graph.",
"This illustrates particularly well the advantageous influence of the compounds of formula (I) on the rheology at low temperatures (40° F., 4° C.) without any marked influence at high temperatures (120° F., 50° C.).",
"The measurements were carried out using a Fann 35 viscometer (from Fann).",
"The tables also indicate the dial readings at different speeds of rotation per minute (rpm).",
"In Tables 2 to 9: PETROFREE LV® (is 2-ethylhexyl octanoate (from Cognis, Germany) PETROFREE LE® is linear alpha-olefin (from Cognis, Germany) PETROFREE® is C8-14 fatty acid 2-ethylhexyl ester (from Cognis, Germany) GELTONE II® is organophilic bentonite (from Baroid, Houston, Tex.)",
"Thinner E1 is Formula I C12/C14 fatty alcohol of the invention containing 2 EO and 4 PO Thinner E2 is Formula I C12/C14 fatty alcohol of the invention containing 5 EO and 4PO TABLE 2 Mud system PETROFREE LV Mud weight, lb/gal 14.0 Oil/water ratio 70/30 Contaminant Drill solids E2, lb/bbl 0 3 5 Temperature, ° F. 40 120 40 120 40 120 Plastic viscosity, cP 118 40 113 34 107 35 Yield point, lb/100 ft2 38 14 25 14 19 13 10 sec gel, lb/100 ft2 16 6 10 6 6 6 10 min gel, lb/100 ft2 22 11 13 8 9 8 Fann 35 dial readings 600 rpm 274 94 251 82 233 83 300 rpm 156 54 138 48 126 48 200 rpm 114 40 97 35 88 35 100 rpm 70 25 56 22 49 22 6 rpm 17 6 10 7 7 6 3 rpm 14 5 7 6 5 5 TABLE 3 Mud system PETROFREE Mud weight, lb/gal 14.0 Oil/water ratio 75/25 Contaminant Excess GELTONE II E2, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 180 51 126 50 Yield point, lb/100 ft2 230 152 19 125 10 sec gel, lb/100 ft2 108 64 10 50 10 min gel, lb/100 ft2 110 66 13 52 Fann 35 dial readings 600 rpm 590 254 271 225 300 rpm 410 203 145 175 200 rpm 336 179 103 149 100 rpm 248 146 59 119 6 rpm 112 79 10 62 3 rpm 100 70 8 58 TABLE 4 Mud system PETROFREE LV Mud weight, lb/gal 16.0 Oil/water ratio 80/20 Contaminant Drill solids E2, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 152 51 142 40 Yield point, lb/100 ft2 62 27 40 19 10 sec gel, lb/100 ft2 22 10 18 10 10 min gel, lb/100 ft2 48 26 22 12 Fann 35 dial readings 600 rpm 366 129 324 99 300 rpm 214 78 182 59 200 rpm 158 59 130 45 100 rpm 98 38 78 30 6 rpm 24 11 16 10 3 rpm 20 9 12 9 TABLE 5 Mud system PETROFREE Mud weight, lb/gal 11.0 Oil/water ratio 70/30 Contaminant Excess GELTONE II E2, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 132 31 88 29 Yield point, lb/100 ft2 54 53 37 53 10 sec gel, lb/100 ft2 33 23 13 26 10 min gel, lb/100 ft2 38 27 17 30 Fann 35 dial readings 600 rpm 318 115 213 111 300 rpm 186 84 125 82 200 rpm 139 71 90 70 100 rpm 91 54 56 55 6 rpm 35 25 15 28 3 rpm 32 21 13 25 TABLE 6 Mud system PETROFREE Mud weight, lb/gal 11.0 Oil/water ratio 70/30 Contaminant Drill solids E2, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 110 34 113 34 Yield point, lb/100 ft2 90 47 73 44 10 sec gel, lb/100 ft2 38 21 27 20 10 min gel, lb/100 ft2 44 24 30 22 Fann 35 dial readings 600 rpm 310 115 299 112 300 rpm 200 81 186 78 200 rpm 157 67 142 64 100 rpm 110 50 95 48 6 rpm 42 23 31 22 3 rpm 38 21 27 19 TABLE 7 Mud system PETROFREE LE Mud weight, lb/gal 16.4 E2, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 173 40 107 43 Yield point, lb/100 ft2 21 9 18 7 10 sec gel, lb/100 ft2 16 8 11 8 10 min gel, lb/100 ft2 19 11 15 11 Fann 35 dial readings 600 rpm 367 89 232 93 300 rpm 194 49 125 50 200 rpm 135 35 88 37 100 rpm 74 22 50 22 6 rpm 12 5 9 6 3 rpm 10 4 7 5 TABLE 8 Mud system PETROFREE LE Mud weight, lb/gal 11.6 E2, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 80 31 56 32 Yield point, lb/100 ft2 25 18 27 16 10 sec gel, lb/100 ft2 12 8 17 9 10 min gel, lb/100 ft2 20 11 23 11 Fann 35 dial readings 600 rpm 185 80 139 80 300 rpm 105 49 83 48 200 rpm 77 37 63 37 100 rpm 46 24 43 24 6 rpm 11 7 14 8 3 rpm 9 6 13 7 TABLE 9 Mud system PETROFREE LV Mud weight, lb/gal 14.0 Oil/water ratio 70/30 Contaminant Drill solids E1, lb/bbl 0 3 Temperature, ° F. 40 120 40 120 Plastic viscosity, cP 118 40 113 35 Yield point, lb/100 ft2 38 14 41 16 10 sec gel, lb/100 ft2 16 6 16 9 10 min gel, lb/100 ft2 22 11 20 11 Fann 35 dial readings 600 rpm 274 94 267 86 300 rpm 156 54 154 51 200 rpm 114 40 114 39 100 rpm 70 25 71 26 6 rpm 17 6 18 8 3 rpm 14 5 14 8 The foregoing description of the invention is intended to be a description of preferred embodiments.",
"Various changes in the details of the described composition and method can be made without departing from the intended scope of this invention as defined by the appended claims."
]
] | Patent_10432787 |
[
[
"Latex products",
"The present invention finds that a detackified natural rubber latex product can be provided by a combination of treatment with a hydrophilic group sealant, coating with a detackifying polymer, halogenation and the like.",
"The present invention also finds that a detackified natural rubber latex product which causes no discoloration of a metallic product surface can be provided by coating it with a detackifying, carboxylated latex vulcanized without using sulfur.",
"The present invention also finds that a detackified natural rubber latex product of controlled protein elution can be provided by chemically modifying protein present in the natural rubber latex with an anionic group, cationic group or the like."
],
[
"1.A detackified natural rubber latex product, characterized in that both surfaces are provided with a detackified, diene-based carboxylated synthetic rubber latex coating layer.",
"2.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that a natural rubber latex is incorporated with a detackifying hydrophilic polymer and/or hydrophilic group sealant.",
"3.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the surfaces of a natural rubber latex product are treated with a hydrophilic group sealant.",
"4.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that a natural rubber latex is incorporated with at least one selected from the group consisting of nonionic polymer and anionic polymer, and cationic polymer and ampholytic polymer which cause no gelation of the natural rubber latex, and further with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"5.The detackified natural rubber latex product with one or both surfaces detackified according to any one of claims 2 to 4, characterized in that an external surface of a natural rubber latex product or a natural rubber latex product incorporated with a hydrophilic group sealant and/or hydrophilic polymer is detackified by providing at least one layer selected from the group consisting of a detackified polymer layer, a halogenation treated layer, a layer treated with a detackifying crosslinking agent of tri- or tetra-valent metallic element, and a layer treated with at least one of a peroxotitania solution, peroxotitania sol, zirconia sol or alumina sol, a layer treated with a hydrophilic group sealant and a layer treated with a carboxyl group sealant.",
"6.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackified polymer coating layer, as set forth in claim 5, on an external surface is a detackifying, diene-based carboxylated synthetic rubber latex coating layer or a detackifying, releasing agent coating layer.",
"7.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying diene-based carboxylated synthetic rubber latex coating layer, as set forth in any one of claims 1, 5 and 6, on an external surface is detackified by incorporating the polymer or a diene-based carboxylated synthetic rubber latex with a hydrophilic group sealant or a carboxyl group sealant.",
"8.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying diene-based carboxylated synthetic rubber latex coating layer, as set forth in claims 1, 5 and 6, on an external surface is detackified by at least one selected from a hydrophilic group sealant and a carboxyl group sealant incorporated in the natural rubber latex.",
"9.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber coating latex layer, as set forth in any one of claims 1, 5 and 6, on an external surface is detackified by treating a surface of the polymer coating layer or diene-based carboxylated synthetic rubber latex coating layer with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"10.A detackified, lubricating, diene-based carboxylated synthetic rubber latex coat or product with one or both surfaces detackified, characterized in that a lubricating, diene-based carboxylated synthetic rubber latex coat or product, which is incorporated with a reactive, cationic compound or the lubricating, diene-based carboxylated synthetic rubber latex coat or product treated with one or more carboxyl group sealants.",
"11.The detackified natural rubber latex product with one or both surfaces detackified according to claim 7, characterized in that an external surface is coated with a detackified, lubricating, diene-based carboxylated synthetic rubber latex incorporated with a reactive, cationic compound.",
"12.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that an internal surface of the natural rubber latex product as set forth in any one of claims 1 to 11 is detackified with at least one selected from a hydrophilic group sealant and a carboxyl group sealant incorporated in the natural rubber latex.",
"13.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that an internal surface of the natural rubber latex product as set forth in any one of claims 1 to 11 is detackified with at least one selected from a hydrophilic group sealant and a carboxyl group sealant incorporated in the detackified polymer coating layer or detackifying, diene-based carboxylated synthetic rubber latex coating layer on an external surface.",
"14.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that an internal surface of the product as set forth in any one of claims 1 to 11 is detackified by providing a detackifying polymer layer, layer treated with detackifying crosslinking agent of tri- or tetra-valent metallic element, or a layer treated with a hydrophilic group sealant or a carboxyl group sealant.",
"15.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer on an internal surface, as set forth in claim 14, is a detackifying, diene-based carboxylated synthetic rubber latex coating layer.",
"16.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in claim 14 or 15, on an internal surface is detackified by incorporating the polymer or the carboxylated synthetic rubber latex with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"17.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in claims 14 or 15, on an internal surface is detackified by coating the internal surface of the polymer coating layer or the carboxylated synthetic rubber latex coating layer with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"18.A detackified natural rubber latex product with one or both surfaces detackified, wherein the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer on an internal surface, as set forth in claims 14 or 15, is detackified with a hydrophilic group sealant or a carboxyl group sealant incorporated in the detackifying polymer coating layer or detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in any one of claims 1 and 5 to 7, on an external surface, or with a hydrophilic group sealant or a carboxyl group sealant incorporated in the natural rubber latex.",
"19.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, nonionic polymer, as set forth in claim 4 or 5, has at least one hydrophilic group selected from the group consisting of hydroxyl (—OH), ether (—O—) and amide (—CONH2—) groups.",
"20.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, anionic polymer, as set forth in claim 4 or 5, has at least one hydrophilic group selected from the group consisting of carboxyl (—COOM), sulfate ester (—OSO3M), sulfonate (—SO2OM), phosphate (—PO3HM or —PO3M2), phosphate ester, —SO2NH2, and —SO2NHCOR groups, where M is hydrogen atom, and alkali metal, ammonia or organoammonium; and R is an alkyl, phenyl which may be substituted or not, or naphthyl group which may be substituted or not.",
"21.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, cationic polymer which causes no gelation of the natural rubber latex, as set forth in claim 4 or 5, has at least one compound selected from the group consisting of amine salt (primary, secondary or tertiary), quaternary ammonium or pyridinium salt, phosphonium salt and sulfonium salt.",
"22.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, ampholytic polymer which causes no gelation of the natural rubber latex, as set forth in claim 4 or 5, has the hydrophilic group as set forth in claims 20 and 21.23.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic polymer, as set forth in any one of claims 4, 5 and 19 to 22, is a water-soluble polysaccharide or derivative thereof.",
"24.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the water-soluble polysaccharide, as set forth in claim 23, is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, ureaphosphate-esterified starch, cationized starch, ampholytic starch, guar gum, phosphate-esterified guar gum, ampholytic guar gum, sodium alginate, carrageenan, locust bean gum, and xanthan gum.",
"25.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic polymer, as set forth in any one of claims 4, 5 and 19 to 22, is water-soluble, water-sensitive or water-dispersible synthetic polymer.",
"26.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic polymer, as set forth in claim 25, is selected from the group consisting of ammonium polyacrylate, ampholytic polyacrylamide, polyethylene oxide, polyvinyl alcohol, cationic polyamide resin, carboxylate-based acrylic copolymer, cationic acrylic copolymer, N-methoxymethylated polyamide modification (water-soluble nylon), acrylate ester copolymer, polyvinyl butyral, and cationic styrene/acrylic acid copolymer.",
"27.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the water-dispersible synthetic polymer, as set forth in claim 25 is selected from the group consisting of polyvinyl acetate, ethylene-vinyl acetate copolymer, styrene/acrylate ester copolymer, styrene/methacrylate ester copolymer, acrylate ester copolymer, alkali-thickened acrylic-based emulsion, methacrylate ester copolymer, vinyl acetate/acrylic acid copolymer, vinyl acetate/acrylate ester copolymer, vinyl acetate/methacrylic acid copolymer, vinyl acetate/methacrylate ester copolymer, polyacrylamide, polymethacrylamide, copolymerized polyamide emulsion, acrylamide-based copolymer, methacrylamide-based copolymer, anionic, cationic and ampholytic modifications of these polymers, polyvinyl butyral emulsion, and polyolefin containing carboxyl group.",
"28.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying, hydrophilic polymer, as set forth in claim 2, is at least one selected from the group consisting of methyl cellulose, locust bean gum, xanthan gum, carboxymethyl cellulose, alginate, carrageenan, and polyamide derivative.",
"29.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18, is a detackifying crosslinking agent of tri- or tetra-valent metallic element.",
"30.The detackified natural rubber latex product with one or both surfaces detackified according to claim 29, characterized in that the detackifying crosslinking agent of tri- or tetra-valent metallic element contains at least selected from the group consisting of aluminum, titanium and zirconium compounds.",
"31.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18 is at least one selected from the group consisting of peroxotitania solution, peroxotitania sol, zirconia sol and alumina sol.",
"32.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18, is a detackifying, hydrophobic, organic crosslinking agent for the hydrophilic polymer as set forth in claim 4 or 5 and/or an auxiliary component of natural rubber latex.",
"33.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying, hydrophobic, organic crosslinking agent, as set forth in claim 32, contains at least one selected from the group consisting of blocked isocyanate, oxazoline and carbodiimide.",
"34.The detackified natural rubber latex product with one or both surfaces detackified according to any one of claims 2 to 18, characterized in that the hydrophilic group sealant or carboxy group sealant contains at least one type of detackifying, hydrogen bond adjustors.",
"35.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying, hydrogen bond adjustor, as set forth in claim 34, is selected from the group consisting of a polyamide compound, polyamide epoxy resin, polyaminepolyurea-based resin and polyamidepolyurea-based resin.",
"36.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or the carboxy group sealant as set forth in any one of claims 2 to 18, and the compound reactive with the carboxyl group in the carboxylated synthetic rubber latex as set forth in claim 10 or 11 are polyamide amine/epihalohydrin condensate, polyamine/epihalohydrin condensate, polyamidepolyurea/epihalohydrin condensate, polyaminepolyurea/epihalohydrin condensate, polyamideaminepolyurea/epihalohydrin condensate, polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, styrene-based polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, cation-modified urea resin, and cation-modified, epoxy-based polyamide resin.",
"37.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18, contains at least one compound selected from the group consisting of monofunctional amine, monofunctional epoxy compound, monofunctional isocyanate, monofunctional blocked isocyanate, alkyl ketene dimer (AKD), alkenyl ketene dimer, alkenyl succinic anhydride (ASA), aliphatic acid anhydride, and isocyanate aziridine derivative.",
"38.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or the carboxy group sealant, as set forth in any one of claims 2 to 18, is a detackifying sizing agent.",
"39.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18 is a detackifying anionic, nonionic, or cationic surfactant.",
"40.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying waterproofing agent, as set forth in any one of claims 2 to 18, acts on a tacky auxiliary component of the natural rubber latex, incorporated hydrophilic nonionic, anionic, cationic or ampholytic polymer, or a polymer coating layer or a carboxylated synthetic rubber latex coating layer.",
"41.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying waterproofing agent, as set forth in claim 40, is a compound having a methylol group or lower alkylated compound thereof, aldehyde-based compound, a compound having an epoxy or chlorohydrin group, a compound having an ethyleneimine group, a polyvinyl butyral-based compound, or a tri- or tetra-valent multi-valent metallic compound.",
"42.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying waterproofing agent, as set forth in claims 40 and 41, is polyamide epoxy resin, branched polyethylene imine, modified polyamine-based resin, polyamide-based resin, ketone resin, alkyl ketene dimer, ammonium zirconium carbonate, or blocked glyoxal resin.",
"43.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18, is a detackifying water repellant.",
"44.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxy group sealant, as set forth in any one of claims 2 to 18, is a detackifying releasing agent.",
"45.A natural rubber latex product of controlled protein elution, characterized by being treated with a compound which can introduce an anionic and/or cationic group in protein in the natural rubber latex.",
"46.The natural rubber latex product of controlled protein elution according to claim 45, characterized in that the compound which can introduce an anionic and/or cationic group in the natural rubber latex is a compound reactive with protein in the natural rubber latex.",
"47.The natural rubber latex product of controlled protein elution according to claim 45, characterized in that the compound which can introduce an anionic and/or cationic group in the natural rubber latex is a fixing compound or compound which can be fixed.",
"48.The natural rubber latex product of controlled protein elution according to claim 46, characterized in that the compound which can introduce an anionic and/or cationic group in the natural rubber latex is reactive dye and derivative of carboxylic anhydride as anionic compounds; polyamideamine/epihalohydrin condensate, polyamine/epihalohydrin condensate, polyamidepolyurea/epihalohydrin condensate, polyaminepolyurea/epihalohydrin condensate, polyamideaminepolyurea/epihalohydrin condensate, polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, styrene-based, polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, cation-modified urea resin, cation-modified epoxy-based polyamide resin, crosslinking agent of multi-valent (trivalent or higher), and peroxotitania solution, peroxotitania sol, zirconia sol and alumina sol as cationic compounds.",
"49.The natural rubber latex product of controlled protein elution according to claim 47, characterized in that the fixing compound which can introduce an anionic and/or cationic group in the natural rubber latex is anionic, ampholytic and/or cationic starch.",
"50.The natural rubber latex product of controlled protein elution according to any one of claims 45 to 48, characterized in that the functional group reactive with protein of the compound which can introduce an anionic group in protein in the natural rubber latex is at least one selected from the group consisting of dichlorotriazine, difluorochlorotriazine, dichloroquinoxaline, monofluorotriazine, β-sulfatoethylsulfone, monochlorotriazine, trichloropyrimidine, carboxypyridino-S-triazine, α-bromoacrylamide, acrylamide, ω-chloroacetyl, epoxy and carboxyl anhydride.",
"51.A natural rubber latex product of controlled protein elution, characterized by being treated with a waterproofing agent (ketone resin) reactive with protein in natural rubber latex under an alkaline condition and capable of fixing the protein.",
"52.A detackified natural rubber latex product of controlled protein elution, characterized by being treated in a manner as set forth in any one of claims 45 to 51, and also in a manner as set forth in any one of claims 1 to 42.53.A producing method of the detackified natural rubber latex product with one or both surfaces detackified, and/or the natural rubber latex product of controlled protein elution, as set forth in any one of claims 1 to 52, characterized by being leaching-treated subsequent to drying at high temperature.",
"54.The detackified natural rubber latex product with one or both surfaces detackified, and/or the natural rubber latex product of controlled protein elution, according to any one of claims 1 to 52, characterized by being a fingerstall, glove, balloon or condom.",
"55.A finger stall of a detackified natural rubber latex with one or both surfaces detackified, and/or a natural rubber latex of controlled protein elution, characterized in that the finger stall of the detackified natural rubber latex and/or natural rubber latex of controlled protein elution as set forth in claim 54 has a shape of being mechanically wound up from a mouth before being released out of a mold."
],
[
"<SOH> BACKGROUND ART <EOH>Latex products of natural rubber, e.g., immersion-processed products (e.g., balloon, glove, fingerstall and condom); extruded products (e.g., rubber yarn and tube); formed products (e.g., balloon and toys); totally rubber products (e.g., rubber sheet, hose and cloth), and rubber-lined products, frequently have tacky surfaces, which may cause deteriorated processability of the products or defective products.",
"In addition, they have disadvantages such as incapability of manufacturing products of a desired product shape.",
"A detackifier has been used to solve these problems.",
"It is generally powdery and referred to as powder.",
"The common powder is of mica, talc, calcium carbonate, white carbon or corn starch.",
"The powder may be transferred to an object which comes into contact with the latex product to possibly cause various problems, when the product is used in the precision area.",
"It may also deteriorate the outer appearances of the latex product.",
"The FDA has issued the regulations on the rubber glove for medical purposes in July 1999, limiting protein elution to 1,200 μg or less and powder quantity to 120 mg or less for each glove.",
"The regulations will be more stringent to limit the powder quantity to 20 mg in and after 2001.The powder quantity is regarded as an important measure against latex-caused allergy, because it plays an important role in allergy sensibilization.",
"Protein in latex of natural rubber will cause no allergy sensibilization, when orally taken, because it is easily decomposed by a digestive juice.",
"However, the powder adsorbs the protein derived from latex of natural rubber at a high concentration during the production process, and is released into the atmosphere while the product is being used.",
"When the powder is inhaled or comes into contact with the skin, the latex protein will be absorbed in a living body, to cause the allergy sensibilization.",
"Therefore, the powder is an important mediator for the latex allergy, and new sensibilization of the latex allergy will be greatly diminished, if a latex product of natural rubber can be free of powder (Ken Yagami, Proceedings for 5 th Latex Allergy Meeting).",
"Thus, making latex products of natural rubber powder-free is an important technical theme.",
"The common tackiness prevention measures other than use of powder include halogenation by the post-chlorination treatment.",
"For examples, U.S. Pat.",
"Nos.",
"3,411,982 and 3,740,262 disclose that a rubber glove has slippery surfaces, when treated for halogenation.",
"U.S. Pat.",
"No.",
"4,304,008 discloses that halogenation facilitates use of the rubber products free of powder.",
"U.S. Pat.",
"No.",
"3,740,262 discloses halogenation of globes to provide powder-free external surface and powder-coated internal surface.",
"Halogenation is a fairly common method of preventing tackiness and blooming by coating the product surface with a thin, halogenated rubber layer, and provides the rubber products with clean, powder-free surface.",
"U.S. Pat.",
"No.",
"4,304,008 discloses a surgical glove comprising natural rubber for the internal layer and halogenated, durable silicone for the external layer, where the internal layer is halogenated to be detackified.",
"U.S. Pat.",
"No.",
"5,284,607, admitting defects involved in halogenation, discloses a method of forming a medical glove using an acid-soluble powder, which is subsequently treated with an acid, e.g., nitric acid, to dissolve the acid-soluble powder and then chlorinated with a bleaching agent.",
"Various improvements are noted in methods of producing rubber products which use powder or substance of particular structure.",
"U.S. Pat.",
"No.",
"4,070,713 discloses a medical glove of two-layered structure with external and internal layers of an elastic material, where particles of zinc oxide, titanium oxide or the like are fast embedded in the internal layer and partly exposed to the inner surface coming into contact with the skin.",
"U.S. Pat.",
"No.",
"4,143,109 discloses the method of producing the above described patent.",
"U.S. Pat.",
"No.",
"5,138,719 discloses a method of producing a powder-free glove, fingerstall and similar products using latex and microcapsules, where the microcapsules are dispersed and disposed in the latex in such a way to increase in concentration towards the inner surface of the product from the outer surface.",
"The microcapsules are present at a sufficiently high concentration on the inner surface to make the surface slippery, facilitating use of the product even in the absence of the powder.",
"U.S. Pat.",
"No.",
"5,881,386 discloses a glove of two-layered structure of polyvinyl chloride and polyester/polyurethane, the inner layer of polyester/polyurethane containing particles of 1 to 75 μm in size.",
"Japanese Patent Laid-Open No.",
"11-12823 discloses a technique for producing a glove which produces less dust for works in clean rooms, where the glove of polyvinylidine chloride paste sol is immersed in an inner surface treatment agent containing particles of 0.1 to 1.5 μm in size.",
"Japanese Patent Laid-Open No.",
"11-61527 discloses a rubber glove easily worn or taken off, provided with a slippery resin layer by immersing the glove in an aqueous dispersing solution containing synthetic rubber latex and an organic filler which are not coagulated in the absence of a coagulating agent contained in the glove body.",
"National Publication of International Patent Application No.",
"9-501983 discloses a silicone-modified powder composition dispersible in water and method of producing the same, describing that the composition can be used as a blocking inhibitor.",
"Recently, latex products coated with various materials have been developed.",
"U.S. Pat.",
"No.",
"4,310,928 provides a powder-free surgical glove coated on the natural rubber surface with oil, fat or lipophilic material dispersed in a coagulated liquid, where the coagulated liquid is incorporated with a surfactant to prevent separation of the oil, fat or lipophilic material.",
"U.S. Pat.",
"Nos.",
"5,780,112 and 5,974,589 disclose a method of adhering a high-density, straight-chain hydrocarbon polymer, in particular polyethylene, to the natural rubber surface with the aid of chlorine generated from acidified hypochlorite, giving the treated latex product which is not tacky although free of powder.",
"National Publication of International Patent Application No.",
"11-507085 discloses a flexible copolymer coating which can be fast adhesive to the rubber product surface and extended without being separated from the rubber surface to which it is bonded, and also discloses an emulsion-based copolymer of a reactive monomer of low surface energy (preferably silicone oligomer), alkyl acrylate and reactive, hard monomer, in consideration of releasability from an immersion mold and easiness of wearing under both dry and wet conditions.",
"A number of methods have been proposed for producing a powder-free glove, which coat the rubber surface with a polymer capable of forming a hydrophilic hydrogel and then cure the polymer layer, e.g., by U.S. Pat.",
"Nos.",
"3,326,742, 3,585,103, 3,607,473, 3,745,042, 3,901,755, 3,925,138, 3,930,076, 3,940,533, 3,966,530, 4,024,317, 4,110,495, and 4,125,477.U.S.",
"Pat.",
"No.",
"4,499,154 discloses a method of producing a talc-free product, where an immersion-processed product is immersed in a natural rubber latex, leached in hot water, impregnated with a diluted acid, treated with water or an aqueous alkali solution to neutralize the surface, immersed in a polymer capable of forming hydrophilic hydrogel (e.g., copolymer of 2-hydroxyethyl methacrylate and methacrylic acid or 2-ethylhexyl acrylate) and a crosslinking agent solution therefor, heated to fix the coating layer to the rubber, treated to vulcanize the rubber, released out of the mold, spread with surfactant-containing silicone, and heated.",
"The patent also discloses that the method improves slipping characteristics of the product for a wet hand, when the coating layer of the hydrogel polymer is crosslinked, and then treated with a cationic surfactant, e.g., long-chain aliphatic amine.",
"This method, although giving a powder-free rubber product, needs many steps to unreasonably push up the production cost, and is impractical for production of a product sensitive to contamination with silicone.",
"U.S. Pat.",
"No.",
"4,575,476 discloses that the product coated with a specific 2-oxyethylmethacrylate-based hydrogel polymer has good slipping characteristics for a dry hand.",
"It also describes that the product surface to come into contact with the skin has improved slipping characteristics for a wet hand, when the hydrogel coating layer is treated with a surfactant (in particular cationic one) and long-chain aliphatic amine, and that tackiness of the surface not coated with the hydrogel is greatly improved when it is treated with a silicone-containing surfactant.",
"U.S. Pat.",
"No.",
"5,688,855 describes that hydrophilicity of the solid surface gives surface lubricity in the presence of water, providing a method of automatically producing a hydrophilicity concentration gradient in the coating layer by coating the rubber product surface with a solution of polymer component capable of forming hydrogel and water-soluble polymer component low in compatibility with the above-described component in a solvent, and evaporating the solvent to separate these components from each other.",
"Japanese Patent Laid-Open No.",
"11-269708 discloses a glove comprising a base layer of rubber or resin laminated on the inner surface with a lubricating layer of collagen-containing rubber or resin.",
"One of the disadvantages of the product produced by the method of coating the rubber surface is the interlayer exfoliation when the rubber is extended.",
"U.S. Pat.",
"No.",
"4,499,154 reinforces adhesion of the coating layer by undercoating the rubber surface with an acid.",
"WO 93/06996-A1 proposes use of a polymer having a repeating structure of a specific ether and ester group as the coating layer.",
"U.S. Pat.",
"No.",
"4,548,844 discloses a method of improving adhesion between the rubber and hydrogel layers by acid treatment, describing that adhesion between these layers is improved when aluminum cations or trivalent or higher cations are applied before the hydrogel polymer, or incorporated in the polymer, conceivably because the hydroxyl or carboxyl group in the hydrogel polymer is bonded to the protein in the rubber latex.",
"Japanese Patent Laid-Open No.",
"6-70942 discloses a multi-layered product composed of the first layer of natural rubber, second layer of natural rubber, poly(acrylamide/acrylic acid) and polyethylene oxide, and third layer of acrylic copolymer and fluorocarbon telomere, claiming that the product can be worn under both dry and wet conditions in the absence of powder.",
"Japanese Patent Laid-Open No.",
"10-95867 discloses a method of producing a powder-free medical glove or the like which is coated with a lubricating composition composed of the first and second components in this order from the wearer's side of the elastomer product.",
"The first composition is composed of at least one compound selected from the group consisting of acetylenediol, organically modified silicone, amino-modified silicone and cationic surfactant, and the second composition of at least one compound selected from the group consisting of cationic surfactant, organically modified silicone, amino-modified silicone and acetylenediol.",
"Japanese Patent Publication No.",
"7-4405 discloses a technique for surface treatment with modified polysiloxane.",
"One of the methods of producing powder-free rubber products coats the rubber product on one side by immersing in latex the mold lined with a coagulating agent, wherein a divalent metallic salt as the coagulating agent (e.g., calcium nitrate) and water-soluble surfactant (preferably nonionic) stable to the metallic salt are incorporated in the coagulating solution to stabilize the latex or resin polymer.",
"This method by itself is not intended to detackify the rubber product, but can detackify it when a releasing agent or detackifier is incorporated as the third component in the coagulating agent composition.",
"U.S. Pat.",
"Nos.",
"3,286,011 and 3,411,982 by Kavalir et al disclose the above techniques.",
"These patents, however, cannot make the product powder-free, because power is used as the releasing agent.",
"It is described that salts of multi-valent metals (e.g., calcium, magnesium and aluminum) can be used as the latex coagulating agent for these patents.",
"The above-described U.S. Pat.",
"No.",
"4,310,928 discloses a technique for producing a surgical glove releasable from an immersion mold using a coagulating agent comprising a coagulating agent solution, e.g., that of calcium nitrate, dispersed with a lipophilic substance.",
"National Publication of International Patent Application No.",
"10-508899 discloses a method of producing a powder-free rubber product by incorporating a coating composition of acrylic-based emulsion copolymer and silicone emulsion in a coagulating agent.",
"The coating composition is produced by copolymerization of a reactive silicone acrylate, alkyl acrylate and hard monomer.",
"It is described that such a composition is known, facilitates releasing when incorporated with a silicone emulsion, and gives the glove showing good wearing characteristics under both dry and wet conditions.",
"EP 640,623 discloses a coagulating agent for natural rubber, composed of a salt-stable polychloroprene or polyurethane and divalent metal salt, and describes that a powder-free rubber glove can be produced by incorporating the coagulating agent with a releasing agent composed of polyethylene wax emulsion and cationic surfactant.",
"Japanese Patent Laid-Open No.",
"11-236466 uses a surfactant, polypropylene wax emulsion or the like as the tacky quenching or releasing agent in place of the above-described polyethylene wax emulsion, describing that it works as the agent to release polychloroprene from the immersion mold, because the cationic surfactant functions to stabilize polychloroprene to be incorporated in the coagulating solution and is more compatible with the immersion mold than with the polymer.",
"Japanese Patent Publication No.",
"2-42082 discloses a coagulating agent composition composed of water incorporated with latex, surfactant and divalent or trivalent metallic salt.",
"National Publication of International Patent Application No.",
"9-511708 employs the Teague process for producing a polyurethane-coated glove, wherein the glove is immersed in an aqueous dispersion or emulsion of polyurethane polymer or copolymer to form the first layer, which is then immersed in a coagulating agent and further in a latex compound to form the second layer.",
"It also discloses a technique for forming a lubricating polymer layer on the second layer.",
"The techniques for powder-free rubber products from novel starting materials are also disclosed.",
"U.S. Pat.",
"No.",
"5,851,683 proposes a special, consecutively copolymerized polymer for a powder-free glove of thermoplastic elastomer for use in clean rooms.",
"These methods of preventing tackiness of latex products are important techniques both from production and purposes of the products, and various ones are proposed.",
"However, they generally need complex steps, and few processes developed so far are simple, effective and economical.",
"It is planned to regulate quantity of protein eluted out of natural rubber latex products in consideration of allergy possibly caused by them, and reduction of the quantity has been demanded.",
"Halogenation is a known process for decomposition of the protein.",
"Moreover, natural rubber latex products have been recently produced by new processes, wherein protein in natural rubber latex is enzymatically decomposed by the aid of protease (disclosed by, e.g., Japanese Patent Laid-Open No.",
"6-56902).",
"These natural rubber latex products involve various disadvantages, e.g., insufficient vulcanization characteristics and strength-related properties.",
"They are tacky as is the case with common natural rubber latex products.",
"The techniques for reducing allergen activity of natural rubber latex products are proposed by, e.g., WO97/08228, wherein protein in natural rubber latex is leached out in the process of producing the product, and protein eluted out in the vicinity of the latex film surface or in the treatment solution is reacted with an epoxy compound, glyoxal or the like.",
"WO97/08228, however, is completely silent on reducing eluted quantity of the proteins planned to be regulated.",
"Moreover, the epoxy compound, glyoxal or the like has mutagenicity, and may cause dermatitis.",
"In addition, it is difficult to detackify a natural rubber latex product, when the tacky substance in leached out to the surface.",
"It is an object of the present invention to provide a novel natural rubber latex product and method of producing the same, in consideration of the problems involved in the current techniques to detackify the product.",
"It is another object to provide a natural rubber latex product which causes no discoloration of the metallic product surface by sulfur used for vulcanization.",
"It is still another object to provide a natural rubber latex product which controls elution of protein and method of producing the same.",
"It is still another object to provide a natural rubber latex product which is free of powder and detackified, and controls elution of protein."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is an oblique view of the immersion type carrier for the present invention.",
"FIG.",
"2 describes a prototype production unit for the fingerstall of the present invention.",
"FIG.",
"3 describes functions of the major parts of the winding-up unit.",
"FIG.",
"4 shows the cross-section of the wound-up finger stall put on a finger.",
"FIG.",
"5 describes the cross-section of the fingerstall wound on the finger.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?",
"The symbols are 1 : chain, 2 : guide rail, 3 : immersion mold, 4 : rod, 5 : guide, 6 : immersion tank, 7 : drying furnace, 8 : winding-up machine, 10 : roll type brush, 11 : film, 12 : fingerstall, 13 : fingertip, 14 : finger, and 15 : wound-up mouth."
],
[
"TECHNICAL FIELD The present invention relates to a novel non-adhesive latex product of natural rubber (NR), and novel method of producing the same.",
"BACKGROUND ART Latex products of natural rubber, e.g., immersion-processed products (e.g., balloon, glove, fingerstall and condom); extruded products (e.g., rubber yarn and tube); formed products (e.g., balloon and toys); totally rubber products (e.g., rubber sheet, hose and cloth), and rubber-lined products, frequently have tacky surfaces, which may cause deteriorated processability of the products or defective products.",
"In addition, they have disadvantages such as incapability of manufacturing products of a desired product shape.",
"A detackifier has been used to solve these problems.",
"It is generally powdery and referred to as powder.",
"The common powder is of mica, talc, calcium carbonate, white carbon or corn starch.",
"The powder may be transferred to an object which comes into contact with the latex product to possibly cause various problems, when the product is used in the precision area.",
"It may also deteriorate the outer appearances of the latex product.",
"The FDA has issued the regulations on the rubber glove for medical purposes in July 1999, limiting protein elution to 1,200 μg or less and powder quantity to 120 mg or less for each glove.",
"The regulations will be more stringent to limit the powder quantity to 20 mg in and after 2001.The powder quantity is regarded as an important measure against latex-caused allergy, because it plays an important role in allergy sensibilization.",
"Protein in latex of natural rubber will cause no allergy sensibilization, when orally taken, because it is easily decomposed by a digestive juice.",
"However, the powder adsorbs the protein derived from latex of natural rubber at a high concentration during the production process, and is released into the atmosphere while the product is being used.",
"When the powder is inhaled or comes into contact with the skin, the latex protein will be absorbed in a living body, to cause the allergy sensibilization.",
"Therefore, the powder is an important mediator for the latex allergy, and new sensibilization of the latex allergy will be greatly diminished, if a latex product of natural rubber can be free of powder (Ken Yagami, Proceedings for 5th Latex Allergy Meeting).",
"Thus, making latex products of natural rubber powder-free is an important technical theme.",
"The common tackiness prevention measures other than use of powder include halogenation by the post-chlorination treatment.",
"For examples, U.S. Pat.",
"Nos.",
"3,411,982 and 3,740,262 disclose that a rubber glove has slippery surfaces, when treated for halogenation.",
"U.S. Pat.",
"No.",
"4,304,008 discloses that halogenation facilitates use of the rubber products free of powder.",
"U.S. Pat.",
"No.",
"3,740,262 discloses halogenation of globes to provide powder-free external surface and powder-coated internal surface.",
"Halogenation is a fairly common method of preventing tackiness and blooming by coating the product surface with a thin, halogenated rubber layer, and provides the rubber products with clean, powder-free surface.",
"U.S. Pat.",
"No.",
"4,304,008 discloses a surgical glove comprising natural rubber for the internal layer and halogenated, durable silicone for the external layer, where the internal layer is halogenated to be detackified.",
"U.S. Pat.",
"No.",
"5,284,607, admitting defects involved in halogenation, discloses a method of forming a medical glove using an acid-soluble powder, which is subsequently treated with an acid, e.g., nitric acid, to dissolve the acid-soluble powder and then chlorinated with a bleaching agent.",
"Various improvements are noted in methods of producing rubber products which use powder or substance of particular structure.",
"U.S. Pat.",
"No.",
"4,070,713 discloses a medical glove of two-layered structure with external and internal layers of an elastic material, where particles of zinc oxide, titanium oxide or the like are fast embedded in the internal layer and partly exposed to the inner surface coming into contact with the skin.",
"U.S. Pat.",
"No.",
"4,143,109 discloses the method of producing the above described patent.",
"U.S. Pat.",
"No.",
"5,138,719 discloses a method of producing a powder-free glove, fingerstall and similar products using latex and microcapsules, where the microcapsules are dispersed and disposed in the latex in such a way to increase in concentration towards the inner surface of the product from the outer surface.",
"The microcapsules are present at a sufficiently high concentration on the inner surface to make the surface slippery, facilitating use of the product even in the absence of the powder.",
"U.S. Pat.",
"No.",
"5,881,386 discloses a glove of two-layered structure of polyvinyl chloride and polyester/polyurethane, the inner layer of polyester/polyurethane containing particles of 1 to 75 μm in size.",
"Japanese Patent Laid-Open No.",
"11-12823 discloses a technique for producing a glove which produces less dust for works in clean rooms, where the glove of polyvinylidine chloride paste sol is immersed in an inner surface treatment agent containing particles of 0.1 to 1.5 μm in size.",
"Japanese Patent Laid-Open No.",
"11-61527 discloses a rubber glove easily worn or taken off, provided with a slippery resin layer by immersing the glove in an aqueous dispersing solution containing synthetic rubber latex and an organic filler which are not coagulated in the absence of a coagulating agent contained in the glove body.",
"National Publication of International Patent Application No.",
"9-501983 discloses a silicone-modified powder composition dispersible in water and method of producing the same, describing that the composition can be used as a blocking inhibitor.",
"Recently, latex products coated with various materials have been developed.",
"U.S. Pat.",
"No.",
"4,310,928 provides a powder-free surgical glove coated on the natural rubber surface with oil, fat or lipophilic material dispersed in a coagulated liquid, where the coagulated liquid is incorporated with a surfactant to prevent separation of the oil, fat or lipophilic material.",
"U.S. Pat.",
"Nos.",
"5,780,112 and 5,974,589 disclose a method of adhering a high-density, straight-chain hydrocarbon polymer, in particular polyethylene, to the natural rubber surface with the aid of chlorine generated from acidified hypochlorite, giving the treated latex product which is not tacky although free of powder.",
"National Publication of International Patent Application No.",
"11-507085 discloses a flexible copolymer coating which can be fast adhesive to the rubber product surface and extended without being separated from the rubber surface to which it is bonded, and also discloses an emulsion-based copolymer of a reactive monomer of low surface energy (preferably silicone oligomer), alkyl acrylate and reactive, hard monomer, in consideration of releasability from an immersion mold and easiness of wearing under both dry and wet conditions.",
"A number of methods have been proposed for producing a powder-free glove, which coat the rubber surface with a polymer capable of forming a hydrophilic hydrogel and then cure the polymer layer, e.g., by U.S. Pat.",
"Nos.",
"3,326,742, 3,585,103, 3,607,473, 3,745,042, 3,901,755, 3,925,138, 3,930,076, 3,940,533, 3,966,530, 4,024,317, 4,110,495, and 4,125,477.U.S.",
"Pat.",
"No.",
"4,499,154 discloses a method of producing a talc-free product, where an immersion-processed product is immersed in a natural rubber latex, leached in hot water, impregnated with a diluted acid, treated with water or an aqueous alkali solution to neutralize the surface, immersed in a polymer capable of forming hydrophilic hydrogel (e.g., copolymer of 2-hydroxyethyl methacrylate and methacrylic acid or 2-ethylhexyl acrylate) and a crosslinking agent solution therefor, heated to fix the coating layer to the rubber, treated to vulcanize the rubber, released out of the mold, spread with surfactant-containing silicone, and heated.",
"The patent also discloses that the method improves slipping characteristics of the product for a wet hand, when the coating layer of the hydrogel polymer is crosslinked, and then treated with a cationic surfactant, e.g., long-chain aliphatic amine.",
"This method, although giving a powder-free rubber product, needs many steps to unreasonably push up the production cost, and is impractical for production of a product sensitive to contamination with silicone.",
"U.S. Pat.",
"No.",
"4,575,476 discloses that the product coated with a specific 2-oxyethylmethacrylate-based hydrogel polymer has good slipping characteristics for a dry hand.",
"It also describes that the product surface to come into contact with the skin has improved slipping characteristics for a wet hand, when the hydrogel coating layer is treated with a surfactant (in particular cationic one) and long-chain aliphatic amine, and that tackiness of the surface not coated with the hydrogel is greatly improved when it is treated with a silicone-containing surfactant.",
"U.S. Pat.",
"No.",
"5,688,855 describes that hydrophilicity of the solid surface gives surface lubricity in the presence of water, providing a method of automatically producing a hydrophilicity concentration gradient in the coating layer by coating the rubber product surface with a solution of polymer component capable of forming hydrogel and water-soluble polymer component low in compatibility with the above-described component in a solvent, and evaporating the solvent to separate these components from each other.",
"Japanese Patent Laid-Open No.",
"11-269708 discloses a glove comprising a base layer of rubber or resin laminated on the inner surface with a lubricating layer of collagen-containing rubber or resin.",
"One of the disadvantages of the product produced by the method of coating the rubber surface is the interlayer exfoliation when the rubber is extended.",
"U.S. Pat.",
"No.",
"4,499,154 reinforces adhesion of the coating layer by undercoating the rubber surface with an acid.",
"WO 93/06996-A1 proposes use of a polymer having a repeating structure of a specific ether and ester group as the coating layer.",
"U.S. Pat.",
"No.",
"4,548,844 discloses a method of improving adhesion between the rubber and hydrogel layers by acid treatment, describing that adhesion between these layers is improved when aluminum cations or trivalent or higher cations are applied before the hydrogel polymer, or incorporated in the polymer, conceivably because the hydroxyl or carboxyl group in the hydrogel polymer is bonded to the protein in the rubber latex.",
"Japanese Patent Laid-Open No.",
"6-70942 discloses a multi-layered product composed of the first layer of natural rubber, second layer of natural rubber, poly(acrylamide/acrylic acid) and polyethylene oxide, and third layer of acrylic copolymer and fluorocarbon telomere, claiming that the product can be worn under both dry and wet conditions in the absence of powder.",
"Japanese Patent Laid-Open No.",
"10-95867 discloses a method of producing a powder-free medical glove or the like which is coated with a lubricating composition composed of the first and second components in this order from the wearer's side of the elastomer product.",
"The first composition is composed of at least one compound selected from the group consisting of acetylenediol, organically modified silicone, amino-modified silicone and cationic surfactant, and the second composition of at least one compound selected from the group consisting of cationic surfactant, organically modified silicone, amino-modified silicone and acetylenediol.",
"Japanese Patent Publication No.",
"7-4405 discloses a technique for surface treatment with modified polysiloxane.",
"One of the methods of producing powder-free rubber products coats the rubber product on one side by immersing in latex the mold lined with a coagulating agent, wherein a divalent metallic salt as the coagulating agent (e.g., calcium nitrate) and water-soluble surfactant (preferably nonionic) stable to the metallic salt are incorporated in the coagulating solution to stabilize the latex or resin polymer.",
"This method by itself is not intended to detackify the rubber product, but can detackify it when a releasing agent or detackifier is incorporated as the third component in the coagulating agent composition.",
"U.S. Pat.",
"Nos.",
"3,286,011 and 3,411,982 by Kavalir et al disclose the above techniques.",
"These patents, however, cannot make the product powder-free, because power is used as the releasing agent.",
"It is described that salts of multi-valent metals (e.g., calcium, magnesium and aluminum) can be used as the latex coagulating agent for these patents.",
"The above-described U.S. Pat.",
"No.",
"4,310,928 discloses a technique for producing a surgical glove releasable from an immersion mold using a coagulating agent comprising a coagulating agent solution, e.g., that of calcium nitrate, dispersed with a lipophilic substance.",
"National Publication of International Patent Application No.",
"10-508899 discloses a method of producing a powder-free rubber product by incorporating a coating composition of acrylic-based emulsion copolymer and silicone emulsion in a coagulating agent.",
"The coating composition is produced by copolymerization of a reactive silicone acrylate, alkyl acrylate and hard monomer.",
"It is described that such a composition is known, facilitates releasing when incorporated with a silicone emulsion, and gives the glove showing good wearing characteristics under both dry and wet conditions.",
"EP 640,623 discloses a coagulating agent for natural rubber, composed of a salt-stable polychloroprene or polyurethane and divalent metal salt, and describes that a powder-free rubber glove can be produced by incorporating the coagulating agent with a releasing agent composed of polyethylene wax emulsion and cationic surfactant.",
"Japanese Patent Laid-Open No.",
"11-236466 uses a surfactant, polypropylene wax emulsion or the like as the tacky quenching or releasing agent in place of the above-described polyethylene wax emulsion, describing that it works as the agent to release polychloroprene from the immersion mold, because the cationic surfactant functions to stabilize polychloroprene to be incorporated in the coagulating solution and is more compatible with the immersion mold than with the polymer.",
"Japanese Patent Publication No.",
"2-42082 discloses a coagulating agent composition composed of water incorporated with latex, surfactant and divalent or trivalent metallic salt.",
"National Publication of International Patent Application No.",
"9-511708 employs the Teague process for producing a polyurethane-coated glove, wherein the glove is immersed in an aqueous dispersion or emulsion of polyurethane polymer or copolymer to form the first layer, which is then immersed in a coagulating agent and further in a latex compound to form the second layer.",
"It also discloses a technique for forming a lubricating polymer layer on the second layer.",
"The techniques for powder-free rubber products from novel starting materials are also disclosed.",
"U.S. Pat.",
"No.",
"5,851,683 proposes a special, consecutively copolymerized polymer for a powder-free glove of thermoplastic elastomer for use in clean rooms.",
"These methods of preventing tackiness of latex products are important techniques both from production and purposes of the products, and various ones are proposed.",
"However, they generally need complex steps, and few processes developed so far are simple, effective and economical.",
"It is planned to regulate quantity of protein eluted out of natural rubber latex products in consideration of allergy possibly caused by them, and reduction of the quantity has been demanded.",
"Halogenation is a known process for decomposition of the protein.",
"Moreover, natural rubber latex products have been recently produced by new processes, wherein protein in natural rubber latex is enzymatically decomposed by the aid of protease (disclosed by, e.g., Japanese Patent Laid-Open No.",
"6-56902).",
"These natural rubber latex products involve various disadvantages, e.g., insufficient vulcanization characteristics and strength-related properties.",
"They are tacky as is the case with common natural rubber latex products.",
"The techniques for reducing allergen activity of natural rubber latex products are proposed by, e.g., WO97/08228, wherein protein in natural rubber latex is leached out in the process of producing the product, and protein eluted out in the vicinity of the latex film surface or in the treatment solution is reacted with an epoxy compound, glyoxal or the like.",
"WO97/08228, however, is completely silent on reducing eluted quantity of the proteins planned to be regulated.",
"Moreover, the epoxy compound, glyoxal or the like has mutagenicity, and may cause dermatitis.",
"In addition, it is difficult to detackify a natural rubber latex product, when the tacky substance in leached out to the surface.",
"It is an object of the present invention to provide a novel natural rubber latex product and method of producing the same, in consideration of the problems involved in the current techniques to detackify the product.",
"It is another object to provide a natural rubber latex product which causes no discoloration of the metallic product surface by sulfur used for vulcanization.",
"It is still another object to provide a natural rubber latex product which controls elution of protein and method of producing the same.",
"It is still another object to provide a natural rubber latex product which is free of powder and detackified, and controls elution of protein.",
"DISCLOSURE OF THE INVENTION The inventors of the present invention have extensively studied to solve the above-described problems.",
"They have investigated various hydrophobicizing agents to make a natural rubber latex product non-hydrophilic and their hydrophobicizing capacity, based on the inventions made by them for detackifying the diene-based carboxylated synthetic rubber latex products (PCT/JP00/03370 and 2000/121767).",
"However, the results are not always satisfactory.",
"Such a treatment brings about some effects, which, however, are insufficient for producing the target powder-free product.",
"Then, they have changed way of thinking, and positively incorporate a hydrophilic substance in natural rubber latex and then treat the rubber for hydrophobicizing, to find that the very tacky natural rubber latex product is easily detackified.",
"More specifically, a film produced by immersing a mold in natural rubber latex shows a strongly tacky substance leached out on the surface, when treated for leaching.",
"The inventors of the present invention consider that this tacky substance is a tacky auxiliary component, e.g., water-soluble protein, derived from a natural rubber latex.",
"Then, they have attempted immersion forming of natural rubber latex, after it is incorporated with carrageenan, considered to have a capacity of reacting with protein (P. M. T. Hansen, J.",
"Dairy Sci., 51(2) 192, 945 (1968)), to find that the surface tackiness is reduced.",
"It is said that carrageenan helps form anionic, hydrophilic, colloidal particles at a pH equivalent to or above the isoelectric point of the protein, by the actions of multi-valent cations, e.g., calcium, lying between negatively charged carrageenan and protein, and that protein forms an insoluble composite with carrageenan at below the isoelectric point.",
"It is therefore considered that carrageenan works to control leaching of the protein to the film surface both in the leaching and drying steps for the natural rubber latex production process.",
"The natural rubber latex product thus produced shows reduced tackiness, partly because of hydrophilicity of incorporated carrageenan itself.",
"Nevertheless, however, it still shows some residual tackiness, and needs a treatment, e.g., use of powder or chlorination, to be detackified.",
"Then, the inventors of the present invention have attempted to apply the techniques, applied for patents by them, for detackifying the diene-based carboxylated synthetic rubber latex products (PCT/JP00/03370 and 2000/121767) to the carrageenan-incorporated natural rubber latex, to find that the product is successfully detackified, which is difficult by the conventional method.",
"More specifically, they have successfully produced a natural rubber latex product, which is detackified in spite of being free of powder, by treating carrageenan as an anionic, hydrophilic polymer and protein or the like as an auxiliary component with a hydrophilic group (which is described later).",
"It is found that a natural rubber latex product showing no tackiness is obtained when a sealant is incorporated in the latex or used for surface treatment of the latex.",
"Next, a polyacrylic acid or acrylic-based copolymer known as a protein modifier is incorporated, to find that each is effective.",
"Then, the inventors of the present invention have studied the above-described effect of detackifying the latex with natural, semi-synthetic and synthetic anionic, hydrophilic polymers, to find that the effective compounds include anionized hydrophilic cellulose derivatives (e.g., carboxymethyl cellulose), anionized starch (e.g., phosphate-esterified starch, anionized starch incorporated with a hydrophobic group, and carboxymethylated starch), and anionized guar gum, alginic acid, pectin, xanthan gum and maleic acid copolymer.",
"In other words, it is considered that, when multi-valent cations, e.g., calcium, are present, they lie between negatively charged polymer having an anionic group and protein to help form the anionic, hydrophilic colloidal particles, as is the case with carrageenan.",
"Therefore, a tacky substance is prevented from leaching out both in the leaching and drying steps.",
"At the same time, treating tacky substances, e.g., anionic, hydrophilic polymer and protein, with a hydrophilic group sealant as the agent for hydrophobicizing natural rubber latex to make it non-hydrophilic or hydrophobic gives a detackified natural rubber latex product without using powder.",
"It is considered that the anionic, hydrophilic polymer controls leaching of protein or the like as an auxiliary component, and, at the same time, provides sites for reaction or adsorption of the hydrophilic group sealant, to make the natural rubber latex product surface non-hydrophilic or hydrophobic, thereby preventing formation of the hydrogen bond and detackifying the product.",
"The hydrophilic group in the anionic, hydrophilic polymer is not limited to carboxyl, but a variety of groups are useful.",
"These include sulfate ester (—OSO3M), sulfonate (—SO2OM), phosphate (—PO3HM or —PO3M2), phosphate ester, —SO2NH2, and —SO2NHCOR groups, wherein M is hydrogen atom, and alkali metal, ammonia or organoammonium, and R is an alkyl, phenyl which may be substituted or not, or naphthyl group which may be substituted or not.",
"Next, the inventors of the present invention have incorporated a nonionic, hydrophilic polymer in place of anionic, hydrophilic polymer in natural rubber latex for the treatment with the hydrophilic group sealant, to confirm that it has the effect similar to that by the anionic, hydrophilic polymer.",
"In other words, a natural rubber latex product can be detackified in spite of being free of powder, when incorporated with methyl cellulose and treated with the hydrophilic group sealant.",
"Then, the inventors of the present invention have conducted the tests with cellulose derivatives, e.g., hydroxypropylmethyl cellulose, hydroxyethylmethyl cellulose and hydroxyethyl cellulose; nonionic hydrophilic synthetic polymers, e.g., polyvinyl alcohol, polyethylene oxide, poly(N-vinyl-2-pyrrolidine) and vinyl ether-based polymers; and natural polysaccharides, e.g., locust bean gum, guar gum, tamarind gum, pullulan, galactomannan, tragacanth gum and dextran, to obtain the similar results.",
"These nonionic, hydrophilic polymers generally have hydroxyl, ether or amide group as the hydrophilic group.",
"The inventors of the present invention have further conducted the tests to incorporate cationic and ampholytic polymers in place of the anionic, hydrophilic polymer in natural rubber latex, which is kept at a pH 10 to 11 with ammonia and dispersed by protein mostly charged negative.",
"It is considered that a cationic polymer, if it can be incorporated, will be directly bonded to protein and phospholipid considered to be the tacky components of natural rubber latex, and effective for detackifying the product.",
"On the other hand, it is also considered that natural rubber latex will be coagulated, when protein dispersing the latex is bonded to the cationic polymer, as predicted by P. M. T. Hansen described earlier.",
"As expected, the natural rubber latex is gelated with cationic polyacrylamide, which is commonly used for waste water treatment, as expected.",
"It is therefore impossible to obtain a natural rubber latex product, although the cationic polymer is weakly cationic and low-molecular-weight type.",
"On the other hand, cationized or ampholytic starch, which is used in the pulp industry, is found to bring about the effect of detackifying a natural rubber latex product similar to that by the anionic, hydrophilic polymer, without excessively increasing the viscosity, and as expected.",
"The cationic group in the cationized starch is mostly of tertiary amine or quaternary ammonium, having a degree of substitution in a range of 0.02 to 0.06, mainly around 0.03 to 0.04.Therefore, the starch is low in degree of cationization.",
"The inventors of the present invention have also conducted the tests with other cationic and ampholytic hydrophilic polymers used in the industry to find that all but few of these polymers tested show the effect of detackifying natural rubber latex without causing gelation.",
"The similar results are obtained with cationic and ampholytic polyacrylamide.",
"The cationic, hydrophilic polymers useful for the present invention include synthetic polymers, e.g., aminoalkyl methacrylate/acrylamide copolymer, polyvinyl pyridium ammonium halide, polyallyl ammonium halide, polyaminomethylacrylamide, polyvinyl imidazoline, polyacrylamide modified by the Mannich reaction, polyacrylamide modified by the Hofmann reaction, polyethyleneimine, polydiallylamine, polypiridium halide, cationized starch, cationized cellulose, cationized polyvinyl alcohol, epoxyamine-based condensate, ionene-based condensate, cationized polymethacrylate ester resin, alkylene diamine/epichlorohydrin polycondensate, cationized polyvinyl pyrrolidone and cationized polyacrylamide; and semi-synthetic polymers, e.g., cationized cellulose, cationized starch and cationized guar gum.",
"The ampholytic, hydrophilic polymers useful for the present invention include ampholytic cellulose, starch, guar gum and polyacrylamide.",
"Whether or not the cationic or ampholytic, hydrophilic polymer gelates natural rubber latex cannot be always predicted, because it depends on type and molecular weight of the polymer, and type, quantity and distribution of the cationic group, among others.",
"However, it can be readily known by adding the polymer to natural rubber latex.",
"Therefore, the cationic and ampholytic, hydrophilic polymers useful for the present invention are limited to those which should not gelate natural rubber latex.",
"The hydrophilic polymer is not necessarily soluble in water.",
"Even a water-dispersible polymer can bring about the effect of detackifying the latex, so long as it is hydrophilic.",
"In particular, natural rubber latex is strongly alkaline, and a water-dispersible polymer may be soluble in alkaline water in natural rubber latex.",
"Moreover, in the case of synthetic polymer, even a water-dispersible one may bring about the effect similar to that provided by a water-soluble one, because the molecule can be freely designed for the polymer.",
"The examples of water-dispersible, synthetic polymers useful for the present invention include polyvinyl acetate, ethylene/vinyl acetate copolymer, styrene/acrylate ester copolymer, styrene/methacrylate ester copolymer, acrylate ester copolymer, methacrylate ester copolymer, vinyl acetate/acrylic acid copolymer, vinyl acetate/acrylate ester copolymer, vinyl acetate/methacrylic acid copolymer, vinyl acetate/methacrylate ester copolymer, polyacrylamide, polymethacrylamide, acrylamide-based copolymer, methacrylamide-based copolymer, and anionic, cationic and ampholytic modifications of these polymers.",
"It is known that each of the above hydrophilic polymers interacts with natural rubber latex chemically, physicochemically and physically, as is seen in the creaming, protective colloidal and thickening phenomena.",
"However, it is considered that the polymer also interacts with the tacky, auxiliary component in the latex, viewed from the controlled elution and leaching of the component in the leaching and drying steps in the latex product production process.",
"It is also considered that a natural rubber latex product is hydrophobicized when a varying hydrophobicizing agent is acted thereon, after being incorporated with the hydrophilic polymer, and that the agent first acts on protein and phospholipid considered as the tacky auxiliary components, which are mostly charged negative.",
"The varying hydrophilic polymer intentionally incorporated in the latex will be the second target to be hydrophobicized.",
"It is considered that the hydrophilic polymer provides the sites for reaction and adsorption of the hydrophilic group sealant to contribute to hydrophobicizing of the natural rubber latex product as a whole.",
"There are various hydrophilic groups to be hydrophobicized; those derived from protein, e.g., carboxyl, amino and thiol, those derived from phospholipids, e.g., phosphate ester; hydrophilic group, e.g., carboxyl; and those derived from the intentionally incorporated with nonionic, anionic, cationic or ampholytic, hydrophilic polymers.",
"The hydrophilic group sealant is an agent which works to hydrophobicize various hydrophilic groups chemically, physicochemically and physically and prevent formation of the hydrogen bond, thereby detackifying the natural rubber latex product.",
"The hydrophilic group sealants include various agents, e.g., hydrophobicizing crosslinking agent showing no tackiness, hydrogen bond modifier showing no tackiness, surfactant showing no tackiness, sizing agent, waterproofing agent and water repellant.",
"The hydrophilic group sealant is not required to hydrophobicize all of these hydrophilic groups, but required to hydrophobicize only to an extent to detackify a natural rubber latex product as a whole.",
"Therefore, many carboxyl group sealants capable of detackifying a natural rubber latex product as a whole function as a hydrophilic group sealant, and so is vice versa that many hydrophilic group sealants capable of detackifying a natural rubber latex product as a whole function as a carboxyl group sealant.",
"Whether an agent has such a function should be judged to see whether it allows natural rubber latex product surfaces to adhere or attach to each other and causes no trouble when the product is used after the products are stored for several months while keeping their surfaces coming into contact with each other.",
"However, it is convenient to concretely judge the function by the tackiness test, conducted in EXAMPLES.",
"The inventors of the present invention have first studied use of a crosslinking agent of tri- or tetra-valent metallic element as the hydrophilic group sealant.",
"More specifically, they have attempted to form an immersion-processed film of natural rubber latex incorporated with an anionic, hydrophilic polymer in the presence of a divalent metallic salt coagulating agent incorporated with water-soluble polyaluminum hydroxide, to find that the film inside surface is detackified.",
"A crosslinking agent of metallic element crosslinks anionic, hydrophilic group by the ionic bond to seal the group and prevent formation of the hydrogen bond, thereby detackifying the latex product.",
"For example, an aluminum salt as the representative tri- or tetra-valent metallic cation follows the Schultz-Hardy law to show a strong coagulating function.",
"Therefore, it is expected to hydrophobicize an anionic, hydrophilic polymer, and found to detackify the tacky, auxiliary components of a natural rubber latex product.",
"These findings suggest that the tacky component of natural rubber latex is anionic.",
"A tetravalent zirconium salt and tri- and tetra-valent titanium salt also have a strong hydrophobicizing effect.",
"Next, the inventors of the present invention have studied to detackify the external surface of the immersion-processed film.",
"More specifically, they have heated the above-described latex film with detackified inner surface, immersed in a water-soluble polyaluminum hydroxide solution, to find that its external surface is detackified with the crosslinked aluminum compound layer thereon.",
"Further, they have heated the above immersion-processed product, released out of the immersion mold, with its both surfaces immersed in an aluminum compound solution, to find that its both surfaces are detackified.",
"The inventors of the present invention have tested, based on the above knowledge, the crosslinked layer formation reactions on the latex surface using various tri- or tetra-valent metallic compounds, to find that the natural rubber latex product with detackified surface can be produced.",
"They have also treated in a similar manner the surface of a peroxotitanium complex, which, although tetravalent, forms a neutral, stable aqueous solution, to find the detackified film is formed on the natural rubber latex film surface without causing interlayer exfoliation.",
"They have further treated the natural rubber latex surface with titania, zirconia and alumina sol, known to form a uniform coating film, to find that the product with detackified, slippery surface can be produced.",
"Natural rubber latex is strongly ammonia-alkaline, and many tri- or tetra-valent crosslinking agents of metallic elements are possibly decomposed by ammonia to form powder of hydroxide or the like on the latex film surface.",
"It is therefore necessary to take a sufficient countermeasure against formation of hydroxide by, e.g., evaporating or eluting out ammonia.",
"A hydroxide, or insoluble or sparingly soluble salt may be incorporated beforehand in natural rubber latex, even though it is a crosslinking agent of tri- or tetra-valent metallic element.",
"The examples of these include aluminum hydroxide, calcium aluminate and satin white.",
"These aluminum compounds, however, tend to gelate latex as time passes.",
"On the other hand, ammonium zirconium carbonate causes no gelation of natural rubber latex in which it is incorporated, and is convenient.",
"PAC, water-soluble polyaluminum hydroxide, peroxotitanium and the like, whose metallic element is tri- or tetra-valent, show greater effect as polymer than as monomer, and these compounds are also crosslinking agents of tri- or tetra-valent metallic elements useful for the present invention.",
"Next, the inventors of the present invention have studied the effect of detackifying a natural rubber latex product with various organic crosslinking agents.",
"Polymers of low intermolecular cohesive energy, such as rubber, will have greatly improved mechanical properties, when their molecules are crosslinked with each other.",
"Natural rubber may not exhibit the inherent rubber characteristics, until it is crosslinked to form the vulcanizate.",
"Crosslinking agents for vulcanizing rubber are mostly of sulfur by far.",
"T. H. Kempermann discusses in detail many non-sulfur crosslinking agents for vulcanization (Rubber Chem.",
"Technol.",
"61, 422, 1988), describing vulcanization by sulfur donor, thiuram, thiourea, bis-mercapto, S—Cl compound, resin, compound having a reactive nitrogen group, compound having a reactive olefin group and peroxide, and ionic vulcanization.",
"Most of them, however, are merely auxiliary in nature or of academic importance.",
"These crosslinking agents for vulcanization cannot detackify a natural rubber latex product.",
"More specifically, natural rubber latex will lose its product value, when vulcanized to an extent of being detackified.",
"The vulcanization methods for the present invention are not limited, and the conventional methods, e.g., vulcanization with the aid of sulfur, peroxide and radioactive ray, can be used.",
"The crosslinking agent necessary for the present invention crosslinks and hydrophobicizes protein and phospholipid considered to be the tacky, auxiliary components of natural rubber latex and a small quantity of hydrophilic polymer intentionally incorporated, thereby preventing formation of the hydrogen bond and detackifying the product.",
"The hydrophilic groups to be crosslinked include carboxyl, amino, hydroxyl and thiol in the case of protein; phosphate ester and carboxyl in the case of phospholipids; and specific hydrophilic group in the case of the hydrophilic polymer.",
"On the other hand, various organic crosslinking agents for improving functions/performances of the rubber product or modification of the resin product pursue improvement of mechanical properties, beginning with resistance to heat and durability, by the crosslinking reactions.",
"The common organic crosslinking agents are frequently used at high temperature of 120° C. or higher, because of priority given to storage and reaction stability.",
"When used at lower temperature for a natural rubber latex product, they may be eluted out from the product surface without reacting the latex, failing to exhibit the effect of detackifying the latex.",
"Moreover, self-crosslinking or multi-functional crosslinking agents may cause other problems, e.g., bonding the product surfaces to each other by the functional group remaining unreacted, and insufficient extent of detackifying the latex product surface.",
"Therefore, the inventors of the present invention have used crosslinking agents reacting at low temperature, e.g., oxazoline-based and carbodiimide-based ones, and treated therewith the latex at high temperature prior to the leaching step, to find that the natural rubber latex product is detackified even with organic crosslinking agents.",
"It is also found that an emulsion type organic crosslinking agent can control itself from eluting out from the external surface of the natural rubber latex product, and exhibits the detackifying effect even when it works to crosslink at higher temperature.",
"Even these crosslinking agents will lose their detackifying effect as time passes, when they are of self-condensing or multi-functional type, because the residual agent bonds the contacting surfaces to each other.",
"Therefore, the organic crosslinking agent exhibiting the detackifying effect is preferably of low-temperature reaction, low-functional or emulsion type.",
"The crosslinking agent for the present invention should hydrophobicize and detackify the crosslinked natural rubber latex product.",
"Such an organic crosslinking agent exhibiting the above functions is referred to as the detackifying, hydrophobicizing organic crosslinking agent in this specification.",
"More specifically, such a crosslinking agent prevents the natural rubber latex product surfaces from adhering or attaching to each other after the products are stored for several months while keeping their surfaces coming into contact with each other.",
"Still more specifically, it is the crosslinking agent which is verified to show no tackiness by the tackiness test, conducted in EXAMPLES.",
"The surface treatment with an organic crosslinking agent can be effected subsequent to the leaching step, which widens a range of applicable agents.",
"Some organic compounds considered to react with various hydrophilic groups bring about the similar effect, although they are not hydrophilic group crosslinking agents.",
"These compounds include glyoxal, benzaldehyde, dimethylol urea, polyamide compounds, polyamidepolyurea compound, polyamidepolyurea/glyoxal condensate, polyaminepolyurea compound, polyamideaminepolyurea compound, polyamideamine compound, polyamideamine/epihalohydrin condensate, polyamideamine/formaldehyde condensate, polyamine/epihalohydrin condensate, polyamine/formaldehyde condensate, polyamidepolyurea/epihalohydrin condensate, polyamidepolyurea/formaldehyde condensate, polyaminepolyurea/epihalohydrin condensate, polyaminepolyurea/formaldehyde condensate, polyamideaminepolyurea/epihalohydrin condensate, and polyamideaminepolyurea/formaldehyde condensate.",
"These compounds are developed for paper as waterproofing agent, sizing agent, printing characteristic improver, wet strength improver and strength improver by incorporating a varying functional or hydrophobicizing group.",
"They have the common feature of controlling the hydrogen bonding in paper.",
"Such a hydrophilic group sealant is referred to as a detackifying, hydrogen bonding adjustor in this specification.",
"Next, the inventors of the present invention have studied monofunctional compounds, e.g., monofunctional epoxy and amine compounds, which can prevent formation of hydrogen bond derived from the hydrophilic group.",
"Being monofunctional, they cannot crosslink the hydrophilic group.",
"As a result, it is confirmed that these compounds, which are considered to bond to and hydrophobicize a hydrophilic group, bring about the effect similar to that by the above-described compounds.",
"Further, the inventors of the present invention have studied sizing agents used in the paper industry as the hydrophilic group sealants.",
"The sizing agents are represented by a rosin-based one, rosin comprising abietic acid as the major ingredient.",
"Rosin coats pulp fibers, by which it exhibits excellent hydrophobicizing effect.",
"It has a large contact angle of 53° with water, and should have a notable effect as a hydrophobicizing agent.",
"It has a very large contact angle of 130°, when bonded to aluminum, to bring about still larger hydrophobicizing effect.",
"These sizing agents have the hydrophilic group sealing effect, as confirmed by the similar tests.",
"The effect of sizing agent is considered to come from physicochemically or physically coating and hydrophobicizing the latex surface.",
"Recently, an alkyl ketene dimer (AKD), alkenyl succinic anhydride (ASA) and cationic sizing agent, among others, have been used as the sizing agents for neutral paper.",
"These sizing agents working under neutral or alkaline conditions, and cationic sizing agents are confirmed to bring about the similar effects.",
"The hydrophobicizing effect by AKD and ASA is generally believed to come from the chemical bond with a hydrophilic group.",
"However, some argue that the compound loses its hydrophilicity as a result of self-decomposition on the fiber surfaces, to hydrophobicize itself as a whole, where the hydrophilic group works as an anchor.",
"Cationic sizing agents, e.g., styrene-based polyamidepolyamine epichlorohydrin resin modified with quaternary amino acid (disclosed by Japanese Patent Laid-Open No.",
"2001-32191), have been developed as the surface sizing agents for paper.",
"These surface sizing agents, in particular cationic-based ones, work to detackify natural rubber latex products.",
"In any case, it is apparent that these sizing agents hydrophobicize the latex surfaces by sealing them chemically, physicochemically or physically.",
"Such a sizing agent is referred to as a detackifying sizing agent in this specification.",
"A waterproofing agent is one of the compounds with improved hydrophobicizing effect and convenience, although functionally similar to the above-described hydrophilic group sealant.",
"These agents are used to waterproof paper coating binders such as starch, protein, casein, PVA, and various types of latex.",
"It is considered, from the objects for which it is used, that a waterproofing agent works as the suitable hydrophilic group sealant for the present invention.",
"In fact, they show good results as confirmed by the similar tests.",
"For example, ammonium zirconium carbonate, which is a crosslinking agent of tetravalent metallic element, is stable when incorporated in natural rubber latex and, being tetravalent, shows excellent detackifying effect, unlike the aluminum-based crosslinking agent of metallic element, which tends to gelate natural rubber latex when directly incorporated therein.",
"There are various compounds useful as waterproofing agents, including compounds having methylol group or lower alkylated compounds thereof, aldehyde-based compounds, compounds having epoxy or chlorohydrin group, compounds having ethyleneimine group, polyvinyl butyral-based compounds, and tri- or tetra-valent multi-valent metallic compounds.",
"Each maker has been developing waterproofing agents one after another for hydrophobicizing and waterproofing purposes.",
"They are also useful for detackifying natural rubber latex products, and are referred to as detackifying waterproofing agents in this specification.",
"There are specific waterproofing agents for specific hydrophilic polymers incorporated.",
"For example, diborate shows a detackfying effect for polyvinyl alcohol.",
"These specific agents are also included in the waterproofing agents useful for the present invention.",
"Water repellants, provided with water repellency, have been also developed.",
"For example, aliphatic amide wax, aliphatic chromium complexes, aluminum stearate, chlorosulfonated polyethylene, ethylene urea-based resin, acrylic-based resin and silicone-based resin have been used as water repellants.",
"They also exhibit a detackifying effect, as do the waterproofing agents.",
"The inventors of the present invention have also conducted the detackifying tests with releasing agents for natural rubber latex products in a manner similar to those with waterproofing agents, to confirm that these agents also work to detackify the products.",
"Releasing agents are used for releasing paper, adhesive tape, process paper and transfer paper.",
"Of these, aqueous or reactive ones are preferable.",
"Non-silicone releasing agents are preferable for precision devices which are sensitive to silicone.",
"The releasing agents capable of detackifying natural rubber latex products are referred to as detackifying releasing agents in this specification.",
"Protein considered as one of the tacky components of natural rubber latex is mostly acidic, and soluble in water or hydrophilic.",
"The inventors of the present invention have considered that the protein in natural rubber latex can provides sites for reaction or adsorption for the hydrophilic group sealant.",
"Therefore, they have produced natural rubber latex products incorporated with various hydrophilic group sealants, e.g., anionic and nonionic surfactants, to find that the thick products are detackified but thinner ones (around 0.1 mm thick) remain tacky.",
"These tacky products can be detackified, when their external surfaces are halogenated or coated with a detackified polymer, as described later.",
"This means that the natural rubber latex product incorporated with the hydrophilic group sealant remains detackified inside.",
"They have considered that the hydrophilic group sealant, itself or bonded to a tacky substance, is eluted out on the external surface of the natural rubber latex product.",
"One of the possible causes for elution of the hydrophilic group sealant is elution or leaching of the protein in the latex, which can provide the reaction or adsorption sites.",
"It is considered that the acidic protein is in condition of fairly easily eluting out in latex, which is at a pH of around 10.5 to 11 in the case of high-ammonia latex and around 10 in the case of low-ammonia latex.",
"Therefore, the inventors of the present invention have considered to reduce content of ammonia in latex.",
"It is desirable to reduce its content upstream of the leaching step, because it is eluted out in this step.",
"They have attempted to treat the natural rubber latex film incorporated with a hydrophilic group sealant in the leaching step after drying it under heating to evaporate ammonia, to find that the detackified natural rubber latex product is obtained without using a hydrophilic polymer.",
"The drying treatment under heating conceivably have the effects of making the tacky substances insoluble or sparingly soluble, increased concentration of the hydrophilic group sealant, and sufficient bonding between the hydrophilic group sealant and tacky substances prior to the leaching step, in addition to evaporation of ammonia.",
"It is also found that the natural rubber latex product is detackified by mere surface treatment with a nonionic, anionic, or cationic surfactant, when drying-treated beforehand.",
"The tests with various other hydrophilic group sealants, e.g., detackifying, hydrophobicizing crosslinking agents, waterproofing agents and sizing agents, have produced the similar results.",
"There are various agents, e.g., the above-described detackifying, hydrophobicizing crosslinking agents, which work as hydrophilic group sealants and are water-dispersible.",
"Many of these hydrophilic group sealants are not eluted out in themselves, even when leaching-treated without undergoing the drying step at high temperature.",
"Moreover, the natural rubber latex product is subjected to a drying step at high temperature anyway before it is completed.",
"Therefore, it is possible to detackify a natural rubber latex product only with a hydrophilic group sealant without drying it at high temperature prior to the leaching step.",
"Next, the inventors of the present invention have studied to detackify the external and internal surfaces separately, known that the internal surface of a natural rubber latex product coming into contact with the mold can be detackified relatively easily, when it is incorporated with a hydrophilic group sealant.",
"More specifically, they have attempted to detackify the external surface by the method, e.g., coating with a detackified polymer, halogenation or crosslinking agent of tri- or tetra-valent metallic element which elutes the treatment agent to only a limited extent in the leaching step, and detackify the internal surface by one of the above-described method.",
"No treatment agent will be eluted out from the internal surface, which comes into contact with the mold but not normally with water.",
"Moreover, the natural rubber latex product is subjected to a drying step at high temperature anyway before it is completed.",
"Therefore, it is not necessary to seriously consider elution of the treatment agent for hydrophobicizing and detackifying the internal surface, which should widen a range of applicable hydrophilic group sealants.",
"The inventors of the present invention have first studied the coating with a detackifying polymer.",
"Coating a natural rubber latex product on both surfaces with the diene-based carboxylated synthetic rubber latex they have invented can make the product detackified.",
"Next, they have studied to provide the detackifying polymer coating layer only on the external surface of the natural rubber latex product, and detackify the internal surface by the above-described various detackifying techniques, e.g., incorporation of a hydrophilic group sealant, to produce the product detackified as a whole, in consideration of the technical complexity and difficulty involved in the inner coating with the diene-based carboxylated synthetic rubber latex.",
"The coating layer of detackifying polymer provided on the internal surface may partly come off, when it is thin, while the product is being released from the mold, to cause defective product.",
"The separated coating layer remaining in the mold will cause serious production problems, when the coagulating solution cannot run over that portion.",
"Moreover, when the coating layer is sufficiently thick, the difference between the polymer coating layer and natural rubber layer in properties are more noted, to cause undesirable phenomena, e.g., interlayer exfoliation.",
"On the other hand, the product with the internal surface coated with a detackifying natural rubber latex layer and external surface coated with a detackifying polymer coating layer can be easily released out of the mold, even when the coating layer is very thin.",
"Moreover, the product free of defects, e.g., interlayer exfoliation, can be produced.",
"A known technique can be used to coat the product with a detackifying polymer, but it is preferable to coat with the diene-based carboxylated synthetic rubber latex developed by the inventors of the present invention.",
"The preferable diene-based carboxylated synthetic rubber latexes include NBR, SBR, CR and MBR.",
"Coating the external surface of a natural rubber latex film with the detackifying, diene-based carboxylated synthetic rubber latex brings about another advantage of reduced pinholes in the product, resulting from double dipping.",
"The technique for detackifying the internal surface of the natural rubber latex product has been described in detail.",
"The technique for the diene-based carboxylated synthetic rubber latex (PCT/JP00/03370) has been applied in such a way that the natural rubber latex film whose internal surface is detackified is immersed in a diluted solution of the detackifying diene-based carboxylated synthetic rubber latex.",
"The external surface is detackified very easily, although the coating layer is very thin, less than 1 PHR.",
"The product shows no quality defects, e.g., interlayer exfoliation.",
"The natural rubber latex product, detackified as well as the one coated with diene-based carboxylated synthetic rubber latex, is obtained when the external surface is coated with a polymer-based, detackifying releasing agent.",
"When a rubber glove is produced, it is turned inside out while being released out of the mold.",
"It has the internal surface coated with a detackifying polymer layer, which, when detackified, can be worn more easily by a hand than a natural rubber latex product.",
"Therefore, it can be worn or taken off more easily than a natural rubber latex product.",
"Therefore, the natural rubber latex product of good wearing characteristics can be obtained without using powder.",
"The detackifying polymer coating layer can be vulcanized in the absence of sulfur.",
"The synthetic rubber latex vulcanized without using sulfur has the external surface negative in the silver plate test, which tests the discoloration reaction between the contacting latex surface and metallic surface.",
"Non-sulfur vulcanization methods are not limited for the present invention, and known ones can be used.",
"These include crosslinking with metals, e.g., zinc oxide, sodium aluminate and aluminum hydroxide, and vulcanization with peroxides.",
"The coating layer is preferably vulcanized to an extent to prevent interlayer separation from the natural rubber latex layer, and to allow it to follow expansion of the natural rubber latex layer.",
"The coating layer may be surface-coated with a polymer solution not intended for vulcanization, because it contributes to the product strength to only a limited extent.",
"Thus, the natural rubber latex product whose external surface is coated with the detackifying polymer layer can have advantages of both natural and synthetic rubber.",
"The natural rubber latex product whose external surface is coated with a detackifying polymer layer can be detackified on both external and internal surfaces by merely incorporating a hydrophilic group sealant without using a hydrophilic polymer.",
"The similar results are obtained when an anionic or nonionic surfactant is used as the hydrophilic group sealant.",
"A highly soluble agent, e.g., anionic or nonionic surfactant, incorporated in natural rubber latex will be eluted out in the leaching step for the production process from the external surface of the immersion-processed product, to make the product tacky as a whole.",
"However, it will not be eluted out from the external surface when it is coated with a detackifying polymer layer, while the internal surface remains detackified because of no surfactant eluted out therefrom.",
"As a result, the product is detackified as a whole.",
"Thus, making the external surface detackified widens a range of the hydrophilic group sealants capable of detackifying the internal surface.",
"The diene-based carboxylated synthetic rubber latex can be detackified by, e.g., incorporating a carboxyl group sealant in the latex, or coating the latex with a carboxyl group sealant.",
"The carboxyl group sealant is discussed in detail in PCT/JP00/03370, described earlier.",
"It is a generic term for those agents, e.g., crosslinking agents of tri- or tetra-valent metal, organic crosslinking agents, various types of hydrophilicity adjustors, and sizing agents which act chemically, physicochemically or physically on the hydrophilic group in the diene-based carboxylated synthetic rubber latex to hydrophobicize and detackify the latex.",
"It is also found that the diene-based carboxylated synthetic rubber latex film has the surface of very high lubricity, when incorporated with various types of reactive cationic compounds, e.g., cationic epichlorohydrin-based resin as one type of the hydrophilicity adjustors.",
"The natural rubber latex product has the detackified external surface of high lubricity, when coated in the similar manner with the diene-based carboxylated synthetic rubber latex incorporated with various types of reactive cationic compounds, e.g., cationic epichlorohydrin-based resin.",
"When a rubber glove is produced, it is turned inside out, the external surface becoming the internal surface.",
"The glove prepared in this manner has an advantage of being easily worn by a hand.",
"The synthetic rubber latex glove of or coated with the diene-based carboxylated synthetic rubber latex incorporated with a reactive cationic compound has the glove film of high lubricity, allowing it to be easily worn by a hand.",
"This means that the diene-based carboxylated synthetic rubber latex glove incorporated with a reactive cationic compound has an independent value.",
"The diene-based carboxylated synthetic rubber latex glove easily worn by a hand can be produced by incorporating a varying reactive cationic compound, e.g., cationic epichlorohydrin-based resin, in the latex.",
"Next, the inventors of the present invention have coated a natural rubber latex film with the diene-based carboxylated synthetic rubber latex incorporated with a carboxyl group sealant and/or hydrophilic group sealant, to find that the natural rubber latex product detackified on both surfaces can be produced, even when the latex is not treated to be detackified.",
"It is considered that the carboxyl group sealant and/or hydrophilic group sealant, incorporated in the detackifying diene-based carboxylated synthetic rubber latex, diffuse into the natural rubber latex layer to make the product detackified as a whole.",
"Furthermore, the inventors of the present invention have provided a thin layer of a common diene-based carboxylated synthetic rubber latex not treated to be detackified over the natural rubber latex whose internal surface is detackified, to find that the detackifying natural rubber latex product is unexpectedly produced.",
"It is considered that the hydrophilic group sealant, incorporated in the natural rubber latex, diffuse into the diene-based carboxylated synthetic rubber latex layer to make the layer detackified.",
"Next, the inventors of the present invention have studied halogenation of the external surface.",
"Halogenation, although possibly causing environmental problems, forms a coating layer of halogenated hydrocarbon in a sense with the halogen atom bonded to the double bond in the rubber molecule on the natural rubber latex product surface.",
"As a result, the halogenated surface of the rubber product is hydrophobicized and detackified while losing the inherent rubber characteristics.",
"The rubber characteristics hinder wearing or taking off of the glove, when it is the rubber product.",
"This is one of the reasons why many natural rubber latex products are halogenated.",
"Halogenation causes many quality and environmental problems.",
"One of the major problems viewed from production is that latex cannot be halogenated on-machine in the mold for both surfaces.",
"Normally, the rubber shape is halogenated after being released out of the mold by a separate step, or its external surface is halogenated on-machine and then the internal surface is halogenated by a separate step after the shape is released out of the mold and turned inside out.",
"Therefore, the halogenation should invariably deteriorate productivity.",
"Therefore, the inventors of the present invention have attempted to develop more efficient halogenation process for natural rubber by detackifying by on-machine halogenation for the external surface and by various detackifying techniques described earlier for the internal surface.",
"More specifically, the natural rubber latex film incorporated with a hydrophilic group sealant is produced, and then halogenated for the external surface only.",
"This produces the detackified natural rubber latex product.",
"The halogenation can be effected on-machine, because it is only for one side.",
"When the product is glove, it has greatly improved wearing/taking-off characteristics, because it is turned inside out while being released out of the mold, the halogenated external surface becoming the internal surface.",
"Thus, the treatment of only one side by halogenation is of high practical value, because the product can be halogenated on-machine and easily worn and taken off.",
"Moreover, the one-side halogenation greatly widens a range of the conditions under which the internal surface can be detackified and applicable detackifying treatment agents, like the coating treatment of the external surface with a detackifying polymer.",
"The third method of detackifying the external surface at low temperature, effected separately from the step of detackifying the internal surface, involves use of a highly reactive, hydrophilic group sealant, e.g., crosslinking agent of tri- or tetra-valent metallic element (e.g., polyaluminum hydroxide, zirconium acetate, zirconium oxychloride or titanium tetrachloride).",
"The treatment agent, itself being charged positive, can be bonded even at low temperature to the external natural rubber latex surface, which is charged negative, causing little problems resulting from elution of the agent in the leaching step.",
"When the crosslinking agent of metallic element is used, however, it is necessary to take some measures, e.g., leaching the latex before treatment with the agent, because, when the agent comes into contact with the film surface while it is highly alkaline and later heated, the powder of hydroxide or the like on the surface may be formed.",
"The internal surface may be detackified in a manner similar to one of the two methods described earlier.",
"The external surface can be detackified with another type of hydrophilic group sealant, needless to say.",
"In such a case, however, it is necessary to allow the hydrophilic group sealant to sufficiently react at high temperature before the leaching step, when the external surface is leached after being treated with the hydrophilic group sealant.",
"The techniques for detackifying natural rubber latex products have been described in detail.",
"The term “detackified” described above does not mean that the surface is not adhesive, but that the surfaces are not adhered to each other to an extent not causing practical problems when they come into contact with each other under pressure while the products are stored for several months, preferably 1 year, even in the absence of powder.",
"In other words, they can be delivered to the markets as the powder-free natural rubber latex products.",
"However, practicality test needs a long time, and it is convenient to determine whether they are detackified or not by the tackiness test.",
"In this specification, those passing the test are regarded as being detackified.",
"The hydrophilic group sealant is the agent which chemically, physicochemically or physically seals the auxiliary components (e.g., protein and phospholipids) considered to cause tackiness of natural rubber latex and the hydrophilic group of the hydrophilic polymer intentionally incorporated in the latex, controls formation of the hydrogen bond, and makes the internal surface of a natural rubber product non-hydrophilic or hydrophobic.",
"More specifically, it is an agent which detackifies the natural rubber latex product surface in the tackiness test conducted in EXAMPLES.",
"The carboxyl group sealant originally means the agent which chemically, physicochemically or physically seals the carboxyl group in the diene-based carboxylated synthetic rubber latex, controls formation of the hydrogen bond derived from the carboxyl group, and detackifies the latex.",
"It is expanded to denote the agent which can contribute to detackifying a natural rubber latex product, because the tacky auxiliary components of natural rubber latex are protein, phospholipids and the like, and anionic substances.",
"The carboxyl group sealant shares fairly many properties with the hydrophilic group sealant.",
"The detackifying crosslinking agent of tri- or tetra-valent metallic element means the crosslinking agent of tri- or tetra-valent metallic element having the detackifying effect.",
"These agents are frequently cationic and water-soluble, but include water-insoluble ones, and also anionic ones, e.g., sodium aluminate and zirconium ammonium carbonate.",
"The detackifying, hydrophobicizing organic crosslinking agent for the present invention is not intended to form a vulcanizate of natural rubber latex, but to chemically, physicochemically or physically seal protein and phospholipids considered to be the tacky auxiliary components of natural rubber latex and the hydrophilic polymer intentionally incorporated in the latex, thereby detackifying the natural rubber latex product.",
"The detackifying hydrogen bond adjustor is an agent for introducing a varying functional group and hydrophobic group to adjust the hydrogen bonds in paper, and used as the printing characteristic improver, wet paper strength improver, waterproofing agent or the like.",
"The detackifying hydrogen bond adjustor is the hydrogen bond adjustor which has a function of detackifying the natural rubber latex product.",
"The detackifying sizing agent is an agent to be incorporated in paper or used for surface treatment of paper to prevent running of ink on paper.",
"The detackifying sizing agent is the sizing agent which has a function of detackifying the natural rubber latex product.",
"The detackifying waterproofing agent is an agent developed to insolubilize water-soluble polymers, e.g., coating binders (e.g., various types of latexes, protein, casein, starch and PVA).",
"The detackifying waterproofing agent is the waterproofing agent which has a function of detackifying the natural rubber latex product.",
"The detackifying water repellant is developed to impart water repellency or waterproofness.",
"The detackifying water repellant is the water repellant which has a function of detackifying the natural rubber latex product.",
"The detackifying releasing agent is an agent used for releasing paper, adhesive tape, process paper, transfer paper and the like.",
"The detackifying releasing agent is the releasing agent which has a function of detackifying the natural rubber latex product.",
"The detackifying surfactant is the agent which acts on natural rubber latex, the hydrophilic polymer intentionally incorporated in natural rubber latex and diene-based carboxylated synthetic rubber latex, to make them non-hydrophilic or hydrophobic.",
"More specifically, it is the surfactant which detackifies internal or external surface of natural rubber latex, as confirmed by the tackiness test.",
"Each of the above agents hydrophobicize chemically, physicochemically and physically protein and phospholipid considered to be the tacky, auxiliary components of natural rubber latex and the hydrophilic polymer intentionally incorporated, thereby preventing formation of the hydrogen bond and detackifying the product.",
"Original purposes for which these agents are developed do not matter, so long as they exhibit the detackifying effect.",
"The hydrophilic group of carboxyl group sealant referred to in this specification is a general term for these agents.",
"The detackifying polymer coating layer includes the coating layer of detackifying diene-based carboxylated synthetic rubber latex and the known polymer coating layer believed to be detackifying.",
"The external surface means the surface which does not come into contact with the mold in the case of the immersion-processed product.",
"When a glove is the product, the external surface is the internal surface of the glove, because it is turned inside out while being released out of the mold.",
"Nevertheless, however, the external surface of the present invention means the surface which does not come into contact with the mold.",
"The internal surface is the surface which comes into contact with the mold.",
"The detackifying wound-up fingerstall is the fingerstall of natural rubber latex which is wound up in the absence of powder (both surfaces are pressed to each other) and can be smoothly wound back when in use.",
"As described above, use of the present invention can easily give the natural rubber latex product detackified on one or both surfaces.",
"The product surfaces are not adhered to each other even when they come into contact with each other under heating during the production process or thereafter, a characteristic which can be used for producing novel products.",
"One example is the fingerstall of detackifying natural rubber latex which is wound up from its mouth on-machine before being released out of the mold.",
"The fingerstall wound up from the mouth has been already developed.",
"For example, referring to FIG.",
"4, the fingerstall 12 put on the fingertip 13 can be worn by simply winding it back on the finger in the arrowed direction, as shown in FIG.",
"5.Its usefulness has been recognized, because it can easily cover the finger.",
"However, a fingerstall as a natural rubber latex product is inherently tacky on both surfaces, and the winding-up type is detackified beforehand with powder or post-treatment of chlorination and then manually wound up.",
"Such a product is rarely used in a factory producing precision processed products, because of difficulty in keeping the products highly clean.",
"On the other hand, the present invention provides a finger stall of natural rubber latex detackified on both surfaces, which can be mechanically wound up on the mold and keep the precision products highly clean.",
"Recently, thinner fingerstalls are increasingly in demand to reduce fatigue of the wearer.",
"A thinner fingerstall, however, is more difficult to wear, and hence thin, powder-free, detackified, clean, wound-up fingerstalls are strongly in demand.",
"The natural rubber latex detackified on both surfaces can be easily made into the fingerstall with a wound-up mouth.",
"When a fingerstall is produced, the upper portion is left tacky without being provided with the hydrophilic group sealant layer or the like, and wound up totally and then wound back in such a way to leave the tacky portion as the wound-up mouth.",
"In the conventional method, it is necessary to provide the wound-up mouth by first winding up only the upper portion of the fingerstall, and then releasing the fingerstall out of the mold in a separate step.",
"The wound-up mouth is greatly in demand for flat products, because it facilitates wearing/taking-off of the fingerstall.",
"A fingerstall can be detackified, after being provided with the wound-up mouth by the conventional method.",
"The wound-up fingerstall described earlier can be provided with the wound-up mouth in a similar manner.",
"Referring to FIG.",
"5, when the fingerstall 12, wound up from the mouth to have the wound-up mouth, is taken off from the finger 14, it can be wound back on the finger easily except for the mouth, which is left tacky unlike the other portion.",
"The fingerstall provided with a wound-up mouth can be easily worn/taken off by picking the mouth by other fingers.",
"Depending on properties of the fingerstall, the wound-up mouth has a function of clamping the finger to keep the fingerstall held thereon.",
"It is possible to produce the wound-up fingerstall having no wound-up mouth by winding up the fingerstall which is detackified over the entire surface.",
"The fingerstall having no wound-up mouth has an advantage of reducing fatigue of the person who wears it for a long time, because the finger is not fastened by the mouth.",
"It is found that quantity of protein eluted out of the natural rubber latex product incorporated with the hydrophilic polymer is generally larger than from the conventional one (Table 26).",
"This will partly prove the controlled leaching of the tacky protein to the surface of the natural rubber latex product incorporated with the hydrophilic polymer during the production process.",
"Nevertheless, however, this is a problem, although quantity of the protein eluted out can be reduced to the normal level by increasing temperature of the leaching step.",
"In an attempt to solve the above problem, the technique described earlier for reducing allergen (WO97/08228) is applied to incorporate an epoxy compound in natural rubber latex.",
"The analysis of the product indicates that the eluted protein quantity is not decreased but conversely increased.",
"The similar results are obtained with the product treated with an organic crosslinking agent, e.g., epoxy compound, for both surfaces (Table 27).",
"The above-described technique is originally developed to reduce allergenic substance by the reaction of allergenic substance eluted out and present in the vicinity of the natural rubber latex film surface with the epoxy compound or the like, on the assumption that allergenic substance is eluted out.",
"It is found that the technique is not applicable to controlling elution of protein, because ε-amino group in protein which these compounds can react with is not necessarily present massively and the reaction products do not always become in-soluble in water.",
"Then, the inventors of the present invention have changed way of thinking, and studied to control elution of protein from the natural rubber latex product by chemical modification of the protein.",
"For example, it is known that amino group in protein greatly changes in coagulation properties, when carboxylated by modifying the amino group in gelatin with phthalic or succinyl compound, because of decreased isoelectric point or changed electrostatic properties (Revised Fundamentals of Photography, Corona-sha, p.153).",
"They have first studied anionization of protein in natural rubber latex.",
"It is believed that natural rubber latex contains protein at around 2%.",
"If it totally remains in the product and then is eluted out therefrom, the eluted protein should reach around 20,000 μg/g.",
"In actuality, however, it reaches only around 100 μg/g.",
"It is known that protein in natural rubber latex is mostly acidic.",
"Therefore, they have considered that anionic protein or protein having carboxyl group is fixed in a natural rubber latex product.",
"They have attempted to incorporate natural rubber latex with a reactive dye as the compound for introducing an anionic group in protein in latex.",
"Such a dye is developed for cellulosic fibers to be dyed in an alkaline or neutral region, and known to react also with protein-based fibers.",
"It should be noted that an anionic group, e.g., sulfonic group, is introduced in the reactive dye, to make it soluble in water.",
"The result is decreased quantity of protein eluted out from the product, as expected.",
"It is considered that the protein is fixed in the natural rubber latex product after reacting with the reactive dye.",
"Fixation of the protein reacting with the reactive dye depends on properties of the reactive group in the dye, number of the reactive groups, easiness of its reaction with protein, its fixation-related properties and so on.",
"The point is that protein in latex is insolubilized as a result of the reaction with the reactive dye.",
"The reactive dye which can fix protein is referred to as the fixing reactive dye.",
"Recently, a variety of reactive dyes have been commercialized by, e.g., developing new functional groups and multi-functional dyes to increase fixation rate of the reactive dye on the fibers.",
"As a result, the reactive dyes suitable for insolubilizing protein in natural rubber latex can be easily selected.",
"It is found that quantity of protein eluted out from natural rubber latex is also decreased, when the latex is incorporated with a compound having a structure of carboxylic anhydride.",
"The similar effect is observed with an ionic starch showing the fixing ability when dissolved at high temperature.",
"For dying fibers with a reactive dye, a cationic fixing agent is used to prevent the dye from coming off from the fibers.",
"The inventors of the present invention have treated natural rubber latex with a cationic, reactive fixing agent after it is incorporated with a reactive dye, to observe that no dye comes off in the leaching step and quantity of protein eluted out of the natural rubber latex product is decreased, as expected.",
"Next, the inventors of the present invention have treated a natural rubber latex product on both surfaces with a cationic reaction type fixing agent, to find that quantity of protein eluted out is decreased unexpectedly.",
"Analysis of the chemical structure of the reaction type dye fixing agent indicates that it is a polyamine epichlorohydrin resin, polyamide polyamine epichlorohydrin resin or the like, by which is meant that it is almost the same as the compound used for detackifying the natural rubber latex product.",
"The natural rubber latex product is prepared again using such an epichlorohydrin resin or the equivalent to measure quantity of protein eluted out.",
"The result is a greatly decreased quantity.",
"It is also found that the quantity is also greatly decreased, when natural rubber latex is directly incorporated with the epichlorohydrin-based compound.",
"Furthermore, it is found that coating the external surface of natural rubber latex with the diene-based carboxylated synthetic rubber latex incorporated with cation-based compound decreases eluted quantity of protein to a very low level.",
"It is considered that the reactive cationic compound reacts with natural rubber latex to introduce the cationic group in the protein and insolubilizes itself, thereby fixing the protein in the natural rubber latex.",
"Therefore, the decreased eluted quantity of protein results from the chemical modification by cationizing the protein in the natural rubber latex.",
"It is also confirmed that eluted quantity of the protein in the natural rubber latex is reduced in the presence of the crosslinking agent of tri- or tetra-valent metallic element, which is reactive with protein and cationic.",
"Furthermore, the inventors of the present invention have attempted to directly incorporate a cationic compound, which is considered to be unreactive with protein in the natural rubber latex.",
"More specifically, they have incorporated a cationic starch, dissolved in water at high temperature, in natural rubber latex to also find decreased quantity of the protein eluted out from the natural rubber latex product.",
"The cationic starch is insoluble in the product at normal temperature, and it is considered that the protein captured by the cationic starch is fixed in the natural rubber latex product.",
"Such a cationic compound, not limited to cationic starch, contains a dispersant insoluble or sparingly soluble in water, or water-soluble cationic compound which is chemically insolubilized by a crosslinking agent or the like.",
"An ampholytic compound, e.g., ampholytic starch, brings about the similar effect.",
"An anion starch also brings about the similar effect, as described earlier.",
"The natural rubber latex product of controlled protein elution, as referred to herein, is treated by leaching to control the elution.",
"Viewed from allergy caused by natural rubber latex, however, some argue to regulate the elution at 100 μg/g as the first target, followed by 50 μg/g as the second target, to prevent latex-caused allergy sensibilization (4th LAF Meeting).",
"Therefore, protein elution is preferably controlled at 50 μg/g or less.",
"This level should be set according to thickness or the like and properties of the product.",
"Some discuss that no allergy sensibilization will be caused by natural latex, when protein elution is controlled at 10 μg/g or less.",
"Some of the natural rubber latex products of controlled protein elution of the present invention show a protein elution level of the order of 10 μg/g, even of the order of several μg/g.",
"The level of protein eluted out from the product of the present invention is considered to be very low, knowing that the level of the protein-free carboxylated NBR latex product sulfur-vulcanized under the same conditions as those for natural rubber latex is 5 μg/g, as shown in Table 40.It is discussed that the JIS method tends to be disturbed by a vulcanization agent or the like to give a higher level of eluted protein (Tomoichi Kanou, et al, Proceedings of 6th Japan Latex Allergy Meeting, 2001, Jul.",
"20).",
"The natural rubber latex product of controlled protein elution is not necessarily powder-free.",
"The product with powder can be used for common purposes.",
"Therefore, the present invention includes the natural rubber latex product of controlled protein and with powder.",
"However, the present invention can give the ideal natural rubber latex product of controlled protein and free of powder for use in production of precision processed products by combining the techniques of detackifying natural rubber latex products and controlling elution of protein.",
"It is also possible to provide the product which causes no discoloration of a metallic surface and is not affected by sulfur for vulcanization by coating it with a layer of detackifying, carboxylated synthetic rubber latex or the like.",
"The present invention provides the following products: 1.A detackified natural rubber latex product, characterized in that both surfaces are provided with a detackified, diene-based carboxylated synthetic rubber latex coating layer.",
"2.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that a natural rubber latex is incorporated with a detackifying hydrophilic polymer and/or hydrophilic group sealant.",
"3.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the surfaces of a natural rubber latex product are treated with a hydrophilic group sealant.",
"4.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that a natural rubber latex is incorporated with at least one selected from the group consisting of nonionic polymer and anionic polymer, and cationic polymer and ampholytic polymer which cause no gelation of the natural rubber latex, and further with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"5.The detackified natural rubber latex product with one or both surfaces detackified according to any one of items 2 to 4, characterized in that an external surface of a natural rubber latex product or a natural rubber latex product incorporated with a hydrophilic group sealant and/or hydrophilic polymer is detackified by providing at least one layer selected from the group consisting of a detackified polymer layer, a halogenation treated layer, a layer treated with a detackifying crosslinking agent of tri- or tetra-valent metallic element, and a layer treated with at least one of a peroxotitania solution, peroxotitania sol, zirconia sol or alumina sol, a layer treated with a hydrophilic group sealant and a layer treated with a carboxyl group sealant.",
"6.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackified polymer coating layer, as set forth in item 5, on an external surface is a detackifying, diene-based carboxylated synthetic rubber latex coating layer or a detackifying, releasing agent coating layer.",
"7.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying diene-based carboxylated synthetic rubber latex coating layer, as set forth in any one of items 1, 5 and 6, on an external surface is detackified by incorporating the polymer or a diene-based carboxylated synthetic rubber latex with a hydrophilic group sealant or a carboxyl group sealant.",
"8.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in items 1, 5 and 6, on an external surface is detackified by at least one selected from a hydrophilic group sealant and a carboxyl group sealant incorporated in the natural rubber latex.",
"9.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying diene-based carboxylated synthetic rubber coating latex layer, as set forth in any one of items 1, 5 and 6, on an external surface is detackified by treating a surface of the polymer coating layer or diene-based carboxylated synthetic rubber latex coating layer with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"10.A detackified, lubricating, diene-based carboxylated synthetic rubber latex coat or product with one or both surfaces detackified, characterized in that a lubricating, diene-based carboxylated synthetic rubber latex coat or product, which is incorporated with a reactive, cationic compound or the lubricating, diene-based carboxylated synthetic rubber latex coat or product treated with one or more carboxyl group sealants.",
"11.The detackified natural rubber latex product with one or both surfaces detackified according to item 7, characterized in that an external surface is coated with a detackified, lubricating, diene-based carboxylated synthetic rubber latex incorporated with a reactive, cationic compound.",
"12.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that an internal surface of the natural rubber latex product as set forth in any one of items 1 to 11 is detackified with at least one selected from a hydrophilic group sealant and a carboxyl group sealant incorporated in the natural rubber latex.",
"13.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that an internal surface of the natural rubber latex product as set forth in anyone of items 1 to 11 is detackified with at least one selected from a hydrophilic group sealant and a carboxyl group sealant incorporated in the detackified polymer coating layer or detackifying, diene-based carboxylated synthetic rubber latex coating layer on an external surface.",
"14.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that an internal surface of the product as set forth in any one of items 1 to 11 is detackified by providing a detackifying polymer layer, layer treated with detackifying crosslinking agent of tri- or tetra-valent metallic element, or a layer treated with a hydrophilic group sealant or a carboxyl group sealant.",
"15.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer on an internal surface, as set forth in item 14, is a detackifying, diene-based carboxylated synthetic rubber latex coating layer.",
"16.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in item 14 or 15, on an internal surface is detackified by incorporating the polymer or the carboxylated synthetic rubber latex with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"17.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in items 14 or 15, on an internal surface is detackified by coating the internal surface of the polymer coating layer or the carboxylated synthetic rubber latex coating layer with at least one selected from a hydrophilic group sealant and a carboxyl group sealant.",
"18.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying polymer coating layer or the detackifying, diene-based carboxylated synthetic rubber latex coating layer on an internal surface, as set forth in items 14 or 15, is detackified with a hydrophilic group sealant or a carboxyl group sealant incorporated in the detackifying polymer coating layer or detackifying, diene-based carboxylated synthetic rubber latex coating layer, as set forth in any one of items 1 and 5 to 7, on an external surface, or with a hydrophilic group sealant or a carboxyl group sealant incorporated in the natural rubber latex.",
"19.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, nonionic polymer, as set forth in item 4 or 5, has at least one hydrophilic group selected from the group consisting of hydroxyl (—OH), ether (—O—) and amide (—CONH2—) groups.",
"20.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, anionic polymer, as set forth in item 4 or 5, has at least one hydrophilic group selected from the group consisting of carboxyl (—COOM), sulfate ester (—OSO3M), sulfonate (—SO2OM), phosphate (—PO3HM or —PO3M2), phosphate ester, —SO2NH2, and —SO2NHCOR groups, where M is hydrogen atom, and alkali metal, ammonia or organoammonium; and R is an alkyl, phenyl which may be substituted or not, or naphthyl group which may be substituted or not.",
"21.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, cationic polymer which causes no gelation of the natural rubber latex, as set forth in item 4 or 5, has at least one compound selected from the group consisting of amine salt (primary, secondary or tertiary), quaternary ammonium or pyridinium salt, phosphonium salt and sulfonium salt.",
"22.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic, ampholytic polymer which causes no gelation of the natural rubber latex, as set forth in item 4 or 5, has the hydrophilic group as set forth in items 20 and 21.23.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic polymer, as set forth in any one of items 4, 5 and 19 to 22, is a water-soluble polysaccharide or derivative thereof.",
"24.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the water-soluble polysaccharide, as set forth in item 23, is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, ureaphosphate-esterified starch, cationized starch, ampholytic starch, guar gum, phosphate-esterified guar gum, ampholytic guar gum, sodium alginate, carrageenan, locust bean gum, and xanthan gum.",
"25.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic polymer, as set forth in any one of items 4, 5 and 19 to 22, is water-soluble, water-sensitive or water-dispersible synthetic polymer.",
"26.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic polymer, as set forth in item 25, is selected from the group consisting of ammonium polyacrylate, ampholytic polyacrylamide, polyethylene oxide, polyvinyl alcohol, cationic polyamide resin, carboxylate-based acrylic copolymer, cationic acrylic copolymer, N-methoxymethylated polyamide modification (water-soluble nylon), acrylate ester copolymer, polyvinyl butyral, and cationic styrene/acrylic acid copolymer.",
"27.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the water-dispersible synthetic polymer, as set forth in item 25 is selected from the group consisting of polyvinyl acetate, ethylene-vinyl acetate copolymer, styrene-acrylate ester copolymer, styrene/methacrylate ester copolymer, acrylate ester copolymer, alkali-thickened acrylic-based emulsion, methacrylate ester copolymer, vinyl acetate/acrylic acid copolymer, vinyl acetate/acrylate ester copolymer, vinyl acetate/methacrylic acid copolymer, vinyl acetate/methacrylate ester copolymer, polyacrylamide, polymethacrylamide, copolymerized polyamide emulsion, acrylamide-based copolymer, methacrylamide-based copolymer, anionic, cationic and ampholytic modifications of these polymers, polyvinyl butyral emulsion, and polyolefin containing carboxyl group.",
"28.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying, hydrophilic polymer, as set forth in item 2, is at least one selected from the group consisting of methyl cellulose, locust bean gum, xanthan gum, carboxymethyl cellulose, alginate, carrageenan, and polyamide derivative.",
"29.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in anyone of items 2 to 18, is a detackifying crosslinking agent of tri- or tetra-valent metallic element.",
"30.The detackified natural rubber latex product with one or both surfaces detackified according to item 29, characterized in that the detackifying crosslinking agent of tri- or tetra-valent metallic element contains at least selected from the group consisting of aluminum, titanium and zirconium compounds.",
"31.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in any one of items 2 to 18 is at least one selected from the group consisting of peroxotitania solution, peroxotitania sol, zirconia sol and alumina sol.",
"32.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in any one of items 2 to 18, is a detackifying, hydrophobic, organic crosslinking agent for the hydrophilic polymer as set forth in item 4 or 5 and/or an auxiliary component of natural rubber latex.",
"33.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying, hydrophobic, organic crosslinking agent, as set forth in item 32, contains at least one selected from the group consisting of blocked isocyanate, oxazoline and carbodiimide.",
"34.The detackified natural rubber latex product with one or both surfaces detackified according to any one of items 2 to 18, characterized in that the hydrophilic group sealant or carboxyl group sealant contains at least one type of detackifying, hydrogen bond adjustors.",
"35.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying, hydrogen bond adjustor, as set forth in item 34, is selected from the group consisting of a polyamide compound, polyamide epoxy resin, polyaminepolyurea-based resin and polyamidepolyurea-based resin.",
"36.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or the carboxyl group sealant as set forth in any one of items 2 to 18, and the compound reactive with the carboxyl group in the carboxylated synthetic rubber latex as set forth in item 10 or 11 are polyamide amine/epihalohydrin condensate, polyamine/epihalohydrin condensate, polyamidepolyurea/epihalohydrin condensate, polyaminepolyurea/epihalohydrin condensate, polyamideaminepolyurea/epihalohydrin condensate, polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, styrene-based polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, cation-modified urea resin, and cation-modified, epoxy-based polyamide resin.",
"37.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in any one of items 2 to 18, contains at least one compound selected from the group consisting of monofunctional amine, monofunctional epoxy compound, monofunctional isocyanate, monofunctional blocked isocyanate, alkyl ketene dimer (AKD), alkenyl ketene dimer, alkenyl succinic anhydride (ASA), aliphatic acid anhydride, and isocyanate aziridine derivative.",
"38.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or the carboxyl group sealant, as set forth in any one of items 2 to 18, is a detackifying sizing agent.",
"39.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in any one of items 2 to 18 is a detackifying anionic, nonionic, or cationic surfactant.",
"40.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying waterproofing agent, as set forth in any one of items 2 to 18, acts on a tacky auxiliary component of the natural rubber latex, incorporated hydrophilic nonionic, anionic, cationic or ampholytic polymer, or a polymer coating layer or a carboxylated synthetic rubber latex coating layer.",
"41.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying waterproofing agent, as set forth in item 40, is a compound having a methylol group or lower alkylated compound thereof, aldehyde-based compound, a compound having an epoxy or chlorohydrin group, a compound having an ethyleneimine group, a polyvinyl butyral-based compound, or a tri- or tetra-valent multi-valent metallic compound.",
"42.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the detackifying waterproofing agent, as set forth in items 40 and 41, is polyamide epoxy resin, branched polyethylene imine, modified polyamine-based resin, polyamide-based resin, ketone resin, alkyl ketene dimer, ammonium zirconium carbonate, or blocked glyoxal resin.",
"43.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in any one of items 2 to 18, is a detackifying water repellant.",
"44.A detackified natural rubber latex product with one or both surfaces detackified, characterized in that the hydrophilic group sealant or carboxyl group sealant, as set forth in any one of items 2 to 18, is a detackifying releasing agent.",
"45.A natural rubber latex product of controlled protein elution, characterized by being treated with a compound which can introduce an anionic and/or cationic group in protein in the natural rubber latex.",
"46.The natural rubber latex product of controlled protein elution according to item 45, characterized in that the compound which can introduce an anionic and/or cationic group in the natural rubber latex is a compound reactive with protein in the natural rubber latex.",
"47.The natural rubber latex product of controlled protein elution according to item 45, characterized in that the compound which can introduce an anionic and/or cationic group in the natural rubber latex is a fixing compound or compound which can be fixed.",
"48.The natural rubber latex product of controlled protein elution according to item 46, characterized in that the compound which can introduce an anionic and/or cationic group in the natural rubber latex is reactive dye and derivative of carboxylic anhydride as anionic compounds; polyamideamine/epihalohydrin condensate, polyamine/epihalohydrin condensate, polyamidepolyurea/epihalohydrin condensate, polyaminepolyurea/epihalohydrin condensate, polyamideaminepolyurea/epihalohydrin condensate, polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, styrene-based, polyamidepolyamine epichlorohydrin resin modified with a quaternary amino group, cation-modified urea resin, cation-modified epoxy-based polyamide resin, crosslinking agent of multi-valent (trivalent or higher), and peroxotitania solution, peroxotitania sol, zirconia sol and alumina sol as cationic compounds.",
"49.The natural rubber latex product of controlled protein elution according to item 47, characterized in that the fixing compound which can introduce an anionic and/or cationic group in the natural rubber latex is anionic, ampholytic and/or cationic starch.",
"50.The natural rubber latex product of controlled protein elution according to any one of items 45 to 48, characterized in that the functional group reactive with protein of the compound which can introduce an anionic group in protein in the natural rubber latex is at least one selected from the group consisting of dichlorotriazine, difluorochlorotriazine, dichloroquinoxaline, monofluorotriazine, β-sulfatoethylsulfone, monochlorotriazine, trichloropyrimidine, carboxypyridino-S-triazine, α-bromoacrylamide, acrylamide, ω-chloroacetyl, epoxy and carboxyl anhydride.",
"51.A natural rubber latex product of controlled protein elution, characterized by being treated with a waterproofing agent (ketone resin) reactive with protein in natural rubber latex under an alkaline condition and capable of fixing the protein.",
"52.A detackified natural rubber latex product of controlled protein elution, characterized by being treated in a manner as set forth in any one of items 45 to 51, and also in a manner as set forth in any one of items 1 to 42.53.A producing method of the detackified natural rubber latex product with one or both surfaces detackified, and/or the natural rubber latex product of controlled protein elution, as set forth in any one of items 1 to 52, characterized by being leaching-treated subsequent to drying at high temperature.",
"54.The detackified natural rubber latex product with one or both surfaces detackified, and/or the natural rubber latex product of controlled protein elution, according to any one of items 1 to 52, characterized by being a fingerstall, glove, balloon or condom.",
"55.A finger stall of a detackified natural rubber latex with one or both surfaces detackified, and/or a natural rubber latex of controlled protein elution, characterized in that the finger stall of the detackified natural rubber latex and/or natural rubber latex of controlled protein elution as set forth in item 54 has a shape of being mechanically wound up from a mouth before being released out of a mold.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is an oblique view of the immersion type carrier for the present invention.",
"FIG.",
"2 describes a prototype production unit for the fingerstall of the present invention.",
"FIG.",
"3 describes functions of the major parts of the winding-up unit.",
"FIG.",
"4 shows the cross-section of the wound-up finger stall put on a finger.",
"FIG.",
"5 describes the cross-section of the fingerstall wound on the finger.",
"The symbols are 1: chain, 2: guide rail, 3: immersion mold, 4: rod, 5: guide, 6: immersion tank, 7: drying furnace, 8: winding-up machine, 10: roll type brush, 11: film, 12: fingerstall, 13: fingertip, 14: finger, and 15: wound-up mouth.",
"BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail.",
"The natural rubber latex for the present invention is not limited, but generally high-ammonium latex and low-ammonium latex.",
"Recently, deprotenized natural rubber latex has been commercialized (e.g., Japanese Patent Laid-Open No.",
"6-56902), and it is also included in the latex useful for the present invention.",
"The method of vulcanizing the natural rubber latex is not limited, and the common vulcanization methods, e.g., those aided by sulfur, peroxide or radioactive ray, can be used.",
"Those latex products for which a measure is required to prevent tackiness include immersion-processed products (e.g., balloon, glove, fingerstall and condom); extruded products (e.g., rubber yarn and tube); formed products (e.g., balloon and toys); totally rubber products (e.g., rubber sheet, hose and cloth), and rubber-lined products, although not limited thereto.",
"The hydrophilic polymer to be incorporated in the natural rubber latex is not limited.",
"A natural, semi-synthetic or synthetic one can be suitably used.",
"The hydrophilic polymer is not necessarily soluble in water.",
"A water-dispersible polymer can be also effective.",
"For synthetic polymer, in particular, a water-dispersible polymer molecular-designed to be soluble in alkaline natural rubber latex can be easily synthesized.",
"A great deal of literature describes hydrophilic polymers, and representative ones include Advanced Technology of Water-Soluble Polymers (edited by Teruo Horiuti, CMC, May 2000)and Chemistry and Technology of Water-Soluble Polymers (edited by Finch, C. A., Plenum Press, 1983).",
"Hydrophilic natural polymers include polysaccharide-, microorganism- and animal-based water-soluble polymers, represented by alginic acid, gum arabic, carrageenan, guar gum, locust bean gum, pectin, tamarind gum, tragacanth gum, starch, xanthan gum, agar, konjaku mannan, galactomannan, dextran, pullulan, curdlan, welan gum and chondroitin sulfuric acid.",
"The representative semi-synthetic, hydrophilic polymers include cellulose-, starch- and alginic acid-based water-soluble polymers.",
"Cellulose-based polymers include ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, hydroxypropyl cellulose, methylhydroxypropyl cellulose, methyl cellulose, and cationized, anionized and ampholytic cellulose.",
"Starch-based polymers include oxidized starch, and starch derivatives (esterified starch (e.g., acetylated and phosphate-esterified starches), etherified starch (e.g., methylated and hydroxyethylated starches), carboxymethylated starch, anionic starch substituted with a hydrophobic group, crosslinked starch, anionized starch, cationized starch, ampholytic starch, among others).",
"Alginic acid-based polymers include propylene glycol alginate, and guar-gum-based polymers include cationized, anionized and ampholytic guar gums.",
"The method of producing the semisynthetic, water-soluble polymer is not limited, and a known method can be used.",
"For example, a starch-based semisynthetic, water-soluble polymer can be produced by the following methods.",
"For example, National Publication of International Patent Application No.",
"10-505135 describes various methods of producing chemically modified starch, including oxidation, phosphorylation, etherification and esterification of starch, and the methods of producing cationized, ampholytic and anionized starch can be also referred to.",
"Japanese Patent Laid-Open No.",
"9-110902 discloses a method of producing starch substituted with a hydrophobic group.",
"P. Molyneux discloses hydrophilic polymers, in particular homopolymers and copolymers for water-soluble, synthetic polymers (“Chemistry and technology of water-soluble polymers”, edited by Finch, C. A., p.1 to 13), raising the following representative hydrophilic synthetic polymers.",
"The hydrophilic or water-soluble homopolymers include water-soluble acrylic-based polymers (polymers of polyacrylic acid, polyacrylate ester, polyacrylamide) and derivatives thereof, nonionic polyacrylamide, anionic polyacrylamide, cationic polyacrylamide, ampholytic polyacrylamide, poly-(N,N-dimethyl-acrylamide, poly-(N-isopropyl-acrylamide), polyaminomethyl acrylamide, polyacrylamide modified by the Mannich reaction, polyacrylamide modified by the Hofmann reaction, polymethacrylic acid and polymethacrylamide), polyimines (polyethyleneimine), polyoxides (polyethylene oxide, polypropylene oxide and polyoxolan), water-soluble vinyl-based polymers (polyethylene sulfonate, polystyrene sulfonate, polyvinyl alcohols and derivatives thereof (polyvinyl alcohol, anion-modified polyvinyl alcohol, cation-modified polyvinyl alcohol and acetal-modified polyvinyl alcohol), polyvinyl amine, polyvinyl methoxyacetal, polyvinyl methyl ether, polyvinyl methyloxazolidone, polyvinylpyrrolidone, poly-4-vinyl-pyridine, poly-4-vinyl-pyridine, poly-4-vinyl-pyridine-N-oxide, poly-4-vinyl-N-alkyl-pyridinium salt, polyvinyl sulfuric acid, polyvinyl imidazoline, carboxyvinyl polymer), water-soluble polyurethane, water-soluble polyester resin, polyamide-based polymer and derivative thereof, and polyamide resin.",
"The representative water-sensitive homopolymers include acrylic-based polymers (polymethyl acrylate, polymethyl methacrylate, poly-2-hydroxyethyl methacrylate and poly-ethylene glycol monomethacrylate), polyoxides (polyoxymethylene, poly-trimethylene oxide and polyacetoaldehyde), vinyl-based polymers (polyvinyl ethylether, polyvinyl acetate, polyvinyl formal and polyvinyl butyral).",
"Water-soluble or water-sensitive copolymers include acrylamide/acrylic acid copolymer, acrylic acid/methyl acrylate copolymer, ethylene oxide/propylene oxide copolymer, maleic anhydride-based copolymer, maleic anhydride/acrylic acid copolymer, maleic anhydride/alkene copolymer, maleic anhydride/styrene copolymer, maleic anhydride/vinyl alkyl ether copolymer, methacrylamide/methacrylic acid copolymer, methacrylic acid/methyl methacrylate copolymer, styrene/styrene sulfonate copolymer, styrene/vinyl pyrrolidone copolymer, vinyl pyrrolidone/vinyl acetate copolymer, vinyl pyrrolidone/dimethylaminoethyl methacrylate copolymer, quaternary vinyl pyrrolidone/dimethylaminoethyl methacrylate copolymer, vinyl pyrrolidone/methacrylamidepropyl/trimethyl ammonium chloride copolymer, vinyl acetal/vinyl alcohol copolymer, vinyl acetate/vinyl alcohol copolymer, vinyl alcohol/vinyl sulfate copolymer, styrene/acrylic resin, ethylene/acrylic acid copolymer, and aminoalkyl methacrylate and acrylamide copolymer thereof.",
"Hydrophilic polymers may be water-dispersible synthetic ones.",
"The examples include polyvinyl acetate, ethylene/vinyl acetate copolymer, styrene/acrylate ester copolymer, styrene/methacrylate ester copolymer, acrylate ester copolymer, methacrylate ester copolymer, vinyl acetate/acrylic acid copolymer, vinyl acetate/acrylate ester copolymer, vinyl acetate/methacrylic acid copolymer, vinyl acetate/methacrylate ester copolymer, polyacrylamide, polymethacrylamide, acrylamide copolymer, methacrylamide copolymer, and anion-, cation- and ampholytic-modifications thereof.",
"These water-dispersible polymers include polymers that dissolve or are designed to dissolve under alkaline conditions when added to natural rubber latex.",
"Hydrophilic polymers can fall into four categories of nonionic, anionic, cationic and ampholytic by their ionic characteristics.",
"Natural rubber latex is charged negative.",
"Therefore, care must be taken, when a hydrophilic polymer is incorporated in latex, not to be gelated.",
"A nonionic and anionic polymer will be rarely gelated, when incorporated in latex.",
"On the other hand, a cationic and ampholytic polymer tends to be gelated, and hence it is necessary to select the polymer causing no gelation.",
"Such a hydrophilic polymer is referred to as the cationic or ampholytic hydrophilic polymer causing no gelation in this specification.",
"Generally speaking, a low-molecular-weight and weakly cationic hydrophilic polymer is suitable, although tendency to gelation varies depending on, e.g., type and molecular weight of polymer, and type and quantity of the cationic group.",
"When incorporated with a hydrophilic polymer, natural rubber latex often increases in viscosity.",
"A moderate increase of viscosity should cause no special problem, and can be coped with by, e.g., dilution of the natural rubber latex.",
"An excessive increase, however, is undesirable.",
"It is necessary to take an adequate measure, e.g., selection of a hydrophilic polymer of low molecular weight.",
"The hydrophilic polymers, in particular natural and semi-synthetic ones, often cause the so-called creaming phenomenon, when incorporated in natural rubber latex.",
"When such a phenomenon occurs, it is necessary to prevent separation of the serum by moving the latex solution.",
"The nonionic, hydrophilic polymer generally has at least one hydrophilic group of hydroxyl (—OH), ether (—O—) or amide (—CONH2—).",
"The anionic, hydrophilic polymer generally has at least one anionic hydrophilic group selected from the group consisting of carboxyl (—COOM), sulfate ester (—OSO3M), sulfonate (—SO2OM), phosphate (—PO3HM or —PO3M2), phosphate ester, —SO2NH2, and —SO2NHCOR groups, wherein M is hydrogen atom, and alkali metal, ammonia or organoammonium; and R is an alkyl, phenyl which may be substituted or not, or naphthyl group which may be substituted or not.",
"However, the anionic, hydrophilic polymer for the present invention is not limited by the anionic, hydrophilic group.",
"The above-described anionic group is introduced as the derivative produced by the known chemical reaction in the presence of an anionic reagent in the case of semi-synthetic polymer, and by the copolymerization with an anionic reagent in the case of synthetic polymer.",
"In the latter case, an anionic group can be introduced by the methods described below for ampholytic polyacrylamide.",
"The examples of the representative anionic, hydrophilic polymers include natural polymers, e.g., gum arabic, carrageenan, pectin, xanthan gum, chondroitin sulfuric acid and alginate; semi-synthetic polymers, e.g., carboxymethyl cellulose, anionized starch (e.g., phosphate-esterified starch and carboxymethylated starch), anionized guar gum.",
"The anionic, synthetic polymers include homopolymer or copolymer of acrylic acid or methacrylic acid; copolymer of acrylic acid and acrylamide polyhydroxycarboxylate; copolymer of acrylic acid or methacrylic acid and monoethylenic monomer (e.g., ethylene, styrene, vinyl ester, acrylate ester and methacrylate ester); copolymer derived from crotonic acid; copolymer containing at least one monomer of maleic acid, fumaric acid, itaconic acid and anhydride thereof, and at least one monomer of vinyl ester, vinyl ether, halogenated vinyl and phenyl vinyl derivative, and acrylic acid and ester thereof; copolymer containing at least one anhydride of maleic acid, citraconic acid and itaconic acid, and at least one monomer of allyl and methallyl esters; and carboxyl-containing polyacrylamide (National Publication of International Patent Application No.",
"No.",
"10-511990).",
"The anionic, hydrophilic polymers for the present invention also include even a polyolefin which has carboxyl group introduced by, e.g., copolymerization of the olefin and composition of a monomer containing an unsaturated carboxylic acid, or oxidation of the polyolefin.",
"The cationic, hydrophilic polymer is characterized by having at least one cationic, hydrophilic group selected from the group consisting of amine salt (primary, secondary or tertiary), quaternary ammonium or pyridinium salt, phosphonium salt and sulfonium salt.",
"The cationic group can be introduced by a known chemical reaction, as is the case with the anionic group.",
"For cationized starch, for example, diethylaminoethyl ether group is introduced by the reaction with 2-diethylaminoethyl chloride hydrochloride, 3-(trimethyl ammonium chloride)-2-hydroxypropyl ether group is introduced by the reaction with 3-chloro-2-hydroxypropyltrimethyl ammonioum chloride as the representative cationic or cation-producing group, or tertiary amino group is introduced by the reaction with a dialkylaminoalkyl halide and made quaternary to produce ammonium (National Publication of International Patent Application No.",
"10-505139).",
"Introduction of the cationic group in a semi-synthetic and synthetic polymer is described later for the ampholytic polymer.",
"The examples of the cationic polymers include cationic polyacrylamide (e.g., aminoalkyl methacrylate and acrylamide copolymers), polyvinyl pyridium ammonium halide, polyallyl ammonium halide, polyaminomethylacrylamide, polyvinyl imidazoline, polyacrylamide modified by the Mannich reaction, polyacrylamide modified by the Hofmann reaction, polyethyleneimine, polydiallylamine, polypiridium halide, cationized starch, cationized cellulose, cationized guar gum, cationized polyvinyl alcohol, epoxyamine-based condensate, ionene-based condensate, cationized polymethacrylate ester resin, alkylene diamine-epichlorohydrin polycondensate, cationized polyvinyl pyrrolidone.",
"The ampholytic, hydrophilic polymer has both anionic and cationic hydrophilic groups described above.",
"The type and production method are not limited.",
"The synthetic polymers include quaternary copolymer of a monomer containing sulfonic acid (or its salt) with monomer containing tertiary amino group; polymers and copolymers of monomers having quaternary ammonium group and sulfonate group; copolymer of a monomer containing carboxylic acid (or its salt) with monomer containing tertiary amino group, including the copolymer made quaternary (e.g., octylacrylamide/butylaminoethyl methacrylate/acrylate ester copolymer); and polymer and copolymer of a monomer containing carboxyl group and quaternary ammonium (e.g., dialkylaminoethyl methacrylate polymer made ampholytic with monochloroacetic acid, disclosed by Japanese Patent 2571980).",
"For semi-synthetic polymers, e.g., ampholytic starch, the starch is treated doubly with a cation and anion modifier.",
"In particular, introduction of a cation group by the aid of a tertiary amino or quaternary ammonium group is combined with introduction of an anion group by the aid of an anionic group, e.g., phosphate, sulfonate, sulfate or carboxyl (National Publication of International Patent Application No.",
"10-505139).",
"Next, for synthetic polymers, introduction of an anionic and cationic group is described taking an ampholytic polyacrylamide as the example, which can be produced by copolymerizing (a) acrylamide or methacrylamide, (b) anionic vinyl monomer, and (c) cationic vinyl monomer.",
"The anionic vinyl monomers include α, β-unsaturated monobasic acids, e.g., (meth)acrylic acid, crotonic acid, (meth)allylcarboxylic acid; α,β-unsaturated dibasic acids, e.g., maleic acid, fumaric acid, itaconic acid and muconic acid; and organic sulfonic acid, e.g., vinyl sulfonic acid, styrene sulfonic acid, 2-acrylamide-2-methylpropane sulfonic acid and (meth)allyl sulfonic acid.",
"One or more of these vinyl monomers can be used, without being limited.",
"The cationic vinyl monomers include vinyl monomers having a tertiary amino group, e.g., N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate and N,N-dimethylaminopropyl (meth) acrylamide; and vinyl monomers containing a quaternary ammonium salt, obtained by reacting a vinyl monomer containing a tertiary amino group with an agent, e.g., methyl chloride, benzyl chloride, dimethyl sulfuric acid or epichlorohydrin which works to make the above monomer quaternary.",
"The other methods of introducing a cationic group include Mannich modification which reacts an anionic polyacrylamide with formalin and a secondary amine, Hofmann modification which reacts an anionic polyacrylamide with hypohalogenic acid, and amide-exchanging reaction with polyamine.",
"The methods of introducing an anionic group include hydrolysis of amide group under an alkaline condition, and sulfomethylation.",
"The hydrophilic polymer is incorporated in natural rubber latex, and normally treated with the hydrophilic group sealant, to hydrophobicize the natural rubber latex product.",
"The hydrophilic group sealant works to hydrophobicize the tacky, auxiliary component (e.g., protein or phospholipids) of the natural rubber latex, to detackify the natural rubber latex product.",
"Incorporation of the hydrophilic polymer is intended to immobilize the tacky, auxiliary component of the natural rubber latex (e.g., protein or phospholipids), to prevent it from leaching to the natural rubber latex product surface.",
"On the other hand, the hydrophilic group in the hydrophilic polymer can form the hydrogen bond, to possibly increase tackiness of the product.",
"Therefore, hydrophobization of the hydrophilic polymer is important, because extent of hydrophilicity and hydrophobicity of the hydrophilic polymer affect detackified extent of the natural rubber latex product.",
"Positive introduction of a hydrophobic group, e.g., anionic starch to introduce the hydrophobic group, is one method.",
"For example, it is necessary to use a polymer made water-soluble by incorporated hydrophobic methyl group (e.g., methyl cellulose) or polymer with controlled extent of the hydrophilic group introduced, in order to control tackiness of the hydrophilic polymer itself.",
"For example, it is known that cationized cellulose can be hydrophobicized by electrostatically bonding thereto a fatty acid salt as an anionic surfactant, to increase its hydrophobicity (Teruo Horiuchi, et al, shougi-shi, 15(1), 83 (1983)).",
"Even the anionic, hydrophilic polymer can show the hydrophobicizing effect in the presence of calcium ion or the like, conceivably because of formation of the chemical bond with a substance having an anionic surfactant activity.",
"It should be noted that natural rubber latex contains an auxiliary component having a hydrophobic group, e.g., phospholipid.",
"Moreover, there is natural rubber latex dispersed with latex by the aid of a surfactant, e.g., deprotenized natural rubber latex.",
"Therefore, some natural rubber latex products can be detackified only with the hydrophilic polymer incorporated in the natural rubber latex, when it is treated in the leaching step after being dried under heating at high temperature to evaporate ammonia incorporated in the natural rubber latex.",
"However, it is difficult to judge whether a specific hydrophilic polymer has a detackifying effect.",
"On the other hand, the judgement can be easily done by the tackiness test, conducted in EXAMPLES.",
"In the present invention, the hydrophilic polymer is specifically referred to as the detackifying, hydrophilic polymer, when the product of the natural rubber latex incorporated only therewith is detackified, as judged by the tackiness test.",
"The examples of the detackifying, hydrophilic polymers include methyl cellulose, locust bean gum, xanthan gum, carboxymethyl cellulose, alginate, carrageenan and polyamide derivative.",
"The hydrophilic group sealant for the present invention is the compound which chemically, physicochemically or physically acts on the hydrophilic group in the natural rubber latex incorporated with the hydrophilic polymer to hydrophobicize the latex, thereby controlling formation of the hydrogen bond derived from the hydrophilic group, hydrophobicizing the natural rubber latex as a whole, and detackifying the natural rubber latex product.",
"The hydrophilic group sealant mainly acts on the tacky, auxiliary components of the natural rubber latex (e.g., protein and phospholipids) and/or the hydrophilic polymer intentionally incorporated to hydrophobicize the latex, but it is still an important function for the sealant to physicochemically or physically hydrophobicize the natural rubber latex itself.",
"The hydrophilic group sealants are represented by, first of all, crosslinking agents of tri- or tetra-valent detackifying metallic element.",
"Such crosslinking agents are not limited, but it is necessary to sufficiently consider stability of the compound and side-effects, e.g., discoloration.",
"The examples of the crosslinking agents of tri- or tetra-valent metallic element useful for the present invention as the ones to be externally added to the latex include water- or alcohol-soluble, trivalent, detackifying metallic element compounds, e.g., salts of aluminum, ferric iron, chromium and thorium, of which aluminum salts (e.g., aluminum chloride, nitrate, sulfate and acetate) are more practically more suitable.",
"Polyaluminum chloride (PAC) and water-soluble polyaluminum hydroxide, being tri- or tetra-valent, are still more suitable.",
"In particular, the latter is effective.",
"A salt of metallic acid can be used, when the metal is ampholytic, and sodium aluminate is one example.",
"The observation suggests that sodium aluminate is converted into aluminum hydroxide on the film covering the latex surface and then crosslinked.",
"The aluminum-based inorganic crosslinking agents to be incorporated in latex beforehand include aluminates of alkali metal (e.g., water-soluble sodium aluminate), aluminates of alkali-earth metal (e.g., sparingly soluble calcium aluminate), and aluminum hydroxide gel.",
"The agents also include various other aluminum compounds, e.g., magnesium methasilicate aluminate, synthesized hydrotalcite, aluminosilica gel and alumino silicate.",
"In other words, these compounds are not dissociated into the ionic aluminum when incorporated, but crosslink the hydrophilic group in the latex with the ions when heated.",
"It is considered that these compounds crosslink the latex by the ions after being converted into aluminum hydroxide.",
"Common crystalline aluminum hydroxide rarely takes part in the crosslinking reaction, but the so-called amorphous aluminum hydroxide does take part in the reaction, when dispersed by, e.g., ball-milling, to have an increased specific surface area.",
"An aluminum-based crosslinking agent, when incorporated in natural rubber latex, may be gelated as time passes, depending on, e.g., type of natural rubber latex or anionic, hydrophilic polymer used.",
"It is therefore necessary to individually consider the pot life or the like of the product.",
"The detackifying compounds of tetravalent metallic element useful for the present invention include zirconium compounds, e.g., zirconium nitrate, zirconium ammonium carbonate, zirconium carbonate W, zirconium ammonium carbonate oxychloride, zirconium oxychloride; trivalent titanium compounds, e.g., titanium trichloride; and tetravalent titanium compounds, e.g., titanium sulfate, titanium tetrachloride, titanium lactate, titanium maleate anhydride and titanium oxalate.",
"Moreover, there are inorganic compounds which detackify latex by forming a uniform film thereon.",
"These include peroxotitania solution, peroxotitanate solution, peroxotitania sol, zirconia sol and alumina sol.",
"Peroxotitania in the form of solution is a titanium oxide having peroxo group (—O—O—), existing as the monomer or polymer.",
"The polymer is generally referred to as peroxotitanate, and soluble in water.",
"Zirconium-doped peroxotitanate or the like in the form of aqueous solution is known as one of the peroxotitanates, and is also included as the one useful for the present invention (Japanese Patent Laid-Open No.",
"7-286114) The commercial products of peroxotitanium complex include Teika's TKS-301 and Sadic's TPA in the form of aqueous solution.",
"Various methods have been proposed for producing peroxotitania sol, zirconia sol and alumina sol, and the commercial products of titania sol include Teika's TKS-203 and Sadic's TO sol.",
"The methods for producing alumina sol are disclosed by, e.g., Japanese Patent Laid-Open Nos.",
"05-02623, 05-024824, 07-291621 and 10-087324, and Nissan Chemical Industries' alumina sol 100, 200 and 520 are known as the commercial products of alumina sol.",
"These compounds have been originally developed as the coating agents to form a uniform film on the metallic surface or the like.",
"They can form chemical bonds, hydrogen bond or the like with various functional groups, and, when used for surface treatment of natural rubber latex, not only form the coating film on the surface but also form a strong bond with the latex film to control exfoliation of the coating film.",
"When the crosslinking agents of tri- or tetra-valent detackifying metallic element are water-soluble metallic salts, the metallic ions of many salts are dissociated to be cationic, becoming reactive with the anionic, hydrophilic group even at low temperature.",
"Moreover, they show a strong coagulating function, following the Schultz-Hardy law.",
"The metallic salts, e.g., sodium aluminate and zirconium ammonium carbonate, are anionic, and can be directly incorporated in latex, because they will not immediately react with latex.",
"The detackifying compounds of tri- or tetra-valent metallic element useful for the present invention also include organic compounds.",
"They are represented by, but not limited to carboxylates, and include aluminum acetate, zirconium acetate, titanium lactate, titanium maleate anhydride, titanium oxalate and titanium butyrate.",
"The second examples of the hydrophilic group sealants are organic crosslinking agents for detackifying/hydrophobicizing the hydrophilic polymer incorporated in the natural rubber latex and/or auxiliary components (e.g., protein and phospholipids) of the latex.",
"Polymers of low intermolecular cohesive energy, such as rubber, will have greatly improved mechanical properties, when their molecules are crosslinked with each other.",
"Natural rubber may not exibit the inherent rubber characteristics, until it is crosslinked to form the vulcanizate.",
"Crosslinking agents for vulcanizing rubber are mostly of sulfur by far.",
"There are various non-sulfur crosslinking agents for vulcanization, including sulfur donor, thiuram, thiourea, bis-mercapto, S—Cl compound, resin, compound having a reactive nitrogen group, compound having a reactive olefin group and peroxide, and ionic agents.",
"However, the natural rubber latex product cannot be detackified by vulcanization with these agents.",
"Any organic crosslinking agent may be used, irrespective of type, so long as it has an effect of detackifying/hydrophobicizing the auxiliary components of the latex or hydrophilic polymer intentionally incorporated in the natural rubber latex.",
"It is however difficult to judge beforehand the properties of the crosslinking agent to be used, e.g., type and number of the functional group therein, and whether or not it has a hydrophobic group, it is self-crosslinking, the decomposed crosslinking agent is detackifying, and it is sufficiently reactive under the natural rubber latex product production conditions to prevent the surfaces of the products being stored from adhering to each other.",
"It is therefore convenient to screen a crosslinking agent capable of detackifying natural rubber latex whether it has a hydrophobicizing effect by the tackiness test.",
"It is necessary to select the detackifying, hydrophobicizing agent from the crosslinking agents of, e.g., epoxy compound; blocked isocyanate, oxazoline-based compound; carbodiimide-based compound; melamine-formaldehyde resin; urea-formaldehyde resin; isocyanate; phenol-formaldehyde resin; glycol and polyol; diamine and polyamine; hexamethoxymethylmelamine; methylol acrylamidemethacry; (Latest Application Technologies of Latex Emulsion, edited by Motoji Okikura, Chunichi-sha, P.323), polyvalent acryloyl compound and polyvalent active ester compound.",
"The polyvalent acryloyl compound is obtained by the dehydration to combine a compound selected from the group consisting of polyhydric alcohol, polyester and polyurethane with acrylic acid through the ester bond, or the ester exchanging reaction between the above-described compound and acrylate ester.",
"The polyvalent active ester compounds specifically include oxalate diester and malonate diester (Japanese Patent Laid-Open No.",
"09-125023).",
"The crosslinking agent which works to seal hydroxyl group in protein or additive (e.g., hydrophilic polymer) is also useful.",
"The organic crosslinking agent for detackifying/hydrophobicizing the hydrophilic group generally needs a fairly high reaction temperature.",
"However, it brings about its inherent effect at around 90 to 120° C. for the present invention, because it is incorporated in a small quantity.",
"The detackifying organic compounds considered to react with the hydrophilic group, although not working as the crosslinking agent for detackifying/hydrophobicizing the hydrophilic group, have the similar effect.",
"These compounds useful for the present invention include glyoxal, polyamide, polyamidepolyurea, polyaminepolyurea, polyamideaminepolyurea, polyamidepolyurea/glyoxal condensate, polyamideamine, polyamideamine/formaldehyde condensate, polyamine/formaldehyde condensate, polyaminepolyurea/formaldehyde condensate, polyamidepolyurea/formaldehyde condensate, polyamideaminepolyurea/formaldehyde condensate, cation-modified urea resin, polyamide epoxy resin, (special)polyaminepolyurea-based resin, (special)polyamidepolyurea-based resin, modified polyamine-based resin, (modified) polyamide-based resin, and amine/polyol reaction product.",
"Many of these compounds are developed for paper as waterproofing agent, printing characteristic improver, wet strength improver and strength improver.",
"They have the common feature of controlling the hydrogen bonding in paper by incorporating a varying functional or hydrophobicizing group.",
"They are effective as are the detackifying, hydrophobicizing organic crosslinking agent under the similar reaction conditions and at a similar content.",
"Of these compounds, those capable of working as the agent for detackifying a natural rubber latex product are referred to as detackifying, hydrogen bonding adjustors in the present invention.",
"The other compounds which can work as the detackifying, hydrogen bonding adjustors include reactive, cationic compounds, e.g., polyamideamine/epihalohydrin condensate, polyamine/epihalohydrin condensate, polyamide polyurea/epihalohydrin condensate, polyaminepolyurea/epihalohydrin condensate, polyamideaminepolyurea/epihalohydrin condensate, tertiary amino group-modified polyamidepolyamine/epichlorhydrin resin, styrene-based tertiary amino group-modified polyamidepolyamine/epichlorhydrin resin, and cation-modified epoxy-based polyamide resin.",
"They also work, when incorporated in carboxylated synthetic rubber latex, to lubricate the latex film surface, and react with protein in natural rubber latex to give the natural rubber latex product of controlled protein elution.",
"The methods of producing the above-described compounds are not limited.",
"Those methods generally employed are described below.",
"A polyamide compound (also referred to as polyamideamine compound) is obtained by the dehydration condensation reaction between an amine compound and compound having carboxyl group.",
"A polyamidepolyurea, polyaminepolyurea, polyamideaminepolyurea and polyamideamine compound are the reaction products of polyalkylenepolyamine, alkylenepolyamine, urea or dibasic carboxylic acid.",
"They may be modified with a small quantity of aldehyde, epihalohydrin, or α,γ-dihalo-β-hydrin.",
"These methods are disclosed by, e.g., Japanese Patent Publication No.",
"59-32597 or Japanese Patent Laid-Open No.",
"4-10097.The polyamideamine/epihalohydrin condensate, polyamideamine/formaldehyde condensate, polyamine/epihalohydrin condensate, polyamine/formaldehyde condensate, polyamidepolyurea/epihalohydrin condensate, polyamidepolyurea/formaldehyde condensate, polyaminepolyurea/epihalohydrin condensate, polyaminepolyurea/formaldehyde condensate, polyamideaminepolyurea/epihalohydrin condensate, polyamideaminepolyurea/formaldehyde condensate are the reaction products of polyalkylenepolyamine, urea, dibasic carboxylic acid, epihalohydrin or formaldehyde.",
"The methods of producing these compounds are disclosed by, e.g., Japanese Patent Publication Nos.",
"52-22982, 60-31948 and 61-39435, and Japanese Patent Laid-Open No.",
"55-127423.Recently, the effects of organohalogen compounds on the environments have been concerned, and the method of producing the polyamidepolyamine epichlorohydrin resin containing a reduced content of these compounds is disclosed by Japanese Patent Laid-Open No.",
"10-152556.Furthermore, various compounds, e.g., epoxy-modified, quaternary epihalohydrin resin (Japanese Patent Laid-Open No.",
"61-252396) and anion-modified epihalohydrin resin (Japanese Patent Laid-Open No.",
"61-281127) of these compounds, have been developed.",
"The method of producing a polyamine epihalohydrin resin is disclosed by, e.g., U.S. Pat.",
"No.",
"3,949,014.The monofunctional compound which reacts with the hydrophilic group of the tacky auxiliary component of natural rubber latex or hydrophilic polymer to hydrophobicize them also has the effect as the hydrophilic group sealant.",
"These compounds specifically include monofunctional amine, isocyanate and blocked isocyanate.",
"Being monofunctional, they cannot form the crosslinked structure; nevertheless, however, they can seal a hydrophilic group.",
"Such a compound preferably has a hydrophobic group, in addition to the functional group involved in the reaction with the hydrophilic group, because such a compound can promote hydrophobicizing of the tacky auxiliary component of natural rubber latex and hydrophilic polymer.",
"These include reactive sizing agents, e.g., those of alkyl ketene dimer (AKD), alkenyl ketene dimer, and alkenyl succinic anhydrides (ASA), and a fatty acid anhydride derivative-based sizing agent, described later.",
"A sizing agent for paper works to hydrophobicize a hydrophilic group in paper to prevent running of ink on paper.",
"It can also detackify the natural rubber latex product, which may be incorporated with a hydrophilic polymer.",
"It conceivably hydrophobicize the hydrophilic group chemically, physicochemically or physically to detackify it.",
"The mechanisms involved in the hydrophobicizing reactions are not fully established.",
"Nevertheless, however, it is developed as a paper hydrophobicizing agent, and has a significant effect and is stable.",
"The paper sizing agent is either incorporated in paper or coating paper, and either is useful for the present invention.",
"Any substance will be useful, whether it is called sizing agent, so long as it brings about the above effect.",
"Of these sizing agents, those which detackify a natural rubber latex product are referred to as the detackifying sizing agents.",
"The sizing agents to be incorporated in paper fall into the general categories of those for acidic, neutral and acidic/neutral conditions (Japanese Patent Laid-Open No.",
"11-61682).",
"The sizing agents for acidic conditions include rosin-based, fatty acid soap, synthetic and petroleum resin agents.",
"The rosin-based sizing agents include those of rosins and derivatives thereof.",
"Rosins include gum, wood and tall oil rosins comprising resin acid as the major ingredient, e.g., abietic, palustric, neoabietic, pimaric, isopimaric or dehydroabietic acid.",
"The rosin derivatives include hydrogenated, disproportionate, polymerized, modified and strengthened rosins, and rosin ester and strengthened rosin ester.",
"The modified rosins include those modified by (alkyl)phenol/formalin resin, xylene resin, aldehyde or styrene.",
"The strengthened rosin is obtained by reacting the above-described rosin with an αβ-unsaturated carboxylic acid under heating.",
"The rosin ester is produced by a known method of esterifying a rosin with a polyhydric alcohol.",
"The strengthened rosin ester is produced by reacting the above-described rosin and/or modified rosin with a known polyhydric alcohol and αβ-unsaturated carboxylic acid consecutively or simultaneously.",
"The fatty acid soap sizing agent includes the one comprising a fatty acid of around 8 to 24 carbon atoms, e.g., palmitic or stearic acid, or a mixture thereof neutralized with an alkali.",
"The synthetic sizing agent includes the one comprising a substituted succinic anhydride (obtained by reacting an oligomer of isobutene dimer or tetramer with maleic anhydride) neutralized with an alkali.",
"The petroleum resin sizing agent includes the one comprising a petroleum resin modified by an unsaturated carboxylic acid, e.g., maleic acid.",
"The petroleum resins include C5-based one obtained by polymerizing a C5 olefin (e.g., 1,3-pentadiene or isoprene), C9-based one obtained by polymerizing a C9 olefin (e.g., coumarone or indene), C5/C9-based one obtained by polymerizing a C5 and C9 olefin, and dicyclopentadiene-based one obtained by polymerizing dicyclopentadiene or a derivative thereof.",
"The sizing agents for neutral conditions include alkyl ketene dimer (AKD)-based, alkenylketene dimer-based and alkenyl succinic anhydride (ASA)-based agents, and rosin-based agent for neutral conditions.",
"The alkyl ketene dimer-based and alkenylketene dimer-based agent can be produced by emulsifying an alkyl ketene dimer and alkenyl ketene dimer, respectively, which are normally produced by treating a chloride of corresponding saturated or unsaturated fatty acid of around 12 to 24 carbon atoms with a base, e.g., triethylamine, for dimerization.",
"The alkenyl succinic anhydride-based sizing agent can be produced by emulsifying an alkenyl succinic anhydride, produced by adding maleic anhydride to an olefin of around 12 to 24 carbon atoms, located at the terminal or inside.",
"The rosin-based sizing agents for neutral conditions include ester of rosin with a polyhydric alcohol, and emulsion of a substance contained in a petroleum resin dispersed in water.",
"The esters of rosin with a polyhydric alcohol include the products containing a rosin ester obtained by the reaction of a rosin with (a) at least one type of compound falling into the category of polyhydric alcohol or with (a) at least one type of compound falling into the category of polyhydric alcohol and (b) at least one compound falling into the category of αβ-unsaturated carboxylic acid or derivative thereof.",
"The known sizing agents for acidic/neutral conditions include cationized fatty acid bisamide-based, cationized petroleum polymer-based, cationized polymer-based and α-hydroxycarboxylic acid-based agents.",
"The cationized fatty acid bisamide-based and cationized petroleum resin-based sizing agents are normally synthesized by reacting a fatty acid or maleic acid adduct with a petroleum resin of around 12 to 24 carbon atoms, respectively, with a polyamine (e.g., diethylenetriamine or triethylenetetramine) or a mixture thereof, and then reacting the product with epichlorohydrin or the like.",
"The cationized polymer-based sizing agent is normally synthesized by the radical copolymerization of a cationic vinyl monomer (e.g., dimethylaminoethyl methacrylate) or hydrophobic monomer (e.g., styrene, acrylonitrile or alkyl (meth)acrylate) in water and/or organic solvent.",
"The α-hydroxycarboxylic acid-based sizing agent is produced by reacting a higher alcohol or amine with an oxyacid, e.g., citric acid.",
"The surface sizing agent is generally composed of the hydrophobic section and anionic section (e.g., carboxyl group) Such a surface sizing agent is obtained by, e.g., copolymerizing a hydrophobic monomer and anionic monomer, e.g.",
"αβ-unsaturated carboxylic acid, αβ-unsaturated dicarboxylic acid or unsaturated sulfonic acid (Japanese Patent Laid-Open No.",
"2000-45197).",
"The specific examples of the surface sizing agent comprising a copolymer of hydrophobic monomer and anionic monomer include styrene/(meth)acrylic acid, styrene/(meth)acrylic acid/(meth)acrylate ester, styrene/maleic acid, styrene/maleic acid/maleate semi-ester, (di)isobutylene/maleic acid and (di)isobutylene/maleic acid/maleate semi-ester copolymers, and salts thereof.",
"The other surface sizing agents include those of alkylketene dimer, alkenyl succinic acid (anhydride), styrene/acrylic acid copolymer, acrylate ester/acrylonitrile copolymer and styrene/dialkylaminoalkyl(meth)acrylate copolymer including the product of its reaction with epihalohydrin (Japanese Patent Laid-Open No.",
"2001-32191).",
"The surfactant is composed of a hydrophilic and hydrophobic group.",
"When the surfactant is coordinated with the tacky auxiliary component of the natural rubber latex or the hydrophilic polymer intentionally incorporated in the latex with the hydrophobic group positioned outside, it should seal the hydrophilic group on the natural rubber latex product surface, and hence hydrophobicize and detackify the surface.",
"Therefore, extent of hydrophobicity of surfactant should determine to what extent the product is detackified.",
"However, whether the surfactant is coordinated with the product surface with the hydrophobic group positioned outside is determined by various factors, e.g., physical and chemical properties of the surfactant itself, properties of the latex, presence of an inorganic salt (e.g., calcium ion), and hydrophilic polymer incorporated.",
"It is therefore difficult to have the general rule.",
"It is therefore necessary to conduct the tackiness test, as in EXAMPLES, to screen the detackifying surfactant.",
"The test results indicate that few surfactants show the detackifying effect, because it is generally leached out in the leaching step and has a tendency of diffusion through the latex film.",
"Nevertheless, however, it can detackify a natural rubber latex product in some circumstances where elution and diffusion of the surfactant are prevented, e.g., when a cationic hydrophilic polymer is incorporated together with an anionic surfactant.",
"The internal surface coming into contact with the mold is not exposed to water in the leaching step.",
"Therefore, separately detackfying the external surface by an adequate method, e.g., coating with a detackifying polymer layer, halogenation-treated layer or layer treated with a detackifying crosslinking agent of tri- or tetra-valent metallic element should greatly widen a range of surfactants which can detackify the internal surface.",
"A general tendency is not observed with the nonionic surfactants, except that the one of high HLB shows a low detackifying effect whereas the amine- and amide-based ones a good detackifying effect.",
"The cationic and ampholytic surfactants are characterized by their cation being bonded to the anion of the carboxyl group through the ionic bond.",
"However, both react chemically with carboxyl group at low temperature, and affect formation of the latex film by the immersion process, when incorporated in the coagulating agent.",
"It is known that the anionic surfactant, when used together with the cationic, hydrophilic polymer, shows an effect of hydrophobicizing the hydrophilic polymer.",
"Therefore, the anionic surfactant in combination with the cationic, hydrophilic polymer should show a fairly good detackifying effect.",
"A surfactant will greatly affect each production step and product properties by its inherent nature, e.g., reducing natural rubber latex viscosity.",
"It is therefore necessary to judge whether a surfactant in question is useful for the present invention or not after confirming its detackifying effect by the tackiness test, and, at the same time, to study its effects on film-making process and product properties.",
"In the coating process for the paper-making industry, starch, protein, casein, varying types of latex, polyvinyl alcohol and so on are used as the adhesives, and a waterproofing agent is used to insolubilize these adhesives by reacting with their hydrophilic groups (e.g., —OH, —CONH, —NH2 and COOH groups).",
"These compounds correspond to protein as the tacky auxiliary component of the natural rubber latex and hydrophilic polymer intentionally incorporated in the natural rubber latex for the present invention.",
"The waterproofing agent reacting with these compounds to hydrophobicize them is considered to be suitable as the agent for detackifying the natural rubber latex product.",
"Waterproofing agents used to be those of formaldehyde, glyoxal, urea formaldehyde resin and melamine formaldehyde resin.",
"However, they have been replaced by the new ones developed and commercialized later, because of various problems caused by them, e.g., unstabilized adhesive compositions, emitting a formaldehyde odor, and discoloration.",
"Waterproofing agents are classified by reactive group involved in the reaction into organic and inorganic agents, the former including compounds having methylol group and lower alkylated compounds thereof (e.g., urea/formaldehyde resin, melamine/formaldehyde resin and polyamidepolyurea/formaldehyde resin), aldehydes (formaldehyde and derivative that liberates thereof, glyoxal, dialdehyde starch, cyclic urea/glyoxal reaction product, blocked glyoxal resin (U.S. Pat.",
"No.",
"4,695,606), glyoxal/polyol reaction product (U.S. Pat.",
"No.",
"4,656,296) and copolymer of acrylamide/glyoxal reaction product), compounds having epoxy or chlorohydrin group (e.g., glycerindiglycidyl ether and polyamide/epoxy resin), compounds having ethyleneimine group (e.g., diphenylethane-bis-4,4′-N,N′-diethylene urea and (branched) polyethyleneimine) and polyvinyl butyral; and the latter including multi-valent metallic compounds (e.g., zirconium ammonium carbonate) (Handbook of Latest Paper Processing, Tec Times, P.469).",
"Those useful for detackifying natural rubber latex, and, at the same time, insolubilizing and waterproofing the tacky auxiliary component and intentionally incorporated hydrophilic group under relatively mild conditions include cycloamide, polyhydric alcohol/carbonyl adduct, (special) polyaminepolyurea-based resin, (special) polyamidepolyurea-based resin, (modified) polyamine-based resin, modified polyamide-based resin, ketone resin, cation-modified urea resin, cation-modified epoxy-based polyamide resin, polyglycidyl ether, blocked glyoxal resin (cyclic urea/glyoxal condensate, cyclic urea/glyoxal/polyol condensate), and amine/polyol reaction product.",
"Of these waterproofing agents, those capable of detackifying a natural rubber latex product are referred to as the detackifying waterproofing agents.",
"Some hydrophilic polymers need specific waterproofing agents.",
"For example, diborate functions as the waterproofing agent for polyvinyl alcohol.",
"These specific waterproofing agents for some hydrophilic polymers also fall into the scope of the present invention (Encyclopaedia of Paper and Paper Processing Agents, Tec Times, P.147).",
"Water repellents have been developed to impart water repellency to paper.",
"Water repellents are not necessarily suitable for detackifying a natural rubber latex product, due to their insufficient hydrophilicity, although they themselves are highly hydrophobic.",
"It is therefore necessary to select the water repellents which can detackify a natural rubber latex product from various ones.",
"These water repellents are referred to as the detackifying water repellents.",
"It is necessary to select the detackifying water repellents from the agents, e.g., fatty acid amide wax, fatty acid/chromium complex, aluminum stearate, chlorosulfonated polyethylene, ethyleneurea-based resins, acrylic resins, and silicone-based resins.",
"In the paper processing industry, lining materials for adhesive labels or the like, e.g., releasing paper, adhesive tape, process paper and transfer paper, are coated with a releasing agent.",
"Releasing agents are broadly classified into two general categories of silicone- and nonsilicone-based ones.",
"The silicone-based agents are basically composed of polydimethyl siloxane as the base polymer and polymethyl hydrogen siloxane as the crosslinking agent.",
"They are sub-classified by crosslinking reaction type into condensing reaction and addition reaction types.",
"Hydroxyl works as the functional group in the polydimethyl siloxane for the former type, and vinyl works as the functional group for the latter type.",
"Polymethyl hydrogen siloxane works as the crosslinking agent for both types.",
"The nonsilicone-based releasing agents, preferable for some purposes, include polymer having a long-chain alkyl group, alkyd resin and acrylic/styrene copolymer, represented by polymer having a long-chain alkyl group.",
"The releasing agent having a long-chain alkyl group is a product of alkylation reaction between a vinyl compound and the polymer.",
"The long-chain alkyl groups are mostly octadecyl.",
"The nonsilicone-based releasing agents are sub-classified into the reaction and non-reaction types, the latter needing no curing step for applying the agent.",
"The reaction type is more preferable for securing the detackifying effect.",
"The known releasing agents of this type include those of adduct of polyethyleneimine and octadecyl isocyanate, adduct of polyvinyl alcohol and isocyanate having a long-chain alkyl group, self-crosslinking acrylic/styrene copolymer.",
"For detackifying a natural rubber latex product, the releasing agent is preferably aqueous.",
"The suitable compounds for introducing an anionic group in protein in natural rubber latex include those working as the dyes reactive under alkaline or neutral conditions.",
"The reactive dye has the structure composed of a colorant base having a water-soluble group and reactive group.",
"The water-soluble groups are generally sulfone group, although not limited thereto.",
"For example, they may be other anionic groups.",
"The reactive dyes, originally developed for dyeing cellulosic fibers, are used also for protein-based fibers, e.g., wool.",
"The reactive dye introduces an anionic group in protein, when it reacts with protein in natural rubber latex.",
"The reactive groups for the reactive dye are not limited.",
"Various reactive groups are known, and are also being developed (Basic Dye chemistry, Sadaji Abeta, Sikisen-sha, P.164).",
"The major reactive groups for the reactive dyes are sulfate ethylsulfone-based, s-triazine-based and pyrimidine-based ones.",
"The basic reactive group is sulfate ethylsulfone for the sulfate ethylsulfone-based one, dichlorotriazine and monochlorotriazine for the s-triazine-based one, and trichloropyrimidine for the pyrimidine-based one.",
"The improved types of the major reactive groups include dissimilar bifunctional ones (e.g., sulfate ethylsulfone/monochlorotriazine, sulfate ethylsulfone/difluorochloropyridine and vinyl sulfone/monochlorotriazine), monochlorotriazine-based group of cyanuric chloride whose second chlorine atom is substituted with a substituent, reactive group substituted with two or more halogenotriazine groups, halogenotriazine group whose chlorine atom is substituted with fluorine, reactive group substituted with two or more monochlorotriazine groups, reactive group with chlorine in monochlorotriazine substituted with nicotinic acid to be reactive in a neutral region, and trichloropyrimidine with one or two chlorine atoms out of its three chlorine atoms are substituted with methyl sulfone group or fluorine atom.",
"Recently, the movements are noted for improving dye fixing characteristics by introducing two or more functional groups or developing new functional groups to reduce loads of dye-containing waste water.",
"N-(sulfate ethyl sulfonylalkyl)-anil group is one example of such groups (Japanese Patent Laid-Open No.",
"7-304981).",
"A reactive dye of high fixing characteristics is considered to have high capacity of fixing protein in natural rubber latex.",
"Such a reactive dye is desirable for production of the natural rubber latex product of controlled elution of protein.",
"Elution of protein may not be completely prevented, even when the reactive dye reacts with protein in natural rubber latex.",
"The eluted protein may be still detected by the protein analysis, which involves vigorous stirring of the sample in the phosphate-buffered physiological saline for 2 hours.",
"In such a case, it is preferable to further treat the natural rubber latex with the so-called fixing agent, as is the case with dyeing with a reactive dye.",
"The fixing agent for the reactive dye, fixing the dye after bonding itself to the anionic group of the dye represented by sulfone group, will also fix protein, when the dye is bonded to the protein.",
"The chemical structures as the basic skeletons of the fixing agents now being used are dicyanodiamide/formalin polycondensate-based, polyamine-based and polycation-based ones, the polycation-based one being prevailing.",
"The commercial agents with the polycation-based structure include dimethylamine/epichlorohydrin polycondensate, dimethyldiallyl ammonium chloride polymer, (di)allyl amine hydrochlorate polymer, quaternary polymer of dialkylaminoethyl methacrylate, diallyl amine salt/sulfur dioxide copolymer, and dimethyldiallyl ammonium chloride/sulfur dioxide copolymer (Senryo Kenkyu, 44, No.2, P.45, 2000).",
"Many of these compounds overlap the above-described cation-based hydrophilic group sealants, and these sealants work also as the fixing agents.",
"Moreover, the crosslinking agents of tri- or tetra-valent metallic elements also function as the fixing agents.",
"A natural rubber latex product is not necessarily colored.",
"Therefore, the compound which is reactive with protein in natural rubber latex and introduces an anionic group therein can reduce elution of the protein from the natural rubber latex product.",
"In other words, the compound which having a reactive group and an anionic group or a functional group which forms an anionic group while having no colorant base also falls into the scope of the present invention.",
"In addition, the compound having anhydrous carboxylic group reacts with protein to introduce the carboxylic group therein, thereby reducing elution of the protein.",
"The water-insoluble, fixing, anionic compound also reduces elution of protein for the rubber product of anionic starch and guar gum.",
"The cationic group reactive with natural rubber latex protein cationizes the protein, fixing it in the natural rubber latex product to reduce its elution.",
"These compounds, e.g., epichlorohydrin-based polycondensates, overlap those compounds useful as the cationic, detackifying, hydrophilic group sealants or fixing agents, indicating that the hydrophobicization leads to fixing of protein and hence to controlled elution of protein.",
"The natural saccharide-related products, e.g., those of starch and guar gum, are frequently molten at high temperature to be used in the form of solution.",
"The starch or the like is not molten at normal temperature, when the product is dried.",
"The natural rubber latex products, incorporated with cationized starch or guar gum to utilize the above nature, show controlled elution of protein, conceivably because the cationized starch or the like fixed on the product electrostatically captures the protein to control its elution even under the analysis conditions.",
"Therefore, the natural rubber latex product incorporated with a compound cationic and insoluble under the analysis conditions is of controlled elution of protein.",
"The cationized polymer will exhibit the effect similar to that of cationized starch, when insolubilized by a known waterproofing agent or the like.",
"There are various methods of producing the detackified natural rubber latex product of the present invention, and are not limited.",
"When the natural rubber latex product is detackified only with the hydrophilic polymer and/or hydrophilic group sealant incorporated therein, it can be produced by the common method.",
"However, the starting material for natural rubber latex is inherently strongly alkaline when incorporated with ammonia, and the tacky auxiliary component (e.g., protein) and the hydrophilic polymer and hydrophilic group sealant to be incorporated are highly soluble in water.",
"Therefore, there is a high possibility that the protein is leached or eluted out.",
"It is therefore desirable to control elution of the tacky component towards the product surface by sufficiently drying the natural rubber latex under heating to evaporate ammonia prior to the leaching step, and, at the same time, by sufficiently reacting the incorporated agent with, or bonding it to, the tacky auxiliary component.",
"The surface of the product already formed into the film can be detackified, when so needed, by bringing one or both surfaces into contact with the hydrophilic group sealant solution to be treated therewith.",
"Some hydrophilic group sealants react with the product very quickly, so that the surface loses tackiness when the latex film is withdrawn from the solution.",
"In some cases, the film needs to be heated after being withdrawn from the solution.",
"In any case, it is desirable to treat the product under heating in order to fully bring about the treatment effect.",
"This surface treatment is applicable to the product produced by the direct immersion process.",
"The internal surface of an immersion-processed product can be detackified by coating the mold with one or more hydrophilic group sealants and the common mono- or di-valent coagulating agent for the external surface, to simultaneously coagulation and treatment of the latex with the sealant(s), when the surface comes into contact with these agents.",
"It is desirable to adopt this treatment for natural rubber latex, even when it can be detackified only with the incorporated detackifying hydrophilic polymer and/or hydrophilic group sealant, because the treatment of the internal surface with the hydrophilic group sealant is a simple procedure.",
"When the external surface of the latex film is to be treated with the hydrophilic group sealant, it can be detackified by immersion of the external surface of the film in a hydrophilic group sealant solution the heat treatment effected in a manner similar to the above.",
"There are three methods for coating the mold with the hydrophilic group sealant.",
"The first method coats the mold with the mixed solution of the common coagulating agent of mono- or di-valent metallic salt for the external surface and one or more hydrophilic group agents of the present invention.",
"The second method coats the mold first with the hydrophilic group sealant of the present invention, and then with the common coagulating agent for the external surface.",
"Immersion of the coated mold in the emulsified latex solution gives the immersion-processed product whose internal surface coming into contact with the mold is detackified.",
"The third method coats the mold with the hydrophilic group sealant as the coagulating agent for the external surface to form the thin film of the hydrophilic group sealant, further coats the thin film with the coagulating agent of mono- or di-valent metallic element for the external surface, and immerses again the coated mold in the latex solution.",
"This method, although capable of detackifying the internal surface, may cause interlayer exfoliation of the product.",
"The natural rubber latex product is generally treated under hydrothermal conditions in the leaching step.",
"This leaches the tacky component of the natural rubber latex to the external surface, and elutes out the incorporated hydrophilic polymer, hydrophilic group sealant and the like from the external surface, to deteriorate the detackifying effect.",
"Therefore, heat treatment at high temperature is frequently required prior to the leaching step for, e.g., evaporation of ammonia.",
"On the other hand, the method which involves no heat-treatment at high temperature can be separately effected for the external surface, which can be easily treated.",
"Halogenation, coating with a detackifying polymer and treatment of the crosslinking agent of tri- or tetra-valent metallic element are some of the methods applicable to the external surface.",
"Each treatment can be effected on-machine.",
"The external surface can be halogenated by the known method.",
"The applicable methods and their effects are described earlier.",
"The treatment with the detackifying polymer immerses the external surface of the product in the detackifying polymer solution and then dries it under heating to make the surface detackified with the coating film.",
"The conventional treatment method to form the coating film is already described in Background Art.",
"To explain the coating treatment with the detackifying diene-based carboxylated synthetic rubber latex, developed by the inventors of the present invention, the external surface can be easily detackified by immersing the external surface in the detackifying diene-based carboxylated synthetic rubber latex solution, diluted to a very low concentration of 5%, and then drying the resultant coating film under heating.",
"The coating film accounts for less than 1 part of the natural rubber latex film, even when it is thin at around 0.1 mm; nevertheless, however, it can sufficiently bring about the detackifying effect.",
"Thickness of the coating film can be freely changed for specific surfaces by changing concentration of the detackifying diene-based carboxylated synthetic rubber latex solution.",
"The diene-based carboxylated synthetic rubber latex needs the carboxyl group sealant to be detackified.",
"This agent can be directly incorporated in the diene-based carboxylated synthetic rubber latex solution or in the natural rubber latex.",
"The natural rubber latex can be also detackified by incorporating the hydrophilic group sealant in the diene-based carboxylated synthetic rubber latex.",
"The hydrophilic group sealants and carboxyl group sealants are functionally interchangeable in many cases.",
"When this is the case, the product can be detackified as a whole by incorporating the hydrophilic group sealant or carboxyl group sealant in the diene-based carboxylated synthetic rubber latex or in the natural rubber latex.",
"The hydrophilic group sealant or carboxyl group sealant may sometimes make the latex unstable, and the extent of unstability is varied depending on properties of each agent or on whether the latex is of the diene-based carboxylated synthetic rubber or natural rubber.",
"Thus, the said sealant is convenient in that the place where the sealant is incorporated can appropriately selected depending on properties of the agent.",
"Another advantage of the coating with the detackifying, carboxylated synthetic rubber latex is that it causes no discoloration of the metallic surface with which the coated natural rubber latex product comes into contact.",
"Therefore, the product is suitably used for handling an electronic part or precision device.",
"When the external surface is to be treated with the crosslinking agent of tri- or tetra-valent metallic element, it can be treated by being immersed in the natural rubber latex solution and then dried under heating.",
"The reaction between a cationic group and the anionic, tacky component is electrostatic in nature, and has an advantage of proceeding at low temperature.",
"However, taking an aluminum compound as the example, it may be converted into aluminum hydroxide in the presence of ammonia, to lose cationic property.",
"In such a case, deteriorated effect or separation of aluminum hydroxide on the surface may result.",
"It is desirable to take an adequate measure against such possibility, e.g., making the immersion solution acidic, elution treatment prior to the immersion treatment, or drying the latex at high temperature to evaporate ammonia.",
"As described above, use of the present invention can easily give the natural rubber latex product detackified on one or both surfaces.",
"The product surfaces are not adhered to each other even when they come into contact with each other under heating during the production process or thereafter, a characteristic which can be used for producing novel products.",
"One example is the fingerstall of detackifying natural rubber latex which is wound up from its mouth on-machine before being released out of the mold.",
"The fingerstall wound up from the mouth has been already developed.",
"For example, referring to FIG.",
"4, the fingerstall 12 put on the fingertip 13 can be worn by simply winding it back on the finger in the arrowed direction F, as shown in FIG.",
"5.Its usefulness has been recognized, because it can easily cover the finger.",
"However, a fingerstall as a natural rubber latex product is inherently tacky on both surfaces, and the winding-up type is detackified beforehand with powder or post-treatment of chlorination and then manually wound up.",
"Such a product is rarely used in a factory producing precision processed products, because of difficulty in keeping the products highly clean.",
"On the other hand, the present invention provides a fingerstall of natural rubber latex detackified on both surfaces, which can be mechanically wound up on the mold and keep the precision products highly clean.",
"Recently, thinner fingerstalls are increasingly in demand to reduce fatigue of the wearer.",
"A thinner fingerstall, however, is more difficult to wear, and hence thin, powder-free, detackified, clean, wound-up fingerstalls are strongly in demand.",
"The natural rubber latex detackified on both surfaces can be easily made into the fingerstall with a wound-up mouth.",
"When a fingerstall is produced, the upper portion is left tacky without being provided with the hydrophilic group sealant layer or the like, and wound up totally and then wound back in such a way to leave the tacky portion as the wound-up mouth.",
"In the conventional method, it is necessary to provide the wound-up mouth by first winding up only the upper portion of the fingerstall, and then releasing the fingerstall out of the mold in a separate step.",
"The wound-up mouth is greatly in demand for flat products, because it facilitates wearing/taking-off of the fingerstall.",
"A fingerstall can be detackified, after being provided with the wound-up mouth by the conventional method.",
"The wound-up fingerstall described earlier can be provided with the wound-up mouth in a similar manner.",
"Referring to FIG.",
"5, when the fingerstall 12, wound up from the mouth to have the wound-up mouth, is taken off from the finger 14, it can be wound back on the finger easily except for the wound-up mouth 15, which is left tacky unlike the other portion.",
"The fingerstall provided with a wound-up mouth can be easily worn/taken off by picking the mouth by other fingers.",
"Depending on properties of the fingerstall, the wound-up mouth has a function of clamping the finger to keep the fingerstall held thereon.",
"It is possible to produce the wound-up fingerstall having no wound-up mouth by winding up the fingerstall which is detackified over the entire surface.",
"The fingerstall having no wound-up mouth has an advantage of reducing fatigue of the person who wears it for a long time, because the finger is not fastened by the mouth.",
"EXAMPLES 1.Preparation of the Starting Materials (1) Preparation of Natural Rubber Latex Natural rubber latex was pre-vulcanized under the following conditions, unless otherwise stated: Starting natural rubber latex: High-ammonium natural rubber latex HA-FELDA LATEX Solid concentration: 60%, pH: almost 10.6 Pre-vulcanization conditions: Sulfur: 0.7 parts Zinc oxide: 1.0 part Zinc di-n-butyldithiocarbamate: 0.6 parts Curing conditions: 40° C. for 24 hours (2) Incorporation of the Hydrophilic Polymer When the hydrophilic polymer was incorporated in the pre-vulcanized natural rubber latex sample, it was dissolved or dispersed in water to have a concentration of around 1 to 2% and added to the latex slowly with stirring.",
"Its content in each EXAMPLE is given in the relevant table.",
"(3) Incorporation of the Hydrophilic Group Sealant When the hydrophilic group sealant was incorporated in the natural rubber latex sample, it was added to the pre-vulcanized natural rubber latex sample.",
"Its content in each EXAMPLE is given in the relevant table.",
"(4) Adjustment of the Natural Rubber Latex Concentration The immersion-processed natural rubber latex was adjusted to contain the natural rubber latex solids at 40.0%, unless otherwise stated.",
"The natural rubber latex film was around 0.10 to 0.13 mm thick, when produced by the coagulation method.",
"(5) Preparation of the Coagulating Solution (or Coagulating Solution Containing the Hydrophilic Group Sealant) The coagulating solution was an aqueous solution, containing calcium nitrate tetrahydrate at 100 g/1000 g, unless otherwise stated.",
"When the internal surface was treated with the hydrophilic group sealant, a given quantity of the sealant was added to the coagulating solution.",
"The concentration of the hydrophilic group sealant in each EXAMPLE is given in the relevant table.",
"(6) Preparation of the Treatment Solution of the Hydrophilic Group Sealant for the External Surface When the external surface of the natural rubber latex film was treated with the hydrophilic group sealant, the treatment solution was diluted with water to have a given hydrophilic group sealant concentration for each EXAMPLE.",
"Its concentration in each EXAMPLE is given in the relevant table.",
"(7) Preparation of the Coating Solution of Detackifying Diene-Based Carboxylated Synthetic Rubber Latex for the External Surface Carboxylated NBR latex was incorporated with 1.5 parts of activated zinc white, 0.25 parts of sodium aluminate (as Al2O3) and 2.5 parts of the carboxyl group sealant, and diluted with water to have the solid latex concentration of 5%, unless otherwise stated.",
"The carboxylated NBR latex and carboxyl group sealant used are shown in each EXAMPLE.",
"2.Formation of the Natural Rubber Latex Film (By Use of Coagulating Agent) The mold was immersed in the above-described coagulating solution containing calcium nitrate tetrahydrate at 100 g/1000 g. The mold held around 0.03 g of the coagulating solution.",
"It was dried, immersed in the natural rubber latex preparation solution for 5 seconds, and withdrawn to form the natural rubber latex film.",
"The dried film was 0.10 to 0.13 mm thick and weighing around 0.3 g. When the internal surface was treated with the hydrophilic group sealant, the above described coagulation solution containing the hydrophilic group sealant was used, to form the natural rubber latex film.",
"3.Post-Treatment of the Formed Natural Rubber Latex Film The natural rubber latex film prepared by the above procedure was heated, and then treated for leaching and post-vulcanization, unless otherwise stated.",
"Treatment temperature and time are shown in each EXAMPLE.",
"4.Treatment of the External Surface of the Formed Natural Rubber Latex Film with the Hydrophilic Group Sealant The natural rubber latex film formed on the mold was heated, and then immersed in the above-described treatment solution of the hydrophilic group sealant for the external surface for 5 seconds, unless otherwise stated.",
"Approximately 0.03 g of the solution was held by the film.",
"It was heated, and then treated for leaching and post-vulcanization.",
"Treatment temperature and time are shown in each EXAMPLE.",
"5.Coating Treatment of the External Surface with the Detackifying Diene-Based Carboxylated Synthetic Rubber Latex The natural rubber latex film formed on the mold was heated, and then immersed in the above-described coating solution of the detackifying diene-based carboxylated synthetic rubber latex for 5 seconds, unless otherwise stated.",
"Approximately 0.05 g of the solution was held by the film.",
"It was heated, and then treated for leaching and post-vulcanization.",
"Treatment temperature and time are shown in each EXAMPLE.",
"6.Chlorination Treatment of the External Surface of the Natural Rubber Latex Film The natural rubber latex film formed on the mold was heated, and then immersed in chlorine water containing chlorine at 0.4% for 5 seconds, to chlorinate the external surface, unless otherwise stated.",
"It was heated, and then treated for leaching and post-vulcanization.",
"Treatment temperature and time are shown in each EXAMPLE.",
"7.Tackiness Test The post-vulcanized natural rubber latex film was wound up on the mold.",
"It was heated at 90° C. for 30 minutes while it was kept wound-up, cooled and then wound back.",
"Extent of detackiness was evaluated according to the four-grade system: O: the film can be easily wound back, O′: the film is slightly difficult to wind back on the way, Δ: the film cannot be wound back on the way, and x: the film cannot be wound back.",
"8.Detackiness Test of the Natural Rubber Latex Film COMPARATIVE EXAMPLE 1 The natural rubber latex film was prepared only from the pre-vulcanized natural rubber latex, and post-vulcanized for the tackiness test.",
"The result was that the film could not be wound back.",
"The post-treatment conditions are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in COMPARATIVE EXAMPLE 1 were: Heating Leaching Post-vulcanization (COMPARATIVE EXAMPLE 1) 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 10 min.",
"COMPARATIVE EXAMPLE 2 The natural rubber latex film was prepared from the pre-vulcanized natural rubber latex incorporated with 0.25 parts of the hydrophilic polymer, and post-vulcanized for the tackiness test.",
"The result was that the film could not be wound back.",
"The post-treatment conditions and hydrophilic polymer incorporated are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in COMPARATIVE EXAMPLE 2 were: Heating Leaching Post-vulcanization (COMPARATIVE EXAMPLE 2) 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 10 min.",
"(Hydrophilic polymer incorporated in the latex) COMPARATIVE EXAMPLE 2 Polyethylene Oxide PEO-8 (SUMITOMO SEIKA CHEMICALS) Properties: Nonionic, White powdery or granular Viscosity (0.5%, 25° C.): 60 mPa.s pH (0.5%)=7.0 EXAMPLES 1 to 13 The natural rubber latex film was prepared from the pre-vulcanized natural rubber latex incorporated with the anionic, hydrophilic polymer, and further with the hydrophilic group sealant, and post-treated for the tackiness test.",
"The result, and the anionic, hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 1.The post-treatment conditions in each EXAMPLE are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization In order to evaluate the effects of the post-treatment, the film was post-treated under two different sets of conditions for the heating and leaching steps: (1) heating at 50° C. for 2 minutes and leaching at 70° C. for 3 minutes, and (2) heating at 90° C. for 5 minutes and leaching at 85° C. for 3 minutes in each of EXAMPLES 1 to 5.Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization (EXAMPLES 1 to 5) (1) 50° C., 2 min.",
"70° C., 3 min.",
"90° C., 5 min.",
"(EXAMPLES 1 to 5) (2) 90° C., 5 min.",
"85° C., 3 min.",
"90° C., 5 min.",
"The film prepared in each of EXAMPLES 1 to 5 was further treated for post-vulcanization at 110° C. for 5 minutes for the tackiness test.",
"Heating Leaching Post-vulcanization (EXAMPLES 6 and 7) 90° C., 5 min.",
"70° C., 5 min.",
"110° C., 5 min.",
"(EXAMPLES 8 to 13) 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 10 min.",
"TABLE 1 Incor- Incor- porated porated Drying Examples Anionic, hydrophilic polymer quantity Hydrophilic group selants quantity prior to Test NO.",
"to be incorporated in the latex (parts) to be incorporated in the latex (parts) leaching results 1 Carboxymethyl cellulose 0.2 Detackifying waterproofing agent: 1.0 50° C. o′ Trade name: CMC DAICEL 1330 Blocked glyoxal resin (Product of 90° C. o (DAICEL CHEMICAL INDUSTRIES) polyamide polyurea glyoxal reaction) Properties: Anionic, White Trade name: Sumirez Resin 5001 powdery, Viscosity (1%, 25° C.): (Sumitomo Chemical Group) 73 cps pH: 6.8, Degree of Properties: Nonionic, Light-colored, etherification: 1.27 transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 2 Carboxymelhyl cellulose 0.2 Detackifying surfactant: 0.4 50° C. o′ Trade name: CMC DAICEL 1330 β-naphthalene sulfonate/ 90° C. o (DAICEL CHEMICAL INDUSTRIES) formalin condensate Properties: Anionic, White Trade name: DEMOL N (Kao Corporation) powdery, Viscosity (1%, 25° C.): Properties: Anionic, Light yellow/ 73 cps pH: 6.8, Degree of brown powdery etherification: 1.27 3 Sodium alginate 0.2 Detackifying, hydrophobicizing, organic 0.4 50° C. o′ Trade name: ALGITEX AG-LL crosslinking agent: Blocked isocyanate 90° C. o (Kimica Corporation) Trade name: Prominate XC-915 (TAKEDA Properties: Anionic, Brown granular, CHEMICAL INDUSTRIES) Viscosity (1%, 20° C.): 45 cps Properties: Nonionic, White emulsion, pH: 7.0 Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 4 Sodium alginate 0.2 Detackifying, reactive sizing agent: 1.0 50° C. o′ Trade name: ALGITEX AG-LL Alkyl ketene dimer 90° C. o (Kimica Corporation) Trade name: Sizepine K-910 Properties: Anionic, Brown granular, (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (1%, 20° C.): 45 cps Properties: Anionic, White emulsion, pH: 7.0 Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 5 Ammonium polyacrylate 0.2 Detackifying sizing agent: Strength- 1.0 50° C. o′ Trade name: ARON A-30 (Toagosei) ened rosin sizing agent 90° C. o Properties: Anionic, Light brown, Trade name: Sizepine E-50 (ARAKAWA viscous liquid, Concentration: 31.1% CHEMICAL INDUSTRIES) Viscosity (30° C.): 9760 cps, Properties: Anionic, Brown, transparent pH (1%, 25° C.): 8.3 liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 6 Carboxylate-based acrylic copolymer 0.2 Detackifying, reactive sizing agent 1.0 90° C. o Trade name: ARON A-7180 (Toagosei) Alkyl ketene dimer Properties: Anionic, Semi-transparent, Trade name: Sizepine K-910 viscous liquid, Concentration: 16.4% (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (25° C.): 20950 cps, Properties: Anionic, White emulsion, pH (25° C.): 9.0 Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 7 Carboxylate-based acrylic copolymer 0.2 Detackifying waterproofing agent: 1.0 90° C. o Trade name: ARON A-7180 (Toagosei) Blocked glyoxal resin (Product of 0 Viscosity (25° C.): 20950 polyamide polyurea glyoxal reaction) cps, pH (25° C.): 9.0 Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 8 Carboxymethyl cellulose 0.25 Detackifying waterproofing agent: 0.5 95° C. o′ Trade name: CELLOGEN F-SB Blocked glyoxal (Cyclic amide (DAI-ICHI KOGYO SEIYAKU) aldehyde condensate) Properties: Anionic, White powdery, Trade name: SUNREZ 700M Viscosity (2%, 25° C.): 200 mPa · s (OMNOVA Solutions) Degree of etherification: 0.9 Properties: Cationic, Brown liquid, Concentration: 45% Viscosity: 25 cps, pH: 6.0 9 Carrageenan 0.25 Detackifying waterproofing agent: 0.5 95° C. o′ Trade name: Soarace WX165 Blocked glyoxal (Cyclic amide (MRC Polysaccharide) aldehyde condensate) Properties: Anionic, White powdery Trade name: Sunrez 700M pH (1.5%): 8.2 (OMNOVA Solutions) Properties: Cationic, Brown liquid, Concentration: 45% Viscosity: 25 cps, pH: 6.0 10 Urea phosphorylated starch 0.25 Detackifying waterproofing agent: 0.25 95° C. o Trade name: MS#4600 Zirconium ammonium carbonate (asZrO2) (Nihon Shokuhin Kako) Trade name: Baycoat 20 Properties: Anionic, Slightly (Nippon Light Metal) yellow powdery Viscosity Properties: Anionic, Slightly yellow (20%, 50° C.): 74 mPa · s, liquid, Concentration: 20% (as ZrO2) pH: 5.5 Viscosity: 8 cps, pH: 9.5 11 Carboxymethyl cellulose 0.25 Detackifying waterproofing agent: 0.15 95° C. o′ Trade name: CELLOGEN PL-15 Zirconium ammonium carbonate (asZrO2) (DAI-ICHI KOGYO SEIYAKU) Trade name: AZ Coat 5800MT (SAN NOPCO) Properties: Anionic, White powdery, Properties: Anionic, Slightly yellow Viscosity (1%, 25° C.): 15 mPa · s liquid, Concentration: 20% (as ZrO2) Degree of etherification: 0.5 Viscosity (25° C.): 10 mPa · s, pH (1%): 9.0 12 Phosphorylated guar gum 0.5 Detackifying waterproofing agent: 0.15 95° C. o Trade name: Mayprofilm 222 Zirconium ammonium carbonate (asZrO2) (Meyhall AG.)",
"Trade name: AZ Coat 5800MT (SAN NOPCO) Properties: Anionic, yellow Properties: Anionic, Slightly yellow powdery Viscosity (2%): liquid, Concentration: 20% (as ZrO2) 45 mPa · s, pH: 8.0 Viscosity (25° C.): 10 mPa · s, pH (1%): 9.0 13 Alkali-thickening type acrylic 0.4 Detackifying, hydrophobicizing cross- 0.8 95° C. o′ emulsion linking agent: Oxazoline-based cross- Trade name: Boncoat 3750 linking agent (DAINIPPON INK AND CHEMICALS) Trade name: Epocross WS-500 Properties: Anionic, Milky white (NIPPON SHOKUBAI) emulsion, Concentration: 23% Properties: Light yellow, transparent Viscosity: 35 cps, pH (1%): 3.0 liquid, Concentration: 38.9% Viscosity (25° C.): 1230 mPa · s, pH: 9.1 EXAMPLES 14 to 21 The pre-vulcanized natural rubber latex was incorporated with the nonionic, hydrophilic polymer, and further with the hydrophilic group sealant.",
"It was formed into the film and post-treated for the tackiness test.",
"The result, and the nonionic, hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 2.The post-treatment conditions in each EXAMPLE are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization In order to evaluate the effects of the post-treatment, the film was post-treated under two different sets of conditions for the heating and leaching steps: (1) heating at 50° C. for 2 minutes and leaching at 70° C. for 3 minutes, and (2) heating at 90° C. for 5 minutes and leaching at 85° C. for 3 minutes in each of EXAMPLES 14 to 19.Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization (EXAMPLES 14 to 19) (1) 50° C., 2 min.",
"70° C., 3 min.",
"90° C., 5 min.",
"(EXAMPLES 14 to 19) (2) 90° C., 5 min.",
"85° C., 3 min.",
"90° C., 5 min.",
"The film prepared in each of EXAMPLES 14 to 19 was further treated for post-vulcanization at 110° C. for 5 minutes for the tackiness test.",
"Heating Leaching Post-vulcanization (EXAMPLE 20) 95° C., 5 min.",
"85° C., 5 min.",
"110° C., 10 min.",
"(EXAMPLE 21) 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"TABLE 2 Incor- Incor- porated porated Drying Examples Nonionic, hydrophilic polymers quantity Hydrophilic group blocking agent quantity prior to Test NO.",
"to be incorporated in the latex (parts) to be incorporated in the latex (parts) leaching results 14 Polyvinyl alcohol 0.2 Detackifying waterproofing agent: 1.0 50° C. o′ Trade name: DENKA POVAL K-05 (DENKI Blocked glyoxal resin (Product of 90° C. o KAGAKU KOGYO KABUSHIKI KAISHA) polyamide polyurea glyoxal Properties: Nonionic, White to light reaction) yellow powdery, Viscosity: 6.0 mPa · s Trade name: Sumirez Resin 5001 pH: 6.0 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 15 Polyvinyl alcohol 0.2 Detackifying surfactant: 0.4 50° C. o′ Trade name: DENKA POVAL K-05 (DENKI β-naphthalene sulfonate/formalin 90° C. o KAGAKU KOGYO KABUSHIKI KAISHA) condensate Properties: Nonionic, White to light Trade name: DEMOL N (Kao Corporation) yellow powdery, Viscosity: 6.0 mPa · s Properties: Anionic, Light yellow/brown pH: 6.0 powdery 16 Methyl cellulose 0.2 Detackifying, hydrophobicizing, organic 0.4 50° C. o′ Trade name: Metolose SM-400 crosslinking agent: Blocked isocyanate 90° C. o (Shin-Etsu Chemical) Trade name: Prominate XC-915 Properties: Nonionic, White powdery (TAKEDA CHEMICAL INDUSTRIES) Viscosity (2%, 20° C.): 436 mPa · s Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 17 Methyl cellulose 0.2 Detackifying, reactive sizing agent: 1.0 50° C. o′ Trade name: Metolose SM-400 Alkyl ketene dimer 90° C. o (Shin-Etsu Chemical) Trade name: Sizepine K-910 Properties: Nonionic, White powdery (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (2%, 20° C.): 436 mPa · s Properties: Anionic, White emulsion, Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 18 Polyethylene oxide 0.2 Detackifying surfactant: Polyoxyethylene 0.4 50° C. o′ Trade name: PEO-8 (SUMITOMO derivative 90° C. o SEIKA CHEMICALS) Trade name: Emulgen A-60 Properties: Nonionic, White powdery (Kao Corporation) or granular, pH (0.5%): 7.0 Properties: Nonionic, Light yellow Viscosity (0.5%, 25° C.): 60 mPa · s liquid pH (5%): 6.5 19 Polyethylene oxide 0.2 Detackifying sizing agent: Alkenyl 1.0 50° C. o′ Trade name: PEO-8 (SUMITOMO succinate 90° C. o SEIKA CHEMICALS) Trade name: Coloparl SS-40 Properties: Nonionic, White powdery (Seiko Chemical Industries) or granular, pH (0.5%): 7.0 Properties: Anionic, Brown liquid, Viscosity (0.5%, 25° C.): 60 mPa · s Concentration: 40.4% Viscosity: 80 cps, pH: 10.4 20 Methyl cellulose 0.25 Detackifying, waterproofing agent: 0.1 95° C. o Trade name: Metolose SM-400 Zirconium ammonium carbonate (asZrO2) (Shin-Etsu Chemical) Trade name: AZ Coat 5800MT (SAN NOPCO) Properties: Nonionic, White powdery Properties: Anionic, Slightly yellow Viscosity (2%, 20° C.): 436 mPa · s liquid, Concentration: 20% (as ZrO2) Viscosity (25° C.): 10 mPa · s, pH (1%): 9.0 21 N-methoxymethylated polyamide 0.25 Detackifying epoxy compound: Glycerol 1.0 95° C. o modification (Water-soluble nylon) polyglycidyl ether Trade name: Toresin Fs-350 Trade name: Denacol EX-313 (Nagase ChemteX Corporation) (Nagase Chemtex Corporation) Properties: Nonionic, Milky white, Properties: Anionic, Light yellow viscous liquid, Concentration: 20% liquid Viscosity (25° C.): 150 mPa · s, Viscosity (30° C.): 350 cps, pH: 7 Epoxy equivalents: 141WPE EXAMPLES 22 to 26 The pre-vulcanized natural rubber latex was incorporated with the cationic or ampholytic, hydrophilic polymer which causes no gelation of the natural rubber latex, and further with the hydrophilic group sealant.",
"It was formed into the film and post-treated for the tackiness test.",
"The result, and the cationic or ampholytic, hydrophilic polymer which causes no gelation of the natural rubber latex and hydrophilic group sealant used in each EXAMPLE are given in Table 3.The post-treatment conditions in each EXAMPLE are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization (EXAMPLES 22 to 26) 95° C., 5 min.",
"85° C., 5 min.",
"110° C., 10 min.",
"TABLE 3 Incorporated Incorporated Examples Cationic or ampholytic, hydrophilic quantity Hydrophilic group blocking agent quantity Test NO.",
"polymers to be incorporated in the latex (parts) to be incorporated in the latex (parts) results 22 Cationized tapioca starch 0.25 Detackifying surfactant: Coconut oil 0.5 ◯ Trade name: Catesize 350 (Nippon NSC) fatty acid sodium sarcosine Properties: Cationic, White powdery Trade name: Neoscope SCN-35 Viscosity (5%, 40° C.): 17 cps (Toho Chemical Industry) Properties: Anionic, Light yellow, transparent liquid, Concentration: 35% pH (1%): 8.2 23 Cationic styrene/acrylic copolymer 0.25 Detackifying waterproofing agent: 0.25 ◯ Trade name: Pearl gum CS (Seiko Zirconium ammonium carbonate (asZrO2) Chemical Industries) Trade name: Baycoat 20 Properties: Cationic, Light-colored, (Nippon Light Metal) transparent, viscous liquid, Properties: Anionic, Slightly yellow Concentration: 20.1% Viscosity (25%): liquid, Concentration: 20% (as ZrO2) 10 cps, pH (2%): 4.3 Viscosity: 8 cps, pH: 9.5 24 Cationic acrylate ester copolymer 0.25 Detackifying waterproofing agent: 0.15 ◯ Trade name: Aquabrid 46753 Zirconium ammonium carbonate (asZrO2) (DAICEL CHEMICAL INDUSTRIES) Trade name: AZ Coat 5800MT (SAN NOPCO) Properties: Cationic, Milky white Properties: Anionic, Slightly yellow emulsion, Concentration: 30% Viscosity liquid, Concentration: 20% (as ZrO2) (23° C.): 320 mPa · s, pH: 6.7 Viscosity (25° C.): 10 mPa · s, pH (1%): 9.0 25 Ampholytic polyacryloamide 0.4 Detackifying sizing agent: Strengthened 1.0 ◯ Trade name: Fixter K-6LS rosin sizing agent (Seiko Chemical Industries) Trade name: Sizepine E-50 Properties: Ampholytic, Light brown, (ARAKAWA CHEMICAL INDUSTRIES) transparent, viscous liquid, Properties: Anionic, Brown, transparent Concentration: 15.2% Viscosity liquid, Concentration: 50.4% Viscosity (25° C.): 680 cps, pH (1.5%): 4.7 (25° C.): 200 cps, pH (5%, 20° C.): 11.0 26 Ampholytic polyacryloamide 0.25 Detackifying waterproofing agent: 0.25 ◯ Trade name: Fixter K-6LS Zirconium ammonium carbonate (asZrO2) (Seiko Chemical Industries) Trade name: Baycoat 20 Properties: Ampholytic, Light brown, (Nippon Light Metal) transparent, viscous liquid, Properties: Anionic, Slightly yellow Concentration: 15.2% Viscosity liquid, Concentration: 20% (as ZrO2) (25° C.): 680 cps, pH (1.5%): 4.7 Viscosity: 8 cps, pH: 9.5 EXAMPLES 27 to 36 The pre-vulcanized natural rubber latex was incorporated with the anionic, hydrophilic polymer, and further surface treated with the hydrophilic group sealant.",
"It was formed into the film for the tackiness test.",
"The result, and the anionic, hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 4.The post-treatment conditions in each EXAMPLE are described below: (Post-treatment) The natural rubber latex film film prepared was surface-treated for both surfaces by the following procedure in the order described below, unless otherwise stated: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 27 to 31 Heating: 50° C., 2 min., Drying: 90° C., 5 min.",
"Leaching: 75° C., 3 min., Post-vulcanization: 90° C., 3 min.",
"The film prepared in each of EXAMPLES 27 to 31 was further treated for post-vulcanization at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLE 32 The film was heated at 50° C. for 2 minutes, and leached at 70° C. for 3 minutes.",
"Then, it was heated at 90° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was dried at 90° C. for 3 minutes, and finally post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLE 33 Heating: 90° C., 5 min., Drying: 90° C., 3 min.",
"Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 34 Heating: 50° C., 2 min., Drying: 90° C., 5 min.",
"Leaching: 70° C., 3 min., Post-vulcanization: 120° C., 5 min.",
"EXAMPLE 35 The film was heated at 50° C. for 3 minutes, and leached at 75° C. for 3 minutes.",
"Then, it was heated at 90° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was post-vulcanized at 95° C. for 5 minutes.",
"EXAMPLE 36 Heating: 50° C., 1 minute, Drying: 90° C., 5 min.",
"Leaching: 75° C., 5 min., Post-vulcanization: 90° C., 12 min.",
"TABLE 4 Incorporated Examples Anionic, hydrophilic polymers quantity Hydrophilic group blocking agent Concen- Test NO.",
"to be incorporated in the latex (parts) for treating both surfaces tration (%) results 27 Carboxymethyl cellulose 0.2 Detackifying sizing agent: Strengthened 0.5 o Trade name: CMC DAICEL 1330 rosin sizing agent (DAICEL CHEMICAL INDUSTRIES) Trade name: Sizepine E-50 Properties: Anionic, White powdery, (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (1%, 25° C.): 73 cps Properties: Anionic, Brown, transparent pH: 6.8, Degree of etherification: 1.27 liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 28 Carboxymethyl cellulose 0.2 Detackifying surfactant: Polyoxyethylene 0.4 o Trade name: CMC DAICEL 1330 derivative (DAICEL CHEMICAL INDUSTRIES) Trade name: Emulgen A-60 (Kao Corporation) Properties: Anionic, White powdery, Properties: Nonionic, Light yellow liquid Viscosity (1%, 25° C.): 73 cps pH (5%): 6.5 pH: 6.8, Degree of etherification: 1.27 29 Sodium alginate 0.2 Detackifying crosslinking agent of metallic 0.5 o Trade name: ALGITEX AG-LL element: Polyaluminum hydroxide (asAl2O3) (Kimica Corporation) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Anionic, Brown granular, Properties: Cationic, Light yellow, Viscosity (1%, 20° C.): 45 cps transparent liquid, Viscosity (30° C.): pH: 7.0 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 30 Sodium alginate 0.2 Detackifying, hydrogen bond adjustor: 0.3 o Trade name: ALGITEX AG-LL Polyamide/polyamine epichlorohydrin resin (Kimica Corporation) Trade name: Sumirez Resin 675 Properties: Anionic, Brown granular, (Sumitomo Chemical Group) Viscosity (1%, 20° C.): 45 cps Properties: Cationic, Brown, transparent pH: 7.0 liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 31 Ammonium polyacrylate 0.2 Detackifying, reactive sizing agent 1.0 o Trade name: ARON A-30 (Toagosei) (waterproofing agent): Alkyl ketene dimer Properties: Anionic, Light brown, Trade name: Sizepine K-287 viscous liquid, Concentration: 31.1% (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (30° C.): 9760 cps, Properties: Cationic, White emulsion, pH (1%, 25° C.): 8.3 Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 32 Carboxymethyl cellulose 1.0 Detackifying crosslinking agent of 0.5 o Trade name: CMC DAICEL 1330 metallic element: Polyaluminum hydroxide (asAl2O3) (DAICEL CHEMICAL INDUSTRIES) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Anionic, White powdery, Properties: Cationic, Light yellow, Viscosity (1%, 25° C.): 73 cps transparent liquid, Viscosity (30° C.): pH: 6.8, Degree of etherification: 1.27 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 33 Carboxymethyl cellulose 1.0 Detackifying crosslinking agent of 0.5 o Trade name: CMC DAICEL 1330 metallic element: Polyaluminum hydroxide (asAl2O3) (DAICEL CHEMICAL INDUSTRIES) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Anionic, White powdery, Properties: Cationic, Light yellow, Viscosity (1%, 25° C.): 73 cps transparent liquid, Viscosity (30° C.): pH: 6.8, Degree of etherification: 1.27 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 34 Carboxymethyl cellulose 0.25 Detackifying, hydrogen bond adjustor: 1.0 o Trade name: CMC DAICEL 1330 Polyamide/polyamine epichlorohydrin resin (DAICEL CHEMICAL INDUSTRIES) Trade name: Sumirez Resin 675 Properties: Anionic, White powdery, (Sumitomo Chemical Group) Viscosity (1%, 25° C.): 73 cps Properties: Cationic, Brown, transparent pH: 6.8, Degree of etherification: 1.27 liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 35 Carboxylate-based acrylic copolymer 0.2 Detackifying, reactive sizing agent 0.5 o Trade name: ARON A-7180 (Toagosei) (waterproofing agent): Alkyl ketene dimer Properties: Anionic, Semi-transparent, Trade name: Sizepine K-287 viscous liquid, Concentration: 16.4% (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (25° C.): 20950 cps, Properties: Cationic, White emulsion, pH (25° C.): 9.0 Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 36 Carboxylate-based acrylic copolymer 0.2 Detackifying, hydrogen bond adjustor: 0.3 o Trade name: ARON A-7180 (Toagosei) Polyamide/polyamine epichlorohydrin resin Properties: Anionic, Semi-transparent, Trade name: Sumirez Resin 6615 viscous liquid, Concentration: 16.4% (Sumitomo Chemical Group) Viscosity (25° C.): 20950 cps, Properties: Cationic, Brown, transparent pH (25° C.): 9.0 liquid, Concentration: 15% Viscosity (25° C.): 40 mPa · s, pH (25° C.): 4.0 EXAMPLES 37 to 49 The pre-vulcanized natural rubber latex was incorporated with the nonionic, hydrophilic polymer, and further surface treated with the hydrophilic group sealant.",
"It was formed into the film for the tackiness test.",
"The result, and the nonionic, hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 5.The post-treatment conditions in each EXAMPLE are described below: (Post-treatment) The natural rubber latex film film prepared was surface-treated for both surfaces by the following procedure in the order described below, unless otherwise stated: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 37 to 42 Heating: 50° C., 2 min., Drying: 90° C., 5 min.",
"Leaching: 85° C., 3 min., Post-vulcanization: 90° C., 3 min.",
"The film prepared in each of EXAMPLES 37 to 42 was further treated for post-vulcanization at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLE 43 The film was heated at 50° C. for 2 minutes, and leached at 70° C. for 3 minutes.",
"Then, it was heated at 90° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was dried at 90° C. for 3 minutes, and finally post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLE 44 Heating: 90° C., 5 min., Drying: 90° C., 3 min.",
"Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 45 Heating: 40° C., 1 minute, Drying: 95° C., 5 min.",
"Leaching: 50° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 46 and 47 Heating: 40° C., 1 minute, Drying: 90° C., 7 min.",
"Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 48 and 49 The film was heated at 50° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was dried at 95° C. for 3 minutes, leached at 85° C. for 1 minute and finally post-vulcanized at 110° C. for 5 minutes.",
"TABLE 5 Incorporated Examples Nonionic, hydrophilic polymers quantity Hydrophilic group blocking agent Concen- Test NO.",
"to be incorporated in the latex (parts) for treating both surfaces tration (%) results 37 Polyvinyl alcohol 0.2 Detackifying sizing agent: Strengthened 0.5 o Trade name: DENKA POVAL K-05 rosin sizing agent (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Sizepine E-50 KAISHA) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Nonionic, While to Properties: Anionic, Brown, transparent light yellow powdery, Viscosity: liquid, Concentration: 50.4% Viscosity 6.0 mPa · s pH: 6.0 (25° C.): 200 cps, pH (5%, 20° C.): 11.0 38 Polyvinyl alcohol 0.2 Detackifying, reactive sizing agent 0.5 o Trade name: DENKA POVAL K-05 (waterproofing agent): Alkyl ketene dimer (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Sizepine K-287 KAISHA) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Nonionic, White to Properties: Cationic, White emulsion, light yellow powdery, Viscosity: Concentration: 20% Viscosity (25° C.): 6.0 mPa · s pH: 6.0 40 cps, pH (20° C.): 3.7 39 Methyl cellulose 0.2 Detackifying surfactant: Polyoxyethylene 0.4 o Trade name: Metolose SM-400 derivative (Shin-Etsu Chemical) Trade name: Emulgen A-60 Properties: Nonionic, White (Kao Corporation) powdery Viscosity (2%, 20° C.): Properties: Nonionic, Light yellow 436 mPa · s liquid pH (5%): 6.5 40 Methyl cellulose 0.2 Detackifying, hydrogen bond adjustor: 0.3 o Trade name: Metolose SM-400 Polyamide/polyamine epichlorohydrin (Shin-Etsu Chemical) resin Properties: Nonionic, White Trade name: Sumirez Resin 675 powdery Viscosity (2%, 20° C.): (Sumitomo Chemical Group) 436 mPa · s Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 41 Polyethylene oxide 0.2 Detackifying crosslinking agent of 0.5 o Trade name: PEO-8 metallic element: Aluminum nitrate (asAl2O3) (SUMITOMO SEIKA CHEMICALS) Properties: Nonionic, White powdery or granular, pH (0.5%): 7.0 Viscosity (0.5%, 25° C.): 60 mPa · s 42 Polyethylene oxide 0.2 Detackifying sizing agent: Alkenyl 0.5 o Trade name: PEO-8 succinate (SUMITOMO SEIKA CHEMICALS) Trade name: Coloparl SS-40 Properties: Nonionic, White (Seiko Chemical Industries) powdery or granular, pH (0.5%): Properties: Anionic, Brown liquid, 7.0 Viscosity (0.5%, 25° C.): Concentration: 40.4% Viscosity: 80 cps, 60 mPa · s pH: 10.4 43 Polyvinyl alcohol 0.25 Detackifying crosslinking agent of 0.5 o Trade name: DENKA POVAL K-05 metallic element: Aluminum nitrate (asAl2O3) (DENKI KAGAKU KOGYO KABUSHIKI KAISHA) Properties: Nonionic, White to light yellow powdery, Viscosity: 6.0 mPa · s, pH: 6.0 44 Polyvinyl alcohol 0.25 Detackifying crosslinking agent of 0.5 o Trade name: DENKA POVAL K-05 metallic element: Aluminum nitrate (asAl2O3) (DENKI KAGAKU KOGYO KABUSHIKI KAISHA) Properties: Nonionic, White to light yellow powdery, Viscosity: 6.0 mPa · s pH: 6.0 45 Polyvinyl alcohol 0.25 Detackifying, reactive sizing agent: 1.0 o Trade name: DENKA POVAL K-05 Alkyl ketene dimer (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Sizepine K-910 KAISHA) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Nonionic, White Properties: Anionic, White emulsion, to light yellow powdery, Viscosity: Concentration: 15% Viscosity (25° C.): 6.0 mPa · s pH: 6.0 6 cps, pH (25° C.): 5.5 46 Polyethylene oxide 0.2 Detackifying, reactive sizing agent: 1.0 o Trade name: PEO-8 Alkyl ketene dimer (SUMITOMO SEIKA CHEMICALS) Trade name: Sizepine K-910 Properties: Nonionic, White (ARAKAWA CHEMICAL INDUSTRIES) powdery or granular, pH (0.5%): Properties: Anionic, White emulsion, 7.0 Viscosity (0.5%, 25° C.): Concentration: 15% Viscosity (25° C.): 60 mPa · s 6 cps, pH (25° C.): 5.5 47 Polyvinyl alcohol 0.2 Internal surface 1.0 o Trade name: DENKA POVAL K-24E Detackifying, reactive sizing agent: (DENKI KAGAKU KOGYO KABUSHIKI Alkyl ketene dimer KAISHA) Trade name: Sizepine K-910 Properties: Nonionic, White (ARAKAWA CHEMICAL INDUSTRIES) to light yellow powdery, Viscosity: Properties: Anionic, White emulsion, 27 mPa · s, pH: 6.0 Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 External surface Detackifying, waterproofing agent: 1.0 Soudium borate 48 Copolymerized polyamide emulsion 0.25 Detackifying crosslinking agent of 1.0 o Trade name: Griltex 2 Suspension metallic element: Alumina sol (asAl2O3) (EMS SHOWA DENKO K.K.)",
"Trade name: Alumina sol 200 Properties: Nonionic, Milky white (Nissan Chemical Industries) aqueous solution, Concentration: 40%, Properties: Cationic, Milky white, Viscosity: 1500 cps, pH: 9.5 colloidal solution, Concentration: 10.1% (as Al2O3) Viscosity (20° C.): 530 mPa · s, pH (20° C.): 4.8 49 Polyvinyl butyral resin emulsion 0.25 Detackifying crosslinking agent of 1.0 o Trade name: Rezem VB-1 metallic element: Alumina sol (asAl2O3) (CHUKYO YUSHI) Trade name: Alumina sol 200 Properties: Nonionic, White liquid, (Nissan Chemical Industries) Concentration: 35% Viscosity Properties: Cationic, Milky white, (25° C.): 20 mPa · s, colloidal solution, Concentration: pH (diluted 10 times): 7.2 10.1% (as Al2O3) Viscosity (20° C.): 530 mPa · s, pH (20° C.): 4.8 EXAMPLES 50 to 53 The pre-vulcanized natural rubber latex was incorporated with the cationic or ampholytic, hydrophilic polymer which causes no gelation of the natural rubber latex, and further surface treated with the hydrophilic group sealant.",
"It was formed into the film for the tackiness test.",
"The result, and the cationic or ampholytic, hydrophilic polymer which causes no gelation of the natural rubber latex and hydrophilic group sealant used in each EXAMPLE are given in Table 6.The post-treatment conditions in each EXAMPLE are described below: (Post-treatment) The natural rubber latex film film prepared was surface-treated for both surfaces by the following procedure in the order described below, unless otherwise stated: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 50 Heating: 40° C., 5 min., Drying: 95° C., 7 min.",
"Leaching: 85° C., 3 minutes, Post-vulcanization: 110° C., 10 minutes EXAMPLES 51 and 53 Heating: 50° C., 5 min., Drying: 95° C., 5 min.",
"Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLE 52 Heating: 40° C., 5 min., Drying: 90° C., 10 min.",
"Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"TABLE 6 Incorporated Examples Cationic or ampholytic, hydrophilic quantity Hydrophilic group selants for Concen- Test NO.",
"polymers to be incorporated in the latex (parts) treating both surfaces tration (%) results 50 Cationic styrene/acrylic copolymer 0.3 Detackifying surfactant: β-naphthalene 1.0 o Trade name: Pearl gum CS(Seiko sulfonate/formalin condensate Chemical Industries) Trade name: DEMOL N (Kao Corporation) Properties: Cationic, Light-colored, Properties: Anionic, Light yellow/brown transparent, viscous liquid, powdery Concentration: 20.1% Viscosity (25%): 10 cps, pH (2%): 4.3 51 Cationic polyamide resin 0.25 Detackifying, hydrogen bond adjustor: 0.5 o′ Trade name: Arafix 255 (ARAKAWA Polyamide/polyamine epichlorohydrin resin CHEMICAL INDUSTRIES) Trade name: Euramine P5600 (Mitsui Chemicals) Properties: Cationic, Brown, Properties: Cationic, Light yellow, transparent liquid, Concentration: 26% transparent liquid, Concentration: 31% Viscosity (25° C.): 183 mPa · s, Viscosity (25° C.): 71.3 mPa · s, pH (20° C.): 3.05 pH (25° C.): 4.5 52 Ampholytic guar gum 0.4 Detackifying, hydrophobicizing, organic 2.0 o Trade name: Meyprobond 120 (SANSHO) crosslinking agent: Blocked isocyanate Properties: Ampholytic, yellow powdery Trade name: Prominate XC-915 (TAKEDA Viscosity (1%, 25° C.): 800 cps, pH: 10 CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 53 Ampholytic polyacryloamide 0.3 Detackifying waterproofing agent: 0.5 o Trade name: Polymerjet 902 (ARAKAWA Branched polyethyleneimine CHEMICAL INDUSTRIES) Trade name: Epomine P-1000 (NIPPON SHOKUBAI) Properties: Ampholytic, Light yellow, Properties: Cationic, Light yellow, slightly turbid liquid transparent, viscous liquid, Concentration: 29.9% Viscosity (25° C.): 1800 mPa · s, Viscosity (25° C.): 633 mPa · s, pH (20° C.): 3.1 pH (5%): 10.6 EXAMPLES 54 and 55 The pre-vulcanized deproteinized natural rubber latex was incorporated with 0.25 parts of the hydrophilic polymer, and further surface treated with the hydrophilic group sealant.",
"It was formed into the film for the tackiness test.",
"The result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 7.The post-treatment conditions and deproteinized natural rubber latex used in each EXAMPLE are described below: (Deproteinized natural rubber latex) SELATEX FDPNR2100 (Sumitomo Rubber Ind.)",
"Solid concentration: 61.7%, pH: almost 10.9 Pre-vulcanization conditions: Sulfur: 0.7 parts Zinc oxide: 1.0 part Zinc di-n-butyldithiocarbamate: 0.8 parts Curing conditions: 40° C. for 48 hours (Post-treatment) The natural rubber latex film film prepared was surface-treated for both surfaces by the following procedure in the order described below, unless otherwise stated: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 54 Heating: 50° C., 5 min., Drying: 95° C., 7 min.",
"Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLES 55 Heating: 95° C., 2 min., Drying: 95° C., 5 min.",
"Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"TABLE 7 Incorporated Examples Hydrophilic polymers to be quantity Hydrophilic group selants for Concen- Test NO.",
"incorporated in the latex (parts) treating both surfaces tration (%) results 54 Carboxymethyl cellulose 0.25 Detackifying, reactive sizing agent 1.0 o Trade name: CMC DAICEL 1330 (waterproofing agent): Alkyl ketene dimer (DAICEL CHEMICAL INDUSTRIES) Trade name: Sizepine K-287 Properties: Anionic, White powdery, (ARAKAWA CHEMICAL INDUSTRIES) Viscosity (1%, 25° C.): 73 cps Properties: Cationic, White emulsion, pH: 6.8, Degree of etherification: 1.27 Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 55 Methyl cellulose 0.25 Detackifying crosslinking agent of 0.5 o Trade name: Metolose SM-400 metallic element: Polyaluminum hydroxide (asAl2O3) (Shin-Etsu Chemical) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Nonionic, White Properties: Cationic, Light yellow, powdery Viscosity (2%, 20° C.): transparent liquid, Viscosity (30° C.): 436 mPa · s 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 EXAMPLES 56 to 62 The pre-vulcanized natural rubber latex was incorporated with the detackifying, hydrophilic polymer.",
"It was formed into the film and post-treated for the tackiness test.",
"The sample whose external surface was coating-treated with the detackifying, carboxylated, synthetic rubber latex and halogenation-treated was also tested by the tackiness test.",
"The tackiness test result, and the detackifying, hydrophilic polymer used in each EXAMPLE are given in Table 8.The post-treatment, external surface coating treatment, carboxylated NBR used, carboxyl group sealant used, and halogenation treatment are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization (EXAMPLES 56 to 62) 90° C., 5 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"(External Surface Coating Treatment Step) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated latex film was 0.1 to 0.13 mm thick, and weighing almost 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 56 to 62 Heating (1): 50° C., 2 min., Heating (2): 90° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) (Carboxyl Group Sealant) Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) (External Surface Halogenation Treatment Step) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in chlorine water→Heating (2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 56 to 62 Heating (1): 50° C., 5 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"Each was further post-vulcanized at 110° C. for 5 min.",
"for the tackiness test.",
"TABLE 8 Test results Incorporated Method of external surface treatment Examples Detackifying, hydrophilic polymers quantity No Coating Haloge- NO.",
"to be incorporated in the latex (parts) treatment treatment nation 56 Methyl cellulose 0.25 o o o Trade name: Metolose SM-400 (Shin-Etsu Chemical) Properties: Nonionic, White powdery Viscosity (2%, 20° C.): 436 mPa · s 57 Locust bean gum 0.2 o o o Trade name: Soar Locust A120F (MRC Polysaccharides) Properties: Nonionic, White powdery Viscosity (1%, 25° C.): 1200 cps 58 Xanthan gum 0.2 o o o Trade name: Soar Xan XG550 (MRC Polysaocharides) Properties: Anionic, White powdery Viscosity (1%, 25° C.): 1500 cps, pH (1%): 7.5 59 Carboxymethyl cellulose 0.2 o o o Trade name: Earnet gum FDM (DAICEL CHEMICAL INDUSTRIES) Properties: Anionic, White powdery Viscosity (1%, 25° C.): 161 cps, pH: 7.5 60 Sodium alginate 0.2 o o o Trade name: ALGITEX AG-LL (Kimica Corporation) Properties: Anionic, Brown granular, Viscosity (1%, 20° C.): 45 cps pH: 7.0 61 Carrageenan 0.2 o o o Trade name: Soar Ace WX165 (MRC Polysaccharides) Properties: Anionic, White powdery pH (1.5%): 8.2 62 Polyamide derivative: Polyoxyethylen 0.5 o o o ealkyl ether Trade name: Elsoft A (Ipposha Oil Industries) Properties: Nonionic, Light yellow, pasty, Concentration: 15% EXAMPLES 63 to 78 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic group sealant.",
"It was formed into the film and post-treated for the tackiness test.",
"The result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 9.The post-treatment for each EXAMPLE is described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization (EXAMPLES 63 to 67) 90° C., 5 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"(EXAMPLE 68) 90° C., 7 min.",
"85° C., 3 min.",
"110° C., 10 min.",
"(EXAMPLES 69 to 71) 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 10 min.",
"(EXAMPLE 72) 38° C., 10 min.",
"70° C., 3 min.",
"110° C., 10 min.",
"(EXAMPLES 73 to 78) 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 10 min.",
"TABLE 9 Incorporated Examples quantity Test NO.",
"Hydrophilic group blocking agents to be incorporated in the latex (parts) results 63 Detackifying waterproofing agent: Blocked glyoxal resin 1.0 o (Product of polyamide polyurea glyoxal reaction) Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 64 Detackifying surfactant: β-naphthalene sulfonate/formalin condensate 0.4 o Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown powdery 65 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 0.4 o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 66 Detackifying, reactive sizing agent: Alkyl ketene dimer 1.0 o Trade name: Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, White emulsion, Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 67 Detackifying sizing agent: Strengthened rosin sizing agent 1.0 o Trade name: Sizepine E-50 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, Brown, transparent liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 68 Detackifying waterproofing agent: Zirconium ammonium carbonate 0.5 o Trade name: Baycoat 20 (Nippon Light Metal) (asZrO2) Properties: Anionic, Slightly yellow liquid, Concentration: 20% (as ZrO2) Viscosity: 8 cps, pH: 9.5 69 Detackifying, hydrophobicizing crosslinking agent: Oxazoline-based crosslinking agent 1.0 o′ Trade name: Epocross WS-500 (NIPPON SHOKUBAI) Properties: Light yellow, transparent liquid, Concentration: 38.9% Viscosity (25° C.): 1230 mPa · s, pH: 9.1 70 Detackifying, hydrogen bond adjustor: Polyaminepolyurea-based resin 1.5 o′ Trade name: Sumirez Resin 703 (Sumitomo Chemical Group) Properties: Weakly cationic, Brown, transparent liquid, Concentration: 50% Viscosity (25° C.): 65 mPa · s, pH (25° C.): 7.0 71 Detackifying, hydrogen bond adjustor: Polyamide polyurea-based resin 1.0 o′ Trade name: Sumirez Resin 302 (Sumitomo Chemical Group) Properties: Nonionic, Light yellow, transparent liquid, Concentration: 60% Viscosity (25° C.): 320 mPa · s, pH (25° C.): 6.8 72 Detackifying, reactive sizing agent: Alkyl ketene dimer 1.0 o′ Trade name: Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, White emulsion, Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 73 Detackifying water repellent: Zirconium-based special wax 0.25 o Trade name: Coat Sizer NZ (DAIWA CHEMICAL INDUSTRIES) Properties: Anionic, Milky white emulsion, Concentration: 30% 74 Detackifying water repellent: Olefin-based resin 0.3 o′ Trade name: Coat Sizer MS-115 (DAIWA CHEMICAL INDUSTRIES) Properties: Anionic, Light brown emulsion, Concentration: 30% pH: 10.0 75 Detackifying water repellent: Special wax 0.5 o′ Trade name: Coat Sizer MN2L (DAIWA CHEMICAL INDUSTRIES) Properties: Anionic, White emulsion, Concentration: 50% pH: 9.0 76 Detackifying water repellent: Styrene/acrylate ester copolymer 0.5 o′ Trade name: Polysol AM-600 (SHOWA HIGHPOLYMER) Properties: Anionic, White emulsion, Concentration: 47.1% Viscosity (23° C.): 46.0, pH: 7.5 77 Detackifying waterproofing agent: Ketone resin 0.5 o Trade name: SI-668 (Nippon PMC corporation) Properties: Nonionic, Slightly white turbid solution, Concentration: 50% Viscosity: 40 cps, pH: 7 78 Detackifying waterproofing agent: Polyamine epichlorohydrin resin 0.5 o Trade name: PA-625 (Nippon PMC corporation) Properties: Weakly cationic, Light brown, transparent liquid, Concentration: 60% Viscosity: 250 cps, pH (20° C.): 7.0 EXAMPLES 79 to 96 The pre-vulcanized natural rubber latex was surface-treated with the hydrophilic group sealant, and formed into the film for the tackiness test.",
"The result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 10.The post-treatment for each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following procedure in the order described below, unless otherwise stated: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 79 to 82 Heating (1): 50° C., 2 min., Heating (2): 90° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 83 Heating (1): 40° C., 5 minutes, Heating (2): 95° C., 5 minutes, Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 84 The film was heated at 95° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was post-vulcanized at 110° C. for 10 minutes.",
"EXAMPLE 85 Heating (1): 95° C., 3 min., Heating (2): 95° C., 10 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLE 86 Heating (1): 95° C., 3 min., Heating (2): 95° C., 7 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLES 87 to 89 Heating (1): 50° C., 5 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLE 90 The film was heated at 50° C. for 2 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 90° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was dried at 90° C. for 5 minutes, and finally post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLE 91 Heating (1): 38° C., 5 min., Heating (2): 38° C., 5 min., Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLES 92 and 93 Heating (1): 40° C., 5 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLES 94 and 95 Heating (1): 50° C., 2 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLE 96 Heating (1): 50° C., 5 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"TABLE 10 Examples Hydrophilic group blocking agents Concen- Hydrophilic group blocking agents Concen- Test NO.",
"for treating the internal surface tration (%) for treating the external surface tration (%) results 79 Detackifying surfactant: 0.5 Detackifying surfactant: 0.5 o β-naphthalene sulfonate/formalin β-naphthalene sulfonate/formalin condensate condensate Trade name: DEMOL N (Kao Corporation) Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown Properties: Anionic, Light yellow/brown powdery powdery 80 Detackifying crosslinking agent of 0.5 Detackifying crosslinking agent of 0.5 o metallic element: Polyaluminum hydroxide (asAl2O3) metallic element: Polyaluminum hydroxide (asAl2O3) Trade name: Paho#2S (Asada Kagaku Kogyo) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 pH: 3.5 81 Detackifying, hydrogen bond adjustor: 0.3 Detackifying, hydrogen bond adjustor: 0.3 o Polyamide/polyamine epichlorohydrin Polyamide/polyamine epichlorohydrin resin resin Trade name: Sumirez Resin 675 Trade name: Sumirez Resin 675 (Sumitomo Chemical Group) (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent Properties: Cationic, Brown, liquid, Concentration: 25% Viscosity transparent liquid, Concentration: (25° C.): 200 mPa · s, pH (25° C.): 4.1 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 82 Detackifying, reactive sizing agent: 0.5 Detackifying, reactive sizing agent: 0.5 o Alkyl ketene dimer Alkyl ketene dimer Trade name: Sizepine K-910 Trade name: Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, White emulsion, Properties: Anionic, White emulsion, Concentration: 15% Viscosity (25° C.): Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 6 cps, pH (25° C.): 5.5 83 Detackifying crosslinking agent of 0.5 Detackifying waterproofing agent: 0.5 o metallic element: Polyaluminum hydroxide (asAl2O3) Zirconium ammonium carbonate (asAl2O3) Trade name: Paho#2S (Asada Kagaku Kogyo) Trade name: AZ Coat 5800MT (SAN NOPCO) Properties: Cationic, Light yellow, Properties: Anionic, Slightly yellow transparent liquid, Viscosity (30° C.): liquid, Concentration: 20% (as ZrO2) 7 cps Concentration: 10.5% (as Al2O3), Viscosity (25° C.): 10 mPa · s, pH: 3.5 pH (1%): 9.0 84 Detackifying crosslinking agent of 0.5 Detackifying crosslinking agent of 0.5 o metallic element: Polyaluminum hydroxide (asAl2O3) metallic element: Polyaluminum hydroxide (asAl2O3) Trade name: Paho#2S (Asada Kagaku Kogyo) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 pH: 3.5 85 Detackifying, hydrogen bond adjustor: 1.5 Detackifying, hydrogen bond adjustor: 1.5 o Polyamide/polyamine epichlorohydrin Polyamide/polyamine epichlorohydrin resin resin Trade name: Sumirez Resin 6615 Trade name: Sumirez Resin 6615 (Sumitomo Chemical Group) (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent Properties: Cationic, Brown, transparent liquid, Concentration: 15% Viscosity liquid, Concentration: 15% Viscosity (25° C.): 40 mPa · s, pH (25° C.): 4.0 (25° C.): 40 mPa · s, pH (25° C.): 4.0 86 Detackifying, hydrophobicizing, organic 2.0 Detackifying, hydrophobicizing, organic 2.0 o crosslinking agent: Blocked isocyanate crosslinking agent: Blocked isocyanate Trade name: Prominate XC-915 Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 160 cps, pH: 6.0 87 Detackifying waterproofing agent: 0.5 Detackifying waterproofing agent: 0.5 o Branched poryethyleneimine Branched poryethyleneimine Trade name: Epomine P-1000 Trade name: Epomine P-1000 (NIPPON SHOKUBAI) (NIPPON SHOKUBAI) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent, viscous liquid, transparent, viscous liquid, Concentration: 29.9% Viscosity (25° C.): Concentration: 29.9% Viscosity (25° C.): 633 mPa · s, pH (5%): 10.6 633 mPa · s, pH (5%): 10.6 88 Detackifying waterproofing agent: 0.5 Detackifying waterproofing agent: 0.5 o′ Branched polyethyleneimine Branched polyethyleneimine Trade name: Epomine SP-018 Trade name: Epomine SP-018 (NIPPON SHOKUBAI) (NIPPON SHOKUBAI) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent liquid, Concentration: transparent liquid, Concentration: 99.8% 99.8% Viscosity (25° C.): 11000 Viscosity (25° C.): 11000 mPa · s, mPa · s, pH (5%): 11.1 pH (5%): 11.1 89 Detackifying, hydrogen bond adjustor: 0.5 Detackifying, hydrogen bond adjustor 0.5 o Polyamide/polyamine epichlorohydrin Polyamide/polyamine epichlorohydrin resin resin Trade name: Euramine P5600 Trade name: Euramine P5600 (Mitsui Chemicals) (Mitsui Chemicals) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent liquid, Concentration: 31% transparent liquid, Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 pH (25° C.): 4.5 90 Detackifying crosslinking agent of 0.5 Detackifying crosslinking agent of 0.5 o metallic element: Polyaluminum hydroxide (asAl2O3) metallic element: Polyaluminum hydroxide (asAl2O3) Trade name: Paho#2S (Asada Kagaku Kogyo) Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 pH: 3.5 91 Detackifying, reactive sizing agent 1.0 Detackifying, reactive sizing agent 1.0 o′ (waterproofing agent): Alkyl ketene (waterproofing agent): Alkyl ketene dimer dimer Trade name: Sizepine K-287 Trade name: Sizepine K-287 (ARAKAWA CHEMICAL INDUSTRIES) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, White emulsion, Properties: Cationic, White emulsion, Concentration: 20% Viscosity (25° C.): Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 40 cps, pH (20° C.): 3.7 92 Detackifying, hydrogen bond adjustor 0.5 Detackifying, hydrogen bond adjustor 0.5 o Polyamidepolyamine epichlorohydrin Polyamidepolyamine epichlorohydrin resin resin Trade name: Euramine P5600 Trade name: Euramine P5600 (Mitsui Chemicals) (Mitsui Chemicals) Properties: Cationic, Light yellow, Properties: Cationic, Light yellow, transparent liquid, Concentration: 31% transparent liquid, Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 pH (25° C.): 4.5 93 Detackifying waterproofing agent: 0.5 Detackifying water repellent: Wax 0.5 o Branched polyethyleneimine emulsion Trade name: Epomine P-1000 Trade name: Coat Sizer MS-365 (NIPPON SHOKUBAI) (DAIWA CHEMICAL INDUSTRIES) Properties: Cationic, Light yellow, Properties: Cationic, White emulsion, transparent, viscous liquid, Concen- Concentration: 60% pH: 7.0 tration: 29.9% Viscosity (25° C.): 633 mPa · s, pH (5%): 10.6 94 Detackifying waterproofing agent: 0.5 Detackifying waterproofing agent: 0.5 o Ketone resin Ketone resin Trade name: SI-668 Trade name: SI-668 (Nippon PMC corporation) (Nippon PMC corporation) Properties: Nonionic, Slightly white Properties: Nonionic, Slightly white turbid solution, Concentration: 50% turbid solution, Concentration: 50% Viscosity: 40 cps, pH: 7 Viscosity: 40 cps, pH: 7 95 Detackifying, reactive sizing agent 1.0 Detackifying waterproofing agent: 0.5 o (waterproofing agent): Alkyl ketene Polyamine epichlorohydrin resin dimer Trade name: PA-625 Trade name: Sizepine K-287 (Nippon PMC corporation) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Weakly cationic, Light brown, Properties: Cationic, White emulsion, transparent liquid, Concentration: 60% Concentration: 20% Viscosity (25° C.): Viscosity: 250 cps, pH (20° C.): 7.0 40 cps, pH (20° C.): 3.7 96 Detackifying releasing agent: 0.5 Detackifying releasing agent: 0.5 o Polyethyleneimine octadecyl isocyanate Polyethyleneimine octadecyl isocyanate adduct adduct Trade name: RP-10W (NIPPON SHOKUBAI) Trade name: RP-10W (NIPPON SHOKUBAI) Properties: Cationic, Milky white Properties: Cationic, Milky white emulsion, Concentration: 18% Viscosity emulsion, Concentration: 18% (25° C.): 98 mPa · s, pH: 8.0 Viscosity (25° C.): 98 mPa · s, pH: 8.0 EXAMPLES 97 to 99 The pre-vulcanized natural rubber latex was treated with the hydrophilic group sealant for the internal surface, and formed into the film.",
"It was then treated with alumina sol, peroxytitania sol or peroxy titania solution for the tackiness test.",
"The result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 11.The post-treatment for each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated for both surfaces by the following procedure in the order described below, unless otherwise stated: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 97 The film was heated at 50° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was dried at 95° C. for 3 minutes, leached at 85° C. for 1 minute, and finally post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLE 98 Heating (1): 50° C., 5 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 99 The film was heated at 50° C. for 5 minutes, and leached at 85° C. for 5 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was dried at 95° C. for 3 minutes, leached at 85° C. for 1 minute and finally post-vulcanized at 110° C. for 5 minutes.",
"TABLE 11 Examples Hydrophilic group blocking agents Concen- Hydrophilic group blocking agents Concen- Test NO.",
"for treating the internal surface tration (%) for treating the external surface tration (%) results 97 Detackifying crosslinking agent Detackifying crosslinking agent of metallic element: Alumina sol of metallic element: Alumina sol 1.0 Trade name: Alumina sol 200 1.0 o Trade name: Alumina sol 200 (asAl2O3) (Nissan Chemical Industries) (asAl2O3) (Nissan Chemical Industries) Properties: Cationic, Milky white, Properties: Cationic, Milky white, colloidal solution, Concentration: colloidal solution, Concentration: 10.1% 10.1% (as Al2O3) Viscosity (20° C.): (as Al2O3) Viscosity (20° C.): 530 mPa · s, pH (20° C.): 4.8 530 mPa · s, pH (20° C.): 4.8 98 Detackifying crosslinking agent of 2.0 Detackifying crosslinking agent of 1.0 o metallic element: Polyaluminum hydroxide (asAl2O3) metallic element: Peroxy titania sol (asTiO2) Trade name: Paho#2S (Asada Kagaku Kogyo) Trade name: TKS-203 Properties: Cationic, Light yellow, (Tayca Corporation) transparent liquid, Viscosity (30° C.): 7 cps Properties: Cationic, Milky white Concentration: 10.5% (as Al2O3), pH: 3.5 emulsion, Concentration: 20.1% (as TiO2) 99 Detackifying crosslinking agent of 2.0 Detackifying crosslinking agent of 1.4 o metallic element: Polyaluminum hydroxide (asAl2O3) metallic element: Peroxy titania (asTiO2) Trade name: Paho#2S (Asada Kagaku Kogyo) solution Properties: Cationic, Light yellow, Trade name: TKC-301 transparent liquid, Viscosity (30° C.): 7 cps (Tayca Corporation) Concentration: 10.5% (as Al2O3), pH: 3.5 Properties: Cationic, yellow, transparent liquid, Concentration: 1.4% (as TiO2) EXAMPLE 100 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic group sealant, and further treated with the hydrophilic group sealant for both surfaces.",
"It was formed into the film for the tackiness test.",
"The result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 12.The post-treatment for each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was dried at 95° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the hydrophilic group sealant solution for treating the external surface for 5 seconds.",
"Furthermore, it was post-vulcanized at 110° C. for 10 minutes.",
"TABLE 12 Incorporated Examples Hydrophilic group blocking agents quantity Hydrophilic group blocking agents Concen- Test NO.",
"to be incorporated in the latex (parts) for treating the surface tration (%) results 100 Detackifying waterproofing agent: 0.5 Internal surface 0.5 o Zirconium ammonium carbonate (asZrO2) Detackifying crosslinking agent (asAl2O3) Trade name: Baycoat 20 of metallic element: Polyaluminum (Nippon Light Metal) hydroxide Properties: Anionic, Slightly Trade name: Paho#2S yellow liquid, Concentration: 20% (Asada Kagaku Kogyo) (as ZrO2) Viscosity: 8 cps, pH: 9.5 Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps,Concentration: 10.5% (as Al2O3), pH: 3.5 External Surface Detackifying waterproofing agent: 0.44 Zirconium ammonium carbonate (asZrO2) Trade name: AZ Coat 5800MT (SAN NOPCO) Properties: Anionic, Slightly yellow liquid, Concentration: 20% (as ZrO2) Viscosity (25° C.): 10 mPa · s, pH (1%): 9.0 EXAMPLES 101 to 110 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer and hydrophilic group sealant.",
"It was formed into the film, and coating-treated with the detackifying, carboxylated NBR for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 13.The post-treatment for, and carboxylated NBR and carboxyl group sealant used in, each EXAMPLE are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated latex film was 0.1 to 0.13 mm thick, and weighing almost 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 101 to 109 Heating (1): 50° C., 2 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"The film prepared in each of EXAMPLES 101 to 109 was further post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLE 110 Heating (1): 38° C., 5 min., Heating (2): 38° C., 5 min., Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) (Carboxyl Group Sealant) Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) TABLE 13 Hydrophilic polymers to Hydrophilic group blocking agents be incorporated in the latex to be incorporated in the latex Incorporated Incorporated Examples quantity quantity Test NO.",
"(parts) (parts) results 101 Carboxymethyl cellulose 0.2 Detackifying surfactant: 0.4 o Trade name: CMC DAICEL 1330 β-naphthalene sulfonate/formalin (DAICEL CHEMICAL INDUSTRIES) condensate Properties: Anionic, White Trade name: DEMOL N (Kao Corporation) powdery, Viscosity (1%, 25° C.): Properties: Anionic, Light yellow/ 73 cps pH: 6.8, Degree of brown powdery etherification: 1.27 102 Carboxymethyl cellulose 0.2 Detackifying, reactive sizing 1.0 o Trade name: CMC DAICEL 1330 agent: Alkyl ketene dimer (DAICEL CHEMICAL INDUSTRIES) Trade name: Sizepine K-910 Properties: Anionic, White (ARAKAWA CHEMICAL INDUSTRIES) powdery, Viscosity (1%, 25° C.): Properties: Anionic, White 73 cps pH: 6.8, Degree of emulsion, Concentration: 15% etherification: 1.27 Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 103 Carboxymethyl cellulose 0.2 Detackifying waterproofing agent: 1.0 o Trade name: CMC DAICEL 1330 Blocked glyoxal resin (Product of (DAICEL CHEMICAL INDUSTRIES) polyamide polyurea glyoxal reaction) Properties: Anionic, White Trade name: Sumirez Resin 5001 powdery, Viscosity (1%, 25° C.): (Sumitomo Chemical Group) 73 cps pH: 6.8, Degree of Properties: Nonionic, Light- etherification: 1.27 colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 104 Polyethylene oxide 0.2 Detackifying surfactant: 0.4 o Trade name: PEO-8 Polyoxyethylene derivative SUMITOMO SEIKA CHEMICALS) Trade name: Emulgen A-60 Properties: Nonionic, White (Kao Corporation) powdery or granular, pH (0.5%): Properties: Nonionic, Light 7.0 Viscosity (0.5%, 25° C.): yellow liquid pH (5%): 6.5 60 mPa · s 105 Polyethylene oxide 0.2 Detackifying, hydrophobicizing, 0.4 o Trade name: PEO-8 organic crosslinking agent: (SUMITOMO SEIKA CHEMICALS) Blocked isocyanate Properties: Nonionic, White Trade name: Prominate XC-915 powdery or granular, pH (0.5%): (TAKEDA CHEMICAL INDUSTRIES) 7.0 Viscosity (0.5%, 25° C.): Properties: Nonionic, White 60 mPa · s emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 106 Polyethylene oxide 0.2 Detackifying, reactive sizing 1.0 o Trade name: PEO-8 agent: Alkyl ketene dimer (SUMITOMO SEIKA CHEMICALS) Trade name: Sizepine K-910 Properties: Nonionic, White (ARAKAWA CHEMICAL INDUSTRIES) powdery or granular, pH (0.5%): Properties: Anionic, White 7.0 Viscosity (0.5%, 25° C.): emulsion, Concentration: 15% 60 mPa · s Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 107 Polyvinyl alcohol 0.2 Detackifying surfactant: 0.4 o Trade name: DENKA POVAL K-05 Polyoxyethylene derivative (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Emulgen A-60 KAISHA) (Kao Corporation) Properties: Nonionic, White Properties: Nonionic, Light to light yellow powdery, Viscosity: yellow liquid pH (5%): 6.5 6.0 mPa · s pH: 6.0 108 Polyvinyl alcohol 0.2 Detackifying waterproofing agent: 1.0 o Trade name: DENKA POVAL K-05 Blocked glyoxal resin (Product of (DENKI KAGAKU KOGYO KABUSHIKI polyamide polyurea glyoxal reaction) KAISHA) Trade name: Sumirez Resin 5001 Properties: Nonionic, White (Sumitomo Chemical Group) to light yellow powdery, Viscosity: Properties: Nonionic, Light- 6.0 mPa · s pH: 6.0 colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 109 Polyvinyl alcohol 0.2 Detackifying sizing agent: 1.0 o Trade name: DENKA POVAL K-05 Alkenyl succinate (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Coloparl SS-40 KAISHA) (Seiko Chemical Industries) Properties: Nonionic, White Properties: Anionic, Brown to light yellow powdery, Viscosity: liquid, Concentration: 40.4% 6.0 mPa · s pH: 6.0 Viscosity: 80 cps, pH: 10.4 110 Urea phosphorylated starch 0.25 Detackifying waterproofing agent: 0.25 o Trade name: MS#4600 Zirconium ammonium carbonate (asZrO2) (Nihon Shokuhin Kako) Trade name: Baycoat 20 Properties: Anionic, Slightly (Nippon Light Metal) yellow Powdery Viscosity (20%, Properties: Anionic, Slightly 50° C.): 74 mPa · s, pH: 5.5 yellow liquid, Concentration: 20% (as ZrO2) Viscosity: 8 cps, pH: 9.5 EXAMPLES 111 to 122 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic group sealant.",
"It was formed into the film, and coating-treated with the detackifying, carboxylated NBR for the external surface.",
"The tackiness test result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 14.The post-treatment for, and carboxylated NBR and carboxyl group sealant used in, each EXAMPLE are described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated latex film was 0.1 to 0.13 mm thick, and weighing almost 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 111 to 115 Heating (1): 50° C., 2 min., Heating (2): 90° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 116 to 120 Heating (1): 50° C., 2 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"The film prepared in each of EXAMPLES 116 to 120 was further post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLES 121 and 122 Heating (1): 38° C., 5 min., Heating (2): 38° C., 5 min., Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) (Carboxyl Group Sealant) Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) TABLE 14 Incorporated Examples quantity Test NO.",
"Hydrophilic group blocking agents to be incorporated in the latex (parts) results 111 Detackifying surfactant: β-naphthalene sulfonate/formalin condensate 0.4 o Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown powdery 112 Detackifying surfactant: Polyoxyethylene derivative 0.4 o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 113 Detackifying sizing agent: Strengthened rosin sizing agent 1.0 o Trade name: Sizepine E-50 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, Brown, transparent liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 114 Detackifying waterproofing agent: Blocked glyoxal resin 1.0 o (Product of polyamide polyurea glyoxal reaction) Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 115 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 0.4 o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 116 Detackifying surfactant: β-naphthalene sulfonate/formalin condensate 0.4 o Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown powdery 117 Detackifying surfactant: Polyoxyethylene derivative 0.4 o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 118 Detackifying, reactive sizing agent: Alkyl ketene dimer 1.0 o Trade name: Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, White emulsion, Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 119 Detackifying waterproofing agent: Blocked glyoxal resin 1.0 o (Product of polyamide polyurea glyoxal reaction) Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 120 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 0.4 o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 121 Detackifying waterproofing agent: Blocked glyoxal 2.0 o (Cyclic amide aldehyde condensate) Trade name: Sumirez 700 M (OMNOVA Solutions) Properties: Cationic, Brown liquid, Concentration: 45% Viscosity: 25 cps, pH: 6.0 122 Detackifying, hydrogen bond adjustor: Polyamide polyurea-based resin 1.0 o Trade name: Sumirez Resin 302 (Sumitomo Chemical Group) Properties: Nonionic, Light yellow, transparent liquid, Concentration: 60% Viscosity (25° C.): 320 mPa · s, pH (25° C.): 6.8 EXAMPLES 123 and 124 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer and/or hydrophilic group sealant.",
"It was formed into the film, and coating-treated with the carboxylated NBR free of the hydrophilic group sealant for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 15.The post-treatment step in each EXAMPLE is described below.",
"The carboxylated NBR coating solution was a mixture of carboxylated NBR latex and Nipol LX-551 (Zeon Corporation) incorporated with 1.5 parts of zinc white and diluted with water to have the solid content of 5%.",
"(Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated latex film was 0.1 to 0.13 mm thick, and weighing almost 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 123 and 124 Heating (1): 50° C., 2 min., Heating (2): 95° C., 5 min., Leaching: 70° C., 3 min., Post-vulcanization: 95° C., 5 min.",
"The film prepared in each of EXAMPLES 123 and 124 was further post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"TABLE 15 Hydrophilic polymers to Hydrophilic group blocking agents be incorporated in the latex to be incorporated in the latex Incorporated Incorporated Examples quantity quantity Test NO.",
"(parts) (parts) results 123 Carboxymethyl cellulose 0.2 Detackifying surfactant: 0.4 o Trade name: CMC DAICEL β-naphthalene sulfonate/ 1330 (DAICEL CHEMICAL INDUSTRIES) formalin condensate Properties: Anionic, White Trade name: DEMOL N powdery, Viscosity (1%, 25° C.): (Kao Corporation) 73 cps pH: 6.8, Degree of Properties: Anionic, Light etherification: 1.27 yellow/brown powdery 124 Not used Detackifying waterproofing agent: 0.5 o Polyamine epichlorohydrin resin Trade name: PA-625 (Nippon PMC corporation) Properties: Weakly cationic, Light brown, transparent liquid, Concentration: 60% Viscosity: 250 cps, pH (20° C.): 7.0 EXAMPLES 125 to 127 The pre-vulcanized natural rubber latex was formed into the film, and coating-treated with the detackifying carboxylated NBR for the external surface.",
"The tackiness test result, and the carboxyl group sealant used in each EXAMPLE are given in Table 16.The post-treatment step and carboxylated NBR used in each EXAMPLE are described below.",
"(Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated latex film was 0.1 to 0.13 mm thick, and weighing almost 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 125 to 127 Heating (1): 50° C., 2 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) TABLE 16 Incorporated Examples Carboxyl group blocking agents to be incorporated in the coating quantity Test NO.",
"solution of diene-based carboxylated synthetic rubber latex (parts) results 125 Detackifying surfactant: Coconut oil fatty acid sodium sarcosine 2.5 o Trade name: Neoscope SCN-35 (Toho Chemical Industry) Properties: Anionic, Light yellow, transparent liquid, Concentration: 35% pH (1%): 8.2 126 Detackifying sizing agent: Strengthened rosin sizing agent 2.5 o Trade name: Sizepine E-50 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, Brown, transparent liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 127 Detackifying waterproofing agent: Zirconium ammonium carbonate 2.5 o Trade name: Baycoat 20 (Nippon Light Metal) (asZrO2) Properties: Anionic, Slightly yellow liquid, Concentration: 20% (as ZrO2) Viscosity: 8 cps, pH: 9.5 EXAMPLES 128 to 140 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer or hydrophilic group sealant, and treated with the hydrophilic group sealant for the internal surface.",
"It was formed into the film, and coating-treated with the detackifying carboxylated NBR for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 17.The post-treatment step, and carboxylated NBR, carboxyl group sealant, hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are described below.",
"(Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 128 to 136 Heating (1): 50° C., 2 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"The film prepared in each of EXAMPLES 128 to 136 was further post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLES 137 and 138 Heating (1): 40° C., 3 min., Heating (2): 40° C., 3 min., Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLE 139 Heating (1): 38° C., 3 min., Heating (2): 38° C., 5 min., Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLE 140 Heating (1): 50° C., 5 min., Heating (2): 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) (Carboxyl Group Sealant) Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) TABLE 17 Hydrophilic polymers or hydrophilic group blocking agents to be incorporated in the latex Hydrophilic group blocking agents Incorporated for treating the internal surface Examples quantity Concen- Test NO.",
"(parts) tration (%) results 128 Carboxymethyl cellulose 0.2 Detackifying surfactant: 0.5 o Trade name: CMC DAICEL 1330 β-naphthalene sulfonate/ (DAICEL CHEMICAL INDUSTRIES) formalin condensate Properties: Anionic, White Trade name: DEMOL N powdery, Viscosity (1%, 25° C.): (Kao Corporation) 73 cps pH: 6.8, Degree of Properties: Anionic, Light etherification: 1.27 yellow/brown powdery 129 Carboxymethyl cellulose 0.2 Detackifying crosslinking agent 0.5 o Trade name: CMC DAICEL 1330 of metallic element: Polyaluminum (asAl2O3) (DAICEL CHEMICAL INDUSTRIES) hydroxide Properties: Anionic, White Trade name: Paho#2S powdery, Viscosity (1%, 25° C.): (Asada Kagaku Kogyo) 73 cps pH: 6.8, Degree of Properties: Cationic, Light etherification: 1.27 yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 130 Carboxymethyl cellulose 0.2 Detackifying, reactive sizing 0.5 o Trade name: CMC DAICEL 1330 agent (waterproofing agent): (DAICEL CHEMICAL INDUSTRIES) Alkyl ketene dimer Properties: Anionic, White Trade name: Sizepine K-287 powdery, Viscosity (1%, 25° C.): (ARAKAWA CHEMICAL INDUSTRIES) 73 cps pH: 6.8, Degree of Properties: Cationic, White etherification: 1.27 emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 131 Polyethylene oxide 0.2 Detackifying surfactant: 0.4 o Trade name: PEO-8 Polyoxyethylene derivative (SUMITOMO SEIKA CHEMICALS) Trade name: Emulgen A-60 Properties: Nonionic, White (Kao Corporation) powdery or granular, pH (0.5%): Properties: Nonionic, Light 7.0 Viscosity (0.5%, 25° C.): yellow liquid pH (5%): 6.5 60 mPa · s 132 Polyethylene oxide 0.2 Detackifying crosslinking agent 0.5 o Trade name: PEO-8 of metallic element: Aluminum (asAl2O3) (SUMITOMO SEIKA CHEMICALS) nitrate Properties: Nonionic, White powdery or granular, pH (0.5%): 7.0 Viscosity (0.5%, 25° C.): 60 mPa · s 133 Polyethylene oxide 0.2 Detackifying, hydrogen bond 0.3 o Trade name: PEO-8 adjustor: Polyamide/polyamine (SUMITOMO SEIKA CHEMICALS) epichlorohydrin resin Properties: Nonionic, White Trade name: Sumirez Resin 675 powdery or granular, pH (0.5%): (Sumitomo Chemical Group) 7.0 Viscosity (0.5%, 25° C.): Properties: Cationic, Brown, 60 mPa · s transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 134 Polyvinyl alcohol 0.2 Detackifying, reactive sizing 0.5 o Trade name: DENKA POVAL K-05 agent: Alkyl ketene dimer (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Sizepine K-910 KAISHA) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Nonionic, White Properties: Anionic, White to light yellow powdery, emulsion, Concentration: 15% Viscosity: 6.0 mPa · s pH: 6.0 Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 135 Polyvinyl alcohol 0.2 Detackifying waterproofing agent: 1.0 o Trade name: DENKA POVAL K-05 Blocked glyoxal resin (Product of (DENKI KAGAKU KOGYO KABUSHIKI polyamide polyurea glyoxal reaction) KAISHA) Trade name: Sumirez Resin 5001 Properties: Nonionic, White (Sumitomo Chemical Group) to light yellow powdery, Properties: Nonionic, Light- Viscosity: 6.0 mPa · s pH: 6.0 colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 136 Polyvinyl alcohol 0.2 Detackifying sizing agent: 0.5 o Trade name: DENKA POVAL K-05 Alkenyl succinate (DENKI KAGAKU KOGYO KABUSHIKI Trade name: Coloparl SS-40 KAISHA) (Seiko Chemical Industries) Properties: Nonionic, White Properties: Anionic, Brown to light yellow powdery, liquid, Concentration: 40.4% Viscosity: 6.0 mPa · s pH: 6.0 Viscosity: 80 cps, pH: 10.4 137 Ampholytic polyacryloamide 0.3 Detackifying waterproofing agent: 0.5 o Trade name: Polymerjet 902 Ketone resin (ARAKAWA CHEMICAL INDUSTRIES) Trade name: SI-668 (Nippon Properties: Ampholytic, Light PMC corporation) yellow, slightly turbid liquid, Properties: Nonionic, Slightly Concentration: 15.4%, Viscosity white turbid solution, Concentration: (25° C.): 1800 mPa · s, 50% Viscosity: 40 cps, pH: 7 pH (20° C.): 3.1 138 Cationic polyamide resin 0.25 Detackifying waterproofing agent: 0.5 o Trade name: Arafix 255 Ketone resin (ARAKAWA CHEMICAL INDUSTRIES) Trade name: SI-668 (Nippon PMC Properties: Cationic, Brown, corporation) transparent liquid, Concentration: Properties: Nonionic, Slightly 26% Viscosity (25° C.): 183 white turbid solution, Concentration: mPa · s, pH (20° C.): 3.05 50% Viscosity: 40 cps, pH: 7 139 Detackifying waterproofing agent: 0.5 Detackifying, reactive sizing 1.0 o Zirconium ammonium carbonate (asZrO2) agent (waterproofing agent): Alkyl Trade name: AZ Coat 5800MT ketene dimer (SAN NOPCO) Trade name: Sizepine K-287 Properties: Anionic, Slightly (ARAKAWA CHEMICAL INDUSTRIES) yellow liquid, Concentration: Properties: Cationic, White 20% (as ZrO2) Viscosity (25° C.): emulsion, Concentration: 20% 10 mPa · s, pH (1%): 9.0 Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 140 Detackifying, hydrogen bond 0.25 Detackifying waterproofing agent: 1.0 o adjustor: Polyamide/polyamine Polyamine epichlorohydrin resin epichlorohydrin resin Trade name: WS-564 (Nippon Trade name: Sumirez Resin 6625 PMC corporation) (Sumitomo Chemical Group) Properties: Cationic, Light amber Properties: Cationic, Brown, liquid, Concentration: 20% transparent liquid, Concentration: Viscosity (25° C.): 50 cps, 25% Viscosity (25° C.): 200 pH (20° C.): 3.7 mPa · s, pH (25° C.): 4.0 EXAMPLES 141 to 152 The pre-vulcanized natural rubber latex was treated with the hydrophilic group sealant for the internal surface.",
"It was formed into the film, and coating-treated with the detackified carboxylated NBR for the external surface for the tackiness test.",
"The test result and the hydrophilic group sealant used in each EXAMPLE are given in Table 18.The post-treatment step, and carboxylated NBR and carboxyl group sealant used in each EXAMPLE are described below.",
"(Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below, unless otherwise stated: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated film was 0.1 to 0.13 mm thick and weighing around 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 141 to 145 Heating (1): 50° C., 2 min., Heating (2): 90° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 146 to 150 The natural rubber latex film formed on the mold was dried at 50° C. for 2 minutes, and leached at 85° C. for 5 minutes.",
"Then, it was heated at 90° C. for 1 minute, and immersed in the coating solution of the detackifying, carboxylated synthetic latex for 5 seconds.",
"Approximately 0.05 g of the solution was held by the film.",
"Furthermore, it was dried at 90° C. for 5 minutes and post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLES 151 and 152 Heating (1): 38° C., 3 min., Heating (2): 38° C., 5 min., Leaching: 70° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) (Carboxyl Group Sealants) (1) Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) The latex was treated with 2.5 parts of the above carboxyl group sealant.",
"The tackiness test results are given in Table 18 as the test results (1).",
"(2) Detackifying waterproofing agent: Polyamidepolyamine epichlorohydrin resin Sumirez Resin 6625 (Sumitomo Chemical Group) The latex was treated with 0.25 parts of the above carboxyl group sealant.",
"The tackiness test results are given in Table 18 as the test results (2).",
"TABLE 18 Test Test Examples Concen- results results NO.",
"Hydrophilic group blocking agents for treating the internal surface tration (%) (1) (2) 141 Detackifying surfactant: β-naphthalene sulfonate/formalin condensate 0.4 o o Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown powdery 142 Detackifying crosslinking agent of metallic element: Polyaluminum hydroxide 0.5 o o Trade name: Paho#2S (Asada Kagaku Kogyo) (asAl2O3) Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 143 Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer 0.5 o o Trade name: Sizepine K-287 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, White emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 144 Detackifying surfactant: Polyoxyethylene derivative 0.4 o o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 145 Detackifying, hydrogen bond adjustor: Polyamide/polyamine epichlorohydrin resin 0.3 o o Trade name: Sumirez Resin 675 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 146 Detackifying surfactant: Polyoxyethylene derivative 0.4 o o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 147 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 1.0 o o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 148 Detackifying, hydrogen bond adjustor: Polyamide/polyamine epichlorohydrin resin 0.3 o o Trade name: Sumirez Resin 675 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 149 Detackifying, monofunctional epoxy compound 1.0 o o Trade name: Denacast EM-103 (Nagase ChemteX Corporation) Properties: Milky white emulsion, Concentration: 40%, Epoxy equivalents: 1463WPE Viscosity (20° C.): 3100 mPa · s, pH: 5.7 150 Detackifying, reactive sizing agent (waterproofing agent): Alkyl ketene dimer 0.5 o o Trade name: Sizepine K-287 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, White emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 151 Detackifying waterproofing agent: Branched polyethyleneimine 0.5 o o Trade name: Epomine P-1000 (NIPPON SHOKUBAI) Properties: Cationic, Light yellow, transparent, viscous liquid, Concentration: 29.9% Viscosity (25° C.): 633 mPa · s, pH (5%): 10.6 152 Detackifying waterproofing agent: Blocked glyoxal resin 2.5 o′ o (Polyhydric alcohol/carbonyl adduct) Trade name: SEQUARES 755 (OMNOVA Solutions) Properties: Cationic, Light yellow, transparent liquid, Concentration: 55% Viscosity (25° C.): 200 cps, pH: 4.5 EXAMPLES 153 and 154 The pre-vulcanized natural rubber latex was treated with the hydrophilic group sealant for the internal surface.",
"It was formed into the film, and external surface was coating-treated with the detackified polymer for the tackiness test.",
"The test result, and the detackified polymer coating agent and hydrophilic group sealant used in each EXAMPLE are given in Table 19.The post-treatment step in each EXAMPLE is described below.",
"(Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in the solution for treating the external surface→Heating (2)→Leaching→Post-vulcanization The treated film was 0.1 to 0.13 mm thick and weighing around 0.3 g. Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 153 and 154 Heating (1): 50° C., 2 min., Heating (2): 90° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"TABLE 19 Hydrophilic group blocking agents Polymer coating agents for for treating the internal surface treating the external surface Examples Concen- Concen- Test NO.",
"tration (%) tration (%) results 153 Detackifying, reactive sizing 1.0 Detackifying waterproofing agent: 5.0 o agent (waterproofing agent): Self-crosslinking type acrylic/ Alkyl ketene dimer styrene copolymer Trade name: Sizspine K-287 Trade name: Sibinol EK-20 (ARAKAWA CHEMICAL INDUSTRIES) (Saiden Chemical Industry) Properties: Cationic, White Properties: Anionic, Milky white emulsion, Concentration: 20% emulsion, Concentration: 39.5% Viscosity (25° C.): 40 cps, Viscosity (30° C.): 2000 pH (20° C.): 3.7 mPa · s, pH: 9.5 154 Detackifying, reactive sizing 1.0 Detackifying releasing agent: 5.0 o agent (waterproofing agent): Long-chain alkyl pendant polymer Alkyl ketene dimer Trade name: peeloil 406 Trade name: Sizepine K-287 (Ipposha Oil Industries) (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, Milky white Properties: Cationic, White emulsion, Concentration: 15% emulsion, Concentration: 20% Viscosity: 500 cps Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 EXAMPLES 155 to 164 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer and hydrophilic group sealant.",
"It was formed into the film, and halogenation-treated for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 20.The post-treatment step in each EXAMPLE is described below.",
"(Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in chlorine water→Heating(2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 155 to 164 Heating (1): 50° C., 2 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"Furthermore, each film was post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"TABLE 20 Hydrophilic polymers to Hydrophilic group blocking agents be incorporated in the latex to be incorporated in the latex Incorporated Incorporated Examples quantity quantity Test NO.",
"(parts) (parts) results 155 Carboxymethyl cellulose 0.2 Detackifying, hydrophobicizing, 0.4 o Trade name: CMC DAICEL 1330 organic crosslinking agent: (DAICEL CHEMICAL INDUSTRIES) Blocked isocyanate Properties: Anionic, White Trade name: Prominate XC-915 powdery, Viscosity (1%, 25° C.): (TAKEDA CHEMICAL INDUSTRIES) 73 cps pH: 6.8, Degree of Properties: Nonionic, White etherification: 1.27 emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 156 Carboxymethyl cellulose 0.2 Detackifying waterproofing agent: 1.0 o Trade name: CMC DAICEL 1330 Blocked glyoxal resin (DAICEL CHEMICAL INDUSTRIES) (Product of polyamide polyurea Properties: Anionic, White glyoxal reaction) powdery, Viscosity (1%, 25° C.): Trade name: Sumirez Resin 5001 73 cps pH: 6.8, Degree of (Sumitomo Chemical Group) etherification: 1.27 Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 157 Carrageenan 0.2 Detackifying surfactant: 0.4 o Trade name: Soarace WX165 Polyoxyethylene derivative (MRC Polysaccharide) Trade name: Emulgen A-60 Properties: Anionic, White (Kao Corporation) powdery pH (1.5%): 8.2 Properties: Nonionic, Light yellow liquid pH (5%): 6.5 158 Carrageenan 0.2 Detackifying sizing agent: 1.0 o Trade name: Soarace WX165 Strengthened rosin sizing agent (MRC Polysaccharide) Trade name: Sizepine E-50 Properties: Anionic, White (ARAKAWA CHEMICAL INDUSTRIES) powdery pH (1.5%): 8.2 Properties: Anionic, Brown, transparent liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 159 Guar gum 0.2 Detackifying, hydrophobicizing, 0.4 o Trade name: Soar Guar RG100 organic crosslinking agent: (MRC Polysaccharide) Blocked isocyanate Properties: Nonionic, White Trade name: Prominate XC-915 powdery Viscosity (1%, 25° C.): (TAKEDA CHEMICAL INDUSTRIES) 1250 cps Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 160 Guar gum 0.2 Detackifying waterproofing agent: 1.0 o Trade name: Soar Guar RG100 Blocked glyoxal resin (MRC Polysaccharide) (Product of polyamide polyurea Properties: Nonionic, White glyoxal reaction) powdery Viscosity (1%, 25° C.): Trade name: Sumirez Resin 5001 1250 cps (Sumitomo Chemical Group) Properties: Nonionic, Light- colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 161 Locust bean gum 0.2 Detackifying surfactant: 0.4 o Trade name: Soar Locust A120F Polyoxyethylene derivative (MRC Polysaccharides) Trade name: Emulgen A-60 Properties: Nonionic, White (Kao Corporation) powdery Viscosity (1%, 25° C.): Properties: Nonionic, Light 1200 cps yellow liquid pH (5%): 6.5 162 Locust bean gum 0.2 Detackifying sizing agent: 1.0 o Trade name: Soar Locust A120F Strengthened rosin sizing agent (MRC Polysaccharides) Trade name: Sizepine E-50 Properties: Nonionic, White (ARAKAWA CHEMICAL INDUSTRIES) powdery Viscosity (1%, 25° C.): Properties: Anionic, Brown, 1200 cps transparent liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 163 Xanthan gum 0.2 Detackifying, monofunctional 1.0 o Trade name: Soar Xan XG550 epoxy compound (MRC Polysaccharides) Trade name: Denacast EM-103 Properties: Anionic, White (Nagase ChemteX Corporation) powdery Viscosity (1%, 25° C.): Properties: Milky white emulsion, 1500 cps, pH (1%): 7.5 Concentration: 40%, Epoxy equivalents: 1463WPE Viscosity (20° C.): 3100 mPa · s, pH: 5.7 164 Xanthan gum 0.2 Detackifying surfactant: 0.4 o Trade name: Soar Xan XG550 β-naphthalene sulfonate/formalin (MRC Polysaccharides) condensate Properties: Anionic, White Trade name: DEMOL N powdery Viscosity (1%, 25° C.): (Kao Corporation) 1500 cps, pH (1%): 7.5 Properties: Anionic, Light yellow/brown powdery EXAMPLES 165 to 174 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic group sealant.",
"It was formed into the film, and halogenation-treated for the external surface.",
"The tackiness test result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 21.The post-treatment step in each EXAMPLE is described below.",
"(Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below, unless otherwise stated: Heating (1)→Immersion in chlorine water→Heating(2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 165 to 168 Heating (1): 50° C., 2 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"Furthermore, each was post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLES 169 to 174 Heating (1): 50° C., 5 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"Furthermore, each was post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"TABLE 21 Incorporated Examples quantity Test NO.",
"Hydrophilic group blocking agents to be incorporated in the latex (parts) results 165 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 0.4 o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 166 Detackifying waterproofing agent: Blocked glyoxal resin 1.0 o (Product of polyamide polyurea glyoxal reaction) Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 167 Detackifying surfactant: Polyoxyethylene derivative 0.4 o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 168 Detackifying sizing agent: Strengthened rosin sizing agent 1.0 o Trade name: Sizepine E-50 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, Brown, transparent liquid, Concentration: 50.4% Viscosity (25° C.): 200 cps, pH (5%, 20° C.): 11.0 169 Detackifying surfactant: β-naphthalene sulfonate/formalin condensate 0.4 o Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown powdery 170 Detackifying, reactive sizing agent: Alkyl ketene dimer 1.0 o Trade name: Sizepine K-910 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Anionic, White emulsion, Concentration: 15% Viscosity (25° C.): 6 cps, pH (25° C.): 5.5 171 Detackifying waterproofing agent: Blocked glyoxal resin 1.0 o (Product of polyamide polyurea glyoxal reaction) Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 172 Detackifying surfactant: Polyoxyethylene derivative 0.4 o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 173 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 0.4 o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 174 Crosslinking agent of detackifying, aqueous resin: Carbodiimide 0.5 o Trade name: CARBODILITE V-02 (Nisshinbo Industries) Properties: Yellow, transparent liquid, Carbodiimide equivalents: 597 Concentration: 40%, pH: 10.1 EXAMPLES 175 to 185 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer, and treated with the hydrophilic group sealant for the internal surface.",
"It was formed into the film, and halogenation-treated for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 22.The post-treatment step in each EXAMPLE is described below.",
"(Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating (1)→Immersion in chlorine water→Heating(2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 175 to 185 Heating (1): 50° C., 2 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"Furthermore, each was post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"TABLE 22 Hydrophilic polymers to be incorporated in the latex Hydrophilic group blocking agents Incorporated for treating the internal surface Examples quantity Concen- Test NO.",
"(parts) tration (%) results 175 Carboxymethyl cellulose 0.2 Detackifying crosslinking agent 0.5 o Trade name: CMC DAICEL 1330 of metallic element: Polyaluminum (asAl2O3) (DAICEL CHEMICAL INDUSTRIES) hydroxide Properties: Anionic, White Trade name: Paho#2S powdery, Viscosity (1%, 25° C.): (Asada Kagaku Kogyo) 73 cps pH: 6.8, Degree of Properties: Cationic, Light yellow, etherification: 1.27 transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 176 Carboxymethyl cellulose 0.2 Detackifying, hydrogen bond adjustor: 0.3 o Trade name: CMC DAICEL 1330 Polyamide/polyamine epichlorohydrin (DAICEL CHEMICAL INDUSTRIES) resin Properties: Anionic, White Trade name: Sumirez Resin 675 powdery, Viscosity (1%, 25° C.): (Sumitomo Chemical Group) 73 cps pH: 6.8, Degree of Properties: Cationic, Brown, etherification: 1.27 transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 177 Carrageenan 0.2 Detackifying surfactant: 0.4 o Trade name: Soarace WX165 Polyoxyethylene derivative (MRC Polysaccharide) Trade name: Emulgen A-60 Properties: Anionic, White (Kao Corporation) powdery pH (1.5%): 8.2 Properties: Nonionic, Light yellow liquid pH (5%): 6.5 178 Carrageenan 0.2 Detackifying, reactive sizing agent 0.5 o Trade name: Soarace WX165 (waterproofing agent) Alkyl ketene (MRC Polysaccharide) dimer Properties: Anionic, White Trade name: Sizepine K-287 powdery pH (1.5%): 8.2 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, White emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 179 Methyl cellulose 0.25 Detackifying crosslinking agent 0.5 o Trade name: Metolose SM-400 of metallic element: Polyaluminum (asAl2O3) (Shin-Etsu Chemical) hydroxide Properties: Nonionic, White Trade name: Paho#2S powdery Viscosity (2%, 20° C.): (Asada Kagaku Kogyo) 436 mPa · s Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 180 Methyl cellulose 0.25 Detackifying, hydrogen bond adjustor: 0.3 o Trade name: Metolose SM-400 Polyamide/polyamine epichlorohydrin (Shin-Etsu Chemical) resin Properties: Nonionic, White Trade name: Sumirez Resin 675 powdery Viscosity (2%, 20° C.): (Sumitomo Chemical Group) 436 mPa · s Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 181 Polyethylene oxide 0.2 Detackifying surfactant: 0.4 o Trade name: PEO-8 Polyoxyethylene derivative (SUMITOMO SEIKA CHEMICALS) Trade name: Emulgen A-60 Properties: Nonionic, White (Kao Corporation) powdery or granular, pH (0.5%): Properties: Nonionic, Light yellow 7.0 Viscosity (0.5%, 25° C.): liquid pH (5%): 6.5 60 mPa · s 182 Polyethylene oxide 0.2 Detackifying, reactive sizing agent 0.5 o Trade name: PEO-8 (waterproofing agent) Alkyl ketene (SUMITOMO SEIKA CHEMICALS) dimer Properties: Nonionic, White Trade name: Sizepine K-287 powdery or granular, pH (0.5%): (ARAKAWA CHEMICAL INDUSTRIES) 7.0 Viscosity (0.5%, 25° C.): Properties: Cationic, White 60 mPa · s emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 183 Xanthan gum 0.2 Detackifying, hydrophobicizing, 1.0 o Trade name: Soar Xan XG550 organic crosslinking agent: Blocked (MRC Polysaccharides) isocyanate Properties: Anionic, White Trade name: Prominate XC-915 powdery Viscosity (1%, 25° C.): (TAKEDA CHEMICAL INDUSTRIES) 1500 cps, pH (1%): 7.5 Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 184 Xanthan gum 0.2 Detackifying sizing agent: Alkenyl 0.5 o Trade name: Soar Xan XG550 succinate (MRC Polysaccharides) Trade name: Coloparl SS-40 Properties: Anionic, White (Seiko Chemical Industries) powdery Viscosity (1%, 25° C.): Properties: Anionic, Brown liquid, 1500 cps, pH (1%): 7.5 Concentration: 40.4% Viscosity: 80 cps, pH: 10.4 185 Ampholytic polyacryloamide 0.3 Detackifying waterproofing agent: 0.5 o Trade name: Polymerjet 902 Branched polyethyleneimine (ARAKAWA CHEMICAL INDUSTRIES) Trade name: Epomine P-1000 Properties: Ampholytic, Light (NIPPON SHOKUBAI) yellow, slightly turbid liquid, Properties: Cationic, Light yellow, Concentration: 15.4%, Viscosity transparent, viscous liquid, (25° C.): 1800 mPa · s, pH Concentration: 29.9% Viscosity (20° C.): 3.1 (25° C.): 633 mPa · s, pH (5%): 10.6 EXAMPLES 186 to 195 The pre-vulcanized natural rubber latex was treated with the hydrophilic group sealant for the internal surface.",
"It was formed into the film, and halogenation-treated for the external surface.",
"The tackiness test result, and the hydrophilic group sealant used in each EXAMPLE are given in Table 23.The post-treatment step in each EXAMPLE is described below.",
"(Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below, unless otherwise stated: Heating (1)→Immersion in chlorine water→Heating(2)→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 186 to 190 Heating (1): 50° C., 5 min., Heating (2): 50° C., 2 min., Leaching: 70° C., 3 min., Post-vulcanization: 90° C., 5 min.",
"Furthermore, each was post-vulcanized at 110° C. for 5 minutes for the tackiness test.",
"EXAMPLES 191 to 195 The natural rubber latex film formed on the mold was dried at 50° C. for 2 minutes, and leached at 85° C. for 5 minutes.",
"Then, it was heated at 90° C. for 1 minute, and immersed in chlorine water (chlorine concentration: 0.4%) for 5 seconds, to halogenate the external surface of the natural rubber latex film.",
"Furthermore, each was dried at 90° C. for 5 minutes and post-vulcanized at 110° C. for 5 minutes.",
"TABLE 23 Concen- Examples tration Test NO.",
"Hydrophilic group blocking agents for treating the internal surface (%) results 186 Detackifying crosslinking agent of metallic element: Polyaluminum hydroxide 0.5 o Trade name: Paho#2S (Asada Kagaku Kogyo) (asAl2O3) Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 187 Detackifying, hydrogen bond adjustor: Polyamide/polyamine epichlorohydrin resin 0.3 o Trade name: Sumirez Resin 675 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 188 Detackifying surfactant: Polyoxyethylene derivative 0.4 o Trade name: Emulgen A-60 (Kao Corporation) Properties: Nonionic, Light yellow liquid pH (5%): 6.5 189 Detackifying, reactive sizing agent (waterproofing agent) Alkyl ketene dimer 0.5 o Trade name: Sizepine K-287 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, White emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 190 Detackifying sizing agent: Alkenyl succinate 0.5 o Trade name: Coloparl SS-40 (Seiko Chemical Industries) Properties: Anionic, Brown liquid, Concentration: 40.4% Viscosity: 80 cps, pH: 10.4 191 Detackifying surfactant: β -naphthalene sulfonate/formalin condensate 0.5 o Trade name: DEMOL N (Kao Corporation) Properties: Anionic, Light yellow/brown powdery 192 Detackifying, hydrophobicizing, organic crosslinking agent: Blocked isocyanate 1.0 o Trade name: Prominate XC-915 (TAKEDA CHEMICAL INDUSTRIES) Properties: Nonionic, White emulsion, Trifunctional group, Molecular weight: 1000 Concentration: 43.3%, Viscosity: 160 cps, pH: 6.0 193 Detackifying waterproofing agent: Blocked glyoxal resin 1.0 o (Product of polyamide polyurea glyoxal reaction) Trade name: Sumirez Resin 5001 (Sumitomo Chemical Group) Properties: Nonionic, Light-colored, transparent liquid, Concentration: 30% Viscosity (25° C.): 32 mPa · s, pH (25° C.): 7.7 194 Detackifying, monofunctional epoxy compound 1.0 o Trade name: Denacast EM-103 (Nagase ChemteX Corporation) Properties: Milky white emulsion, Concentration: 40%, Epoxy equivalents: 1463WPE Viscosity (20° C.): 3100 mPa · s, pH: 5.7 195 Detackifying, reactive sizing agent (waterproofing agent) Alkyl ketene dimer 0.5 o Trade name: Sizepine K-287 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, White emulsion, Concentration: 20% Viscosity (25° C.): 40 cps, pH (20° C.): 3.7 EXAMPLES 196 to 199 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic group sealant and/or hydrophilic polymer.",
"It was formed into the film, and treated with the hydrophilic group sealant reactive at low temperature for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 24.The post-treatment step and hydrophilic group sealant reactive at low temperature for treating the external surface in each EXAMPLE are described below.",
"(Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 196 and 198 Heating: 50° C., 2 min., Drying: 90° C., 7 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLES 197 and 199 Heating: 38° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"(Hydrophilic group sealant reactive at low temperature for the external surface) Detackifying crosslinking agent of metallic element: Zirconyl acetate Zircosol ZA (DAIICHIKIGENSO KAGAKUKOGYO) Properties: Cationic, Slightly brown aqueous solution Concentration: 15.18% (as ZrO2), pH: 3.4 The above-described sealant was diluted with water to have a ZrO2 concentration of 1% for use in each EXAMPLE.",
"TABLE 24 Hydrophilic polymers to Hydrophilic group blocking agents be incorporated in the latex to be incorporated in the latex Incorporated Incorporated Examples quantity quantity Test NO.",
"(parts) (parts) results 196 Not used Detackifying, hydrogen bond adjustor: 1.5 o Polyamide polyurea-based resin Trade name: Sumirez Resin 703 (Sumitomo Chemical Group) Properties: Weakly cationic, Brown, transparent liquid, Concentration: 50% Viscosity (25° C.): 65 mPa · s, pH (25° C.): 7.0 197 Urea phosphorylated starch 0.25 Detackifying waterproofing agent: 0.25 o Trade name: MS#4600 Zirconium ammonium carbonate (asZrO2) (Nihon Shokuhin Kako) Trade name: Baycoat 20 Properties: Anionic, (Nippon Light Metal) Slightly yellow powdery Properties: Anionic, Slightly Viscosity (20%, 50° C.): yellow liquid, Concentration: 20% 74 mPa · s, pH: 5.5 (as ZrO2) Viscosity: 8 cps, pH: 9.5 198 Not used Detackifying waterproofing agent: 0.5 o Polyamine epichlorohydrin resin Trade name: PA-625 (Nippon PMC corporation) Properties: Weakly cationic, Light brown, transparent liquid, Concentration: 60% Viscosity: 250 cps, pH (20° C.): 7.0 199 Ampholytic polyacryloamide 0.4 Detackifying sizing agent: 1.0 o Trade name: Fixter K-6LS Strengthened rosin sizing agent (Seiko Chemical Industries) Trade name: Sizepine E-50 Properties: Ampholytic, Light (ARAKAWA CHEMICAL INDUSTRIES) brown, transparent, viscous Properties: Anionic, Brown, liquid, Concentration: 15.2%, transparent liquid, Concentration: Viscosity (25° C.): 680 cps, 50.4% Viscosity (25° C.): 200 pH (1.5%): 4.7 cps, pH (5%, 20° C.): 11.0 EXAMPLES 200 to 203 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer or hydrophilic group sealant, and treated with the hydrophilic group sealant for the internal surface.",
"It was formed into the film, and treated with the hydrophilic group sealant reactive at low temperature for the external surface.",
"The tackiness test result, and the hydrophilic polymer and hydrophilic group sealant used in each EXAMPLE are given in Table 25.The post-treatment step and hydrophilic group sealant reactive at low temperature for treating the external surface in each EXAMPLE are described below.",
"(Post-reatment) The natural rubber latex film film prepared was treated by the following steps in the order described below, unless otherwise stated: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 200 The natural rubber latex film formed was heated at 38° C. for 5 minutes, and leached at 70° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the treating solution of the hydrophilic group sealant for the external surface for 5 seconds.",
"Furthermore, it was post-vulcanized at 110° C. for 10 minutes.",
"EXAMPLE 201 Heating: 95° C., 3 min., Drying: 95° C., 10 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"EXAMPLES 202 and 203 Heating: 40° C., 4 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 10 min.",
"(Hydrophilic group sealant reactive at low temperature for treating the external surface) EXAMPLE 200 Detackifying crosslinking agent of metallic element: Polyaluminum hydroxide Paho#2S (Asada Kagaku Kogyo) The above-described sealant was diluted with water to have an Al2O3 concentration of 0.5% for use in each.",
"EXAMPLES 201 to 203 Detackifying crosslinking agent of metallic element: Zirconyl acetate Zircosol ZA (DAIICHIKIGENSO KAGAKUKOGYO) The above-described sealant was diluted with water to have a ZrO2 concentration of 1% for use.",
"TABLE 25 Hydrophilic polymers to be incorporated in the latex Hydrophilic group blocking agents Incorporated for treating the internal surface Examples quantity Concen- Test NO.",
"(parts) tration (%) results 200 Not used Detacking crosslinking agent of 0.5 o metallic element: Polyaluminum (asAl2O3) hydroxide Trade name: Paho #2S (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, Transparent liquid, Viscosity (30° C.): 7 cps, Concentration: 10.5% (as Al2O3), pH: 3.5 201 Not used Detackifying, hydrogen bond adjustor: 1.5 o Polyamide/polyamine epichlorohydrin resin Trade name: Sumirez Resin 6615 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 15% Viscosity (25° C.): 40 mPa · s, pH (25° C.): 4.0 202 Cationized tapioca starch 0.25 Crosslinking agent of detackifying, 0.5 o′ Trade name: Catesize 350 aqueous resin: Carbodiimide (Nippon NSC) Trade name: CARBODILITE V-02 Properties: Cationic, White (Nisshinbo Industries) powdery Viscosity (5%, 40° C.): Properties: Yellow, transparent 17 cps liquid, Carbodiimide equivalents: 597 Concentration: 40%, pH: 10.1 203 Carboxymethyl cellulose 0.25 Detackifying, hydrogen bond adjustor: 1.0 o Trade name: CMC DAICEL 1330 Polyamide/polyamine epichlorohydrin (DAICEL CHEMICAL INDUSTRIES) resin Properties: Anionic, White Trade name: Sumirez Resin 675 powdery, Viscosity (1%, 25° C.): (Sumitomo Chemical Group) 73 cps pH: 6.8, Degree of Properties: Cationic, Brown, etherification: 1.27 transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.1 9.Quantitative Analysis of Protein Eluted protein was quantitatively analyzed in accordance with JIS T-9010 (Testing method for biological safety of rubber products); 3.6 Colorimetry based on the BCA method for water-soluble protein.",
"10.Analysis of Protein in Natural Rubber Latex Film COMPARATIVE EXAMPLE 3 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer, formed into the film, and post-treated for analysis of protein.",
"The analysis result and the hydrophilic polymer used are given in Table 26.The post-treatment step is described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in COMPARATIVE EXAMPLE 3 were: Heating Leaching Post-vulcanization 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"TABLE 26 Incorporated Protein Hydrophilic polymers to be quantity content incorporated in the latex (parts) (ppm) Carboxymethyl cellulose 0.25 140 Trade name: CMC DAICEL 1330 (DAICEL CHEMICAL INDUSTRIES) Properties: Anionic, White powdery, Viscosity (1%, 25° C.): 73 cps pH: 6.8, Degree of etherification: 1.27 Carboxylate-based acrylic copolymer 0.25 60 Trade name: ARON A-7180 (Toagosei) Properties: Anionic, Semi-transparent, viscous liquid, Concentration: 16.4% Viscosity (25° C.): 20950 cps, pH (25° C.): 9.0 Polyamide derivative: Polyoxyethylen 0.25 66 ealkyl ether Trade name: Elsoft A (Ipposha Oil Industries) Properties: Nonionic, Light yellow, pasty, Concentration: 15% Methyl cellulose 0.4 247 Trade name: Metolose SM-400 (Shin-Etsu Chemical) Properties: Nonionic, White powdery Viscosity (2%, 20° C.): 436 mPa · s Locust bean gum 0.5 120 Trade name: Soar Locust A120F (MRC Polysaccharides) Properties: Nonionic, White powdery Viscosity (1%, 25° C.): 1200 cps Alkyl acetalized polyvinyl alcohol 0.5 140 Trade name: Eslec KW-3 (SEKISUI CHEMICAL) Properties: Nonionic, Transparent, viscous liquid, Concentration: 20% Viscosity: 3500 mPa · s, pH: 6 COMPARATIVE EXAMPLE 4 The pre-vulcanized natural rubber latex was incorporated or treated for both internal and external surfaces with an epoxy compound.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result and the epoxy compound are given in Table 27.The post-treatment step is described below: (Post-treatment) When the natural rubber latex was incorporated with the epoxy compound: The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in COMPARATIVE EXAMPLE 4 were: Heating Leaching Post-vulcanization 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"When the natural rubber latex was surface-treated with the epoxy compound: The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in COMPARATIVE EXAMPLE 4 were: Heating: 50° C., 2 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"TABLE 27 Protein content Epoxy compound (ppm) Glycerol polyglycidyl ether When 215 incorporated: Trade name: Denacol EX-313 Incorporated (Nagase ChemteX Corporation) Properties: Anionic, Light yellow quantity liquid (parts) Viscosity (25° C.): 150 mPa · s, 1.0 Epoxy equivalents: 141WPE When used for 103 surface treatment Concentration (%) 2.0 COMPARATIVE EXAMPLE 5 The pre-vulcanized natural rubber latex was formed into the film, and post-treated for analysis of protein.",
"The analysis result is given in Table 28.The post-treatment step is described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below, unless otherwise stated: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in COMPARATIVE EXAMPLE 5 were: Heating Leaching Post-vulcanization (Leaching treatment at lower temperature) 50° C., 8 min.",
"70° C., 3 min.",
"110° C., 5 min.",
"(Leaching treatment at higher temperature) 95° C., 5 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"(Chlorination of both internal and external surfaces) The natural rubber latex film prepared was heated at 50° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 5 minutes, released out of the mold, and immersed in chlorine water (chlorine concentration: 0.4%) for 3 minutes, to halogenate both surfaces.",
"Then, it was leached at 85° C. for 1 minute and finally post-vulcanized at 110° C. for 5 minutes.",
"COMPARATIVE EXAMPLE 6 The diene-based carboxylated NBR latex was pre-vulcanized under the same conditions as those for the above-described natural rubber latex.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result is also given in Table 28.The post-treatment step and diene-based carboxylated NBR latex used are described below: (Post-treatment) The NBR latex film prepared was heated at 95° C. for 5 minutes, leached at 85° C. for 3 minutes, and finally post-vulcanized at 110° C. for 5 minutes.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) TABLE 28 Measured Comparative protein Example No.",
"Remarks content (ppm) 5 Leaching at lower temperature 125 as the post-treatment step Leaching at higher temperature 75 as the post-treatment step Chlorination on both surfaces 10 as the post-treatment step 6 The diene-based carboxylated NBR, 5 pre-vulcanized under the same conditions as those for the natural rubber latex, and leached at higher temperature as the post-treatment step EXAMPLES 204 to 210 The pre-vulcanized natural rubber latex was incorporated with the anionic group introducing compound.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result and the anionic group introducing compound used in each EXAMPLE are given in Table 29.The post-treatment step is described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"TABLE 29 Measured Incorporated protein Examples quantity content NO.",
"Anionic group introducing compounds to be incorporated in the latex (parts) (ppm) 204 Sulfate ethyl sulfone/monochlorotriazine-based bifunctional reactive dye 0.5 36.0 Trade name: Sumifix Supra Brilliant yellow 3GF (Sumitomo Chemical Group) 205 Sulfate ethyl sulfone/monochlorotriazine-based multi-functional reactive dye 0.5 24.0 Trade name: Sumifix HF yellow 3R gran (Sumitomo Chemical Group) 206 Dichlorotriazine-based reactive dye 0.5 25.0 Trade name: Procion yellow MX-3R (BASF Corporation) 207 Carboxypyridinio-S-triazine-based reactive dye 0.5 38.0 Trade name: Kayacelon React Yellow CN-4G (NIPPON KAYAKU) 208 α-Bromo acrylamide-based reactive dye 0.5 34.9 Trade name: Lanasol Yellow 4G (Ciba Specialty Chemicals) 209 Alkenyl succinic anhydride 0.25 45.0 Trade name: Coloparl Z-100 (Seiko Chemical Industries) Properties: Brown, transparent liquid, Viscosity (25° C.): 200 cps 210 Urea phosphorylated starch 0.25 39.0 Trade name: MS#4600 (Nihon Shokuhin Kako) Properties: Anionic, Slightly yellow powdery Viscosity (20%, 50° C.): 74 mPa · s, pH: 5.5 EXAMPLES 211 to 213 The pre-vulcanized natural rubber latex was incorporated with the anionic group introducing compound, and treated with the cationic group introducing compound for both surfaces.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result and the anionic group and cationic group introducing compounds used in each EXAMPLE are given in Table 30.The post-treatment step is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following steps in each EXAMPLE in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 211 Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 212 and 213 Heating: 50° C., 2 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"TABLE 30 Reactive dyes to be incorporated in the latex Cationic group introducing compounds Measurement Incorporated for treating both surfaces result Examples quantity Concen- protein NO.",
"(parts) tration (%) content (ppm) 211 Sulfate ethyl sulfone/monochlorotriazine- 0.5 Polyamine epichlorohydrin resin 1.0 17.5 based bifunctional reactive dye Trade name: CIBAFIX E Trade name: Sumifix Supra Brilliant (Ciba Specialty Chemicals) yellow 3GF (Sumitomo Chemical Group) Properties: Cationic, Light yellow liquid, Concentration: 20% pH (5% solution): 5 212 Sulfate ethyl sulfone/monochlorotriazine- 0.25 Polyamine epichlorohydrin resin 1.0 18.5 based multi-functional reactive dye Trade name: WS-564 (Nippon Trade name: Sumifix HF yellow 3R gran PMC corporation) (Sumitomo Chemical Group) Properties: Cationic, Light amber liquid, Concentration: 20% Viscosity (25° C.): 50 cps, pH: 3.7 213 Sulfate ethyl sulfone/monochlorotriazine- 0.5 Crosslinking agent of metallic 1.0 12.5 based bifunctional reactive dye element: Polyaluminum hydroxide (asAl2O3) Trade name: Sumifix HF yellow 3R gran Trade name: Paho#2S (Asada (Sumitomo Chemical Group) Kagaku Kogyo) Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 EXAMPLES 214 to 217 The pre-vulcanized natural rubber latex was treated with the cationic group introducing compound for both surfaces.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result and the cationic group introducing compound used in each EXAMPLE are given in Table 31.The post-treatment step in each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLES 214 and 216 Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 215 and 217 Heating: 50° C., 2 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"TABLE 31 Cationic group introducing compounds for treating both surfaces Analysis result Examples Concentration protein content NO.",
"(%) (ppm) 214 Polyamine epichlorohydrin resin 1.0 37.5 Trade name: CIBAFIX E (Ciba Specialty Chemicals) Properties: Cationic, Light yellow liquid, Concentration: 20% pH (5% solution): 5 215 Crosslinking agent of metallic element: Polyaluminum hydroxide 2.0 14.0 Trade name: Paho#2S (Asada Kagaku Kogyo) (asAl2O3) Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps, Concentration: 10.5% (as Al2O3), pH: 3.5 216 Polyamide/polyamine epichlorohydrin resin 0.5 15.0 Trade name: Euramine P5600 (Mitsui Chemicals) Properties: Cationic, Light yellow, transparent liquid, Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 217 Polyamine epichlorohydrin resin 2.0 12.5 Trade name: WS-564 (Nippon PMC corporation) Properties: Cationic, Light amber liquid, Concentration: 20% Viscosity (25° C.): 50 cps, pH: 3.7 EXAMPLES 218 to 223 The pre-vulcanized natural rubber latex was incorporated with the cationic group introducing compound.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result and the cationic group introducing compound used in each EXAMPLE are given in Table 32.The post-treatment step in each EXAMPLE is described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"TABLE 32 Cationic group introducing compounds to be incorporated in the latex Incorporated Analysis result Examples quantity protein content No (parts) (ppm) 218 Polyamide/polyamine epichlorohydrin resin 0.25 17.5 Trade name: Sumirez Resin 6625 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.0 219 Polyamidepolyamine resin epichlorohydrin modification 0.25 35.0 Trade name: Wet Master GT-360 (Toho Chemical Industry) Properties: Cationic, Light yellow/brown, slightly white turbid liquid, Concentration: 59.2% Viscosity (25° C.): 173 mPa · s, pH: 4.3 220 Styrene-based tertiary amino group-modified polyamide/polyamine epichlorohydrin resin 0.25 32.5 Trade name: Polymaron 360 (ARAKAWA CHEMICAL INDUSTRIES) Properties: Cationic, Light yellow, slightly white turbid liquid, Concentration: 20.4% Viscosity: 14.5 mPa · s, pH: 4.9 221 Zirconium ammonium lactate or zirconium ammonium citrate 0.25 35.0 Trade name: SEQUAREZ 82 (OMNOVA Solutions) Properties: Cationic, Transparent white solution, Concentration: 27% Viscosity: 17 mPa · s, pH: 7.5 222 Cationized tapioca starch 0.25 28.0 Trade name: Cato308 (Nippon NSC) Properties: Cationic, White powdery 223 Ampholytic starch 0.25 35.0 Trade name: Optibond 3282 (Nippon NSC) Properties: Ampholytic, White powdery EXAMPLE 224 The pre-vulcanized natural rubber latex was incorporated with the waterproofing agent reactive with and capable of fixing protein in the natural rubber latex under an alkaline condition.",
"It was formed into the film, and post-treated for analysis of protein.",
"The analysis result and the waterproofing agent used in this EXAMPLE are given in Table 33.The post-treatment step is described below: (Post-treatment) The natural rubber latex film prepared was treated by the following steps in the order described below: Heating→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating Leaching Post-vulcanization 95° C., 7 min.",
"85° C., 3 min.",
"110° C., 5 min.",
"TABLE 33 Waterproofing agent to be incorporated in the latex Incorporated Analysis result Example quantity protein content No.",
"(parts) (ppm) 224 Ketone resin 0.25 34.0 Trade name: SI-668 (Nippon PMC corporation) Properties: Nonionic, Slightly white turbid solution, Concentration: 50% Viscosity: 40 cps, pH: 7 EXAMPLES 225 to 229 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer, and surface-treated with the cationic group introducing compound.",
"It was formed into the film for the tackiness test and analysis of protein.",
"The results, and the hydrophilic polymer and cationic group introducing compound used in each EXAMPLE are given in Table 34.The Post-treatment step in each EXAMPLE is described below: (Treatment) The natural rubber latex film prepared was treated by the following steps in the order described below for both surfaces, unless otherwise stated: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 225 Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 226 Heating: 50° C., 2 minutes, Drying: 95° C., 5 minutes, Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 227 The natural rubber latex film was dried at 50° C. for 1 minute, and leached at 75° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the treating solution of the hydrophilic group sealant for the external surface for 5 seconds.",
"Furthermore, it was dried at 95° C. for 5 minutes, leached at 75° C. for 1 minute, and finally post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLES 228 and 229 The natural rubber latex film was heated at 50° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the treating solution of the hydrophilic group sealant for the external surface for 5 seconds.",
"Furthermore, it was dried at 95° C. for 3 minutes, leached at 85° C. for 1 minute, and finally post-vulcanized at 110° C. for 5 minutes.",
"TABLE 34 Hydrophilic polymers to Measured be incorporated in the latex Cationic group introducing compounds protein Incorporated for treating both surfaces content (ppm) Tackiness Examples quantity Concen- protein test NO.",
"(parts) tration (%) content (ppm) results 225 Carboxylate-based acrylic copolymer 0.25 Polyamide/polyamine epichlorohydrin 1.0 22.5 o Trade name: ARON A-7180 (Toagosei) resin Properties: Anionic, Semi-transparent, Trade name: Euramine P5600 (Mitsui viscous liquid, Concentration: 16.4% Chemicals) Viscosity (25° C.): 20950 cps, Properties: Cationic, Light yellow, pH (25° C.): 9.0 transparent liquid, Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 226 Alkyl acetalized polyvinyl alcohol 0.5 Polyamide/polyamine epichlorohydrin 2.0 17.0 o Trade name: Eslec KW-3 resin (SEKISUI CHEMICAL) Trade name: Sumirez Resin 6625 Properties: Nonionic, Transparent, (Sumitomo Chemical Group) viscous liquid, Concentration: 20% Properties: Cationic, Brown, Viscosity: 3500 mPa · s, pH: 6 transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.0 227 Hydrogen bond adjustor, Polyamide 0.25 Internal surface 2.5 21.0 o derivative Polyoxyethylen ealkyl Crosslinking agent of metallic (asAl2O3) ether element: Polyaluminum hydroxide Trade name: Elsoft A Trade name: Paho#2S (Ipposha Oil Industries) (Asada Kagaku Kogyo) Properties: Nonionic, Light Properties: Cationic, Light yellow, pasty, Concentration: 15% yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 External surface 1.0 Polyamide/polyamine epichlorohydrin resin Trade name: Sumirez Resin 6625 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.0 228 Copolymerized polyamide emulsion 0.25 Internal surface 1.0 16.2 o Trade name: Griltex 2 Suspension Crosslinking agent of metallic (asAl2O3) (EMS SHOWA DENKO K.K.)",
"element: Polyaluminum hydroxide Properties: Nonionic, Milky white Trade name: Paho#2S aqueous solution, Concentration: 40% (Asada Kagaku Kogyo) Viscosity: 1500 cps, pH: 9.5 Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 External surface 1.0 Crosslinking agent of metallic (asAl2O3) element: Alumina sol Trade name: Alumina sol 200 (Nissan Chemical Industries) Properties: Cationic, Milky white, colloidal solution, Concentration: 10.1% (as Al2O3) Viscosity (20° C.): 530 mPa · s, pH (20° C.): 4.8 229 Polyvinyl butyral resin emulsion 0.25 Internal surface 1.0 15.0 o Trade name: Rczcm VB-1 Crosslinking agent of metallic as Al2O3 (CHUKYO YUSHI) element: Polyaluminum hydroxide Properties: Nonionic, White Trade name: Paho#2S liquid, Concentration: 35% (Asada Kagaku Kogyo) Viscosity (25° C.): 20 mPa · s, Properties: Cationic, Light pH (diluted 10 times): 7.2 yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 External surface 1.0 Crosslinking agent of metallic as Al2O3 element: Alumina sol Trade name: Alumina sol 200 (Nissan Chemical Industries) Properties: Cationic, Milky white, colloidal solution, Concentration: 10.1% (as Al2O3) Viscosity (20° C.): 530 mPa · s, pH (20° C.): 4.8 EXAMPLES 230 to 232 The pre-vulcanized natural rubber latex was incorporated with the reactive dye, and surface-treated with the cationic group introducing compound.",
"It was formed into the film for the tackiness test and analysis of protein.",
"The results, and the reactive dye and cationic group introducing compounds used in each EXAMPLE are given in Table 35.The post-treatment step in each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 230 Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 231 to 232 Heating: 50° C., 2 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"TABLE 35 Reactive dyes to be Measured incorporated in the latex Cationic group introducing compounds protein Incorporated for treating both surfaces content (ppm) Tackiness Examples quantity Concen- protein test NO.",
"(parts) tration (%) content (ppm) results 230 Sulfate ethyl sulfone/monochloro- 0.5 Polyamine epichlorohydrin resin 1.0 17.5 o triazine-based bifunctional reactive Trade name: CIBAFIX E dye (Ciba Specialty Chemicals) Trade name: Sumifix Supra Brilliant Properties: Cationic, Light yellow yellow 3GF (Sumitomo Chemical Group) liquid, Concentration: 20% pH (5% solution): 5 231 Sulfate ethyl sulfone/monochloro- 0.25 Polyamine epichlorohydrin resin 1.0 18.5 o triazine-based multi-functional Trade name: WS-564 (Nippon reactive dye PMC corporation) Trade name: Sumifix HF yellow 3R Properties: Cationic, Light amber gran (Sumitomo Chemical Group) liquid, Concentration: 20% Properties: Viscosity (25° C.): 50 cps, pH: 3.7 232 Sulfate ethyl sulfone/monochloro- 0.5 Detackifying crosslinking agent 1.0 12.5 o triazine-based bifunctional reactive of metallic element: Polyaluminum (asAl2O3) dye hydroxide Trade name: Sumifix Supra Brilliant Trade name: Paho#2S yellow 3GF (Sumitomo Chemical Group) (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 EXAMPLES 233 to 236 The pre-vulcanized natural rubber latex was surface-treated with the cationic group introducing compound.",
"It was formed into the film for the tackiness test and analysis of protein.",
"The results and the cationic group introducing compound used in each EXAMPLE are given in Table 36.The post-treatment step in each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: EXAMPLE 233 Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLES 234 to 235 Heating: 50° C., 2 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"EXAMPLE 236 The natural rubber latex film was heated at 50° C. for 5 minutes, and leached at 85° C. for 5 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the treating solution of the hydrophilic group sealant for the external surface for 5 seconds.",
"Furthermore, it was dried at 95° C. for 3 minutes, leached at 85° C. for 1 minute, and finally post-vulcanized at 110° C. for 5 minutes.",
"TABLE 36 Cationic group Cationic group Measured introducing compounds for introducing compounds for protein treating the external surface treating the external surface content (ppm) Tackiness Examples Concen- Concen- protein test NO.",
"tration (%) tration (%) content (ppm) results 233 Polyamide/polyamine epichloro- 0.5 Polyamide/polyamine epichloro- 0.5 15.0 o hydrin resin hydrin resin Trade name: Euramine P5600 Trade name: Euramine P5600 (Mitsui Chemicals) (Mitsui Chemicals) Properties: Cationic, Light yellow, Properties: Cationic, Light transparent liquid, Concentration: yellow, transparent liquid, 31% Viscosity (25° C.): Concentration: 31% Viscosity 71.3 mPa · s, pH (25° C.): 4.5 (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 234 Polyamine epichlorohydrin resin 2.0 Polyamine epichlorohydrin 2.0 12.5 o Trade name: WS-564 (Nippon resin PMC corporation) Trade name: WS-564 (Nippon Properties: Cationic, Light amber PMC corporation) liquid, Concentration: 20% Properties: Cationic, Light Viscosity (25° C.): 50 cps, pH: 3.7 amber liquid, Concentration: 20% Viscosity (25° C.): 50 cps, pH: 3.7 235 Crosslinking agent of metallic 2.0 Crosslinking agent of metallic 2.0 14.0 o element: Polyaluminum hydroxide (asAl2O3) element: Polyaluminum hydroxide (asAl2O3) Trade name: Paho#2S Trade name: Paho#2S (Asada Kagaku Kogyo) (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, Properties: Cationic, Light transparent liquid, Viscosity yellow, transparent liquid, (30° C.): 7 cps Concentration: Viscosity (30° C.): 7 cps 10.5% (as Al2O3), pH: 3.5 Concentration: 10.5% (as Al2O3), pH: 3.5 236 Crosslinking agent of metallic 2.0 Crosslinking agent of metallic 1.4 16.3 o element: Polyaluminum hydroxide (asAl2O3) element: Peroxy titania sol (asTiO2) Trade name: Paho#2S Trade name: TKC-301 (Asada Kagaku Kogyo) (Tayca Corporation) Properties: Cationic, Light yellow, Properties: Cationic, Yellow transparent liquid, Viscosity transparent liquid, (30° C.): 7 cps Concentration: Concentration: 1.4% (as TiO2) 10.5% (as Al2O3), pH: 3.5 EXAMPLES 237 and 238 The pre-vulcanized natural rubber latex was incorporated with the cationic group introducing compound, and treated with the cationic group introducing compound for both surfaces.",
"It was formed into the film, and post-treated for the tackiness test and analysis of protein.",
"The results, and the cationic compound and cationic group introducing compound used in each EXAMPLE are given in Table 37.The post-treatment step in each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in each EXAMPLE were: Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"TABLE 37 Cationic group introducing compounds to Measured be incorporated in the latex Cationic group introducing compounds protein Incorporated for treating the external surface content (ppm) Tackiness Examples quantity Concen- protein test NO.",
"(parts) tration (%) content (ppm) results 237 Cationized starch 0.25 Polyamide/polyamine epichloro- 0.5 14.0 o Trade name: Cato308 hydrin resin (Nippon NSC) Trade name: Euramine P5600 Properties: Cationic, (Mitsui Chemicals) White powdery Properties: Cationic, Light yellow, transparent liquid, Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 238 Cationized starch 0.25 Polyamine epichlorohydrin resin 1.0 27.5 o Trade name: Cato308 Trade name: WS-564 (Nippon (Nippon NSC) PMC corporation) Properties: Cationic, Properties: Cationic, Light White powdery amber liquid, Concentration: 20% Viscosity (25° C.): 50 cps, pH: 3.7 EXAMPLE 239 The pre-vulcanized natural rubber latex was incorporated with the waterproofing agent reactive with and capable of fixing protein in the natural rubber latex under an alkaline condition.",
"It was surface-treated with the cationic group introducing compound, formed into the film, and post-treated for the tackiness test and analysis of protein.",
"The analysis result, and the waterproofing agent and cationic group introducing compound used in this EXAMPLE are given in Table 38.The post-treatment step in each EXAMPLE is described below: (Post-treatment) The natural rubber latex film film prepared was treated by the following steps in the order described below: Heating→Immersion in the solution for treating the external surface→Drying→Leaching→Post-vulcanization Post-treatment temperature and treatment time in this EXAMPLE were: Heating: 50° C., 5 min., Drying: 95° C., 5 min., Leaching: 85° C., 3 min., Post-vulcanization: 110° C., 5 min.",
"TABLE 38 Waterproofing agents to be Cationic group Measured incorporated in the latex introducing compounds protein Incorporated for treating both surfaces content (ppm) Tackiness quantity Concen- protein test NO.",
"(parts) tration (%) content (ppm) results 239 Ketone resin 0.25 Polyamide/polyamine epichloro- 0.5 25 o Trade name: SI-668 hydrin resin (Nippon PMC corporation) Trade name: Euramine P5600 Properties: Nonionic, (Mitsui Chemicals) Slightly white turbid Properties: Cationic, Light solution, Concentration: yellow, transparent liquid, 50% Viscosity: 40 cps, pH: 7 Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 EXAMPLES 240 to 246 The pre-vulcanized natural rubber latex was incorporated with the hydrophilic polymer or cationic group introducing compound, and treated with the cationic group introducing compound for the internal surface.",
"It was formed into the film, and coating-treated with the detackifying, carboxylated, NBR or halogenation-treated for the external surface, for the tackiness test and analysis of protein.",
"The results, and the hydrophilic polymer and cationic group introducing compound used in each EXAMPLE are given in Table 39.The external surface coating treatment step, carboxylated NBR used, carboxyl group sealant used, and halogenation treatment step are described below: (External Surface Coating Treatment Step) The natural rubber latex film prepared was heated at 50° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 1 minute, and immersed in the external surface coating solution for 5 seconds.",
"Furthermore, it was dried at 95° C. for 3 minutes, leached at 85° C. for 1 minute, and finally post-vulcanized at 110° C. for 5 minutes.",
"The coating solution of the carboxylated NBR latex was incorporated only with 0.25 parts of the carboxyl group sealant, and diluted with water to have the solid latex concentration of 5%.",
"(Carboxylated NBR Latex) Nipol LX-551 (Zeon Corporation) (Carboxyl Group Sealant) Detackifying, hydrogen bond adjustor: Polyamidepolyamine epichlorohydrin resin Sumirez Resin 6625 (Sumitomo Chemical Group) (External Surface Halogenation Treatment Step) The natural rubber latex film prepared was heated at 50° C. for 5 minutes, and leached at 85° C. for 3 minutes.",
"Then, it was heated at 95° C. for 5 minutes, and immersed in chlorine water (chlorine concentration: 0.4%) for 3 minutes.",
"Furthermore, it was leached at 85° C. for 1 minute, and finally post-vulcanized at 110° C. for 5 minutes.",
"EXAMPLE 247 The prototype fingerstall production unit shown in FIG.",
"2 was constructed, based on the immersion type carrier shown in FIG.",
"1 (Japanese Patent Laid-Open No.",
"07-329084), and used to produce the fingerstalls in a manner similar to the procedure above-described in each EXAMPLE.",
"In the immersion type carrier shown in FIG.",
"1, the chain 1 moves along the guide rail 2 to carry the immersion mold 3, and the rod 4 moves along the guide 5 to move the immersion mold 3 in the vertical direction.",
"Referring to FIG.",
"2, the immersion mold 3, when passing over the immersion tank 6, moves downwards to be immersed in the tank 6.The immersion tanks are prepared for each of the coagulating liquid, latex liquid, leaching water and external surface treatment solution.",
"They are replaced with each other, as required, for the immersion or leaching treatment.",
"On completion of the immersion or leaching treatment, the immersion mold 3 is passed to the drying furnace 7 in which it is dried.",
"The guide 5 is adjusted in such a way to prevent the immersion mold 3 from moving downwards and coming into contact with the winding machine 8, while the machine 8 is not in service.",
"The immersion mold 3 is set immobilized, as required, during the immersion, drying or leaching step for a given treatment time.",
"The winding machine 8 winds up the film 11 from the immersion mold 3 on which it is set by rotating the roll-shaped brushes (FIG.",
"3) 10 disposed obliquely and passing the immersion mold 3 between them.",
"On completion of the latex film forming process, the film is wound up and released from the immersion mold 3 on which it is set by passing the mold 3 through the winding machine 8.Each fingerstall wound up is dried at 90° C. for 30 minutes to finish the production step.",
"The fingerstall thus produced can be easily put on a finger.",
"TABLE 39 Hydrophilic polymers or cationic group introducing compounds to Cationic group be incorporated in the latex introducing compounds Measured Incor- for treating the protein porated internal surface Eexternal content (ppm) Tackiness Examples quantity Concen- surface protein test NO.",
"(parts) tration (%) treatment content (ppm) results 240 Alkyl acetalized polyvinyl 0.5 Polyamide/polyamine 2.0 coated 16.5 ◯ alcohol epichlorohydrin resin halogenized 19.1 Trade name: Eslec KW-3 Trade name: Sumirez Resin (SEKISUI CHEMICAL) 6625 (Sumitomo Chemical Properties: Nonionic, Group) Transparent, viscous liquid, Properties: Cationic, Concentration: 20% Viscosity: Brown, transparent liquid, 3500 mPa · s, pH: 6 Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.0 241 Copolymerized polyamide 0.25 Crosslinking agent of 1.0 coated 18.7 ◯ emulsion metallic element: (asAl2O3) halogenized 17.0 Trade name: Griltex 2 Polyaluminum hydroxide Suspension (EMS SHOWA Trade name: Paho#2S DENKO K.K.)",
"(Asada Kagaku Kogyo) Properties: Nonionic, Properties: Cationic, Milky white aqueous solution, Light yellow, transparent Concentration: 40% liquid, Viscosity (30° C.): Viscosity: 1500 cps, pH: 9.5 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 242 Cationized starch 0.25 Polyamide/polyamine 0.5 coated 2.4 ◯ Trade name: Cato308 epichlorohydrin resin halogenized 9.2 (Nippon NSC) Trade name: Euramine P5600 Properties: Cationic (Mitsui Chemicals) White powdery Properties: Cationic, Light yellow, transparent liquid, Concentration: 31% Viscosity (25° C.): 71.3 mPa · s, pH (25° C.): 4.5 243 Polyamide/polyamine 0.25 Polyamine epichlorohydrin 1.0 coated 2.5 ◯ epichlorohydrin resin resin halogenized 8.3 Trade name: Sumirez Resin Trade name: WS-564 6625 (Sumitomo Chemical (Nippon PMC corporation) Group) Properties: Cationic, Light Properties: Cationic, amber liquid, Concentration: Brown, transparent liquid, 20% Viscosity (25° C.): Concentration: 25% Viscosity 50 cps, pH: 3.7 (25° C.): 200 mPa · s, pH (25° C.): 4.0 244 Not used Crosslinking agent of 2.0 coated 11.3 ◯ metallic element: (asAl2O3) halogenized 15.0 Polyaluminum hydroxide Trade name: Paho#2S (Asada Kagaku Kogyo) Properties: Cationic, Light yellow, transparent liquid, Viscosity (30° C.): 7 cps Concentration: 10.5% (as Al2O3), pH: 3.5 245 Not used Polyamide/polyamine 2.0 coated 9.5 ◯ epichlorohydrin resin halogenized 10.1 Trade name: Sumirez Resin 6625 (Sumitomo Chemical Group) Properties: Cationic, Brown, transparent liquid, Concentration: 25% Viscosity (25° C.): 200 mPa · s, pH (25° C.): 4.0 246 Not used Zirconium oxychloride 1.0 coated 9.9 ◯ (asZrO2) halogenized 11.7 Industrial Applicability The present invention provides a detackified natural rubber latex product.",
"The invention also provides the natural rubber latex product from which protein present in the natural rubber latex is eluted out to only a limited extent.",
"The present invention also provides the natural rubber latex product which causes no discoloration of the metallic product surface with which it comes into contact, when it is to be used for handling a precision device or the like, by coating it, as required, with a synthetic rubber latex layer vulcanized without using sulfur."
]
] | Patent_10432879 |
[
[
"Acoustic communication system",
"There is described a number of encoders for encoding a data signal within an audio signal.",
"In some of the encoders, the audio signal is separated into a tonal part and a residual part, and the data signal is shaped based on the residual part.",
"In other encoders, the data signal is separated into a tonal part and a residual part, and the data signal is combined with the audio signal independence upon the residual part.",
"In other encoders, the rate at which the data is encoded within the audio signal is varied in dependence upon the audio signal.",
"There are also described various decoders associated with the described encoders."
],
[
"1-161.",
"(canceled) 162.An apparatus for shaping a data signal for embedding within an audio signal, the apparatus comprising: a first receiver operable to receive the data signal; a second receiver operable to receive the audio signal; a processor operable to process the audio signal to generate a residual signal representative of a difference between a tonal component of the received audio signal and the received audio signal; and a shaper operable to shape the spectrum of the data signal in dependence upon the residual signal to form a shaped data signal.",
"163.An apparatus according to claim 162, wherein the processor comprises: a modeller operable to model the audio signal to generate a tonal signal representative of a tonal component of the audio signal; and a subtractor operable to differentiate the tonal signal and the audio signal to generate the residual signal.",
"164.An apparatus according to claim 163, wherein the modeller comprises: a time series modeller operable to apply a time series model to the audio signal in order to generate a plurality of coefficients indicative of the audio signal; and a synthesiser operable to synthesise the tonal signal using the generated coefficients.",
"165.An apparatus according to claim 164, wherein the time series modeller is operable to perform linear predictive coding in order to generate said coefficients.",
"166.An apparatus according to claim 164, further comprising a modifier operable to modify the time series model in order to vary the number of coefficients generated by said time series modeller.",
"167.An apparatus according to claim 166, in which the modifier comprises a user interface, wherein the modifier is operable to vary the number of generated coefficients in accordance with a user signal received via the user interface.",
"168.An apparatus according to claim 166, in which the modifier comprises a tonality determiner operable to determine a level of tonality of the audio signal, wherein the modifier is operable to vary the number of generated coefficients in dependence upon the determined level of tonality.",
"169.An apparatus according to claim 162, wherein the data signal has a data signal spectrum and the shaper comprises: a spectrum analyser operable to analyse the spectrum of the residual signal to generate a plurality of frequency-dependent coefficients; a scaling factors generator operable to generate a plurality of scaling factors using the plurality of frequency-dependent coefficients; and an adjuster operable to adjust the data signal spectrum using the plurality of scaling factors to generate the shaped data signal.",
"170.An apparatus according to claim 169, wherein the spectrum analyser is a first spectrum analyser and is operable to generate a first plurality of frequency-dependent coefficients, and the adjuster comprises: a second spectrum analyser operable to analyse the spectrum of the data signal to generate a second plurality of frequency-dependent coefficients; a scaler operable to scale the second plurality of frequency-dependent coefficients using the plurality of scaling factors to generate a plurality of scaled coefficients; and a synthesiser operable to synthesise the shaped data signal using the plurality of scaled coefficients.",
"171.An apparatus according to claim 170, wherein the first and second spectrum analysers are operable to perform a Fourier transform.",
"172.An apparatus according to claim 170, wherein the first and second spectrum analysers are operable to perform a wavelet transform.",
"173.An apparatus according to claim 170, wherein the scaling factors generator is operable to perform a psycho-acoustic analysis of the first plurality of frequency dependent coefficients.",
"174.An apparatus according to claim 173, wherein the scaling factors generator comprises a psycho-acoustic filter for performing the psycho-acoustic analysis.",
"175.An apparatus according to claim 174, wherein the psycho-acoustic filter has a variable bandwidth, and wherein said scaling factors generator comprises an adjuster operable to vary the variable bandwidth of the psycho-acoustic filter.",
"176.An apparatus according to claim 175, wherein said adjuster comprises a user interface and a setter operable to set the bandwidth of the psycho-acoustic filter in accordance with a user input received via the user interface.",
"177.An apparatus according to claim 175, wherein the adjuster comprises: a tonality determiner operable to determine a level of tonality of the residual signal; and an adjuster operable to set the bandwidth of the psycho-acoustic filter in dependence upon the determined level of tonality.",
"178.An apparatus according to claim 162, wherein the data signal has a data signal spectrum, the residual signal has a residual signal spectrum, and the shaper is arranged to shape the data signal spectrum to match the residual signal spectrum.",
"179.An apparatus according to claim 162, wherein the shaper is arranged to shape the data signal so that the temporal profile of the shaped data signal substantially matches the temporal profile of the residual signal.",
"180.An apparatus according to claim 162, further comprising a data spreader operable to spread the data signal to generate a spread data signal having a spread spectrum, wherein the shaper is operable to shape the spread spectrum in dependence upon the residual signal to form the shaped data signal.",
"181.An apparatus according to claim 180, in which the data spreader comprises a first pseudo-noise code generator operable to generate a first pseudo-noise code, wherein the data spreader is operable to perform direct sequence spread spectrum encoding using the first pseudo-noise code.",
"182.An apparatus according to claim 181, wherein the data signal comprises a sequence of data symbols, and wherein the data spreader is operable to combine each data symbol of the data signal with at least part of the first pseudo-noise code.",
"183.An apparatus according to claim 181, wherein the data signal comprises a sequence of data symbols, wherein the data spreader further comprises a second pseudo-noise code generator operable to generate a second pseudo-noise code which is different from the first pseudo-noise code, and wherein the data spreader is operable to represent each data symbol of the data signal by a code sequence from either the first pseudo-noise code or the second pseudo-noise code in dependence upon the value of the data element.",
"184.An apparatus according to claim 183, wherein the second pseudo-noise code generator is operable to generate a second pseudo-noise code which is substantially orthogonal to the first pseudo-noise code.",
"185.An apparatus for embedding a data signal within an audio signal, the apparatus comprising: a first receiver operable to receive the data signal; a second receiver operable to receive the audio signal; a processor operable to process the audio signal to generate a residual signal representative of a difference between a tonal component of the received audio signal and the received audio signal; a shaper operable to shape the spectrum of the data signal in dependence upon the residual signal to form a shaped data signal; and a signal combiner operable to combine the shaped data signal and the audio signal to generate a modified audio signal.",
"186.An apparatus according to claim 185, wherein the signal combiner comprises an adder operable to add the shaped data signal and the audio signal to form the modified audio signal.",
"187.An apparatus according to claim 186, wherein the adder comprises a weighted adder operable to perform a weighted addition in accordance with at least one weighting factor.",
"188.An apparatus according to claim 187, wherein the signal combiner further comprises an adjuster operable to vary the at least one weighting factor.",
"189.An apparatus according to claim 188, wherein the adjuster comprises: a first power monitor operable to determine a power level of the audio signal; a second power monitor operable to determine a power level of the shaped data signal; and a setter operable to set the at least one weighting factor in dependence upon the determined power levels of the audio signal and the shaped data signal.",
"190.An apparatus according to claim 189, wherein the setter is operable to set the at least one weighting factor so that, after weighting, the ratio of the power level of the shaped data signal and the power level of the audio signal is above a predetermined value.",
"191.An apparatus according to claim 189, wherein the setter is operable to set the at least one weighting factor so that, after weighting, the ratio of the power level of the shaped data signal and the power level of the modified audio signal is above a predetermined value.",
"192.An apparatus according to claim 188, wherein the adjuster comprises a user interface for receiving a user signal indicative of the at least one weighting factor.",
"193.An apparatus according to claim 185, wherein the signal combiner comprises: a first adder operable to add the shaped data signal and the residual signal to form a modified residual signal; and a second adder operable to add the modified residual signal and the audio signal to form the modified audio signal.",
"194.An apparatus according to claim 193, wherein the first adder comprises a weighted adder operable to perform a weighted addition of the shaped data signal and the residual signal in dependence upon at least one weighting factor.",
"195.An apparatus according to claim 194, wherein the first adder further comprises an adjuster operable to vary the at least one weighting factor.",
"196.An apparatus according to claim 195, wherein the adjuster comprises a user interface for receiving a user signal indicative of the at least one weighting factor.",
"197.An apparatus according to claim 195, wherein the adjuster comprises: a tonality monitor operable to determine a level of tonality of the audio signal; and a setter operable to set the at least one weighting factor in dependence upon the determined level of tonality of the audio signal.",
"198.An apparatus according to claim 195, wherein the adjuster comprises: a first power level monitor operable to determine a power level of the residual signal; a second power level monitor operable to determine a power level of the shaped data signal; and a setter operable to set the at least one weighting factor in dependence upon the determined power levels of the audio signal and the shaped data signal.",
"199.An apparatus according to claim 198, wherein the setter is arranged to set the at least one weighting factor so that the ratio of the power level of the shaped data signal and the power level of the residual signal is above a predetermined value.",
"200.An apparatus for embedding a data signal within an audio signal, the apparatus comprising: a first receiver operable to receive the data signal; a second receiver operable to receive the audio signal; a processor operable to process the audio signal to generate a residual signal representative of a difference between a tonal component of the received audio signal and the received audio signal; a shaper operable to shape the spectrum of the data signal in dependence upon the residual signal to form a shaped data signal; and a signal generator operable to generate a tonal signal representative of the tonal component of the received audio signal; and a signal combiner operable to combine the shaped data signal and the tonal signal to generate a modified audio signal.",
"201.An apparatus according to claim 200, wherein the signal combiner comprises an adder operable to add the shaped data signal and the tonal signal to form the modified audio signal.",
"202.An apparatus according to claim 200, wherein the signal combiner comprises: a first adder operable to add the shaped data signal and the residual signal to form a modified residual signal; and a second adder operable to add the modified residual signal and the tonal signal to form the modified audio signal.",
"203.An apparatus according to claim 202, wherein the first adder comprises a weighted adder operable to perform a weighted addition of the shaped data signal and the residual signal in dependence upon at least one weighting factor.",
"204.An apparatus according to claim 203, wherein the first adder further comprises an adjuster operable to vary the at least one weighting factor.",
"205.An apparatus according to claim 204, wherein the adjuster comprises a user interface for receiving a user signal indicative of the at least one weighting factor.",
"206.An apparatus according to claim 204, wherein the adjuster comprises: a tonality monitor operable to determine a level of tonality of the residual signal; and a setter operable to set the at least one weighting factor in dependence upon the determined level of tonality of the audio signal.",
"207.An apparatus according to claim 204, wherein the adjuster comprises: a first power monitor operable to determine a power level of the residual signal; means for determining a power level of the shaped data signal; and a setter operable to set the at least one weighting factor in dependence upon the determined power levels of the audio signal and the shaped data signal.",
"208.An apparatus according to claim 207, wherein the setter is arranged to set the at least one weighting factor so that the ratio of the power level of the shaped data signal and the power level of the residual signal is above a predetermined value.",
"209.A method of shaping a data signal for embedding within an audio signal, the method comprising: receiving the data signal; receiving the audio signal; processing the received audio signal to generate a residual signal representative of a difference between a tonal component of the received audio signal and the received audio signal; and shaping the data signal in dependence upon the residual signal to form a shaped data signal.",
"210.A storage device storing instructions including instructions for causing a programmable processing apparatus to become operable to perform a method according to claim 209.211.A signal conveying instructions including instructions for causing a programmable processing apparatus to become operable to perform a method according to claim 209."
],
[
"This invention relates to an acoustic communication system in which a data signal is conveyed by acoustic waves.",
"International patent publication WO 98/32248 describes an acoustic communication system for updating price data displayed on supermarket shelves.",
"In this communication system, acoustic signals are transmitted within the audible frequency range at a power level at which the acoustic signals are virtually inaudible to people within the supermarket.",
"An embodiment of the present invention provides a novel encoding technique for encoding a data signal within an electrical signal which is subsequently converted into a corresponding acoustic signal.",
"Another embodiment of the present invention provides an alternative application for an acoustic communication system in which data is transmitted using acoustic waves and components for the application.",
"Exemplary embodiments of the invention will now be described with reference to the accompanying drawings, in which: FIG.",
"1 schematically shows a signalling system for communicating a data signal to a cellular phone via the audio track of a television signal and for downloading information from the internet to the cellular phone in accordance with the data signal; FIG.",
"2 schematically shows an encoder which forms part of the signalling system illustrated in FIG.",
"1; FIG.",
"3 is a plot comparing the power spectrum of a typical audio track of a television signal with that of a modulated data signal with and without spread spectrum encoding; FIG.",
"4 schematically shows a shaping unit which forms part of the encoder illustrated in FIG.",
"2; FIG.",
"5 is a plot of a power spectrum corresponding to the sensitivity of a human ear with and without the presence of a narrowband tone; FIG.",
"6 schematically shows a cellular phone which forms part of the signalling system illustrated in FIG.",
"1; FIG.",
"7A is a schematic block diagram showing the functional configuration of a processor which forms part of the cellular phone illustrated in FIG.",
"6 when receiving and processing a data signal; FIG.",
"7B is a flow chart illustrating the steps performed by the cellular phone shown in FIG.",
"6 to receive and process a data signal; FIG.",
"8 schematically shows a scaling unit for a first alternative encoder to the encoder illustrated in FIG.",
"2; FIG.",
"9 is a plot illustrating a portion of the audio track, a signal output by the shaping unit illustrated in FIG.",
"4 using the audio track, and a signal output by the shaping unit illustrated in FIG.",
"8 using the audio track; FIG.",
"10 schematically shows a second alternative encoder to the encoder illustrated in FIG.",
"2; FIG.",
"11 is a schematic block diagram showing the functional configuration of a processor of a first alternative cellular phone to the cellular phone illustrated in FIG.",
"6 when demodulating a signal encoded by the second alternative encoder illustrated in FIG.",
"10; FIG.",
"12 schematically shows an audio mixer of a third alternative encoder to the encoder illustrated in FIG.",
"2; FIG.",
"13A is a timing diagram illustrating a typical audio track; FIG.",
"13B is a timing diagram illustrating the predictable portion of the audio track illustrated in FIG.",
"13A; FIG.",
"13C is a timing diagram showing the non-predictable portion of the audio track illustrated in FIG.",
"13A; FIG.",
"13D is a timing diagram showing a spread spectrum data signal; FIG.",
"13E is a timing diagram showing the spread spectrum data signal illustrated in FIG.",
"13D after it has been shaped to approximate the non-predictable portion of the audio track as illustrated in FIG.",
"13C; FIG.",
"13F is a timing diagram showing a modified audio track obtained by combining the predictable portion of the audio track as illustrated in FIG.",
"13B with the shaped spread spectrum signal as shown in FIG.",
"13E; FIG.",
"14 schematically shows an audio mixer for a fourth alternative encoder to the encoder illustrated in FIG.",
"2; FIG.",
"15 schematically shows an audio mixer for a fifth alternative encoder to the encoder illustrated in FIG.",
"2; FIG.",
"16 schematically shows a sixth alternative encoder to the encoder illustrated in FIG.",
"2; FIG.",
"17 is a schematic block diagram showing in more detail a data rate determining unit which forms part of the sixth alternative encoder illustrated in FIG.",
"16; FIG.",
"18 schematically shows a second alternative cellular phone to the cellular phone illustrated in FIG.",
"6; FIG.",
"19 schematically shows a decoder which forms part of the second alternative cellular phone illustrated in FIG.",
"18; FIG.",
"20 shows in more detail an acquisition unit which forms part of the decoder illustrated in FIG.",
"19; FIG.",
"21 shows in more detail a normalisation circuit which forms part of the acquisition unit illustrated in FIG.",
"20; FIG.",
"22 shows in more detail an averaging circuit which forms part of the normalisation circuit illustrated in FIG.",
"21; FIG.",
"23 is a plot of the output of the normalisation circuit of the acquisition unit illustrated in FIG.",
"20 in the presence of a single-path signal; FIG.",
"24 is a plot of the output of a cross-correlator which forms part of the acquisition unit shown in FIG.",
"20; FIG.",
"25 shows in more detail the components of a processor used in the decoder illustrated in FIG.",
"19; FIG.",
"26 shows in more detail a correlate and compare unit which forms part of the decoder illustrated in FIG.",
"19; FIG.",
"27 shows in more detail a power comparator which forms part of the correlate and compare unit illustrated in FIG.",
"26; FIG.",
"28 schematically shows a first alternative signalling system in which data is communicated between two cellular phones via acoustic signals; FIG.",
"29 is a block diagram showing in more detail a cellular phone of the first alternative signalling system illustrated in FIG.",
"28; FIG.",
"30 schematically shows a second alternative signalling system in which a cellular phone encodes a control signal, which varies in accordance with the telephone number of a caller, within a ring signal and a toy detects the ring signal and responds in accordance with the control signal; FIG.",
"31 schematically shows a cellular phone which forms part of the second alternative signalling system illustrated in FIG.",
"30; FIG.",
"32 is schematic block diagram showing the functional configuration of a processor of the cellular phone illustrated in FIG.",
"31 when receiving an incoming call; FIG.",
"33 schematically shows a toy which forms part of the second alternative signalling system illustrated in FIG.",
"30; FIG.",
"34 schematically shows a third alternative signalling system in which a data signal is conveyed between a computer and a toy via an acoustic signal emitted by a cellular telephone; FIG.",
"35 is a schematic block diagram showing the functional configuration of a processor of a cellular phone for the third alternative signalling system illustrated in FIG.",
"34 when processing a received control signal; FIG.",
"36 schematically shows a first alternative server for the signalling system illustrated in FIG.",
"1; FIG.",
"37 schematically shows a fourth alternative signalling system in which a control signal is communicated to a toy via the audio track of a television signal; FIG.",
"38 is a schematic block diagram of a data rate determining unit of an encoder which forms part of the fourth alternative signalling system; FIG.",
"39A is a timing diagram showing a typical audio track; FIG.",
"39B is a timing diagram showing a typical data signal; FIG.",
"39C is a timing diagram showing a modified data signal generated by modifying the data signal illustrated in FIG.",
"39B in accordance with the audio track illustrated in FIG.",
"39A; and FIG.",
"40 schematically shows an alternative encoder for the fourth alternative signalling system illustrated in FIG.",
"36.FIG.",
"1 schematically illustrates a first embodiment of the invention in which a data signal F(t), generated by a data source 1, is encoded within an audio track from an audio source 3 by an encoder 5 to form a modified audio track for a television program.",
"In this embodiment, the data signal F(t) conveys a Uniform Resource Locator (URL) identifying a web page, accessible via the Internet, associated with the television program.",
"The modified audio track output by the encoder 5 is then combined with the corresponding video track, from a video source 7, by a signal generator 9 to form a television signal conveying the television program.",
"In this embodiment the data source 1, the audio source 3, the video source 7 and the encoder 5 are all located in a television studio and the television signal is broadcast by a conventional broadcast network 11 using a radio frequency (RF) signal 13.The RF signal 13 is detected by a television aerial 15 which directs the television signal to a conventional television 17.The television 17 has a display (not shown) for showing the video track and a loudspeaker (not shown) for outputting the modified audio track as an acoustic signal 19.In this embodiment, a cellular phone 21 (sometimes referred to as a mobile phone) detects the acoustic signal 19 emitted by the television 17 using a microphone 23 which converts the detected acoustic signal into a corresponding electrical signal.",
"The cellular phone 21 then decodes the electrical signal to recover the data signal F(t).",
"The cellular phone 21 also has conventional components such as a loudspeaker 25, an antenna 27 for communicating with a cellular telecommunications network, a display 29, a keypad 31 for entering numbers and letters, and menu keys 33 for accessing menu options.",
"In this embodiment, the cellular phone 21 is able to access the Internet using the Wireless Application Protocol (WAP).",
"After the data signal F(t) has been recovered by the cellular phone 21, in response to a user requesting further information about the television program being shown by the television 17 by selecting a menu option using the menu buttons 33, the cellular phone 21 transmits to a base station 35, via RF signals 37, a request to download the web page corresponding to the URL conveyed by the data signal F(t).",
"The base station 35 forwards the request, via a telecommunications network 39, to the server 41 identified in the URL conveyed by the data signal F(t).",
"The server 41 retrieves from a database 43 the requested web page and transmits the requested web page to the cellular phone 21 via the telecommunications network 39 and the base station 35.The user is then able to read the web page on the display 29 and, for example, access further web pages referenced in the requested web page or make a transaction to buy a product associated with the television program.",
"As those skilled in the art will appreciate, an advantageous feature of the above-described signalling system is that conventional television broadcast networks, televisions, cellular communications networks and web pages can be used, although a novel encoder 5 and a novel cellular phone 21 are required.",
"FIG.",
"2 shows in more detail the main components of the encoder 5 of the first embodiment.",
"The data signal F(t) is input to a forward error correction (FEC) unit 51 which separates the data signal F(t) into blocks of 7 data bits and encodes each block of seven data bits using a (15,7) block code, in particular a BCH (Bose, Chaudhuri and Hocquenghem) code, to form a block of 15 data bits.",
"Those skilled in the art will appreciate that the extra bits added by the BCH code enable errors in transmission to be detected and corrected.",
"In this embodiment, the FEC unit 51 also adds a synchronisation bit (hereafter referred to as the SYNC bit) having a value of “0” at the beginning of each block of 15 data bits to generate a modified data signal M(t) formed by 16-bit frames of data.",
"In this embodiment, the modified data signal M(t) is a logic signal which is generated at approximately 7.8 bits per second.",
"An advantageous feature of the first embodiment is that a spread spectrum encoding technique is used to spread the energy of the modified data signal M(t) over a wide range of frequencies.",
"This has the effect of making the data signal less noticeable in the acoustic signal 19 emitted by the television 17.In particular, if the modified data signal M(t) is directly combined with the audio track without such coding, then it is more likely to be heard by a viewer of the television 17.In this embodiment, direct sequence spread spectrum (DSSS) encoding is used to spread the energy of the data signal over a wide band of frequencies.",
"In order to perform the DSSS encoding, a first pseudo-noise code generator 53a is used to generate a pseudo-noise code PN0 and a second pseudo-noise code generator 53b is used to generate a pseudo-noise code PN1.As those skilled in the art of telecommunications will appreciate, pseudo-noise codes are binary codes which appear to be completely random in nature, but which are in fact deterministic, i.e.",
"they can be reproduced.",
"In particular, these codes are generated by exclusive-OR (XOR) feedback from synchronously clocked registers.",
"By continually clocking the registers, the pseudo-noise code is cyclically reproduced.",
"The number of registers, the registers used in the feedback path and the initialisation state of the registers determines the length of the code and the specific code produced.",
"In this embodiment, the pseudo-noise code generators 53 have 12 registers and generate pseudo-noise codes having 4095 bits (which will hereinafter be referred to as chips using the standard nomenclature in the art to distinguish the bits of the pseudo-noise code from the bits of the data signal to be spread) in a stream with no sequence of more than 12 chips repeated in the 4095 chips.",
"Such a pseudo-noise code is conventionally referred to as a 12-bit code after the number of registers used to generate it.",
"At the end of each stream of 4095 chips a binary 0 is added to make the total length of the stream 4096 chips.",
"In this embodiment, the pseudo-noise codes PN0 and PN1 are orthogonal to each other and therefore if they are sequentially input chip by chip into respective inputs of an XOR gate then another pseudo-noise sequence is output by the XOR gate.",
"The output of the first pseudo-noise code generator 53a is connected to a non-inverting input of a first AND gate 55a while the output of the FEC unit 51 is connected to an inverting input of the first AND gate 55a.",
"The output of the FEC unit 51 is also connected to a non-inverting input of a second AND gate 55b and the output of the second pseudo-noise code generator 53b is connected to a non-inverting input of the second AND gate 55b.",
"The outputs of the first and second AND gates 55 are connected together to generate a spread data signal I(t) which corresponds to PN0 when the value of the modified data signal M(t) is 0 and corresponds to pseudo-noise code PN1 when the value of the modified data signal M(t) is 1.In this embodiment, each pseudo-noise code is generated at a rate of 2000 chips per second so that each data bit is multiplied by a sequence of 256 chips and each data frame is multiplied by 4096 chips.",
"As each data frame corresponds to a complete cycle of the pseudo-noise codes PN0 and PN1, the SYNC bit, which has a data value of “0”, is always represented by the same 256 chip sequence of the pseudo-noise code PN0.This allows a decoder to synchronise itself with a received chip sequence more easily.",
"The logic signal I(t) is input to a modulator 57 which uses phase shift keying to modulate a 2 kHz carrier signal generated by a local oscillator (not shown) in accordance with the value of the logic signal I(t).",
"In particular, the carrier signal is a digital signal having a sample rate of 8 kHz and a 16-bit value per sample.",
"Each chip of the logic signal I(t) therefore modulates a sequence of four samples of the carrier signal.",
"In this embodiment, the modulator 57 includes a re-sampling circuit (not shown) which re-samples the modulated signal at the sampling rate of the audio track, which in this embodiment is 22.05 kHz, to generate a modulated signal G(t).",
"The effect of the spread spectrum encoding is illustrated in FIG.",
"3 which shows a typical audio signal 71 in the frequency range of 0 to 6 kHz with, as is normally the case, the power being predominantly concentrated at the lower frequencies.",
"FIG.",
"3 also shows a modulated data signal 73 which would result if no spreading was carried out and the modified data signal M(t) was directly modulated by the modulator 57.As shown, this modulated data signal 73 is a narrow band signal centred at approximately 2 kHz and having a peak power significantly above the power level of the audio signal 71 at that frequency.",
"However, if spreading is performed as well as modulating, a spread signal 75 is obtained which has a power spectrum with a main band spread between 0 and 4 kHz and harmonic bands at higher frequencies.",
"As the power of the spread signal 75 is distributed over a wider range of frequencies than for the modulated signal 73, the peak power level is significantly reduced.",
"For many applications the spread signal 75 is not noticeable to a listener or is heard only as a background white noise.",
"Further, the majority of the energy of the main band is in a frequency range for which most conventional television loudspeakers work satisfactorily.",
"There is, therefore, no requirement for a user to obtain a new television set to take advantage of the invention.",
"Returning to FIG.",
"2, the modulated signal G(t) and the audio track are input to an audio mixer 59 where they are combined to form the modified audio track.",
"In particular, in the audio mixer 59 the modulated signal G(t) and the audio track are input to a shaping unit 61 which analyses the power spectrum of the audio track and modifies the power spectrum of the modulated signal G(t) in dependence upon the power spectrum of the audio track in order to generate a shaped signal S(t) which is less obtrusive when combined with the audio track.",
"FIG.",
"4 shows in more detail the main components of the shaping unit 61.The audio track, which in this embodiment is generated at 22,050 samples per second with each sample having a 16-bit value, is input to a first Fast Fourier Transform (FFT) unit 81 which splits the audio track into blocks each having 2048 samples applying conventional overlapping and windowing techniques.",
"The FFT unit 81 measures for each block the energy in 2048 frequency sub-bands to generate 2048 coefficients which are output to a scaling unit 83.The scaling unit 83 applies a psycho-acoustic algorithm to generate scaling factors for the 2048 frequency sub-bands.",
"In particular, the scaling unit includes a psycho-acoustic filter which outputs, for each frequency sub-band of each block of samples, a scaling factor based on the energy in that and neighbouring frequency sub-bands for that block of samples and for preceding and succeeding neighbouring blocks of samples.",
"Using a psycho-acoustic algorithm allows higher signal levels to be used than if a simple scaling algorithm was applied because it takes into account the dynamic variation of sensitivity of the human ear in the presence of sounds.",
"This will be explained further with reference to FIG.",
"5 which shows the sensitivity of a typical human ear for different frequencies (in other words, the minimum sound levels for different frequencies which can be heard by a typical human ear) without any background noise (the plot referenced as 101) and in the presence of a narrow band signal 103 (the dashed plot referenced as 105).",
"As can be seen from FIG.",
"5, the ability of the human ear to distinguish sound in the frequency range of the narrow band signal 103 and in a range of frequencies both above and below the frequency range of the narrow band signal 103 is significantly reduced.",
"There are therefore audio signals which cannot be heard by the human ear in the presence of the narrow band signal 103, even though they would be heard if the narrow band signal 103 is not present.",
"In this embodiment, the psycho-acoustic algorithm uses channel descriptors, which are stored in a memory 85, to approximate how the audio track will be modified by the broadcast network 11 and television 17 when determining the scaling factors for each of the 2048 frequency sub-bands.",
"Psycho-acoustic algorithms have been previously investigated for data compression, since sounds which would not be discerned by a listener can be removed from an audio data stream.",
"Further details of psycho-acoustic encoding can be found in the paper “Transform Coding of Audio Signals Using Perceptual Noise Criteria” by James D. Johnston, IEEE Journal on Selected Areas in Communications, Vol.",
"6, No.",
"2, February 1988, whose contents are hereby incorporated by reference.",
"The modulated signal G(t) is input to a second FFT unit 87 which, in the same manner as the FFT unit 81, splits the modulated signal G(t) into blocks of 2048 samples and generates 2048 coefficients corresponding to the energy in 2048 frequency sub-bands for each block.",
"In this embodiment, the 2048 frequency sub-bands for the second FFT unit 87 match the 2048 frequency sub-bands of the first FFT unit 81.The 2048 coefficients output by the second FFT unit 87 and the 2048 scaling factors output by the scaling unit 83 are input to a multiplier 89 where the coefficient for each frequency sub-band of the modulated signal G(t) is multiplied by the corresponding scaling factor for that frequency sub-band determined from the audio track.",
"The scaled coefficients output by the multiplier 89 are then input to an Inverse Fast Fourier Transform (IFFT) unit 91 which generates the shaped signal S(t) from the scaled coefficients, taking into account the windowing and overlapping.",
"Returning to FIG.",
"2, the audio track is also input to a time delay unit 63 which delays the audio track for a period of time corresponding to the time required for the shaping unit 61 to generate the shaped signal S(t) using the audio track.",
"The audio track output from the time delay unit 63 and the shaped signal S(t) are input to an adder 65 which performs a simple linear adding operation to combine them to form the modified audio track.",
"The purpose of the time delay unit 63 is to ensure that each portion of the audio track is combined in the adder 65 with the portion of the shaped signal S(t) which has been shaped in accordance with the power spectrum of that portion of the audio track.",
"As described above, the modified audio track is combined with a video track to form a television signal which is then broadcast over a conventional television network 11.An aerial 15 detects the broadcast television signal 13 and directs a corresponding electrical signal to a television 17 which outputs an acoustic signal 19 corresponding to the modified audio track using a loudspeaker (not shown).",
"The output acoustic signal 19 is then detected by the microphone 23 of the cellular phone 21.FIG.",
"6 shows in more detail the main components of the cellular phone 21 of the first embodiment.",
"As shown, the microphone 23 of the cellular phone 21 converts the detected acoustic signal 19 into a corresponding electrical signal H(t) which is input to an anti-aliasing filter 111.The filtered signal output by the anti-aliasing filter 111 is input to an analog-to-digital converter (ADC) 113 which converts the filtered signal into a corresponding digital signal D(t) using a sampling rate of 8 kHz.",
"The output of the ADC 113 is connected to an AUDIO_IN input of a processor 115.Digital signals output via an AUDIO_OUT output of the processor 115 are input to a digital-to-analog converter (DAC) 117 which converts the digital signals into corresponding analog signals which are then amplified by an amplifier 119 before being output as acoustic waves by the loudspeaker 25.A DISP_OUT output of the processor 115 is connected to the display 29 while a KEY_IN input to the processor 115 is connected to the keyboard 31.An RF_OUT output of the processor 115 is connected to a radio frequency (RF) processor unit 121 which processes baseband digital signals output by the processor 115 to form RF signals for broadcast via the antenna 27.The RF processor unit 121 also processes incoming RF signals received via the antenna 27 to form baseband signals which are input to an RF_IN input of the processor 115.A random access memory (RAM) 123, a non-volatile random access memory (NVRAM) 125 and a read only memory (ROM) 127 are also connected to the processor 115.The NVRAM 125 stores data associated with the user of the cellular phone 21, for example a phone book listing the names and associated phone numbers of commonly called people, and the RAM 123 provides working space for use during the operation of the processor 115.The ROM 127 stores routines which control the operation of the processor 115.In particular, the ROM 127 stores routines which enable the processor to operate in three different modes.",
"In a call mode, which is activated when the cellular phone 21 is used during a telephone call, acoustic signals received by the microphone 23 are converted by the processor 115 into baseband signals which are output to the RF processor unit 121 and incoming baseband signals from the RF processor unit 121 are converted into audio signals which are output by the loudspeaker 25.In a standby mode, which is the default mode when no telephone call is being made, the processor 115 waits for either an RF signal initiating a call to be detected by the antenna 27 or for one of the keys of the keyboard 31 to be pressed.",
"In the standby mode the processor 115 does not process acoustic signals detected by the microphone 23.The call mode and standby modes are conventional modes of operation for a cellular phone.",
"In the third mode, hereafter called the monitor mode, the processor 115 processes acoustic signals 19 received by the microphone 23 to recover any data signal embedded within the acoustic signal 19 in addition to performing the operations of the standby mode.",
"The operation of the processor 115 in the monitor mode will now be described in more detail with reference to FIGS.",
"7A and 7B.",
"FIG.",
"7A is a schematic block diagram showing the functional configuration of the processor 115 during the monitor mode and FIG.",
"7B shows a flow chart illustrating the main steps performed by the processor 115 in the monitor mode.",
"The monitor mode is initiated, in step S1, by the user selecting a menu option using the menu keys 33.Once the monitor mode has been initiated, the processor 115 receives, in step S3, the digital signal D(t) conveying audio data from the microphone 23 via the AUDIO_IN input of the processor 115.The digital signal D(t) is then processed by a demodulator 130 which demodulates, in step S5, the digital signal D(t) using conventional digital signal processing techniques.",
"The demodulated digital signal is then processed by a despreading module 131 which despreads, in step S7, the audio data.",
"This despreading involves monitoring for the chip sequence corresponding to the SYNC bit which, when detected, is processed to determine the timing of the received chip sequence and the slight frequency offset between the chip rate of the received chip sequence and the chip rate of the pseudo-noise codes generated within the cellular phone 21.In general, there will be a slight frequency offset which arises from three main causes.",
"The first cause is that many transmission media slightly vary the transmission rate of the audio track, thereby varying the chip rate of the chip sequence conveyed by the audio track.",
"The second cause is that Doppler effects can occur, for example due to movement of the cellular phone 21, which affects the chip rate in the detected signal.",
"Finally, the third main cause is that there is always a slight difference between the clock frequencies used to generate the pseudo-noise codes in the encoder 5 and the cellular phone 21.The despreading module 131 is then able to determine from the received chip sequence transitions between the pseudo-noise codes PN0 and PN1, thereby recovering the modified data signal.",
"The recovered modified data signal is then processed by a data signal regenerator 132 which recovers, in step S9, the original data signal F(t) by removing the synchronisation bit and decoding the remaining (15,7) BCH code, thereby recovering the URL associated with the television programme.",
"After the URL has been recovered, the data signal regenerator 132 outputs, in step S11, a signal to a signal generator 133 which sends a control signal to a display driver 134 which in turn outputs a drive signal to the display 29 causing the display 29 to show a message received indicator.",
"The message received indicator enables the user of the cellular phone 21 to see that further information about the television program is available from the internet.",
"The data signal regenerator 132 also stores the URL in the RAM 123.The processor 115 then waits, in step S13, for the user to request further information.",
"If the user does not request further information within a preset time, the routine returns to step S3.If, however, the user selects the menu option for requesting further information then this selection is input to the keyboard interface 135 which sends a signal to a browser 136 causing the browser 136 to access, in step S15, the web page corresponding to the URL conveyed by the original data signal F(t).",
"In particular, the browser retrieves the URL from the RAM 123 and outputs a baseband signal, via the RF_OUT output, which is converted to an RF signal by the RF processor unit 121 and is broadcast by the antenna 27.Subsequently, a RF signal conveying web page data for the IP address is detected by the antenna 27 and converted to a baseband signal by the RF processor unit 121, and the baseband signal is input to the RF_IN input of the processor 115.In the processor 115, the web page data is processed by the browser 136 which sends the web page data to the display driver 134 which in turn outputs, in step S17, a drive signal causing the display 29 to display the received web page.",
"Those skilled in the art will appreciate that processors typically used in digital cellular phones are well suited to be programmed to perform the digital signal processing required in the monitor mode.",
"As described above, in the first embodiment a cellular phone 21 retrieves from the acoustic signal 19 corresponding to the audio track of a television program an IP address for a web page associated with that program so that the user of the cellular phone can download additional information about the television program.",
"By using spread spectrum encoding techniques to generate a spread signal and by shaping the spread signal in dependence upon the power spectrum of the audio track, the data embedded within the audio track can be hidden from a listener.",
"An advantage of encoding the data signal within the acoustic signal is that the bandwidth required to electronically transmit the audio track after the data signal has been encoded therein is no more than that required to transmit the audio track by itself.",
"In the first embodiment, the shaping unit 61 analyses the frequency spectrum of the modulated signal G(t) to generate a set of frequency-dependent coefficients which are then scaled by scaling factors determined by analysing, using a Fast Fourier Transform, the frequency spectrum of a segment of the audio track.",
"In this way, the shaping unit 61 is able to identify peaks of the audio track in the frequency domain and scale the frequency spectrum of the spread signal G(t) accordingly.",
"A second embodiment will now be described, with reference to FIGS.",
"8 and 9, in which the shaping unit of the first embodiment is replaced by an alternative shaping unit which performs a wavelet transform instead of a Fast Fourier Transform.",
"The remaining components of the signalling system of the second embodiment are the same as those for the first embodiment and will not therefore be described again.",
"FIG.",
"8 shows the main components of the shaping unit 141 of the second embodiment.",
"As shown, the audio track is input to a first wavelet transform (WT) unit 143 which splits the audio track into blocks having 2048 samples.",
"For each block, the first WT unit 143 decomposes the sequence of samples into a linear superposition of 2048 weighted basis functions, with the basis functions forming a wavelet family.",
"As those skilled in the art will appreciate, the basis functions of a wavelet family differ in both the frequency and the time domain.",
"The weighting coefficients therefore convey both time domain and frequency domain information.",
"The first WT unit 143 then outputs the 2048 weighting coefficients for the basis functions to a scaling unit 145 which applies a psycho-acoustic algorithm, using channel descriptors from a channel descriptor memory 147, to generate for each wavelet basis function a corresponding scaling factor.",
"The modulated signal G(t) is input to a second WT unit 149 which splits the modulated signal G(t) into blocks of 2048 samples and decomposes each block, using the same wavelet basis functions as the first WT unit 143, to generate 2048 weighting coefficients.",
"The second WT unit 149 outputs the 2048 weighting coefficients to a multiplier 151 where each weighting coefficient is multiplied by the scaling factor determined by the scaling unit 145 for the corresponding wavelet function using the audio track.",
"The 2048 scaled coefficients output by the multiplier 151 are input to an inverse wavelet transform (IWT) unit 153 which synthesises a scaled signal S(t) by superposing the family of wavelet basis functions weighted by the respective scaled coefficients output by the multiplier 151.Those skilled in the art will appreciate that by carefully selecting the family of wavelet basis functions, bearing in mind the expected profile of the audio track, the number of weighting coefficients required in order to synthesise the shaped signal S(t) satisfactorily is no more than the number required using a FFT.",
"For some families of wavelet basis functions, it is even possible to use fewer coefficients than are required for a FFT.",
"The time localisation of the wavelet functions is advantageous for shaping the modulated signal G(t) because the profile of a block of the modulated signal G(t) can be shaped in the time domain as well as in the frequency domain.",
"This will be explained further with reference to FIG.",
"9 which shows a block of samples of the audio track (the plot referenced 157), a FFT-shaped signal 159 generated by analysing the block of the audio track using a FFT, and a WT-shaped signal 161 generated by analysing the segment of the audio track using a wavelet transform.",
"As shown in FIG.",
"9, the power spectrum of the audio track is close to zero for the second half of the block of samples.",
"When using FFT analysis, this zero amplitude can only be achieved by destructive interference between the basis functions (i.e.",
"sine and cosine waves), which will depend upon the exact phase of each basis function.",
"However, when using a Fast Fourier Transform the coefficients generated for the modulated signal G(t) have magnitudes which are determined only by the frequency spectrum of the block of the audio track and contain no phase information.",
"The destructive interference required to obtain near-zero power will not therefore generally occur and the FFT-shaped signal 159 does not follow the temporal profile of the audio track.",
"On the other hand, as the wavelet basis functions are localised in time, when using a wavelet transform it is not necessary to rely upon destructive interference to produce zero amplitude and therefore the WT-shaped signal 161 more closely matches the temporal profile of the audio track.",
"The time localisation obtainable using wavelet analysis also has the advantage that psycho-acoustic analysis can be performed in the time domain within a single block of samples.",
"In particular, a similar psycho-acoustic effect to that described above for the frequency domain, in which frequency components which would otherwise be audible can be masked by neighbouring frequency components, also exists in the time domain in that after a loud sound stops, the human ear does not immediately recover the sensitivity indicated by the plot 101 in FIG.",
"5.Therefore, it is possible to increase the amplitude of the modulated signal G(t) immediately after a peak in the sound of the audio track without it becoming overly noticeable to a listener.",
"In the first and second embodiments a 2 kHz carrier signal is modulated using phase shift keying in accordance with the logic signal I(t) to produce a spread signal G(t) having a power spectrum with a main band spread between 0 and 4 kHz.",
"However, the energy spectrum of the audio track could be concentrated in a frequency range away from 2 kHz, in which case the power level of the shaped signal (and hence the signal to noise ratio) needs to be low if the data signal is not to be noticed by a listener.",
"A third embodiment will now be described with reference to FIGS.",
"10 and 11 in which the encoder of the first embodiment is replaced by an alternative encoder in which the frequency of the carrier wave is varied in accordance with the frequency spectrum of the audio track, and the software stored in the ROM of the cellular phone is modified to account for this variation in frequency during decoding.",
"The remaining components of the signalling system of the third embodiment are the same as those of the first embodiment and will not therefore be described again.",
"FIG.",
"10 shows the main components of the encoder 163 of the third embodiment.",
"In FIG.",
"10, components which are the same as corresponding components of the encoder of the first embodiment have been referenced with the same numerals and will not be described again.",
"As shown, the audio track is input to a spectrum analyser 164 which monitors the frequency spectrum of the audio track.",
"In particular, the spectrum analyser 164 monitors the energy in 2048 frequency sub-bands, each corresponding to 10 Hz, and determines the set of 200 adjacent frequency sub-bands which has the greatest energy.",
"The spectrum analyser 164 then outputs a control signal to a modulator 165 to adjust the frequency of the local oscillator so that the carrier signal is in the centre of the frequency range corresponding to the identified set of frequency sub-bands.",
"The modulated signal G(t) output by the modulator 165 is then shaped and combined with the audio track to form the modified audio track in the same manner as described above in the first embodiment.",
"In this embodiment, the cellular phone is substantially as described in the first embodiment except that when the processor is configured for the monitor mode, the demodulator 130 shown in FIG.",
"7A is in effect replaced by the demodulator module 166 shown in FIG.",
"11.As shown, the digital signal D(t) received at the AUDIO-IN input is processed by a spectrum analyser 167 which determines, in the same manner as the spectrum analyser 164, the frequency spectrum of the digital signal D(t) and outputs a signal identifying the centre frequency.",
"The digital signal D(t) is also delayed by a time delay module 168 and then processed by a demodulator 169 whose carrier frequency is controlled by the signal output by the spectrum analyser 167 to match the centre frequency.",
"The purpose of the time delay unit 168 is to ensure that each part of the digital signal D(t) is demodulated using the carrier frequency calculated for that part.",
"The demodulated signal output by the demodulator 169 is processed by the despreading module 131 and the process continues as described with reference to FIGS.",
"7A and 7B.",
"In this embodiment, the carrier frequency is varied so that the main band of the modulated signal G(t) is moved to a frequency range in which the energy of the audio track is relatively large.",
"This has the effect that the scaling factors determined by the shaping unit 61 are generally larger than if the carrier frequency was kept constant.",
"In this way, the power of the shaped signal S(t) is increased.",
"In the first to third embodiments, the modulated signal G(t) is combined with the audio track after having been scaled in order to reduce its noticeability in the modified audio track.",
"A fourth embodiment will now be described with reference to FIGS.",
"12 and 13 in which the encoder of the first embodiment is replaced by an alternative encoder which removes part of the audio track, shapes the modulated signal G(t) to approximate the removed part of the audio track, and then adds the shaped signal to the remaining part of the audio track.",
"The remaining components of the signalling system of the fourth embodiment are the same as those for the first embodiment and will not therefore be described again.",
"FIG.",
"12 shows the main components of the audio mixer 171 of the fourth embodiment.",
"As shown, the audio track is input to a linear predictive coding (LPC) unit 173 which uses conventional linear predictive coding techniques to analyse the audio track.",
"Linear predictive coding takes advantage of the fact that for many sounds the power at any instant is strongly dependent on the power in preceding instants in order to generate a small number of linear prediction (LP) coefficients, sometimes called predictors, from which the sound can be reproduced.",
"In particular, a block of samples is analysed to determine the LP coefficients a1, a2 .",
".",
".",
"an which, when used in the equation: s(k)=a1s(k−1)+a2s(k−2)+ .",
".",
".",
"+ans(k−n)+ξ(k) (1) where s(k) is the value of the k-th sample, give the smallest set of error values ξ(k) for the block.",
"In this embodiment, the LPC unit 173 splits the audio track into blocks of 2048 samples and determines, using a maximum likelihood algorithm, a set of 10 LP coefficients for each block.",
"These 10 LP coefficients model (through equation (1) above) the tonal components of the audio track to be regenerated.",
"The determined LP coefficients are output by the LPC unit 173 and input to a conventional synthesis unit 175 which for each sample synthesises, using the LP coefficients and equation (1) above, a predicted sample value corresponding to s(k)−ξ(k).",
"The sequence of predicted values output by the synthesis unit 175 form a tonal part P(t) of the audio track.",
"The audio track is also input to a time delay unit 177 which introduces a time delay corresponding to the processing time required to analyse the audio track in the LPC unit 173 and to synthesize the tonal part P(t) in the synthesis unit 175.The output of the time delay unit 177 and the output of the synthesis unit 175 are input to a subtractor 179 in which the tonal part P(t) is subtracted sample by sample from the actual audio track so that the subtractor 179 outputs a noise part N(t) which corresponds to the error values ξ(k), in other words the random part of the audio track (sometimes referred to as the residue, the residual part or the atonal part).",
"The noise part N(t) is input to a first FFT unit 181 which splits the noise part N(t) into blocks each having 2048 samples, using conventional windowing and overlapping techniques, and outputs for each block 2048 frequency-dependent coefficients corresponding to the energy in 2048 frequency sub-bands.",
"Similarly, the modulated signal G(t) is input to a second FFT unit 183 which, in the same manner as the first FFT unit 181, splits the modulated signal G(t) into blocks of 2048 samples and generates 2048 coefficients corresponding to the energy in 2048 frequency sub-bands for each block.",
"The 2048 coefficients output by the second FFT unit 183 are input to a multiplier 185.The 2048 coefficients output by the first FFT unit 181 are input to a scaling unit 187 which calculates and outputs 2048 scaling factors which are input to the multiplier 185 where the co-efficient for each frequency sub-band of the modulated signal G(t) is multiplied by the corresponding scaling factor for that frequency sub-band determined from the noise part N(t).",
"The scaling unit 187 applies a scaling algorithm to calculate the scaling factors so that when the scaled coefficients output by the multiplier 189 are input to an IFFT unit 189, which generates a shaped signal S(t) using the scaled coefficients, the shaped signal S(t) approximates the noise part N(t).",
"The tonal part P(t) output by the synthesis unit 175 is also input to a time delay unit 191 which delays the tonal part P(t) by a time corresponding to the time required to generate and process the noise part N(t) and to scale the modulated signal G(t) using the noise part N(t) to generate the shaped signal S(t).",
"The output of the time delay unit 191 and the shaped signal S(t) are then input to an adder 193 which performs a linear adding operation to generate the modified audio track.",
"In this way, the noise part N(t) of the audio track is replaced by a version of the modulated signal G(t) which has been shaped to approximate the noise part N(t) of the audio track in order to form the modified audio track.",
"FIGS.",
"13A to 13F are plots which illustrate the signals at different points in the audio mixer 171.FIG.",
"13A shows the audio track which is input to the audio mixer 171.FIG.",
"13B shows the tonal part P(t) which is output by the synthesis unit 175 and FIG.",
"13C shows the noise part N(t) which is obtained by subtracting the tonal part P(t) from the audio track.",
"FIG.",
"13D shows the modulated signal G(t) input to the audio mixer 171 and FIG.",
"13E shows the shaped signal S(t) output by the IFFT unit 191.FIG.",
"13F shows the modified audio track formed by adding the tonal part P(t) shown in FIG.",
"13B and the shaped signal S(t) shown in FIG.",
"13E.",
"A comparison of FIGS.",
"13A and 13F shows that the profile of the modified audio track is approximately the same as the profile of the original audio track.",
"In the fourth embodiment, the noise part of the audio track, which is particularly associated with hiss-like sounds, is removed from the audio track and replaced by the shaped signal S(t).",
"A fifth embodiment of the invention will now be described with reference to FIG.",
"14 in which the encoder of the first embodiment is replaced by an encoder which modifies the relative amplitudes of the audio track and the shaped signal in dependence upon the level of tonality of the audio track.",
"The remaining components of the fifth embodiment are identical to those of the first embodiment and will not therefore be described again.",
"FIG.",
"14 shows the main components of the audio mixer 195 of the fifth embodiment.",
"As shown, the audio track is input to a first FFT unit 197 which splits the audio track into blocks of 2048 samples using conventional overlapping and windowing techniques and, for each block, determines the energy in 2048 frequency sub-bands to generate 2048 coefficients which are output to a scaling unit 199 and a spectrum analyser 201.The scaling unit 199 applies the same scaling algorithm as that used in the fourth embodiment to generate 2048 scaling factors which are output to a multiplier 203.The modulated signal G(t) is input to a second FFT unit 205 which, in the same manner as the first FFT unit 197, splits the modulated signal G(t) into blocks of 2048 samples and for each block generates 2048 coefficients corresponding to the energy in 2048 frequency sub-bands which match the frequency sub-bands for the first FFT unit.",
"The 2048 coefficients output by the second FFT unit 205 are input to the multiplier 203 where each co-efficient is multiplied by the scaling factor, output by the scaling unit 199, for its respective frequency sub-band.",
"The 2048 scaled coefficients output by the multiplier 203 are input to an IFFT unit 207 which generates the shaped signal S(t) from the scaled coefficients.",
"The spectrum analyser 201 determines the level of tonality of each block of the audio track from the 2048 coefficients output by the first FFT unit 197.In particular, this is achieved by statistically analysing the 2048 coefficients to determine if a tonal signal is present, which will be indicated by peaks in a small number of frequency sub-bands, or if no tonal signal is present in which case the frequency coefficients for the sub-bands will vary more randomly.",
"The spectrum analyser 201 then outputs a signal indicative of the level of tonality which, in this embodiment, is a four bit binary number which varies from 0000 for when the audio track is almost purely noise-like to 1111 when the audio track is almost purely tonal.",
"The audio track is also input to a time delay unit 209 which delays the audio track for a time corresponding to the time required to analyse a portion of the audio track and generate the shaped signal S(t) for that portion.",
"The output of the time delay unit 209, the shaped signal S(t), and the four bit binary number output by the spectrum analyser 201 are input to respective input ports of a variable combiner 211.In the variable combiner 211, the delayed audio track is input to a variable amplifier 213a where it is multiplied by a gain factor G determined from the four bit number output by the spectrum analyser 201 and the shaped signal S(t) is input to a second variable amplifier 213b where it is multiplied by a gain factor of (1-G).",
"The outputs of the first and second variable amplifiers 213 are then added together by an adder 215 to generate the modified audio track.",
"In the fifth embodiment, the audio track and the shaped signal S(t) are weighted in accordance with the level of tonality of the audio track and then added together to form the modified audio track.",
"In particular, the more noise-like the audio track the greater the proportion of the modified audio track which is formed by the shaped signal S(t).",
"Further, in the fourth embodiment the noise-like part of the audio track is removed and replaced by the shaped signal S(t).",
"However, a problem with the fourth and fifth embodiments is that if the audio track is very tonal then the noise-like part is small and the shaped signal S(t) cannot be added at sufficiently high signal levels for it to be reliably decoded in the decoder.",
"Therefore, if the audio track is very tonal, the psycho-acoustic shaping technique described in the first embodiment is preferred.",
"A sixth embodiment will now be described with reference to FIG.",
"15 in which the encoder of the first embodiment is replaced by an alternative encoder which combines the psycho-acoustic shaping technique described in the first embodiment, the linear predictive coding technique described in the fourth embodiment and the weighting technique described in the fifth embodiment.",
"In the encoder of the sixth embodiment, a user is able to set parameters which determine the nature of the encoding.",
"This is advantageous because the masking ability of the audio track is subjective and therefore better masking is generally obtained if the user can set the encoding parameters than if the encoding parameters were automatically set.",
"The remaining components of the sixth embodiment are identical to those of the first embodiment and will not therefore be described again.",
"FIG.",
"15 shows the main components of the audio mixer 217 of the sixth embodiment.",
"Components which are the same as corresponding components in the audio mixer of the fourth embodiment have been referenced with the same numerals and will not be described again.",
"As shown, the audio track is input to the LPC unit 173 which determines and outputs LP coefficients corresponding to the tonal part P(t) for sequential blocks of 2048 samples.",
"In this embodiment, a user interface 218 is provided via which a user can set the number of LP coefficients used in the LPC unit 173.In response to the user setting the number of LP coefficients, the user interface 218 outputs a control signal to the LPC unit 173 which causes the LPC unit 173 to use the selected number of LP coefficients.",
"As in the fourth embodiment, the noise part N(t) is obtained by inputting the LP coefficients output by the LPC unit 173 to a synthesis unit 175 in order to generate the tonal part P(t), and then subtracting the tonal part P(t) from the audio track using the subtracter 179.In this embodiment, the noise part N(t) is input, via a time delay unit 219, to a first input port of a variable combiner 220 as well as to the first FFT unit 181.The coefficients output by the first FFT unit 181 are input to a scaling unit 221 which applies a psycho-acoustic algorithm, using channel descriptors from a channel descriptors memory 222, to generate 2048 scaling factors which are input to the multiplier 185 to multiply respective ones of the 2048 coefficients output by the second FFT unit 183 to generate the scaled coefficients for forming the shaped signal S(t).",
"In this embodiment, the user is able to set the level of the psycho-acoustic encoding via the user interface 218.In particular, the scaling unit applies a psycho-acoustic filter and, in response to the user setting the level of the psycho-acoustic encoding, the user interface outputs a control signal which determines the bandwidth of the psycho-acoustic filter (i.e.",
"the number of neighbouring frequency sub-bands and blocks of data samples whose energy is taken into account when determining the scaling factors).",
"If the highest level of psycho-acoustic encoding is desired, then the bandwidth is set so that the scaling unit 221 effectively acts in the same manner as the scaling unit of the first embodiment.",
"If, however, the lowest level of psycho-acoustic encoding is desired, then the bandwidth is set so that the scaling unit 221 effectively acts in the same manner as the scaling unit of the fourth embodiment.",
"The shaped signal S(t) output by the IFFT unit 189 is input to a second input port of the variable combiner 220.In this embodiment, a user is able to select using the user interface 218 the gain factor G used in the variable combiner 220, in response to which the user interface 218 outputs a signal to a third input port of the variable combiner 220.In the variable combiner 220, the delayed noise part N(t) is input to a variable amplifier 223a where it is multiplied by the gain factor G and the shaped signal S(t) is input to a second variable amplifier 223b where it is multiplied by a gain factor (1-G).",
"The output of the first and second variable amplifiers 223 are then added together by an adder 224 to form a modified noise part N′(t).",
"The purpose of the time delay unit 219 is to ensure that each portion of the noise part N(t) is combined in the variable combiner 220 with the portion of the shaped signal S(t) which was shaped using that portion of the noise part N(t).",
"The tonal part P(t) output by the synthesis unit 175 is input, via a time delay unit 225, to the adder 195 along with the output of the variable combiner 220.The adder 195 performs a simple linear addition to generate the modified audio track.",
"The purpose of the time delay unit 225 is to ensure that the tonal part P(t) in synchronised with the modified noise part N′(t).",
"In this embodiment, the audio mixer 217 allows a user to adjust the encoding in accordance with the tonality of the audio track to improve the masking of the data by the audio track.",
"For example, if there is only a small atonal portion to the audio track, the user can reduce the number of LP coefficients used by the LPC unit 173 in order to increase the energy in the noise part N(t) because less of the audio track is modelled by the LPC unit 173.Reducing the number of LP coefficients also has the effect of increasing the tonal features within the noise part N(t).",
"In the extreme case, the number of LP coefficients can be set to 0 so that the noise part N(t) is the same as the audio track.",
"As the tonal features within the noise part N(t) increase, the user can set the scaling unit 221 to apply a wider bandwidth psycho-acoustic filter to take advantage of the masking ability of these tonal features.",
"The user can subsequently set the gain factor G of the variable combiner to 0.9 so that 90% of the modified noise part N′(t) is formed by the noise part N(t) and 10% of the modified noise part N′(t) is formed by the shaped signal S(t).",
"If, however, the audio track is predominantly atonal, then the user can increase the number of coefficients used by the LPC unit 173, decrease the bandwidth of the psycho-acoustic filter applied by the scaling unit 221 and set the value of the gain factor G of the variable combiner 220 to zero so that the modified noise part N′(t) is identical to the shaped signal S(t).",
"In this embodiment the user can adjust the number of coefficients of the LPC unit 173, the bandwidth of the psycho-acoustic filter and the gain factor G freely.",
"This allows the user to determine, for a required signal to noise ratio, the set-up for which the modified audio track is, in the opinion of the user, most similar to the original audio track.",
"In the first to sixth embodiments, the rate at which the data signal F(t) is conveyed by the modified audio track is constant.",
"However, the ability of the audio track to hide the data signal F(t) is better during loud portions of the audio track than during quiet portions of the audio track.",
"A seventh embodiment of the invention will now be described with reference to FIGS.",
"16 to 27 in which the encoder and the cellular phone of the first embodiment are replaced by an alternative encoder, which varies the rate at which data is encoded within the modified audio track depending upon the power spectrum of the audio track, and an alternative cellular phone.",
"The remaining components of the seventh embodiment are the same as the corresponding components in the first embodiment and will therefore not be described again.",
"FIG.",
"16 shows the main components of the encoder 231 of the seventh embodiment.",
"As shown, the data signal F(t) is input to an FEC unit 233 which separates the data signal F(t) into segments of 7 data bits and applies a (15,7) BCH code to generate a modified data signal M′(t).",
"In this embodiment, unlike the first embodiment, the FEC unit 233 does not add a synchronisation bit.",
"The modified data signal M′(t) and the audio track are input to a data rate determining unit 235 which monitors the audio track and outputs either one, two or three data streams in dependence upon the power spectrum of the audio track.",
"The data rate determining unit 235 will now be described in more detail with reference to FIG.",
"17.As shown in FIG.",
"17, in the data rate determining unit 235 the modified data signal M′(t) is input to a first-in first-out (FIFO) memory 261 which acts as a cache memory.",
"The audio track is input to a spectrum analyser 263 which determines the power in the 0 to 5 kHz portion of the audio track and outputs a signal indicative of the determined power to an output signal generator 265 which has three data output ports and three enable signal output ports.",
"The output signal generator 265 determines from the signal from the spectrum analyser 263 if the power in the 0 to 5 kHz portion of the audio track is above a first predetermined level, between the first predetermined level and a second predetermined level which is greater than the first predetermined level, or above the second predetermined level.",
"If the output signal generator 265 determines that the power level is below the first predetermined level, then the output signal generator 265 outputs a single data stream.",
"In particular, the output signal generator 265 outputs from a first data output port a data stream DAT1 by sequentially retrieving data bit by bit from the FIFO memory 261, apart from every sixteenth bit of the data stream DAT 1 which is set to a data value of 0 to act as a SYNC bit.",
"The output signal generator 265 also sets the output EN1 of a first enable output port to a value of 1, and sets the output EN2 of a second enable output port and the output EN3 of a third enable output port to a value of 0.If the output signal generator 265 determines that the power level is between the first and second predetermined levels, then the output signal generator 265 outputs two parallel data streams DAT1, DAT2.In particular, the output signal generator 265 retrieves data from the FIFO memory 261 two bits at a time and outputs one of the retrieved bits through the first data output port to form data stream DAT1 and the other retrieved bit through the second data output port to form data stream DAT2, except that for every sixteenth pair of bits output the value of the data stream DAT1 is set to 0 in order to act as a SYNC bit and the output signal generator retrieves a single data bit from the FIFO memory 261 for the data stream DAT2.The SYNC bit is only added to the data stream DAT1 because the chip rate and timing information for the data stream DAT1 will also apply to the data stream DAT2.The output signal generator also sets respective outputs EN1, EN2 of the first and second enable signal output ports to a value of 1 and the output EN3 of the third enable signal output port to a value of 0.In this way, two parallel data streams are output from the data rate determining unit 235.If the output signal generator 265 determines that the power level is above the second predetermined level, then the output signal generator 265 outputs three parallel data streams through the data output ports.",
"In particular, the output signal generator 265 retrieves data from the FIFO memory 261 three bits at a time and outputs one bit through the first data output port to form data stream DAT1, one bit through the second data output port to form data stream DAT 2, and one bit through the third serial data port to form a data stream DAT3.Again, the value of every sixteenth bit of the data stream DAT1 is set to 0 to provide a SYNC bit.",
"The output signal generator 265 also sets the outputs EN1, EN2 and EN3 of the first to third enable signal output ports to a value of 1.In this way, three parallel data streams are output by the data rate determining unit 235.Returning to FIG.",
"16, the encoder 231 includes six pseudo-noise code generators (not shown) which respectively generate first to sixth pseudo-noise codes PN0 to PN5 which are input to non-inverting inputs of respective ones of six AND gates 237a to 237f.",
"Each data stream is input to a respective pair of the AND gates 237, in the manner shown in FIG.",
"2, where they are combined (when the respective pair of AND gates are enabled by the enable signal) with a respective pair of the pseudo-noise codes PN0 to PN 5.The data streams output by the first and second AND gates 237a,237b are input to a first adder 239a where they are added together to generate a first logic signal I1(t), which corresponds to the pseudo-noise code PN0 when the data value of the data stream DAT1 is 0 and corresponds to the pseudo-noise code PN1 when the data value of the data stream DAT1 is 1.Similarly, the data streams output by the third and fourth AND gates 237c,237d are input to a second adder 239b where they are added together to generate a second logic signal I2(t) and the data streams output by the fifth and sixth AND gates 237e,237f are input to a third adder 239c to generate a third logic signal I3(t).",
"In this embodiment, the first to third data streams DAT1, DAT2 and DAT3 are all output by the data rate determining unit at a rate of 10.7 bits per second and the first to sixth pseudo-noise code generators generate the first to sixth pseudo-noise codes PN0-PN5 respectively at a rate of 1,378 chips per second.",
"Further, in this embodiment the first to sixth pseudo-noise code generators generate 11-bit codes with a binary 0 added after each sequence of 2047 chips to form a repeating sequence of 2048 chips.",
"By using the same data rates for the data streams DAT1-DAT3 and the same chip rates for the pseudo-noise codes PN0-PN5, the processing required in the decoder to recover the original data signal is significantly simplified.",
"The first logic signal I1(t) is input to a first modulator 241a together with the output EN1 from the data rate determining unit 235.When the output EN1 has a value of 1, the modulator 241 uses phase shift keying to modulate a 2.76 kHz carrier signal in accordance with the value of the logic signal I(t) to generate a modulated signal G1(t).",
"In this embodiment, the carrier signal is a digital signal having a sample rate of 22.05 kHz and the modulated signal G1(t) is a sequence of 16-bit numbers.",
"Similarly, the second logic signal I2(t) is input to a second modulator 241b which, when the output EN2 has a value of 1, modulates a 2.76 kHz carrier signal in accordance with the value of the second logic signal I2(t) to generate a second modulated signal G2(t) and the third logic signal I3(t) is input to a third modulator 241c which, when the output EN3 has a value of 1, uses phase shift keying to modulate a 2.76 kHz carrier signal in accordance with the value of the logic signal I3(t) to generate third modulated signal G3(t).",
"If the enable signal applied to a modulator 241 is zero, the modulator outputs a null signal.",
"The first to third modulated signals G(t) are input to a 16-bit adder 243 which adds the 16-bit values of the first to third modulated signals G(t) for each sample to generate a final modulated signal G4(t).",
"The audio track is also input to a time delay unit 245 and the output of the time delay unit 245 is input to a shaping unit 247 along with the final modulated signal G4(t) to generate the shaped signal S(t).",
"The shaping unit 247 is identical to the shaping unit described with reference to FIG.",
"4 in the first embodiment and therefore will not be described again.",
"The purpose of the time delay unit 245 is to introduce a time delay corresponding to the time taken for the data rate determining unit 235 to monitor the audio track and output the first to third data streams and for the AND gates 237, adders 239, modulator 241 and 16-bit adder 243 to generate the final modulated signal G4(t).",
"In this way, the final modulated signal G4(t) is shaped using the same portion of the audio track as was monitored to determine the data rate.",
"As described in the first embodiment, the shaping unit 247 shapes the final modulated signal G4(t) so that it is less noticeable when, after having been combined with the audio track, it is output as an acoustic signal.",
"In this embodiment, however, the modulated signal G4(t) is pre-processed to contain more data when combined with loud portions of the audio track than when combined with quiet portions of the audio track.",
"The audio track output by the time delay unit 245 is also input to a second time delay unit 249, which delays the audio track for a period of time corresponding to the time required for the shaping unit 247 to generate the shaped signal S(t).",
"The audio track output by the time delay unit 249 and the shaped signal S(t) are then input to an adder 251 which performs a simple linear adding operation to combine them to form the modified audio track.",
"The modified audio track is then combined with the video track to generate a television signal which is broadcast in the same manner as for the first embodiment.",
"The broadcast television signal is then detected by a conventional television set which outputs the modified audio track as an acoustic signal.",
"The cellular phone 261 of the seventh embodiment will now be described with reference to FIGS.",
"18 to 27 in which components which are identical to those of the cellular phone of the first embodiment have been referenced with the same numerals and will not be described again.",
"The main difference between the cellular phone 261 of the seventh embodiment and the cellular phone 21 of the first embodiment is that the cellular phone 261 of the seventh embodiment includes a decoder 263 to recover the original data signal F(t) from the digital signal D(t) output by the ADC 113 rather than using a software routine run by a central processor 265.The recovered data signal F(t) output by the decoder 263 is then input to an input DATA_IN input of the processor 265.FIG.",
"19 shows in more detail the main components of the decoder 263.In this embodiment, the decoder 263 uses coherent detection, in which a received data stream is synchronously multiplied by the pseudo-noise codes PN0-PN5, to perform despreading.",
"It is, however, necessary to ensure that the chip sequence in the digital signal D(t) and the chip sequences of the pseudo-noise codes PN0-PN5 are time-synchronised.",
"To achieve an initial synchronisation, the digital signal D(t) is input to an acquisition unit 271 which generates timing signals which are input to a processor 273 which then generates the pseudo-noise codes PN0-PN5 in accordance with the timing signals.",
"In this embodiment, the processor 273 first determines from the timing signals any slight frequency offset between the chip rate of the chip sequence in the digital signal D(t) and the chip rate of the pseudo-noise codes PN0-PN5, and then outputs a signal S indicative of the frequency offset to a re-sampling circuit 275.The digital signal D(t) is input to the re-sampling circuit 275 where it is stored in blocks of 8192 samples.",
"The re-sampling circuit re-samples the 8192 samples in each block at a rate determined by the signal S output by the processor 273 so that the pseudo-noise codes conveyed by the re-sampled digital signal R(t) have the same chip rate as the pseudo-noise codes generated by the processor 271.For example, if the determined frequency offset indicates a chip rate of 1392 Hz, which corresponds to an increase of 1% over the nominal chip rate of 1378 Hz, then the re-sampling rate is set at 22.2705 Hz to allow for the additional chips present.",
"The re-sampled data is determined in the re-sampling circuit 275 from the 8192 stored samples using interpolation techniques to give, for the exemplary 1% increase in chip rate, 8274 samples.",
"For the re-sampled data, each chip will correspond to sixteen samples and therefore each data bit will correspond to 2048 samples.",
"FIG.",
"20 shows in more detail the main components of the acquisition unit 271, the timing signals output by the acquisition unit 271 to the processor 273, and the signals output by the processor 273.As shown in FIG.",
"20, the samples of the digital signal D(t) output by the ADC 113 are input sequentially to a series of four digital matched filters 291a to 291d which are arranged so that after passing through the first matched filter 291a the samples are output via the cascade output (indicated in FIG.",
"20 by a) of the first matched filter 291a and input to the second matched filter 291b and so on.",
"Each matched filter 291 has 512 taps so that the four matched filters 291 have a sequence of 2048 taps, and the matched filters 291 are matched to the chip sequence of the pseudo-noise code PN0 corresponding to the SYNC bit.",
"The reason why a single matched filter having 2048 taps is not used rather than the four series connected matched filters 291 will now be described.",
"In particular, if a single large matched filter was used in order to detect the SYNC bit, and if the rate at which the code PN0 is generated is different to the chip rate in the received electrical signal D(t), then this lack of synchronisation will lead to a severe degradation in the peak score output by the matched filter.",
"This is because a large single matched filter performs the correlation over a larger time window and consequently the effects of the lack of synchronisation can build up over a longer period of time.",
"In contrast, by using a number of smaller matched filters connected in series, the time window over which each of the matched filters performs the correlation is much smaller than that of the larger single matched filter.",
"Hence, the lack of synchronisation will cause less degradation for each of the individual smaller matched filters.",
"As a result, larger frequency offsets between the chip rate in the digital signal D(t) and the chip rate of the pseudo-noise code PN0 can be tolerated by using the four matched filters 211 rather than a single matched filter.",
"The score output by each of the matched filters 291 (which is indicated by output b and which is updated at each clock pulse as the samples of D(t) are clocked through the matched filters) is input to a corresponding one of four normalisation circuits 293a to 293d.",
"The normalisation circuits 293 provide a normalised output for a wide dynamic signal range of the digital signal D(t).",
"This enables the output of the normalisation circuit to be analysed by a simple thresholding operation.",
"FIG.",
"21 shows schematically the contents of each normalisation circuit 293.As shown, the current score from the corresponding matched filter 291 is input to a time delay unit 301 where it is delayed for 256 clock periods, which corresponds to the time taken for the samples of the digital signal D(t) to propagate halfway through the corresponding one of the matched filters 291.The current score is also input to an averaging circuit 303 which uses the current score to update a running total of the last 512 scores.",
"The output of the time delay unit 301 is then input to a divider 305 which divides the delayed score by the current value of the running total, to produce the normalised output.",
"The above processing makes the normalisation circuit particularly well suited to systems where a spread spectrum signal is hidden in an acoustic signal, because the acoustic signal will typically vary over a large dynamic range.",
"FIG.",
"22 shows in more detail the contents of the averaging circuit 303.As shown, the current score is input to a time delay unit 311, where it is delayed for 512 clock periods, and a first adder 313 where the inverse of the time delayed score is added to the current score.",
"The output of the first adder 313 is then input to a second adder 315 which adds it to the current value of the running total (delayed by one clock cycle) output by the time delay unit 317, to generate a new current value of the running average which is used by the divider circuit 305.In this way, the value of each new sample is added to the running total while the value of the sample 512 clock periods before the new sample is subtracted.",
"FIG.",
"23 shows a typical output of one of the normalisation circuits 293, when two consecutive 16-bit frames pass through the corresponding matched filter 291.In FIG.",
"23 reference timings 321a to 321c are illustrated which are separated by 32768 clock periods (nominally corresponding to the time required for the samples corresponding to one 16-bit frame to pass through the matched filter).",
"The period between two adjacent reference timings 321 will hereinafter be referred to as a frame period.",
"A first peak 323 in the normalised score, corresponding to a first SYNC bit, occurs a time τ1 after the nearest preceding reference timing 321a, while a second peak 325, corresponding to a second SYNC bit, occurs a time τ2 after the nearest preceding reference timing 321b.",
"If there is no frequency offset in the chip rates, then τ1 is equal to τ2 (since in 32768 clock periods the samples corresponding to a 16-bit frame will pass completely through the four matched filters 291) and the matched filters 291a-291d all output peaks at the same time.",
"However, if there is a frequency offset in the chip rates, then there will be a timing offset τoff, defined by τ2−τ1, between the peaks in neighbouring frames which is dependent on the frequency offset.",
"Further, a frequency offset in the chip rates will lead to the peaks output by the four matched filters 211a to 211d not occurring simultaneously.",
"However, the timing offset (τoff) for the output of each of the normalisation circuits 293 should be identical.",
"In this embodiment, the acquisition unit 271 makes use of this, in order to quantify the frequency offset and hence to correct for it.",
"The way in which this is done will now be described.",
"As shown in FIG.",
"20, in this embodiment, the output of each normalisation circuit 293 is input to a corresponding cross-correlator 295a to 295d where it is cross-correlated with the output from the same normalisation circuit for the immediately preceding frame.",
"This is achieved by passing the output score from each normalisation unit 293 through a corresponding time delay unit 297a to 297d which delays the scores by one frame period (i.e.",
"32768 clock periods).",
"The output from the normalisation circuit 293 is then cross correlated with the corresponding delayed output, by the cross-correlator 295.In this embodiment, a maximum frequency offset corresponding to twelve clock samples is anticipated.",
"Therefore, the cross-correlators 295 only look for a cross-correlation peak over a range of time offsets between the two frames, varying between a twelve sample lead and a twelve sample lag.",
"This results in a significant reduction in the amount of processing required by the cross-correlators 295.FIG.",
"24 shows a typical output of one of the cross-correlators 295.The x-axis corresponds to the time offset between the two frames output by the normalisation circuit 293 and the y-axis corresponds to the score output by the cross-correlator 295.A cross-correlation peak 331 occurs at a time offset τoff which is equal to τ2−τ1.As mentioned above, the time offset for each of the matched filters 291a-291d should be identical and therefore the position of the cross-correlation peak 331 in the output of each of the cross-correlators 295 should be the same.",
"The outputs of the four cross-correlators 295 are therefore added together by an adder 299 and the output of the adder 299, labelled OFFSET in FIG.",
"20, is input to the processor 273.The processor 273 then calculates the frequency offset (from τoff and the size of the correlation window of the matched filters 291) and sends the signal S indicative of the frequency offset to the re-sampling circuit 275.Once the frequency offset has been removed, it is then necessary to synchronise the pseudo-noise codes PN0-PN5 generated by the processor 273 with the chip sequence in the digital signal D(t).",
"In this embodiment, this is achieved by inputting the output scores Ai, Bi, Ci and Di from the four normalisation circuits 293 directly into the processor 273 which determines, from the largest peak present in the four outputs, the timing of the chip sequence in the detected electrical signal D(t).",
"The processor 273 then uses the determined timing when generating the pseudo-noise codes PN0-PN5 to ensure that they are synchronised with the chip sequence within the digital signal D(t).",
"In this embodiment, the processor 273 is a microprocessor based system which is schematically illustrated in FIG.",
"25.As shown, the processor 273 includes an interface circuit 335 for interfacing a central processing unit (CPU) 337 with the normalised scores Ai, Bi, Ci and Di output from the normalisation circuits 293, for interfacing the CPU 337 with the adder 299, and for outputting the pseudo-noise codes PN0-PN5.As shown in FIG.",
"25, the interface circuit 335 also receives a signal (TRACK) which is used in a tracking operation which will be described in more detail below.",
"In carrying out the calculations described above, the processor 273 processes the values received from the interface circuit 335 in accordance with predetermined instructions stored in a program memory 339.A working memory (RAM) 341 is also provided for use by the CPU 337 when performing calculations.",
"A user interface 343 is also provided to allow a user to adjust the settings of the processor 273, for example in order to change or alter the program instructions stored in the program memory 339 so that the decoder can be reconfigured.",
"Returning to FIG.",
"19, the output of the re-sampling circuit 275 is input to first to third correlate and compare units 279a to 279c.",
"The first correlate and compare unit 279a correlates the data output by the re-sampling circuit with the first and second pseudo noise codes PN0,PN1 and then compares the results of these two correlations.",
"Similarly, the second correlate and compare unit 279b correlates the data output by the re-sampling circuit 275 with the third and fourth pseudo noise codes PN2,PN3 and compares these two correlations and the third correlate and compare unit 279c correlates the data output by the re-sampling circuit 275 with the fifth and sixth pseudo noise codes PN4,PN5 and compares the results of these two correlations.",
"FIG.",
"26 shows in more detail the main components of the first correlate and compare unit 279a (the second and third correlate and compare units 279b, 279c have the same structure but different inputs).",
"AS shown in FIG.",
"21, the first correlate and compare unit 279a has three channels which have been labelled late, on-time and early.",
"As will be explained below, the three channels enable the time synchronisation to be tracked while data other than the SYNC bit is being transmitted.",
"The data signal R(t) is input into each of the three channels of the correlate and compare unit 279 and in each channel it is separately multiplied by PN0 and PN1.In the late channel, the data signal R(t) is input to a first multiplier 351a, where it is multiplied by PN1 time-delayed by two clock periods by a first time delay unit 353a, and to a second multiplier 351b, where it is multiplied by PN0 time delayed by two clock periods by a second time delay unit 353b.",
"Similarly, in the on-time channel the data signal R(t) is input to a third multiplier 351c, where it is multiplied by PN1 time-delayed by one clock period by a third time delay unit 353c, and to a fourth multiplier 351d, where it is multiplied by PN0 time-delayed by one clock period by a fourth time delay unit 353d.",
"In the early channel, the data signal R(t) is input to a fifth multiplier 351e, where it is multiplied by PN1, and to a sixth multiplier 351f, where it is multiplied by PN0.When the data signal R(t) is multiplied by PN1, if the chip sequence in the data signal R(t) corresponds to PN1, then a narrow band signal at about the carrier frequency of 1378 Hz will be generated.",
"Similarly, when the data signal R(t) is multiplied by PN0, if the chip sequence of the signal R(t) matches PN0, then a narrow band signal at the carrier frequency will be generated.",
"In this way, for each channel, if the received data bit has a value of 1, then the output of the first, third and fifth multipliers 351a, 351c, 351e will contain a narrow band signal at the carrier frequency and, because PN1 and PN0 are orthogonal, the output of the second, fourth and sixth multipliers 351b, 351d, 351f will not contain the narrow band signals.",
"Similarly, if the received data bit has a value of 0, then the output of the second, fourth and sixth multipliers 351b, 351d, 351f will contain the narrow band signal at the carrier frequency and the output of the first, third and fifth multipliers 351a, 351c, 351e will not.",
"The outputs of the two multipliers 351 in each channel are input to a corresponding one of three power comparators 355a to 355c, one of which is shown in more detail in FIG.",
"27.As shown, in the power comparator 355 the outputs of the two multipliers 351 are input to respective bandpass filters 361a and 361b which are centred on the carrier frequency.",
"The output of each bandpass filter 361 is then input to a respective power monitor 363a, 363b which determines the power of the signal output from the corresponding bandpass filter 361.As mentioned above, when the received data bit is a “1”, the output from the power monitor 363a should be greater than the output from the power monitor 363b.",
"In contrast, when the received data bit is a “0”, the output from the power monitor 363b should be greater than the output from the power monitor 363a.",
"Therefore, the outputs from the power monitors 363 are input to a comparator 365 which outputs a value which varies in dependence upon the difference between the outputs of the two power monitors 363.In this embodiment, the output from the power monitor 363a is input to the positive terminal of the comparator 365 and the output from the power monitor 363b is input to the negative terminal of the comparator 365.Therefore, if the received data bit is a “1”, then the output of the comparator 365 will be a large positive value, and if the received data bit is a “0”, then the output of the comparator 355 will be a large negative value.",
"In this embodiment, as the encoder 263 continuously outputs the data stream DAT1, when decoding the digital signal D(t) corresponding to the modified audio track the first correlate and compare unit 279a will, provided PN0 and PN1 are time-synchronised with the received chip sequence, output a continuous sequence of either positive or negative values indicative of data bits “1” and data bits “0” respectively.",
"However, the encoder 263 only outputs the second data stream DAT2 and the third data stream DAT3 for the louder portions of the audio track.",
"In this embodiment, if the data stream DAT3 is not present then in the third correlate and compare unit 279c neither the output of the fifth multiplier 351e nor the output of the sixth multiplier 351f will contain a narrow band signal at the carrier frequency and the output of the power comparators 355 for all three channels will be approximately zero volts.",
"Similarly, if the data stream DAT2 is not present then the outputs from the second correlate and compare units 279b will be approximately zero volts.",
"Returning to FIG.",
"19, the output of the on-time channel of the first to third correlating compare units 279 are input to a data regeneration circuit 281 which determines which of the data streams DAT1, DAT2 and DAT3 are present, removes the SYNC bits and decodes the (15,7) BCH code to regenerate the original data signals.",
"The output of the on-time channel of each correlate and compare unit 279 is also input, together with the outputs of the late and early channels of each correlate and compare unit 279, into an analysis unit 285.The analysis unit 285 determines which of the channels provides the largest output, which indicates the channel for which there is the best match between the timing of chip sequence in the data signal R(t) and in the pseudo-noise codes PN0 to PN5.If the late channel provides the largest output, then analysis unit 285 sends a signal (on the control line labelled TRACK) to the processor 273 indicating that the clock should skip a sample so that the on-time channel once more produces the largest output.",
"Similarly, if the early channel produces the largest output, then the analysis unit 285 outputs a signal to the processor 273 which causes the clock 277 to make a double sample so that the on-time channel once more produces the largest output.",
"In this way, a tracking operation is accomplished in which time synchronisation of PN0 to PN5 with a chip sequence encoded in the data signal R(T) is checked on a sample-sample basis and, if necessary, the timing of PN0 to PN5 is adjusted to correct for a reduction in synchronisation.",
"In the first to seventh embodiments, a data signal conveying a URL identifying a website associated with a television program is input to an encoder in which the data signal is spread and mixed with the audio track for the television program prior to the television program being broadcast over a conventional television network.",
"A cellular phone then recovers the URL from the acoustic signals output by a television tuned to the television program so that the user of the cellular phone, if desired, can download the web page to the cellular phone.",
"The first to seventh embodiments describe different techniques for encoding the data signal within the audio track.",
"Alternative embodiments will now be described in which the encoder is incorporated within a cellular phone so that a data stream can be encoded within an acoustic signal emitted by the cellular phone.",
"FIG.",
"28 illustrates an eighth embodiment of the invention in which a first cellular phone 401_1 emits an acoustic signal 403, having encoded therein user data associated with the user of the first cellular phone 401_1, from a loudspeaker 25_1.In this embodiment, the user data includes the name of the user and the telephone number of the first cellular phone 401_1.The emitted acoustic signal 403 is detected by a second cellular phone 401_2, using a microphone 23_2, and the second cellular phone 401_2 then decodes and stores the user data.",
"In particular, the name of the user of the first cellular phone 401_1 is stored along with the telephone number of the first cellular phone 401_1 in a “phone book” stored in the second cellular phone 401_2.Similarly, the second cellular phone 401_2 emits an acoustic signal conveying user data associated with the user of the second cellular phone 401_2 from a loudspeaker 25_2 for detection and decoding by the first cellular phone 401_1.It will be appreciated that transmitting user data in this manner removes the requirement of inputting names and phone numbers via the keypad 31 of the cellular phone 401, which is generally a slow and awkward process because the keys of the keypad 31 are small.",
"FIG.",
"29 schematically illustrates the main components of the cellular phone 401 of the eighth embodiment.",
"In FIG.",
"29, components which are identical to corresponding components of the cellular phone of the first embodiment have been referenced by the same reference numerals and will not be described again.",
"As shown, in this embodiment the processor 411 of the cellular phone 401 has an AUDIO_OUT output and a DATA_OUT output which are connected to respective inputs of an encoder 413.In this embodiment, the only difference between the encoder 413 and the encoder of the first embodiment is that the modulator of the encoder 413 does not include a re-sampling circuit because the digital signal output via the AUDIO-OUT output of the processor 411 has a sample rate of 8 kHz.",
"The output of the encoder 413 is connected to the input of the DAC 117.In this way, user data output via the DATA_OUT output of the processor 411 is encoded within an audio track output by the AUDIO_OUT output to form a modified audio track.",
"In the cellular phone 401 of this embodiment, the digital signal D(t) output from the ADC 113 is input to a decoder 415 as well as the AUDIO_IN input of the processor 411.The decoder 415 is identical to the decoder of the seventh embodiment and will not therefore be described again.",
"The decoder 415 recovers user data encoded within the digital signal D(t) and outputs the recovered user data to a DATA_IN input of the processor 411.In this way, user data conveyed by an acoustic signal from another cellular phone 401 can be decoded for processing by the processor 411.In this embodiment, the user data associated with the user of the cellular phone 401 is stored in the NVRAM 125 and the ROM 127 stores a sub-routine which, in response to the user selecting a menu option using the menu keys 33, causes the processor 411 to output, via the DATA_OUT output, the user data and to output, via the AUDIO_OUT output, a preset audio sequence.",
"The purpose of the preset audio sequence is to provide an audible indication to the user that the data transfer is taking place.",
"In the encoder 413, the user data is spread, modulated and shaped as described in the first embodiment before being added to the preset audio sequence to form a modified audio sequence.",
"The modified audio sequence output by the encoder 413 is input to the DAC 117 where it is converted into an analogue signal which is amplified by the amplifier 119 and output as an acoustic wave by the loudspeaker 25.When the cellular phone 401 detects, using the microphone 23, an acoustic wave conveying user data, the user data is decoded by the decoder 415 and input, via the DATA_IN input, to the processor 411.In response to the user data being input, the processor 411 runs a sub-routine stored in the ROM 127 which extracts the user name and phone number from the user data and stores the extracted user name and phone number as an entry in a look-up table within the NVRAM 125.In this embodiment, the NVRAM 125 can store up to 100 entries which form an electronic phone book.",
"In the eighth embodiment, data is transferred between two cellular phones 401 via an acoustic communications link.",
"However, cellular phones could also use an acoustic communications link to transmit data signals to other electronic devices.",
"A ninth embodiment will now be described with reference to FIGS.",
"30 to 33 in which a cellular phone encodes a control signal within an emitted acoustic signal; a toy detects the acoustic signal, recovers the control signal and responds in accordance with the control signal in a manner discernable to humans.",
"As shown in FIG.",
"30, a caller 423 uses a telephone handset 427, connected to a telecommunications network 429, to phone the user (not shown) of the cellular phone 421.The call is routed by the telecommunications network to a base station 431, which covers the area in which the cellular phone 421 is currently located, and the base station 431 outputs a RF signal 433 which is detected by the antenna of the cellular phone 421.Included in the RF signal 433 is identification data identifying the phone number of the handset 427.Those skilled in the art will appreciate that such identification data is sent in many conventional telephone systems.",
"In this embodiment, the cellular phone 421 determines from the incoming RF signal 433 the telephone number of the handset 427, retrieves from a look-up table a control signal identifier associated with the determined telephone number, and encodes the control signal corresponding to the control signal identifier within a ring tone which is output as an acoustic signal 435.In this embodiment, the acoustic signal 435 is detected by a microphone 437 in an electronic toy 425.As shown in FIG.",
"30, the electronic toy 425 includes two arms 439a, 439b and a face formed by: a nose 441; two eyes 443a, 443b; an articulated mouth 445; and two articulated eyebrows 447a, 447b.",
"The electronic toy 425 also includes a loudspeaker 449.The toy 425 decodes the control signal within the ring tone emitted by the cellular phone 421 and responds by outputting a sound via the loudspeaker 449 and by moving the mouth 445 and the eyebrows 447 to from an expression in accordance with the recovered control signal.",
"FIG.",
"31 shows the main components of the cellular phone 421 of the ninth embodiment.",
"In FIG.",
"31, components which are identical to corresponding components of the cellular phone of the first embodiment have been referenced by the same reference numerals and will not be described again.",
"As shown, the processor 461 has a DATA_OUT output which is connected to a first input of an encoder 463 and the AUDIO_OUT output of the processor 461 is connected to a second input of the encoder 463.In this embodiment, the encoder 463 is identical to the encoder of the eighth embodiment.",
"The encoder 463 encodes a data signal output via the DATA_OUT output of the processor 461 within an audio signal output via the AUDIO_OUT output of the processor 461 to form a modified audio signal which is converted into an analog signal by the DAC 117, and the analog signal is then amplified by the amplifier 119 prior to being converted into an acoustic signal by the loudspeaker 25.FIG.",
"32 shows the functional configuration of the processor 461 when a signal initiating a telephone call is first received via the antenna 27.As shown, the received signal is input to a ring signal generator 471 which generates a ring signal which is output via the AUDIO_OUT output of the processor 461.The received signal is also input to a phone number extractor 473 which determines from the received signal the telephone number of the caller.",
"The determined telephone number is processed by a response retriever 475 which accesses a response identifier corresponding to the determined telephone number from a response storage region 477 of the NVRAM 125.As shown, the response storage region 477 is in the form of a look-up table which stores a plurality of telephone numbers along with corresponding response identifiers.",
"In this embodiment, there are three possible responses which have response identifiers A, B and C respectively.",
"When the response retriever 475 has retrieved a response identifier from the response storage region 477, the retrieved response identifier is processed by a data signal generator 479 which generates a corresponding control signal which is output via the DATA_OUT output of the processor 461.As described above, the acoustic signal 435 is detected by a microphone 437 of an electronic toy 425 which converts the acoustic signal into an electrical signal.",
"FIG.",
"33 schematically shows the main components of the electronic circuitry within the toy 425.As shown, the electrical signal output by the microphone 437 is input to an anti-aliasing filter 491 before being converted into a digital signal by a DAC 493.The digital signal output by the DAC 493 is input to a decoder 495 which recovers the control signal.",
"In this embodiment, the decoder 495 is identical to the decoder for the eighth embodiment.",
"The recovered control signal is input to a processor 497 which is connected to a RAM 499, which provides working memory, and a ROM 501 which stores routines which are executed in response to the control signal.",
"A first output of the processor 497 is connected to a first motor drive circuit 503 which outputs drive signals to an eyebrows motor 505 for causing movement of the articulated eyebrows 447.A second output of the processor 497 is connected to a second motor drive circuit 507 which outputs drive signals to a mouth motor 509 for causing movement of the articulated mouth 445.A third output of the processor 497 is connected, via a DAC 511 and an amplifier 513, to a loudspeaker 515.In operation, receipt of a control signal activates a corresponding routine stored in the ROM 501 associated with the control signal which causes the processor 497 to output a signal to the motor drive circuit 503, causing the motor drive circuit 503 to drive the eyebrows motor 505 to move the articulated eyebrows 447, and outputs a signal to the second motor drive circuit 507 causing the second motor drive circuit 507 to output a drive signal to the mouth motor 509 to move the articulated mouth 445.In this way, the desired expression corresponding to the control signal is formed.",
"Further, an audio signal associated with the control signal is output by the processor 497 to the DAC 511 where it is converted into an analog signal which is then amplified by the amplifier 513 before being output as an acoustic signal by the loudspeaker 449.As shown in FIG.",
"30, the control signal associated with the user 423 causes the facial expression of the toy 425 to become a frown, and the audio signal associated with the user 423 is a groan.",
"In the ninth embodiment, a cellular phone determines a control signal associated with the telephone number of a caller and combines the control signal with the ring tone for detection by the toy 425.However, control signals could also be transmitted to a cellular phone over a telecommunications network for transmission by the cellular phone as an acoustic signal for subsequent detection and processing by an electronic device.",
"A tenth embodiment will now be described with reference to FIGS.",
"34 and 35 in which the facial expression of, and sounds output by, the toy 425 of the ninth embodiment are altered in accordance with control signals which are transmitted, using the Short Message Service (SMS) defined in the Global System for Mobile communication (GSM) specification, over the telecommunications network 429 to a cellular phone.",
"In FIG.",
"34, components which are identical to corresponding components in FIG.",
"30 have been referenced with the same numerals and will not be described again.",
"As shown in FIG.",
"34, a computer system 521 is connected to a modem 523 via which the computer system 521 sends signals over the telecommunications network 429.In response to a user of the computer system 521 indicating a control signal to be sent to the cellular phone 525, a corresponding control signal identifier is output, using the modem 523, in the form of an SMS message to the telecommunications network 429 which routes the SMS message to the base station 431 covering the location of the cellular phone 525.The base station 431 then emits an RF signal 433 conveying the SMS message for detection by the cellular phone 525.In this embodiment, the hardware components of the cellular phone 525 are identical to those of the cellular phone described with reference to FIG.",
"31 for the ninth embodiment.",
"However, in this embodiment the ROM in the cellular phone 525 includes a routine which, in response to an SMS message conveying a control signal identifier, retrieves the control signal identifier from the SMS message and outputs the corresponding control signal and a preset audio signal via the DATA_OUT and AUDIO_OUT outputs of the processor respectively.",
"FIG.",
"35 shows the functional configuration of the processor of the cellular phone when processing a received SMS message conveying a control signal identifier.",
"The SMS message is processed by a SMS message analyser 531 which retrieves the control signal identifier.",
"The SMS message analyser then causes an audio signal generator 535 to output the preset audio signal via the AUDIO_OUT output of the processor and a data signal generator 533 to output the control signal corresponding to the control signal identifier from the processor via the DATA_OUT output.",
"The encoder then combines the control signal with the preset audio signal to form a modified audio signal which is output as the acoustic signal 435.In this embodiment, the preset audio signal is only used to alert the user of the cellular phone that a control signal is being sent.",
"In this embodiment, the toy 425 detects the acoustic signal 435, and responds in the same manner as described in the ninth embodiment.",
"Modifications and Further Embodiments In the first to tenth embodiments, an acoustic data channel is used to either input data into or output data from a cellular phone.",
"The addition of an acoustic data channel to a cellular phone has many advantages, for example: 1.The cellular phone is already equipped with a microphone and a loudspeaker which can be used for the acoustic data channel.",
"Therefore, the weight of the cellular phone does not need be significantly increased.",
"2.The processors typically used in most digital cellular phones are well suited to be programmed to carry out software routines for encoding and/or decoding data within the acoustic data channel.",
"3.Acoustic communication techniques are well suited for short range communication, particularly as they are not subject to the same regulatory requirements as RP communications techniques.",
"4.There are already established networks for distributing acoustic signals, for example television and radio networks.",
"5.Many people carry a cellular phone on their person virtually all the time.",
"It therefore provides an ideal communication route for data which are dependent upon the location of a person.",
"6.Using an acoustic link to cellular phones enables data to be targeted to people within a specific location, for example a particular building via a public address system within the building, or listeners to a particular audio source, for example a particular radio network.",
"This is advantageous both to the data supplier, because data is supplied efficiently, and to the user of the cellular phone, because only data which is likely to be useful is received.",
"7.Using an acoustic communication technique to input data into a cellular phone is generally more convenient than inputting data using the keys of the cellular phone because the keys are generally small.",
"As those skilled in the art will appreciate, some of the above-described advantages also arise with other portable telephones, such as cordless telephones which communicate with a base station directly connected to a public switched telephone network (PSTN), or even a standard telephone which is directly connected to a PSTN.",
"Those skilled in the art will also appreciate that the telephone could be incorporated as part of another device, for example a personal digital assistant (PDA).",
"As described above in the first to seventh embodiments, an acoustic data channel is particularly well suited to so-called “smart” phones which have some data processing capability in addition to voice call handling capability because the acoustic communication techniques can be used, for example, to control the accessing of data or to output signals in accordance with received data.",
"In the first to seventh embodiments, the cellular phone is able to download data from the internet using the Wireless Application Protocol.",
"Other types of “smart” phone include those employing the i-MODE system, “Java phones” which have incorporated therein the Java 2 Platform Micro Edition (J2ME), and the future 3G cellular phones.",
"In the first to seventh embodiments, a user is able to download a web page from the internet about a television programme using a cellular phone.",
"In particular, a URL for the web page is encoded within the audio track of the television programme.",
"In this way the acoustic data channel is used to establish a link between the cellular phone and the web page.",
"The URL encoded within the television programme could relate to a web page specifically mentioned in the television programme as a source of further information.",
"Alternatively, the URL could be for a “hidden” web page whose existence is not generally broadcast and therefore can only be accessed by suitably enabled cellular phones.",
"For example, a feature film could have encoded within its soundtrack a URL for a web page having cast interviews etc.",
"Those skilled in the art will appreciate that another form of web page identifier could be encoded within the audio track instead of the URL.",
"For example, a combination of the Internet Protocol address of the server storing the web page and index data identifying the desired web page to the server could be used.",
"In an alternative embodiment, a proprietary web page identifier is encoded within the audio track, and on receiving an acoustic signal conveying a proprietary web page identifier, the cellular phone accesses a database storing a look-up table associating a plurality of proprietary web page identifiers with associated IP addresses and/or URLS.",
"In this way, the data location of the web page associated with the received proprietary web page identifier is determined.",
"The database could be located either in the cellular phone or at a remote server which the cellular phone accesses using the cellular telecommunications network.",
"If the database is stored in the cellular phone, the database can be periodically updated, for example by sending new data using a SMS message.",
"If the proprietary database is located at a remote server, on receiving a proprietary web page identifier the remote server can either send the corresponding IP address/URL back to the cellular phone or forward to the corresponding IP address/URL a request to download data directly to the cellular phone.",
"An advantage of using a remote server to store the database of proprietary web page identifiers and associated IP addresses is that the number of times the server is accessed by the user of the cellular phone can be monitored.",
"This enables the television programme maker to assess the usefulness of encoding the web pages within the television programme.",
"Further, by monitoring the number of times the user of the cellular phone accesses the server, a loyalty scheme can be established in which the user receives rewards, for example free gifts, for frequent use.",
"In particular, if the user accesses the database a predetermined number of times then a reward is given.",
"This encourages the user of the cellular phone to make use of the server.",
"For the first to seventh embodiments, the television signal need not be broadcast using a transmitter but could be sent to the television set along a cable network or via a satellite.",
"It will also be appreciated that the same techniques could be applied to a radio signal, whether broadcast using a transmitter or sent along a cable network.",
"Further these techniques can be applied to a point-to-point communication system as well as broadcast systems.",
"In addition, conventional encryption techniques could be used so that the television or radio signal could only be reproduced after processing by decryption circuitry.",
"As another alternative, the television signal could be stored on a video cassette, a digital versatile disk (DVD), hard disk or the like.",
"In this way, no signal is transmitted through the atmosphere or through a cable network but rather the television signal is stored on a recording medium which is subsequently played to a user on the user's television set.",
"Similarly, an audio signal could be stored on an audio cassette, compact disc (CD) or the like.",
"Further, the audio track could be stored as a computer file, for example an MP3 file, on a hard disk or the like.",
"In the first to seventh embodiments, the monitor mode is activated by the user of the cellular phone.",
"In other embodiments, the monitor mode is activated in response to an external stimulus rather than by the user of the cellular phone.",
"For example, the monitor mode could be activated by a received SMS message.",
"Alternatively, the cellular phone could be configured so that the functionality of the monitor mode is continuously activated.",
"As described above, in the monitor mode a cellular phone monitors for any data signals conveyed by acoustic signals detected by the microphone.",
"In an embodiment, in order to save power during periods when no data signals are detected, the cellular phone can operate in a “power-save” mode in which the cellular phone only periodically checks for data signals rather than continuously checking for data signals.",
"For example, the cellular phone could check for data signals for a period of three seconds and, if no data signals are detected, the cellular phone then waits ten seconds before checking again.",
"The power-save mode could be activated automatically by the cellular phone if no data signal is received for a predetermined time period.",
"Alternatively, the power-save mode could be activated by a user or by an external stimulus.",
"In the first to seventh embodiments, the web page whose URL is detected within the audio track of a television programme is accessed by the user of the cellular phone selecting a menu option using menu keys.",
"The accessing of the web page could also be initiated by an external stimulus, for example a command sent using the acoustic data channel.",
"In some applications, the web page is automatically accessed by the cellular phone without requiring any input from the user or an external stimulus, in which case the cellular phone need not store the URL or other web page identifier.",
"For example, in an embodiment a sequence of web page identifiers is encoded within the audio track of a television programme and a cellular phone detects the sequence of web page identifiers and automatically downloads the corresponding web pages.",
"In this way, the cellular phone can display web pages which vary in synchronism with the content of the television programme.",
"Such automatic accessing could be activated in response to a user instruction or an external stimulus, or alternatively the cellular phone could be pre-configured for automatic accessing.",
"Preferably, the user is able to disable the automatic accessing if the user considers the data being downloaded to be irrelevant.",
"In an alternative embodiment, the user of the cellular phone is able to store a received web page identifier in a NVRAM within the cellular phone for future access by selecting an appropriate menu option.",
"In the described embodiments, the user inputs an instruction to the cellular phone by accessing a menu option.",
"Examples of user instructions are activating the monitor mode or power-save mode, accessing a web page corresponding to a received web page identifier or instructing the cellular phone to access automatically the web page corresponding to a received web page identifier.",
"Instead of or in addition to such a “soft key” arrangement for entering instructions, the cellular phone could include one or more “hard keys” associated with the acoustic data channel, i.e.",
"dedicated keys provided on the cellular phone which are each associated with a corresponding instruction.",
"These “hard keys” could be either pre-configured during manufacture of the cellular phone or programmed by the user of the cellular phone.",
"In an alternative embodiment, the cellular phone includes voice recognition software so that instructions can be entered by a user speaking an associated word or phrase into the microphone of the cellular phone.",
"In the first to seventh embodiments, the data encoded within the audio track of a television programme relates to a web page.",
"Alternatively, the data encoded within the audio track of a television programme could be related to, for example, the title of a musical track being played on the television.",
"This title can then be displayed by the cellular phone.",
"Alternatively, telephone numbers could be encoded within the audio track of a television programme, for example a “prize” telephone number could be encoded which can be automatically dialled during a broadcast to enter a competition.",
"The acoustic data channel could also be used to transmit gaming information to the cellular phone.",
"For example, for computer games played on the cellular phone which have a multi-level structure with each level being accessed by a password, the passwords could be encoded within an audio track so that they can be downloaded to the cellular phone, via the acoustic data channel, in order to allow access to respective levels.",
"The audio track could be, for example, the audio track of a film associated with the computer game so that people who have seen the film can have access to one or more levels of the computer game.",
"Alternatively clues or tips related to a computer game could be encoded within an audio track.",
"In another alternative application, the data signal F(t) encoded within the audio track of a television programme or a radio programme could convey audience survey data identifying the programme being watched and/or listened to by the user of the cellular phone.",
"The use of a cellular phone for audience survey is advantageous for three main reasons.",
"Firstly, the user of a cellular phone tends to carry the cellular phone on their person.",
"The collected audience survey data is therefore associated with a user, rather than a television set or radio as in most automated audience survey systems.",
"Secondly, the cellular phone is able to send the audience survey data for the user automatically, using the cellular communications network, to a remote database where audience survey data from many users is collated and analysed.",
"This provides a simple mechanism for collecting the audience survey data without requiring user involvement.",
"Thirdly, by incorporating the audience survey operation within a cellular phone which the user normally has either on their person or nearby, the user is less aware of the audience survey being performed than if the user has to carry a dedicated audience survey device about on their person, even if the user knows that the cellular phone is collecting audience survey data.",
"This alleviates a common problem with audience surveys that users alter their viewing and/or listening habits in view of the audience survey.",
"In a preferred embodiment of the audience survey application, as each item of audience survey data is received by the cellular phone it is immediately downloaded, via the cellular telecommunications network, to an audience survey station where it is stored in an audience survey database.",
"In this embodiment, the data signal is only a channel identifier for the radio station being listened to or the television channel being watched rather than an identifier for a particular programme.",
"FIG.",
"36 shows the main components of the audience survey station 551 for this preferred embodiment.",
"The audience survey station 551 receives an audience survey signal, via a telecommunications network, conveying the phone number of the cellular phone and the channel identifier.",
"The received audience survey signal is input to a controller 553 which retrieves the channel identifier and, using a time signal from a time signal generator 555, determines from a programme database 557 the programme currently being watched by the user of the cellular phone.",
"In particular, the programme database stores the programmes for each channel identifier for each time and therefore the programme being watched can be determined from the time signal and channel identifier.",
"The controller 553 then stores, in the audience survey database 559, data corresponding to the user, who is identified by the telephone number of the cellular phone, and the television programme being watched by the user.",
"In this way, an almost instantaneous result for the audience survey is produced.",
"The acoustic data channel could also be used for opinion polling.",
"For example, a television programme about a subject could have encoded within its audio track questions about the subject which are displayed by the cellular phone.",
"The user is then able to input an answer to the cellular phone which automatically forwards the answer to a remote site where it is collated along with answers from other cellular phones.",
"An advantage of such opinion polling is that the user of the cellular phone only has to press a button to answer a question and therefore it requires less effort by the user than other telephone opinion polls in which the user has to dial a telephone number, wait for connection and then give a response.",
"This will encourage more people to participate in the opinion poll.",
"For the above example where the opinion poll is associated with a television programme, the result can be given during the television programme.",
"This is an example of how the acoustic data channel can be used to “close the loop” from a broadcaster to a viewer/listener (via a media broadcast) and from the viewer/listener back to the broadcaster (via a telecommunications network).",
"In another application, data for a quiz is transmitted to the cellular phone using the acoustic data channel.",
"For example, a question is broadcast and detected by a plurality of cellular phones.",
"The respective users of the cellular phones input their answers which are transmitted, via the cellular communications network, back to the originator of the question.",
"A prize could then be awarded for the fastest answer.",
"In preferred embodiments, the data transmitted by the cellular phone is enhanced by adding user profile data stored in the cellular phone.",
"This is particularly useful for the audience survey and opinion polling applications described above because the collected data can be analysed in accordance with user characteristics contained in the user profile data.",
"For example, if the user profile data specified the age of the user then the audience survey station could determine the age profile of the audience of a television/radio programme.",
"Those skilled in the art will appreciate that cellular phones which can determine their position are known.",
"For example, the location of a cellular phone can be determined to within five kilometres by identifying the base station of the cellular communications network which is handling the RF communications with the cellular phone.",
"More precise techniques are known, for example the Enhanced Observed Time Difference (EOTD) technique which is described in U.S. Pat.",
"No.",
"6,094,168, whose contents are hereby incorporated by reference, and can determine the position of the cellular phone to within about one hundred metres.",
"Alternatively, the cellular phone could use a Global Positioning System (GPS) or other satellite based systems to determine its position to within about ten metres by analysing RF signals received from satellites.",
"Therefore, data transmitted by the cellular phone can be enhanced with location data calculated by the cellular phone instead of, or in addition to, the user profile data.",
"In this way, for example, an audience survey station could determine the geographical distribution of the audience for a television/radio programme.",
"An advantage of sending enhanced data including data which is either stored in the cellular phone, for example the user profile data, or calculated by the cellular phone, for example location data, is that no additional effort is required by the user.",
"In a further alternative application, the encoder is incorporated within a public address system, for example in an airport or a railway station, and the text of an announcement made over the public address system is encoded within the acoustic signal conveying the announcement.",
"Then, if a user does not hear the announcement, the text of the announcement can be displayed to the user by the user's cellular phone.",
"In a further application, the data signal added to a broadcast audio track includes a source identifier and a time stamp.",
"The user of a cellular phone including a decoder for decoding the data signal is then able to transmit, via the cellular communications network, the source identifier and the time stamp to a remote server which stores a database identifying for each combination of source identifier and time stamp the title of the music being played.",
"The remote server then transmits the title associated with the received source identifier and time stamp to the cellular phone which displays the title to the user of the cellular phone.",
"For example, if a radio station is playing a song having a data signal encoded therein with a source identifier identifying the radio station and a time stamp identifying the track number of the song being played (e.g.",
"the tenth song played by that radio station that day), then the user can download from the remote server the title of the song.",
"Alternatively, the audio track may be a song in a film and the source identifier identifies the film and the time stamp identifies where in the film the song is being played.",
"It will be appreciated that this application could also be performed by any device having a decoder for decoding data sent over an acoustic data channel, a data link via which the device is able to access a telecommunications network, and a display for displaying the title of the song.",
"In an embodiment, the device is removably connectable to a personal computer which is in turn connected to a telecommunications network via a modem.",
"In the first to seventh embodiments, a data signal is encoded within an audio track of a television programme in a television studio.",
"This encoding need not, however, occur prior to the broadcasting of the television signal.",
"For example, the data signal could be carried within the vertical blanking intervals of the video track and either a television set or a “set-top box” could then extract the data signal from the video track and encode it within the audio track.",
"The encoder need not, therefore, be in the television studio but could also, for example, be in a television or a set-top box.",
"The acoustic data channel could also be used to transmit messages which are either broadcast or selectively addressed to particular cellular phones.",
"In an embodiment, the cellular phone includes an acoustic-only mode in which the RF output is disabled but the phone is still able to detect and analyse acoustic signals to recover data.",
"This is particularly advantageous for transmitting messages in areas where, for safety reasons, cellular phones cannot normally be used because of their RF emissions.",
"Examples of such areas include airports, petrol stations and hospitals.",
"The acoustic data channel for transmitting messages could be formed by any commonly encountered audio source.",
"For example, the audio track of a television/radio programme or the output of a public address system could be used.",
"In a particular application, the public address system within a shop is used to transmit message data about, for example, special offers available in the shop.",
"The ability to encode data which is specific to a particular geographical area is useful.",
"For example, a television programme could be broadcast by several different television companies covering different geographical areas.",
"The data encoded within the television programme could therefore be varied from area to area.",
"In this way, if the data is associated with contact addresses, then the data encoded within the television programme for each television station could be associated with only the contact addresses within the geographical area covered by the television station.",
"This is advantageous because it reduces the amount of data sent to the user by automatically removing data which is unlikely to be relevant.",
"The data encoded within the audio track could also be used to control the operation of the cellular phone.",
"For example, in locations such as cinemas, aeroplanes and petrol stations where cellular phones should not be used, a data signal can be broadcast to the cellular phone which either switches the cellular phone off or puts it into a silent mode as appropriate.",
"In one application, the data encoded within the audio track is for a ring tone for the cellular phone, which responds by storing the ring tone data in a NVRAM (or other type of non-volatile memory) within the cellular phone.",
"The ring tone generator of the cellular phone can then generate a ring tone corresponding to the stored ring tone data.",
"In an embodiment, a recorded song has encoded therein data for a ring tone sounding like a segment of the song, and the cellular phone decodes the ring tone so that the ring tone of the cellular phone can be set to sound like the song.",
"In another application, the data for an icon is encoded within the audio track for decoding by the cellular phone.",
"The icon can then either be displayed by the cellular phone or included in data messages to other cellular phones.",
"The acoustic data channel could be used to transmit small application files for the cellular phone.",
"For example, for “Java phones” small application files commonly referred to as APPlets (sometimes referred to as MIDlets) could be received by the cellular phone via the acoustic data channel.",
"The acoustic data channel could also be used to trigger an application file which has already been downloaded onto the cellular phone.",
"For example, a Java phone could have stored an APPlet for displaying an animation sequence and the APPlet could be activated, and the animation sequence accordingly displayed, in response to a trigger signal received via the acoustic data channel.",
"Applications files could also be downloaded into the cellular phone corresponding to computer characters whose behaviour varies in response to a user's actions.",
"In particular, a “behaviour” algorithm within the application file determines the actions of the computer character on the basis of inputs by the user of the cellular phone.",
"The user is therefore able to nurture the behaviour of the computer character.",
"Such computer characters are known, for example Tamagochi.",
"The acoustic data channel could then be used to convey control data which modify the behaviour algorithm of a computer character so that the behaviour of the computer character reacts to events occurring around the user of the cellular phone.",
"For example, a horror film could have control data encoded within its sound track which makes the computer character behave in a manner indicative of being agitated or scared.",
"In this way, the computer character is able to react to external events.",
"In all the above applications and embodiments, a single acoustic data channel is provided for a cellular phone.",
"However, more than one acoustic data channel could be provided.",
"For example, if spread spectrum encoding is used then different pseudo-noise codes could be allocated to each acoustic data channel (i.e.",
"a code division multiple access (CDMA) arrangement).",
"Application files containing pseudo-noise codes for establishing an acoustic data channel and configuration instructions for responding to data received via that acoustic data channel could also be downloaded into the cellular phone.",
"For example, the acoustic data channel corresponding to an application file could be associated with a particular company who send proprietary web page identifiers via the acoustic data channel, and the cellular phone responds by forwarding the proprietary web page identifiers to a server operated by the company.",
"A cellular phone could have more than one of these application files stored therein, with the user being able to select one of the application files, and therefore the pseudo-noise codes used for decoding a detected acoustic signal, using a menu.",
"In an embodiment, a acoustic control channel is provided which uses pseudo-noise codes which are programmed within the cellular phones during manufacture.",
"Subsequently, an application file containing pseudo-noise codes for establishing an additional acoustic data channel and configuration instructions for responding to data received via the additional data channel, are transmitted over the acoustic control channel.",
"In an alternative embodiment, the application file is downloaded via the cellular communications network.",
"A problem with transmitting data acoustically is that the data transmission rates are generally slow, especially if it is desired to minimise the obtrusiveness of the acoustic data signal to a listener.",
"For any application which requires the downloading of a significant amount of data, for example an APPlet file, this problem can be at least partially solved by transmitting a web page address, which requires significantly less data, for a web page from which the data is downloadable, using for example the Wireless Applications Protocol, via the cellular communications network.",
"Alternatively the data could be downloaded using e-mail.",
"Further, it is not essential that the data is downloaded from a web page because it could be downloaded from any information source accessible via the cellular communications network.",
"As the bandwidth required to transmit an electrical signal conveying an original audio track modified by having a data signal encoded therein is no more than required for transmitting an electrical signal conveying the original audio track, during a call a cellular phone could encode a data signal within the outgoing RF signals conveying voice data for receipt by another cellular phone which retrieves the data signal from the received voice data.",
"The data signal could, for example, correspond to a business card.",
"In all the above described applications and embodiments, the cellular phone or other telephone apparatus comprises either an encoder for encoding a data signal within an audio track, or a decoder for decoding a data signal within an electrical signal corresponding to a detected acoustic signal or both an encoder and a decoder.",
"However, this is not essential because a conventional telephone apparatus could be used which either receives from a remote device having an encoder, via a telecommunications network, an audio track which already has a data signal encoded within it for outputting by the telephone apparatus as an acoustic signal, or could transmit, via the telecommunications network, an electrical signal corresponding to a detected acoustic signal to a remote device having a decoder.",
"The eighth, ninth and tenth embodiments describe systems in which a cellular phone encodes a data signal within an acoustic signal emitted by the cellular phone for detection by an electronic device.",
"In the eighth and tenth embodiments, the data signal is encoded in a preset audio sequence whose only purpose is to alert the user that a data transfer is taking place.",
"This preset audio sequence is therefore not essential and the spread data signal could be directly output.",
"In the eighth embodiment, user data is transmitted between two cellular phones using the acoustic data channel.",
"Alternatively, the acoustic data channel could be used to enable two cellular phones to interact with each other.",
"In an embodiment, an interactive game is provided which allows two players to play against each other using their respective cellular phones with data being transmitted between the cellular phones using the acoustic data channel.",
"For example, the interactive game could be a chess game which has the advantage that only a small amount of data need be transmitted between the two cellular phones.",
"In the ninth and tenth embodiments, the control signals are used to activate corresponding routines within the electronic device.",
"However, those skilled in the art will appreciate that the control signals could themselves convey the routines which are implemented by the electronic device.",
"For example, the control signal could convey information enabling a speech synthesiser located within the electronic device to produce a desired sound, for example a word or phrase.",
"In the ninth and tenth embodiments, the control signals emitted by the cellular phone are used to control a toy.",
"Those skilled in the art will appreciate that the control signals could control devices other than toys.",
"For example, the cellular phone could be used as a remote control device so that by inputting instructions into the cellular phone, acoustic control signals are output for controlling a television, a home entertainment system, an airconditioning unit or another home appliance.",
"The cellular phone can also be used to store credits, in which case a portion of either the RAM or NVRAM is dedicated to storing a value indicative of the number of credits held by the user of the cellular phone.",
"These credits can be downloaded into the cellular phone either via the conventional cellular communications network or via the acoustic data channel.",
"The cellular phone can then output, via the acoustic data channel, control signals to vending appliances in order to purchase goods.",
"The credits stored in the cellular phone could be accrued on a loyalty basis.",
"For example, each time a user of the cellular phone goes to a particular cinema chain, a device within the cinema sends, via the acoustic data channel, a credit to the cellular phone, which in response increments the number of stored credits by one.",
"When the number of credits stored in the cellular phone reaches a predetermined number the cellular phone can emit a control signal, via the acoustic data channel, which reduces the price of a cinema ticket.",
"The cellular phone could be used to store an electronic boarding card for an airflight.",
"Alternatively, the cellular phone could receive electronic tickets, via the acoustic data channel, for other public transport systems such as rail or coach.",
"The cellular phone could also emit a signal, via the acoustic data channel, which controls ticket barriers provided in a public transport system when the cellular phone is storing an electronic ticket.",
"The cellular phone could also interact via the acoustic data channel with a network device connected to a network other than a cellular communications network, for example a Bluetooth network.",
"In this way, the cellular phone can communicate with remote devices via networks other than the cellular communications network.",
"In some of the above embodiments, SMS messages are used to convey data to or from the cellular phone.",
"Other alternative RF data transmission techniques could be used, for example EMS messaging and MMS messaging.",
"Those skilled in the art will appreciate that the novel encoding and decoding techniques described in the first to tenth embodiments could also be used in applications which do not involve a telephone.",
"For example, the described encoding and decoding techniques could be used in a toy system such as that described in International Patent Publication WO 01/10065, the contents of which are hereby incorporated by reference.",
"FIG.",
"37 shows the main components of such a toy system.",
"In FIG.",
"37, the television broadcast system is identical to that of the first embodiment and has therefore been referenced using the same numerals as in the first embodiment.",
"Further, the electronic toy is identical to that of the ninth and tenth embodiments and has therefore been referenced using the same numerals as in the ninth and tenth embodiments.",
"In this toy system, the data signal F(t) is a control signal for the electronic toy 425 and, on detecting the acoustic signal conveying the control signal, the electronic toy 425 alters its expression and outputs an associated sound.",
"In another embodiment, the electronic toy 425 further includes a user interface, for example a keyboard, via which a user can input information into the electronic toy.",
"This allows the user to interact with a television programme or the like via the electronic toy.",
"For example, the television programme may be a quiz show and when a question is broadcast, the answer could be digitally encoded within the audio track corresponding to the question so that it can be decoded by the decoder within the electronic toy.",
"The user then enters an answer to the question into the electronic toy, via the user interface, and the electronic toy compares the user's answer with the answer recovered from the audio track.",
"The electronic toy then responds in dependence upon whether the user's answer is correct or not by, for example, smiling if the answer is correct and frowning if the answer is not.",
"Preferably, the electronic toy is able to send the user's answer to a question to the programme maker of the quiz show via a telecommunications network.",
"For example, if the television programme is broadcast on a digital network the user's answer can be directly sent over the same network as used for broadcasting the television programme.",
"If the electronic toy includes a timer for timing how long the user took to enter the answer, then details of this time could be sent to the programme maker of the quiz show which could then award a prize to the person who responds the quickest with the correct answer.",
"It will be appreciated that for this type of three-way interaction (i.e.",
"between a television programme, an electronic toy and a user), the electronic toy could be replaced by any object having suitable circuitry for decoding data sent using the acoustic data channel and for processing the data received via the acoustic data channel and the user interface.",
"Although hardware encoders are used in the described embodiments, those skilled in the art will appreciate that the function of these hardware encoders could also be performed by a computer apparatus running appropriate software.",
"Similarly, the function of the software decoder of the first to sixth embodiments could be implemented in a hardware decoder and the function of the hardware decoder of the seventh to tenth embodiments could be implemented by a software decoder.",
"The invention therefore also extends to computer programs, particularly computer programs on or in a carrier, adapted for putting the invention into practice.",
"The program may be in the form of source code, object code, a code intermediate source and object code such as in partially compiled form, or in any other form suitable for use in the implementation of the processes according to the invention.",
"The carrier may be any entity or device capable of carrying the program.",
"For example, the carrier may comprise a storage medium such as a ROM, for example a CD-ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disk or hard disk.",
"Further, the carrier may be a transmissible carrier such as an electrical or optical signal which may be conveyed via electrical or optical cable or by radio or other means.",
"When the program is embodied in a signal which may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means.",
"Alternatively, the carrier may be an integrated circuit in which the program is embedded, the integrated circuit being adapted for performing, or for use in the performance of, the relevant processes.",
"Those skilled in the art will appreciate that the invention can be applied to cellular phones which do not conform with the GSM specification.",
"Further, web page data can be retrieved using protocols other than the Wireless Application Protocol.",
"The first to seventh embodiments describe seven different encoders.",
"Those skilled in the art will appreciate that any of these seven encoders could be used in the above described further embodiments and applications.",
"In an embodiment, the data signal generator and the encoder are in physically separated units, and the data signal is input to an input port of the encoder unit.",
"In an alternative embodiment, the encoder and the decoder could be housed in the same unit (i.e.",
"share a common housing).",
"Those skilled in the art will appreciate that the wavelet transforms used in the second embodiment can replace the Fourier transforms used in the third to tenth embodiments.",
"In the third embodiment, the carrier frequency used in the modulator is varied so that the centre frequency of the spread data signal is positioned in a relatively high energy portion of the audio track.",
"In particular, the energy in 2048 frequency sub-bands is monitored and the carrier frequency of the modulator varied accordingly.",
"In an alternative embodiment, the number of frequency sub-bands could be reduced, for example to eight, in order to reduce the amount of processing required.",
"In the decoder for the third embodiment, the received audio track is analysed to determine the carrier frequency used in the modulator in the encoder.",
"However, if the encoder uses a small number of frequency sub-bands, and therefore only a small number of carrier frequencies are possible, then in an alternative embodiment the demodulator in the decoder passes the received audio track through a number of separate channels with each channel using a respective one of the possible carrier frequencies, and subsequently determines which channel provides the strongest signal in order to recover the data signal.",
"This has the advantage over determining the carrier frequency from the received audio track that if the audio track is altered by the communication channel between the encoder and the decoder then the data signal is still correctly recovered.",
"In the fourth embodiment, a linear predictive coding (LPC) algorithm is used to separate the audio track into a tonal part, whose values are determinable from each other, and a noise part, whose values are apparently random.",
"Alternatively, other auto-regressive algorithms could be used to isolate a random part of the audio track which can be at least partially replaced by a shaped spread spectrum signal.",
"Further, the frequency spectrum of the audio track could be analysed to identify tonal components which are subsequently removed to separate the tonal part and the noise part.",
"In the fifth embodiment, the tonality of the audio track is determined by analysing the frequency spectrum of the audio track.",
"Alternatively, the tonality could be determined by applying a time series model, for example an LPC model, having a fixed number of coefficients and determining the power in the modelled signal corresponding to the coefficients.",
"In the sixth embodiment, the number of LP coefficients, the level of the psycho-acoustic encoding and the gain factor G used in the variable combiner are selected by a user.",
"Alternatively, one or two or all three of these variables could be automatically set by the encoder.",
"For example, the number of LP coefficients used could be determined from the tonality of the audio track.",
"In the sixth embodiment, the noise part N(t) and the shaped signal S(t) are input to a variable combiner, and the output of the variable combiner is added to the tonal part P(t) to form the modified audio track.",
"Alternatively, the original audio track could be input to the variable combiner instead of the noise part N(t), as in the fourth embodiment, so that the output of the variable combiner forms the modified audio track and the LPC analysis is only used for shaping the modulated signal G(t).",
"In other words, the modulated signal G(t) is shaped to approximate the noise part N(t) of an audio track to form a shaped signal S(t), and then the shaped signal S(t) is added directly to the audio track in a variable combiner.",
"In an embodiment, the gain factor G for the variable combiner is automatically set so that the signal to noise ratio of the data signal in the modified audio track is within a predetermined range, for example between −10 and −15 dB.",
"The signal to noise ratio can be calculated by inputting the shaped data signal S(t) into a first power monitor, inputting the audio track into a second power monitor, and then dividing the power in the shaped data signal by the total power level obtained by adding the power in the audio track and the power in the shaped data signal.",
"Alternatively, the signal to noise ratio could be calculated by dividing the power in the shaped data signal by the power in the audio track.",
"Those skilled in the art will appreciate that for some applications the described shaping techniques for shaping the data signal to reduce the obtrusiveness of the data signal in the modified audio track are not required because it is not essential for the audio track to be reproduced at high quality.",
"In addition, the encoder of the sixth embodiment could be modified to remove the variable combiner, in which case the signal shaped using the psycho-acoustic algorithm is added directly to the tonal part of the audio track.",
"For automated encoders which do not require a user input, the encoding can be carried out in real-time and therefore is suitable for, for example, real-time addition to television broadcasts or public address announcements.",
"In the seventh embodiment, the encoder separates a single data signal into one or more data streams dependent upon the ability of the audio track to hide the data signals.",
"In particular, in the seventh embodiment the additional data streams are sent in parallel with the bit rate of each data stream being the same regardless of the number of data streams.",
"Alternatively, the data rate could be varied by increasing the bit rate for a single data stream while maintaining the same chip rate so that fewer chips are used per bit.",
"In an embodiment, for higher data rate transmission the bit rate is doubled and only the first half of the pseudo-noise code sequences PN0 and PN1 of the seventh embodiment is used to spread the data bits.",
"This has the advantage that a high data rate decoder could be provided which, although it would only be able to decode data transmitted at the higher data rate, only needs to store the first half of each of the pseudo-noise codes PN0 and PN1.In another embodiment, for higher data rate transmission the bit rate is doubled and for each sequence of two bits one is represented by the first half of one of the pseudo-noise codes sequences PN0 and PN1 and the other is represented by the second half of one of the pseudo-noise code sequences PN0 and PN1.This has the advantage that the pseudo-noise codes are sequentially repeated over their maximal length rather than having to be reset for every new bit.",
"In applications where an electronic device responds to the data signal encoded within the modified audio track, if the response of the electronic device is required to be synchronised with the audio track then the variable data rate techniques described above could disrupt this synchronisation.",
"In order to allow the decoder to recover synchronisation of the data signal and the audio track, the encoder could encode timing data in the data signal indicative of the required synchronisation.",
"An embodiment of such an encoder will now be described with reference to FIG.",
"38 which illustrates an alternative data rate determining unit 571 to replace the data rate determining unit 235 of the seventh embodiment.",
"In FIG.",
"38 components which are identical to corresponding components in the seventh embodiment have been referenced with the same numerals.",
"As shown in FIG.",
"38, in the data rate determining unit 571 the audio track is input to a spectrum analyser 573 which monitors the power of the audio track within the frequency range of 1 to 5 kHz.",
"In particular, the average power level in the monitored frequency band over a time period of five seconds is determined.",
"The spectrum analyser 573 outputs a signal which varies in accordance with the determined average power level to a timing encoder 575 and the output generator 265.The modified data stream M′(t) is input to the timing encoder 575 via the FIFO memory 261.If the average power level output by the spectrum analyser 573 is high, then the data rate determining unit 571 uses the opportunity to send as much data as possible and therefore time synchronisation between the data signal and the audio track is lost.",
"However, the timing encoder 575 determines, using the average power level output by the spectrum analyser 573, the amount of this synchronisation loss and encodes timing data within the modified data signal M′(t) which enables a decoder to retrieve time synchronisation between the data signal and the audio track.",
"The output generator then outputs either one, two or three data streams as described in the seventh embodiment.",
"The operation of the data rate determining unit 571 will now be explained with reference to FIGS.",
"39A to 39C.",
"FIG.",
"39A shows an audio track having a large first peak 577a followed by a small second peak 577b and then a large third peak 577c.",
"FIG.",
"39B shows a data signal which is synchronised with the audio track illustrated in FIG.",
"39A and has a series of three peaks 578a to 578b.",
"The first and third data signal peaks 578a, 578c coincide with the large first and third audio track peaks 577a, 577c which effectively mask by the first and third data signal peaks 578a, 578c.",
"However, the second data signal peak 577b which cannot effectively mask the second data signal peak 578b.",
"Therefore, as illustrated in FIG.",
"39C which shows the data signal output by the data rate determining unit 571, the data for the second data signal peak 578b is output at the same time as the data for the first data signal peak 578a, in a first peak 579a, along with timing data to enable a decoder to recover timing synchronisation between the data signal and the audio track.",
"When combined with the audio track, the first peak 579a is masked by the first audio signal peak 577a.",
"In an alternative embodiment, synchronisation between the data signal and the audio track is maintained using timing data as described above, but the acoustic signal only conveys a single data stream whose chip rate is constant but whose volume is varied in dependence on the audio track.",
"Decoding is more straightforward if the chip rate is maintained constant for the different data rates because it is easier to maintain synchronisation between the received chip sequence and the pseudo-noise codes generated in the decoder.",
"However, the chip rate could be varied in order to vary the bandwidth of the spread signal so that it is better masked by the audio track.",
"In an embodiment, the encoder varies the chip rate in accordance with the tonality of the audio track.",
"If the audio track is very tonal then the encoder uses a low chip rate and varies the carrier frequency used during modulation, as described in the third embodiment, so that the spread data signal is masked within the tonal region of the audio track, while if the audio track has a broader frequency spectrum then the encoder uses a high chip rate.",
"In this embodiment, the encoder determines the chip rate and carrier frequency used by analysing the audio track.",
"Alternatively, in the decoder the received audio track could be passed through a number of channels with a respective different one of the possible chip rates being used in each channel, with the decoder then determining which channel provides the largest signal.",
"In the encoder of the tenth embodiment, the electronic toy 425 outputs a sound and alters its facial expression in accordance with a data signal encoded within the audio track of a television signal predominantly in the frequency range 0 to 4 kHz.",
"This frequency range was chosen because nearly all loudspeakers work well in this frequency range.",
"However, many loudspeakers work well up to much higher frequencies.",
"An alternative encoder 581 will now be described with reference to FIG.",
"40 in which only the data for controlling the sound output by the electronic toy 425 is centred in the frequency range 0 to 4 kHz, and the data for controlling the facial expression of the electronic toy 425 is centred in the frequency range 4 to 8 kHz.",
"This is advantageous if, for example, the audio track is particularly quiet and is therefore unable to hide data for both controlling the facial expression and the output sound in the 0 to 4 kHz frequency band.",
"In FIG.",
"40, components which are identical to corresponding components in FIG.",
"2 have been referenced with the same numerals.",
"As shown in FIG.",
"40, a first data signal F1(t) for controlling the sound output by the electronic toy 425 and a second data signal F2(t) for controlling the facial expression of the electronic toy 425 are input to respective channels of the encoder.",
"The first data signal F1(t) is modified by a first FEC unit 51a, spread using first and second pseudo-noise codes PN0, PN1, and then modulated using a carrier frequency of 2 kHz by a first modulator 57 to form a first modulated signal G1(t).",
"Similarly, the second data signal F2(t) is modified by a second FEC unit 51b, spread using third and fourth pseudo-noise codes PN2, PN3, and then modulated using a carrier frequency of 6 kHz by a second modulator 583 to form a second modulated signal G2(t).",
"The first and second modulated signals are then added by an adder 585 before being input to the shaping unit 61.When the modified audio track output by the encoder 581 illustrated in FIG.",
"40 is converted into an acoustic signal by a loudspeaker, if the loudspeaker works satisfactorily up to 7 kHz then the electronic toy will both output a sound and alter its facial expression.",
"However, if a lower quality loudspeaker is used then the electronic toy will only output a sound because it is unable to decode the data signal for altering the facial expression.",
"Those skilled in the art will appreciate that the encoder 581 shown in FIG.",
"40 is particularly useful in applications where the first data signal F1(t) is a main data signal, which conveys the most important information, and the second data signal F2(t) is an auxiliary data signal, which conveys optional information.",
"In this way the main data signal is reliably decoded even if the auxiliary data signal is not.",
"Further, this concept can be extended to three or more information channels.",
"For example, information could be transmitted in three separate frequency bands centred on 2 kHz, 5.5 kHz and 9 kHz respectively.",
"If plural information channels are used, then the decoder could include a switch which determines which of the information channels is decoded.",
"In an alternative embodiment, N data signals are input to an encoder.",
"The encoder analyses the audio track to determine its ability to hide data, and based on this analysis encodes M data signals (where M is less than or equal to N) of the N data signals within the audio track, with the number M varying as the ability of the audio track to hide data varies.",
"Those skilled in the art will also appreciate that the alternative decoding techniques described in WO 01/10065 can be used in the first to tenth embodiments.",
"In the described embodiments, the data signal F(t) is continuously added to the audio track.",
"Alternatively, the encoder could identify the portions of the audio track which are better suited to hiding the data signal and then only encode data in those identified portions.",
"In an embodiment, the audio track is input to a spectrum analyser which determines the tonality of the audio track (for example the spectrum analyser 201 of the fifth embodiment) and only adds the data signal in the portions of the audio track for which the tonality is below a predetermined level.",
"In another embodiment, the encoder determines which portions are better suited to hide the data signal based on the power of the audio track, while in yet another embodiment the encoder determines the portions based on a combination of the power and tonality.",
"As described above, in some applications control data for synchronising an operation of a cellular phone with a broadcast audio signal, for example for a television program or a radio program, is encoded within the audio signal and the acoustic wave corresponding to the audio signal is detected by a cellular phone which decodes the control data and responds with the required synchronisation.",
"A problem with such synchronised systems is that sometimes the audio track at the moment the response by the cellular phone is required is too quiet to hide the control data effectively.",
"This problem can be alleviated by sending the control data in advance during a louder portion of the audio track along with timing data indicating the timing when the cellular phone should respond in accordance with the control data.",
"Then, in the quieter portion of the audio track when the response takes place, no data need be transmitted.",
"The timing data need not be transmitted simultaneously with control data, but could be transmitted separately.",
"For example, at the beginning of a data signal conveying a sequence of control data, timing data could be transmitted indicating that the response to each item of control data is to be delayed for a preset period of time.",
"Although the time delay is not optimised for each item of control data, sending timing data in this way reduces the total amount of data which needs to be sent.",
"In the described embodiments, the data signal is spread over audible frequencies using DSSS encoding.",
"As those skilled in the art will appreciate, signals encoded using DSSS encoding can be decoded in two main ways.",
"In one way, the encoded signal is synchronously multiplied by the same pseudo-noise code which was used to encode the signal, a technique commonly referred to as coherent detection.",
"In the other way, a correlator such as a matched filter is used to correlate the encoded signal with the pseudo-noise code which was used to encode the signal, a technique commonly referred to as incoherent detection.",
"In the described embodiments, a data bit whose value is “0” is represented by a pseudo-noise code PN0 and a data bit whose value is “1” is represented by a pseudo-noise code PN1.Those skilled in the art will appreciate that if coherent decoding is used, then the pseudo-noise code PN0 can be the inverse of pseudo-noise code PN1.In an embodiment, a first plurality of pseudo-noise codes are provided for representing a data bit whose value is “0” and a second plurality of pseudo-noise codes are provided for representing a data bit whose value is “1”.",
"The choice of which pseudo-noise code is used to encode a data bit can then be based upon which pseudo-noise code produces a data signal which is the least perceptible when added to an audio track.",
"Subsequently, the decoder could either analyse the audio track to determine which pseudo-noise code had been used or decode the received audio track using all possible pseudo-noise codes.",
"Decoding using all possible pseudo-noise codes has the advantage that the decoding is more robust if the audio track is altered by the communication channel between the encoder and the decoder.",
"However, if there are a large number of pseudo-noise codes then analysing the audio track to determine which pseudo-noise code was used requires less processing power.",
"The described embodiments utilise a SYNC bit which is added to the data signal to help the decoder decode the data signal.",
"Preferably, the SYNC bit is added to the audio track with a higher signal level than the other data bits of the data signal to improve the ability of the decoder to decode the data signal.",
"Further, if the signal level of the SYNC bit is increased, the decoder is able to reduce the number of chips within the chip sequence corresponding to the SYNC bit which are being monitored to achieve synchronisation.",
"Those skilled in the art will appreciate that the signal level of the SYNC bit could be increased by requiring the signal-to-noise ratio of the data signal in the modified audio track to be greater for a portion of the data signal corresponding to a SYNC bit than for the rest of the data signal.",
"In some of the described embodiments, the signal level of the data within the audio track is varied, for example by varying the number of data streams or by amplifying the SYNC bits.",
"In these embodiments, preferably the signal level is smoothly or gradually varied, for example following a Gaussian function, because this reduces the obtrusiveness of the data within the audio track.",
"The energy of a data signal can be spread over a wide range of frequencies by using techniques other than DSSS encoding.",
"For example, an Orthogonal Frequency Division Modulation (OFDM) technique can be used in which, for example, 256 narrow-band orthogonal carriers carry identical data.",
"These 256 narrow-band carriers are evenly distributed in the frequency range of 1 to 5 kHz and thus spreading of the energy of the data signal is achieved.",
"The original data signal can then be reconstructed by demodulating the recombining each of the narrow-band signals.",
"It will be appreciated by a person skilled in the art that still further techniques could be used to spread the energy of the data signal.",
"For example, frequency hopping could be used in which the frequency of the modulated data signal is changed in a random manner.",
"Although spread spectrum encoding is preferred because it reduces the noticeability of the acoustic data channel to a listener, it is not an essential feature of the invention.",
"The acoustic data channel could also be formed, for example, by using a dedicated narrow frequency range.",
"Alternatively, a data signal could be encoded within an audio track by systematically modifying either frequency or time information within the audio track.",
"For example, an echo modulation scheme could be used in which an echo signal is added with a time delay which is varied in accordance with a data signal.",
"Alternatively, a critical band encoding technique could be used in which each data value is associated with a respective set of narrow-band frequencies.",
"In another embodiment, the acoustic data channel uses an ultrasonic link which has the advantage that it is not audible to the user of the cellular phone.",
"In the first to eighth embodiments, phase shift keying is used to modulate the spread data signal.",
"Those skilled in the art will appreciate that other modulation schemes, for example frequency shift keying or quadrature amplitude modulation, could be used instead.",
"For ease of explanation, the data signal in the described embodiments has been encoded within a single channel audio track.",
"However, the data signal could also be encoded within a stereo audio track having two or more channels.",
"The data signal can be added synchronously to more than one channel of the stereo audio track or with a time offset of, for example, 150 ms between the channels.",
"Introducing a time offset has the advantage of adding an additional level of time diversity, thereby enabling a more robust regeneration of the data signal because there is less chance of losing data due to background noise (i.e.",
"noise which is not the modified audio track).",
"Alternatively, two different broadband signals could be generated with each one being added to a respective channel of the stereo audio track.",
"Alternatively, for a multi-channel audio track, the data signal need not ne encoded in every channel of the audio track.",
"For example, for a television programme in which one channel of the audio track carries voice data and another channel of the audio track carries background music data, the data signal could be added only to the channel carrying background music data.",
"In some embodiments, a psycho-acoustic algorithm is used to reduce the obtrusiveness of the data signal within the modified audio track.",
"However, a psycho-acoustic algorithm is not essential and could be replaced by a simpler algorithm which requires less processing power.",
"Those skilled in the art will appreciate that conventional equalisation techniques, for example using a rake receiver, can be applied in the decoder to improve the bit error rate in the presence of multi-path components or frequency response impairments.",
"Further, an automatic gain control circuit could be included at the input of the decoder.",
"The precise values of the bit rates, chip rates, sampling rates and modulation frequencies described in the detailed embodiments are not essential features of the invention and can be varied without departing from the invention.",
"Further, while in the described embodiments the data signal is a binary signal, the data signal could be any narrow band signal, for example a modulated signal in which frequency shift keying has been used to represent a “1” data bit by a first frequency and a “0” data bit as second different frequency.",
"Further, those skilled in the art will appreciate that the order in which the spreading, modulating and shaping is performed in the encoder can be varied.",
"Although digital signal processing techniques have been described as the preferred implementation of the invention, analogue processing techniques could be used instead.",
"As those skilled in the art will appreciate, the sampling rate of 22.05 kHz matches that used for one channel of a compact disc and therefore the encoders and decoders described for these embodiments are suitable for use in systems where a data signal is conveyed by an audio track recorded on a compact disc.",
"A sampling rate of 44.1 kHz could also be used for recording the data signal on a compact disc, and a sampling rate of 48 kHz could be used for recording a data signal on a DVD.",
"It will be appreciated that the term audio track refers to an electrical signal which is intended to be reproduced as a corresponding acoustic signal by a loudspeaker in the audible range of frequencies, which typically spans from 20 Hz to 20,000 Hz.",
"The duration of the audio track can be short, for example the ring tone of a cellular phone or doorbell, or long, for example the soundtrack to a movie.",
"The cellular phone used in the above embodiment could be replaced by a different cellular communication device such as a pager.",
"Further, the cellular phone could be replaced by a different mobile internet device, such as a laptop computer, a portable web browser or a personal digital assistant (PDA)."
]
] | Patent_10432889 |
[
[
"Electronic Data Storage Medium",
"The invention concerns an electronic data storage medium including a body (30) made from insulated plastic material which has two main parallel sides, a semiconductor component (34) and a number of external electrical contact pads flush with the first main side (30a) of the body.",
"The body (30) includes a recess (32) opening out onto the first main side (30a).",
"The storage medium also includes a number of conducting elements (28), each with a first end (a) flush with the first main side to form an external contact pad, a second end (b) arranged in the said recess and an intermediate part (c) buried in the body; and A number of connecting elements (38) to electrically connect the terminals of the semiconductor component to the second ends of the conducting elements."
],
[
"1.An electronic data storage medium including a body made from insulated plastic material which has two main parallel sides, a semiconductor component and a number of external electrical contact pads flush with the first main side of the body, wherein, the said body includes a recess opening out onto the first main side, in which the semiconductor component is fixed, the said storage medium also includes: a number of conducting elements, each conducting element having a first end flush with the first main side to form an external contact pad, a second end arranged in the said recess and an intermediate part buried in the body; and a number of connecting elements to electrically connect the terminals of the semiconductor component to the second ends of the conducting elements; the volume of the said recess not occupied by the said semiconductor component being filled by an insulating material.",
"2.The electronic data storage medium according to claim 1, wherein each electrical conducting element has the shape of a folded conducting strip, the two ends being approximately parallel and the intermediate part being inclined, the said recess being arranged between the second ends of the conducting elements forming the external contact pads.",
"3.The electronic data storage medium according to claim 1, wherein each electrical conducting element has the shape of a blanked part, the two ends being approximately parallel and the intermediate part being inclined and having a shape not rectilinear with the said ends, the said recess being offset with respect to the portion of the main side of the body containing the first ends of the electrical conducting elements.",
"4.The electronic data storage medium according to claim 1, wherein the said body has the shape of a right parallelepiped with two main sides parallel and an edge.",
"5.The electronic data storage medium according to claim 4, wherein the said body has aligned hollows separating a first body portion containing the said semiconductor component and the said external contact pads, and a second portion, the said hollows enabling the two body portions to be separated.",
"6.The electronic data storage medium according to claim 4, wherein the said body has near its edge elements to clip onto another part.",
"7.A method of manufacturing an electronic data storage medium including a body, a semiconductor component, external electrical contact pads and electrical connecting elements between the said component and the said external contact pads, said method comprising: supplying a mould, whose cavity has the shape of the body to be produced, the said cavity having one main side flat, the said mould having a core projecting into the said main side; arranging a number of electrical conducting elements in the said cavity, each having a first end applied against the said main side, to form an external contact pad, a second end applied against the said core and an intermediate part; injecting a hot thermoplastic material under pressure into the said cavity so that the said material fills the entire volume of the cavity not occupied by the said conducting elements; demoulding the part so produced, to obtain the body of the data storage medium with a recess, the first ends of the conducting elements being flush with the main side, the second ends of the conducting elements being flush with the wall of the said recess, the intermediate part of the conducting elements being buried in the thermoplastic material; fixing the said semiconductor component in the said recess; electrically connecting the second ends of the conducting elements to the terminals of the semiconductor component; and filling the volume of the said recess not occupied by the said semiconductor component with an insulating material."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>To manufacture these types of electronic data storage media, the technique most often used is as follows: in a first step, a data storage medium body is created with the required external shape, and including a recess.",
"In a second step, an electronic module is produced, composed of the semiconductor component which is fixed on a support, this support itself defining the external electrical contact pads connected to the semiconductor component.",
"Lastly, the electronic module is fixed in the recess of the body of the electronic data storage medium.",
"These various steps and in particular the realization of the electronic module are relatively complex and therefore generate relatively high costs, whereas the manufacture of the electronic data storage medium must have a global manufacturing cost as low as possible so that this cost has no significant impact on the service that can be obtained using the electronic data storage medium.",
"Also, the electronic module is “relatively large” and it is difficult to fix in the recess of the body of the electronic data storage medium."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The first objective of this invention is to supply an electronic data storage medium which can be manufactured at a lower cost whilst offering the same quality.",
"To reach this objective, according to the invention, the electronic data storage medium includes a body made from insulated plastic material which has two main parallel sides, a semiconductor component and a number of external electrical contact pads flush with the first main side of the body.",
"It is characterized in that: the said body includes a recess opening out onto the first main side, in which the semiconductor component is fixed, the said storage medium also includes: a number of conducting elements, each conducting element having a first end flush with the first main side to form an external contact pad, a second end arranged in the said recess and an intermediate part buried in the body; and a number of connecting elements to electrically connect the terminals of the semiconductor component to the second ends of the conducting elements; the volume of the said recess not occupied by the said semiconductor component being filled by an insulating material.",
"We can see that in the electronic data storage medium defined above, the conducting elements intended to form external electrical contact pads and some of the electrical connecting assembly between the semiconductor component and the external contact pads are partly buried in the plastic body of the data storage medium, thereby fastening them and positioning them with respect to the body of the data storage medium, this body including a recess in which the semiconductor component alone can be easily fixed.",
"After fitting the electrical connecting elements, the recess is filled with a plastic material such as an epoxy resin to obtain the final shape of the electronic data storage medium.",
"According to a preferred mode of realization, the electrical data storage device is characterized in that each electrical conducting element has the shape of a blanked part, the two ends of this part being approximately parallel and the intermediate part being inclined and having a non rectilinear shape, the recess being offset with respect to the portion of the main side of the body containing the first ends of the electrical conducting elements which form the external electrical contact pads.",
"According to this preferred mode of realization, the recess intended to receive the semiconductor component is offset with respect to the location of the external electrical contact pads.",
"It is therefore possible to use a large semiconductor component whilst giving all the external electrical contact pads the configuration which is provided for in particular by the ISO standards concerning bank cards or cards for fixed telephones.",
"Another objective of this invention is to supply a mode of realization of an electronic data storage medium which reduces the cost of manufacture whilst producing electronic data storage media which have the same characteristics as with the modes of realization of the prior art.",
"To reach this objective according to the invention, the method of manufacturing an electronic data storage medium including a body, a semiconductor component, external electrical contact pads and electrical connecting elements between the said component and the said external contact pads, is characterized in that it includes the following steps: a mould is supplied, whose cavity has the shape of the body to be produced, the said cavity having one main side flat, the said mould having a core projecting into the said main side; in the said cavity a number of electrical conducting elements are arranged, each having a first end applied against the said main side, to form an external contact pad, a second end applied against the said core and an intermediate part; a hot thermoplastic material is injected under pressure into the said cavity so that the said material fills the entire volume of the cavity not occupied by the said conducting elements; the part so produced is demoulded, to obtain the body of the data storage medium with a recess, the first ends of the conducting elements being flush with the main side, the second ends of the conducting elements being flush with the wall of the said recess, the intermediate part of the conducting elements being buried in the thermoplastic material; the said semiconductor component is fixed in the said recess; the second ends of the conducting elements are connected electrically to the terminals of the semiconductor component; and the volume of the said recess not occupied by the said semiconductor component is filled with an insulating material.",
"Other features and advantages of the invention will appear on reading the description which follows of several modes of realization of the invention given as non-limiting examples.",
"The description refers to the attached figures, on which:"
],
[
"FIELD OF THE INVENTION This invention concerns an electronic data storage medium of the type with a body of insulating material and a semiconductor component fixed in the said body and a mode of realization of such a storage medium.",
"An electronic data storage medium is considered to be a portable object composed of a body generally made from plastic material in which is fixed a semiconductor component formed by electronic circuits which can store data and generally process it.",
"The semiconductor component is connected electrically to external electrical contact pads to make the electrical link between the semiconductor component and the circuits of a reading and writing device into which the storage medium is inserted.",
"The reading and writing device is associated with various devices controlled by the reader, these devices may be ticket distributors, payment terminals, fixed telephones or GSM type portable telephones.",
"The term electronic memory cards or bank cards is used for the storage media used in payment terminals or in fixed telephones, these cards having a right parallelepiped body and reduced thickness whose dimensions are defined by standards.",
"For portable telephones, the so-called SIM cards may have various types of body shape adapted to the readers associated with the portable telephones.",
"This invention concerns the various types of electronic data storage media defined above.",
"BACKGROUND OF THE INVENTION To manufacture these types of electronic data storage media, the technique most often used is as follows: in a first step, a data storage medium body is created with the required external shape, and including a recess.",
"In a second step, an electronic module is produced, composed of the semiconductor component which is fixed on a support, this support itself defining the external electrical contact pads connected to the semiconductor component.",
"Lastly, the electronic module is fixed in the recess of the body of the electronic data storage medium.",
"These various steps and in particular the realization of the electronic module are relatively complex and therefore generate relatively high costs, whereas the manufacture of the electronic data storage medium must have a global manufacturing cost as low as possible so that this cost has no significant impact on the service that can be obtained using the electronic data storage medium.",
"Also, the electronic module is “relatively large” and it is difficult to fix in the recess of the body of the electronic data storage medium.",
"SUMMARY OF THE INVENTION The first objective of this invention is to supply an electronic data storage medium which can be manufactured at a lower cost whilst offering the same quality.",
"To reach this objective, according to the invention, the electronic data storage medium includes a body made from insulated plastic material which has two main parallel sides, a semiconductor component and a number of external electrical contact pads flush with the first main side of the body.",
"It is characterized in that: the said body includes a recess opening out onto the first main side, in which the semiconductor component is fixed, the said storage medium also includes: a number of conducting elements, each conducting element having a first end flush with the first main side to form an external contact pad, a second end arranged in the said recess and an intermediate part buried in the body; and a number of connecting elements to electrically connect the terminals of the semiconductor component to the second ends of the conducting elements; the volume of the said recess not occupied by the said semiconductor component being filled by an insulating material.",
"We can see that in the electronic data storage medium defined above, the conducting elements intended to form external electrical contact pads and some of the electrical connecting assembly between the semiconductor component and the external contact pads are partly buried in the plastic body of the data storage medium, thereby fastening them and positioning them with respect to the body of the data storage medium, this body including a recess in which the semiconductor component alone can be easily fixed.",
"After fitting the electrical connecting elements, the recess is filled with a plastic material such as an epoxy resin to obtain the final shape of the electronic data storage medium.",
"According to a preferred mode of realization, the electrical data storage device is characterized in that each electrical conducting element has the shape of a blanked part, the two ends of this part being approximately parallel and the intermediate part being inclined and having a non rectilinear shape, the recess being offset with respect to the portion of the main side of the body containing the first ends of the electrical conducting elements which form the external electrical contact pads.",
"According to this preferred mode of realization, the recess intended to receive the semiconductor component is offset with respect to the location of the external electrical contact pads.",
"It is therefore possible to use a large semiconductor component whilst giving all the external electrical contact pads the configuration which is provided for in particular by the ISO standards concerning bank cards or cards for fixed telephones.",
"Another objective of this invention is to supply a mode of realization of an electronic data storage medium which reduces the cost of manufacture whilst producing electronic data storage media which have the same characteristics as with the modes of realization of the prior art.",
"To reach this objective according to the invention, the method of manufacturing an electronic data storage medium including a body, a semiconductor component, external electrical contact pads and electrical connecting elements between the said component and the said external contact pads, is characterized in that it includes the following steps: a mould is supplied, whose cavity has the shape of the body to be produced, the said cavity having one main side flat, the said mould having a core projecting into the said main side; in the said cavity a number of electrical conducting elements are arranged, each having a first end applied against the said main side, to form an external contact pad, a second end applied against the said core and an intermediate part; a hot thermoplastic material is injected under pressure into the said cavity so that the said material fills the entire volume of the cavity not occupied by the said conducting elements; the part so produced is demoulded, to obtain the body of the data storage medium with a recess, the first ends of the conducting elements being flush with the main side, the second ends of the conducting elements being flush with the wall of the said recess, the intermediate part of the conducting elements being buried in the thermoplastic material; the said semiconductor component is fixed in the said recess; the second ends of the conducting elements are connected electrically to the terminals of the semiconductor component; and the volume of the said recess not occupied by the said semiconductor component is filled with an insulating material.",
"Other features and advantages of the invention will appear on reading the description which follows of several modes of realization of the invention given as non-limiting examples.",
"The description refers to the attached figures, on which: BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a vertical cross section of an injection mould which can be used to manufacture the data storage medium; FIGS.",
"2a to 2c are vertical cross sections of the storage device illustrating the various steps of its manufacture; FIG.",
"3 is a partial plan view of a finished data storage medium; FIG.",
"4 is a plan view of a first realization variant of the data storage medium; FIG.",
"5 is a plan view of a second realization variant of the data storage medium; and FIG.",
"6 is a partial vertical cross section of a third realization variant of the data storage medium.",
"DETAILED DESCRIPTION Referring firstly to FIGS.",
"1 to 3, we will describe a preferred mode of realization of the electronic data storage medium.",
"FIG.",
"1 shows a vertical cross section of an injection mould which can be used to manufacture the body of the data storage medium.",
"The mould 10 is formed from a lower fixed part 12 and an upper mobile part 14 which define a hollow or cavity.",
"The cavity 16 has the external shape of the body of the data storage device and has two main flat parallel sides 18 and 20 and an outer edge 22 defined by parts 12 and 14 of the mould.",
"The mould also has a injection nozzle 24 for the plastic material used to make the body of the data storage medium.",
"In the main side 20 of the mould cavity 16 a core 26 is mounted, projecting into the main side 20 to form a recess in the body obtained by moulding.",
"Before injecting the plastic material through the nozzle 24, conducting elements such as 26 and 27 are positioned, intended to form the external electrical contact pads of the storage medium and part of the connection between the semiconductor components and the external contact pads.",
"In this mode of realization, the conducting elements 26 and 27 include a first end a, a second end b and an intermediate part c which are obtained for example by folding a metal strip.",
"The ends a and b are parallel whereas the intermediate part c is inclined.",
"The mould 10 includes means not represented to press the ends a of the conducting elements 26 and 28 against the main wall 20 of the cavity 16 and means to press the second ends b against the end side 26a of the core 26.These support means can either be vacuum systems or electromagnetic systems.",
"It must be pointed out that the intermediate parts c of the conducting elements 27 and 28 are kept away from the side wall 26b of the core 26.Means are also provided to maintain the positioning of the conducting elements 27 and 28 in the mould cavity.",
"The thermoplastic material is injected into the mould cavity 16 via the injection nozzle(s) 24.The material is injected hot and under pressure.",
"The injection material can be for example ABS or other similar materials.",
"If ABS is used, the injection temperature lies preferably between 270 and 290° C. whereas the mould and the core are kept at a temperature of preferably between 10 and 50° C. After solidification of the moulding material, the part so produced is demoulded.",
"It is represented on FIG.",
"2a.",
"It consists of a moulded part 30 forming the body of the data storage medium which of course has the shape of the mould cavity 16 with the recess 32 formed by the core 26.The conducting elements 27 and 28 are fastened to the body 30 especially due to the fact that their intermediate parts c are buried in the plastic material.",
"However, the first ends of the conducting elements a are flush with the main side 30a of the data storage medium whereas the second ends b are flush with the bottom 32a of the recess.",
"In the next step represented by FIG.",
"2b, the semiconductor component 34 is fixed on the bottom 32a of the recess 32 by any suitable means.",
"The semiconductor component includes terminals 36 in its upper side 34a.",
"The electrical connection between the terminals 36 of the semiconductor components and the ends b of the conducting elements 27, 28 are made with soldered conducting wires such as 38.To carry out this step, the “flip-chip” technique can also be used.",
"This technique consists of making a conducting bump on each terminal of the semiconductor chip.",
"These bumps are fixed directly on the ends b of the conducting elements 28.This avoids the use of conducting wires.",
"We can see that in this case, the side 34a of the semiconductor chip with the terminals 36 is turned towards the bottom 32a of the recess 32.Preferably, a layer of insulating material is first applied on side 34a of the chip between the conducting bumps, this layer having approximately the same thickness as the bumps.",
"In the last step illustrated by FIG.",
"2c, the volume of the recess 32 not occupied by the semiconductor component 34 is filled with an insulating plastic material 40 which may, for example, be an epoxy resin.",
"The upper side 40a of this material may possibly be machined on the surface so that this upper side is flush in the upper side 30a of the body of the data storage medium.",
"FIG.",
"3 represents a plan view of part of the body 30 of the data storage medium.",
"This figure shows the ends a of the conducting elements 27 and 28.Preferably, the conducting elements 27 and 28 are arranged in two parallel rows of four conducting elements.",
"These ends a form the external electrical contact pads 42 and 44 of the data storage medium.",
"This figure also shows the upper side 40a of the plastic material filling the recess 32.FIG.",
"4 represents a plan view of a realization variant of the electronic data storage medium if this storage medium forms a SIM card of modifiable dimensions.",
"The body of the storage medium 50 which is obtained by moulding, as previously explained, has a first portion 52 which contains the semiconductor component 34 and a second portion 54, these two portions being connected by snap-off areas 56 made from the same plastic material.",
"To provide the mechanical strength of the body of the data storage medium, during the injection moulding, it would be possible to insert metal grids such as 56 and 58 in the mould cavity.",
"The internal sides of the two parts of the mould are preformed to obtain the hollows defining the snap-off areas 56.Preferably, the card body obtained by moulding has the dimensions of an ISO type card, i.e.",
"rectangular shape with dimensions approximately 8.5 mm×55 mm.",
"From the card so obtained, a card with the shape represented on FIGS.",
"4 and 5 can be obtained, either by planning, during the moulding, pre blanking slots or by blanking the card body after the moulding step.",
"In the mode of realization represented on FIG.",
"4, the external electrical contact pads 42 and 44 are arranged each side of the recess 32 containing the semiconductor component 34.This arrangement is only suitable if the semiconductor component 34 has relatively reduced dimensions so that connecting elements can be fitted between the terminals of the semiconductor component and the external contact pads represented on FIG.",
"2.This is the configuration which is presently used for bank cards or cards for fixed telephones.",
"FIG.",
"5 represents a realization variant allowing the use of a semiconductor component 60 of larger dimensions.",
"According to this mode of realization, the recess 62 made in the body 64 to take the semiconductor component 60 is offset with respect to the area 66 of the body, flush with the external electrical contact pads 68 and 70.To obtain this offset, the electrical conducting elements 72 and 74 placed in the mould have a special shape.",
"Each conducting element 72 or 74 has a first end a′, a second end b′ and an intermediate part c′.",
"The first and second ends a′ and b′ are identical with the ends a and b of FIG.",
"2 or 4.However, the intermediate part c′ is blanked from a conducting sheet so as to produce a non rectilinear shape (U shape) to obtain the offset between the recess 62 and the external contact pads 68 and 70 formed by the ends a′ of the conducting elements 72 and 74.We may also consider that the intermediate parts c′ of the conducting elements 72 and 74 are perpendicular to the ends a′ and b′.",
"With this arrangement, the spacing e between the two rows of external contact pads 68 and 70 provided for by the standards can be respected whilst creating a space e′ between the second ends b′ of the conducting elements which is compatible with the dimensions of the semiconductor components 60.FIG.",
"6 shows a third mode of realization of the card body.",
"During the moulding operation, a special shape is planned for the cavity in order to realize a projection 80 of the card body 82 parallel to one edge of the card body.",
"This projection forms a male clipping part, for example, in the shape of a dovetail.",
"The card body 82 can then be fixed on an extension 84 of the card body so that the body of the card so obtained has greater dimensions.",
"The extension 84 includes, for example, a groove 86, also in the shape of a dovetail, to clip mechanically onto the male part 80."
]
] | Patent_10432926 |
[
[
"Display memory, driver circuit, display, and cellular information apparatus",
"A display memory able to reduce power consumption, able to generate graphics at a high speed, and not needing memory mapping, a driver circuit, a display using the driver circuit, and a portable information apparatus, wherein a CPU read circuit is connected to one bit line of a display memory 7, a display read circuit is connected to the other bit line, a write circuit is connected to both bit lines, the CPU read circuit and write circuit are assigned to the access from the CPU, the display read circuit is assigned to the display screen display, and further the access from the CPU and the reading to the display screen are assigned to different two level periods of a clock signal of the memory and independently controlled.",
"Further, a drive power supply of the display memory is divided and a drive power supply voltage is supplied to the display memory for every memory cell or for every plurality of memory cells."
],
[
"1.A display memory for storing pixel data to be supplied to pixels of a display, comprising: at least one pair of bit lines; at least one column of memory cells each having a first storage node and a second storage node able to hold states of a complementary first level and second level; a first read circuit for reading the stored data of said first storage node output to one bit line of said pair of bit lines; and a second read circuit for reading the stored data of said second storage node output to the other bit line of said pair of bit lines.",
"2.A display memory as set forth in claim 1, wherein said second read circuit inverts and outputs the level of the stored data of said second storage node output to said other bit line.",
"3.A display memory as set forth in claim 2, further comprising a write circuit for outputting the data of said first level and second level to said first and second storage nodes of said memory cells to each the pair of bit lines and writing the data into said memory cells.",
"4.A display memory as set forth in claim 3, wherein said memory comprises: a controlling means for controlling the operation of said display memory, a write port including at least one said write circuit, a first read port including at least one said first read circuit, and a second read port including at least one said second read circuit; said first read port supplies the data stored in said memory cell to said display; said second read port reads the data from said memory cell and outputs the same to said controlling means; and said write port writes the data from said controlling means into said memory cell.",
"5.A display memory as set forth in claim 4, wherein, in a first level period of a clock signal of said display memory, said first read port performs a first access for outputting the data read via said first read circuit to said display, and in a second level period of the clock signal of said display memory, said second read port and said write port perform a second access for outputting the data read via said second read circuit to said controlling means and inputting the write data to be written into said memory cell from said controlling means.",
"6.A display memory as set forth in claim 3, wherein: said memory comprises a bit selecting means for selecting the memory cell into which the data is to be written, and said write circuit outputs the data of said first level and second level at said first and second storage nodes of the memory cell selected by said bit selecting means to each of the pair of bit lines of the memory cell to be written.",
"7.A display memory as set forth in claim 3, wherein said memory comprises: a drive use power supply voltage source for said display memory and a switching device for selectively connecting a power supply voltage supply end of at least one memory cell and said drive use power supply voltage source.",
"8.A display memory as set forth in claim 5, wherein: signal terminals for said first access are arrayed at one side part of said display memory, signal terminals for said second access are arrayed in the other side part different from that one side part, and a first interface for said first access and a second interface for said second access are connected to said first access use signal terminals and said second access use signal terminals of said display memory while sandwiching said display memory therebetween.",
"9.A display memory as set forth in claim 8, wherein: said first interface has a first line latch for storing one line's worth of image data in a horizontal direction of pixels arrayed in said matrix, said write port outputs said one line's worth of data to the selected bit line via the first line latch, and said second read port outputs said one line's worth of data from said display memory to said controlling means.",
"10.A display memory as set forth in claim 8, wherein: said second interface has a second line latch for storing one line's worth of image data in the horizontal direction of pixels arrayed in a matrix, and said first read port outputs said one line's worth of data from said display memory to said display via the second line latch.",
"11.A display memory as set forth in claim 8, wherein, in said display, a plurality of pixel cells are arrayed in a matrix, in said display memory, a plurality of memory cells are arrayed in a matrix corresponding to the matrix array of said plurality of pixel cells, in each memory cell, the pixel data for driving the corresponding pixel cell of the matrix of said display is stored by said write port, and said first read port latches the image data in units of lines and supplies the same to the pixels of the corresponding line of said display.",
"12.A driver circuit for driving pixels arrayed in a matrix of a display by signals corresponding to image data stored in a display memory, wherein said display memory comprises: at least one pair of bit lines; at least one column of memory cells each having a first storage node and a second storage node able to hold states of a complementary first level and second level; a first read circuit for reading the stored data of said first storage node output to one bit line of said pair of bit lines; and a second read circuit for reading the stored data of said second storage node output to the other bit line of said pair of bit lines.",
"13.A driver circuit as set forth in claim 12, wherein, said second read circuit inverts and outputs the level of the stored data of said second storage node output to said other bit line.",
"14.A driver circuit as set forth in claim 13, wherein said display memory further comprises a write circuit for outputting the data of said first level and second level to said first and second storage nodes of said memory cells to each the pair of bit lines and writing the data into said memory cells.",
"15.A driver circuit as set forth in claim 14, wherein said display memory comprises: a controlling means for controlling the operation of said display memory, a write port including at least one said write circuit, a first read port including at least one said first read circuit, and a second read port including at least one said second read circuit; said first read port supplies the data stored in said memory cell to said display; said second read port reads the data from said memory cell and outputs the same to said controlling means; and said write port writes the data from said controlling means into said memory cell.",
"16.A driver circuit as set forth in claim 15, wherein, in a first level period of a clock signal of said display memory, said first read port performs a first access for outputting the data read via said first read circuit to said display, and in a second level period of the clock signal of said display memory, said second read port and said write port perform a second access for outputting the data read via said second read circuit to said controlling means and inputting the write data to be written into said memory cell from said controlling means.",
"17.A driver circuit set forth in claim 14, wherein: said display memory comprises a bit selecting means for receiving a write control signal and selecting the memory cell into which the data is to be written, and said write circuit outputs the data of said first level and second level at said first and second storage nodes of the memory cell selected by said bit selecting means to each of the pair of bit lines of the memory cell to be written.",
"18.A driver circuit as set forth in claim 14, wherein said display memory comprises: a drive use power supply voltage source for said display memory and a switching device for selectively connecting a power supply voltage supply end of at least one memory cell and said drive use power supply voltage source.",
"19.A driver circuit as set forth in claim 16, wherein: signal terminals for said first access are arrayed at one side part of said display memory, signal terminals for said second access are arrayed in the other side part different from that one side part, and a first interface for said first access and a second interface for said second access are connected to said first access use signal terminals and said second access use signal terminals of said display memory while sandwiching said display memory therebetween.",
"20.A driver circuit as set forth in claim 19, wherein: said first interface has a first line latch for storing one line's worth of image data in a horizontal direction of pixels arrayed in said matrix, said write port outputs said one line's worth of data to the selected bit line via the first line latch, and said second read port outputs said one line's worth of data from said display memory to said controlling means.",
"21.A driver circuit as set forth in claim 19, wherein: said first line latch stores for every pixel write control data for designating the pixel data to be written into said display memory in the pixel data latched in said first line latch, and said write port writes the pixel data latched at said first line latch designated by the write control data into said display memory.",
"22.A driver circuit as set forth in claim 19, wherein, in said display, a plurality of pixel cells are arrayed in a matrix, in said display memory, a plurality of memory cells are arrayed in a matrix corresponding to the matrix array of said plurality of pixel cells, in each memory cell of said display memory, the pixel data for driving the corresponding pixel cell of the matrix of said display is stored by said write port, and said first read port latches the image data in units of lines and supplies the same to the pixels of the corresponding line of said display.",
"23.A driver circuit as set forth in claim 22, wherein each image data in the one line of said display's worth of image data latched by said first line latch is stored in said display memory as image data for driving a corresponding pixel in the pixels of the corresponding line of said display.",
"24.A driver circuit as set forth in claim 19, wherein: said second interface has a second line latch for storing one line's worth of image data in the horizontal direction of pixels arrayed in a matrix, and said first read port outputs said one line's worth of data from said display memory to said display via the second line latch.",
"25.A driver circuit as set forth in claim 24, wherein a bit width of said second line latch is the same as a bit width of one line's worth of image data in the horizontal direction of said pixels arrayed in a matrix.",
"26.A driver circuit as set forth in claim 24, wherein said second interface further comprises: a selection circuit for sequentially selecting R, G, B data included in the image data held in said second line latch and converting said image data to time divided signals and digital/analog converting means for converting digital signals to analog signals, said selection circuit outputs the time divided signals obtained by time division of the R, G, B data included in said image data to said digital/analog converting means, and said digital/analog converting means convert the time divided signals to the analog signals and supply the same to said display.",
"27.A driver circuit as set forth in claim 26, wherein said selection circuit selects the R, G, B data included in the pixel data held in said line latch asynchronously to the clock signal of said display memory and converts them to time divided signals.",
"28.A driver circuit for driving pixels arrayed in a matrix of a display by signals corresponding to pixel data supplied from a controlling means and stored in the display memory, comprising: a line latch for storing one line's worth of pixel data in a horizontal direction of said pixels arrayed in a matrix and a driving means for writing the data supplied from said controlling means into said display memory via said line latch in units of said one line's worth of the image data, reading the image data from said display memory, and outputting the same to said controlling means.",
"29.A driver circuit as set forth in claim 28, wherein said driving means stores the image data in said line latch up to the amount of one line, then writes the same into said display memory at one time.",
"30.A driver circuit as set forth in claim 28, wherein said driving means outputs one line's worth of the image data in the horizontal direction of said pixels arrayed in a matrix at one time from said display memory to said line latch.",
"31.A driver circuit as set forth in claim 28, wherein said driving means stores each pixel data in one line's worth of pixel data of said pixels arrayed in a matrix held in said line latch in said display memory as pixel data for driving a corresponding pixel in pixels of a corresponding line among said pixels arrayed in a matrix.",
"32.A driver circuit as set forth in claim 28, wherein, said line latch stores for every pixel write control data for designating the pixel data to be written into said display memory in the pixel data held in said line latch, and said driving means writes the pixel data held in said line latch designated by the write control data into said display memory.",
"33.A driver circuit for driving pixels arrayed in a matrix of a display by signals corresponding to pixel data supplied from a controlling means and stored in the display memory, comprising: a line latch for storing one line's worth of pixel data in a horizontal direction of said pixels arrayed in a matrix and an outputting means for reading said image data from said display memory via said line latch in units of said one line's worth of the image data and outputting the same to the corresponding pixels of said display.",
"34.A driver circuit as set forth in claim 33, wherein a bit width of said line latch is the same as a bit width of one line's worth of image data in the horizontal direction of said pixels arrayed in a matrix.",
"35.A driver circuit as set forth in claim 32, wherein said outputting means performs a first access for outputting the image data stored in said display memory to said pixels in a first level period of a clock signal of said display memory, and said controlling means performs a second access for reading the image data stored in said display memory and writing the data to be written into said display memory in a second level period of the clock signal of said display memory.",
"36.A driver circuit as set forth in claim 32, wherein said circuit further comprises: a selection circuit for sequentially selecting R, G, B data included in the image data held in said line latch and converting said image data to time divided signals and digital/analog converting means for converting digital signals to analog signals, said selection circuit outputs the time divided signals obtained by time division of the R, G, B data included in said image data to said digital/analog converting means, and said digital/analog converting means convert the time divided signals to the analog signals and supply the same to said display.",
"37.A driver circuit as set forth in claim 36, wherein said selection circuit selects the R, G, B data included in the pixel data held in said line latch asynchronously to the clock signal of said display memory and converts them to time divided signals.",
"38.A display comprising: a display screen wherein pixels are arrayed in a matrix; a scanning circuit for scanning said pixel matrix by each row and supplying voltage to a selected row; a driver circuit for outputting signals corresponding to image data to said pixels; and a display memory for storing said image data, wherein said display memory has at least one pair of bit lines, at least one column of memory cells each having a first storage node and a second storage node able to hold states of a complementary first level and second level, a first read circuit for reading the stored data of said first storage node output to one bit line of said pair of bit lines, and a second read circuit for reading the stored data of said second storage node output to the other bit line of said pair of bit lines.",
"39.A display as set forth in claim 38, wherein said second read circuit inverts and outputs the level of the stored data of said second storage node output to said other bit line.",
"40.A display as set forth in claim 39, wherein said display memory further comprises a write circuit for outputting the data of said first level and second level to said first and second storage nodes of said memory cells to each the pair of bit lines and writing the data into said memory cells.",
"41.A display as set forth in claim 39, wherein said display memory comprises: a controlling means for controlling the operation of said display memory, a write port including at least one said write circuit, a first read port including at least one said first read circuit, and a second read port including at least one said second read circuit; said first read port supplies the data stored in said memory cell to said display; said second read port reads the data from said memory cell and outputs the same to said controlling means; and said write port writes the data from said controlling means into said memory cell.",
"42.A display as set forth in claim 41, wherein, in a first level period of a clock signal of said display memory, said first read port performs a first access for outputting the data read via said first read circuit to said display, and in a second level period of the clock signal of said display memory, said second read port and said write port perform a second access for outputting the data read via said second read circuit to said controlling means and inputting the write data to be written into said memory cell from said controlling means.",
"43.A display as set forth in claim 40, wherein: said display memory comprises a bit selecting means for receiving a write control signal and selecting the memory cell into which the data is to be written, and said write circuit outputs the data of said first level and second level at said first and second storage nodes of the memory cell selected by said bit selecting means to each of the pair of bit lines of the memory cell to be written.",
"44.A display as set forth in claim 40, wherein said display memory comprises: a drive use power supply voltage source for said display memory and a switching device for selectively connecting a power supply voltage supply end of at least one memory cell and said drive use power supply voltage source.",
"45.A display as set forth in claim 42, wherein: signal terminals for said first access are arrayed at one side part of said display memory, signal terminals for said second access are arrayed in the other side part different from that one side part, and a first interface for said first access and a second interface for said second access are connected to said first access use signal terminals and said second access use signal terminals of said display memory while sandwiching said display memory therebetween.",
"46.A display as set forth in claim 45, wherein: said first interface has a first line latch for storing one line's worth of image data in the horizontal direction of pixels arrayed in a matrix, and via said first line latch, said write port outputs said one line's worth of data to a selected bit line and said second read port outputs said one line's worth of data from said display memory to said controlling means.",
"47.A display as set forth in claim 45, wherein: said first line latch stores for every pixel write control data for designating the pixel data to be written into said display memory in the pixel data latched by said first line latch, and said write port writes the pixel data designated by the write control data into said display memory.",
"48.A display as set forth in claim 45, wherein, in said display, a plurality of pixel cells are arrayed in a matrix, in said display memory, a plurality of memory cells are arrayed in a matrix corresponding to the matrix array of said plurality of pixel cells, in each memory cell of said display memory, the pixel data for driving the corresponding pixel cell of the matrix of said display is stored by said write port, and said first read port latches the image data in units of lines and supplies the same to the pixels of the corresponding line of said display.",
"49.A display as set forth in claim 48, wherein each image data in the one line of said display's worth of image data latched by said first line latch is stored in said display memory as image data for driving a corresponding pixel in the pixels of the corresponding line of said display by said write port.",
"50.A display as set forth in claim 45, wherein: said second interface has a second line latch for storing one line's worth of image data in the horizontal direction of pixels arrayed in a matrix, and said first read port outputs said one line's worth of data from said display memory to said display via the second line latch.",
"51.A display as set forth in claim 50, wherein a bit width of said second line latch is the same as a bit width of one line's worth of image data in the horizontal direction of said pixels arrayed in a matrix.",
"52.A display as set forth in claim 51, wherein: said second interface further has: a selection circuit for sequentially selecting R, G, B data included in the image data held in said second line latch and converting said image data to time divided signals and digital/analog converting means for converting digital signals to analog signals; said selection circuit outputs the time divided signals obtained by time division of the R, G, B data included in said image data to said digital/analog converting means; and said digital/analog converting means convert the time divided signals to the analog signals and supply the same to said display.",
"53.A display as set forth in claim 52, wherein said selection circuit selects the R, G, B data included in the pixel data held in said second line latch asynchronously to the clock signal of said display memory and converts them to time divided signals.",
"54.A display comprising: a display screen wherein pixels are arrayed in a matrix; a scanning circuit for scanning said pixel matrix by each one row and supplying a voltage to a selected row; a driver circuit for outputting signals corresponding to image data to said pixels; and a display memory for storing said image data, wherein said driver circuit has: a line latch for storing one line's worth of image data in a horizontal direction of said pixels arrayed in a matrix and a driving means for writing the data supplied from said controlling means into said display memory or reading the image data from said display memory via said line latch in units of said one line's worth of the image data and outputting the same to said controlling means.",
"55.A display as set forth in claim 54, wherein said driving means stores the image data in said line latch up to the amount of one line, then writes the same into said display memory at one time.",
"56.A display as set forth in claim 54, wherein said driving means outputs one line's worth of the image data in the horizontal direction of said pixels arrayed in a matrix at one time from said display memory to said line latch.",
"57.A display as set forth in claim 54, wherein said driving means stores each pixel data in one line's worth of pixel data of said pixels arrayed in a matrix held in said line latch in said display memory as pixel data for driving a corresponding pixel in pixels of a corresponding line among said pixels arrayed in a matrix.",
"58.A display as set forth in claim 54, wherein: said line latch stores for every pixel write control data for designating the pixel data to be written into said display memory in the pixel data latched in said line latch, and said driving means writes the pixel data held in said line latch designated by the write control data into said display memory.",
"59.A display comprising: a display screen wherein pixels are arrayed in a matrix; a scanning circuit for scanning said pixel matrix by each row and supplying a voltage to a selected row; a driver circuit for outputting signals corresponding to the image data supplied from the controlling means to said pixels; and a display memory for storing said image data, wherein said driver circuit has: a line latch for storing one line's worth of image data in a horizontal direction of pixels arrayed in said matrix state and an outputting means for reading said image data from said display memory via said line latch in units of said one line's worth of image data and supplying the same to corresponding pixels of said display.",
"60.A display as set forth in claim 59, wherein a bit width of said line latch is the same as a bit width of one line's worth of image data in the horizontal direction of said pixels arrayed in a matrix.",
"61.A display as set forth in claim 59, wherein: said outputting means performs a first access for outputting the image data stored in said display memory to said pixels in a first level period of a clock signal of said display memory, and said controlling means performs a second access for reading the image data stored in said display memory and writing the data to be written into said display memory in a second level period of the clock signal of said display memory.",
"62.A display as set forth in claim 59, wherein: said driver circuit further comprises: a selection circuit for sequentially selecting R, G, B data included in the image data held in said line latch and converting said image data to time divided signals and digital/analog converting means for converting digital signals to analog signals; said selection circuit outputs the time divided signals obtained by time division of the R, G, B data included in said image data to said digital/analog converting means; and said digital/analog converting means convert the time divided signals to the analog signals and supply the same to said display.",
"63.A display as set forth in claim 62, wherein said selection circuit selects the R, G, B data included in the pixel data held in said line latch asynchronously to the clock signal of said display memory and converts them to time divided signals.",
"64.A portable information comprising: a display wherein a plurality of pixel cells are arrayed in a matrix and a display memory for storing pixel data to be supplied to pixel cells of said display, wherein said display memory has: a controlling means for controlling the operation of said display memory, a plurality of memory cells, each having a first storage node and a second storage node able to hold states of a complementary first level and second level, arrayed in a matrix corresponding to the matrix array of said plurality of pixel cells, a first read port for reading the stored data of said first storage node of each memory cell, a second read port for reading the stored data of said second storage node of each memory cell, a write port for writing pixel data for driving corresponding pixel cells of the matrix of said display into said memory cells, a first line latch for storing one line's worth of pixel data in the horizontal direction of said pixel cells arrayed in a matrix, and a second line latch for storing one line's worth of image data in the horizontal direction of said pixel cells arrayed in a matrix; said write port outputs said one line's worth of data to a plurality of said memory cells via said first line latch; said first read port latches the image data in said second line latch in units of lines and outputs the same to corresponding pixel cells of said display; and said second read port outputs said one line's worth of data to said controlling means via said first line latch."
],
[
"<SOH> BACKGROUND ART <EOH>Liquid crystal displays are being widely used as display systems of mobile phones and PDAs (Personal Digital Assistants) and other portable information devices by making use of their light weight, thinness, low power consumption, and other features.",
"Further, due to the spread of mobile phones and the Internet, the displays of portable information devices are being required to be further enlarged in size, offer color, and otherwise be improved in quality and are being strongly required to be ultra-low in power consumption for realizing long hours of usage.",
"In liquid crystal drivers, therefore, it has become important to realize lower power consumption while handling larger screens and color.",
"In conventional liquid crystal drivers, the power consumption of the logic circuits inside the LSI has been lowered by a variety of methods, but if dealing with the enlargement of size of screens or colorization and other improvements in image quality, the number of drive devices increases, so the power consumption rises accordingly.",
"In order to realize lower power consumption, the method of building a display memory (also referred to as a “frame memory”) into a liquid crystal driver has been employed.",
"This eliminates the need for a controller memory for transfer of display data, slashes the number of parts, and realizes a reduction of the power consumption.",
"Further, a new drive system may be employed to reduce the power consumption.",
"Concerning this subject, for example, Japanese Unexamined Patent Publication (Kokai) No.",
"7-64514 discloses a liquid crystal driver having a built-in general purpose memory realizing high speed and lower power and a liquid crystal display using that driver.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"2000-293144 discloses a liquid crystal display device using a liquid crystal driver with a built-in memory generating graphics with a low power consumption and at a high speed and able to reduce the load of the CPU.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"7-281634 discloses a liquid crystal display using a liquid crystal driver with a built-in memory achieving lower power consumption and realizing high speed graphic drawing access.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"7-230265 realizes a liquid crystal drive device improving the means of supply of power and having a built-in memory with a low power consumption and a large capacity.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"7-175445 discloses a technique achieving lower power consumption and higher speed graphic drawing without lowering the operating efficiency of the system by building into the liquid crystal driver a display memory accessible by a general purpose memory interface.",
"In the layout of an LSI of a liquid crystal driver having a built-in conventional display memory, however, the interface has terminals at one side of the general purpose memory cells, so general purpose interface signal interconnects must be detoured around them.",
"Power is taken for the amount of those interconnects.",
"Further, a conventional display memory uses data buses, address buses, and control signal buses for display and graphics drawing and requires bus arbitration.",
"Due to this, if the number of accesses for display is large, the time for the drawing is reduced.",
"Further, in the conventional system, the memory is accessed from the CPU for every group of pixels.",
"Therefore, for example, when desiring to store one screen's worth of data from the CPU into the memory, (one screens's worth of number of pixels)/(number of pixels in group of pixels) of write operations to the memory are required, so the number of times of operation of the memory was large.",
"The operating power consumption of the memory is proportional to the number of times of read/write operations, therefore consequently an increase of the power consumption is induced.",
"Further, when transferring display data from the memory to the liquid crystal panel, one horizontal line on the display screen's worth of the display data was simultaneously output, but the data was read from the memory for this purpose not in amounts of one horizontal line at one time, but in amounts of an output data line of the liquid crystal driver.",
"For example, when desiring to display one screen's worth of data stored in a memory on an LCD display screen, (one screen's worth of number of pixels)/(group of pixels) of read operations of the memory become necessary, so there is the disadvantage that power of the amount of the number of times of access is consumed.",
"Further, in the conventional system, the operation has to be performed at the high frequency of the memory.",
"No margin can be given to the access time of the CPU.",
"Therefore, there is a disadvantage that this is not suited display of a moving picture requiring quick switching of the screen.",
"Further, when using a conventional memory, the images of the memory array and the pixel array of the liquid crystal are not the same, so it is necessary to calculate where a pixel is in the memory at the time of drawing.",
"Further, a conventional display memory rewrites all data to be written at one time when writing data.",
"Accordingly, when there is a data which is not desired to be changed in the data to be written at one time, a so-called read-modify-write system which reads out the data in advance before rewriting the data, modifies the bits to be rewritten while masking the data not desired to be rewritten, and then writes the data into the memory is employed.",
"For this reason, there were the disadvantages that the number of operation was large and power was consumed.",
"Further, conventionally, when outputting image data stored in a display memory to a digital/analog converter (DAC), since RGB data corresponding to the three primary colors of the color cannot be output in a time division manner, the outputs of the display memory were directly connected to DACs in one-to-one correspondence.",
"In this way, conventionally, since a DAC was necessary for every RGB data, the number of DACs was large and an increase of the power consumption was induced.",
"In order to reduce the power consumption of the DACs, the settling time must be adjusted.",
"Since the operating speeds of the DACs and the display memory are different, they must be separately controlled.",
"Depending on the characteristics of the DACs, the phases of the input signals must be adjusted.",
"Conventionally, however, when outputting the data of the display memory to the DACs, the timing for outputting the RGB data is fixed.",
"The phase of the data cannot be freely changed to match with the characteristics of the DACs, so this necessity could not be coped with.",
"Further, in order to lower the power consumption of a liquid crystal display, there is the method of lowering the power supply voltage.",
"When the operating power supply voltage becomes smaller than 3.0V, however, malfunctions occur.",
"Further, for a method of supplying power considering power conservation, there is a partial display mode used in a standby screen of mobile phones, but in this partial display mode, although nothing is displayed on the screen, leakage current of the memory cells still flows, so there is the disadvantage of consumption of power."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a view of the overall configuration of a display according to the present invention.",
"FIG.",
"2 is a circuit diagram of a concrete example of the configuration of a memory cell of a display memory according to a first embodiment.",
"FIG.",
"3 is a view of the configuration of principal parts of a driver circuit according to the first embodiment.",
"FIGS.",
"4A to 4 F are timing charts of the operation of the display memory according to the first embodiment of the present invention.",
"FIG.",
"5 is a view of the configuration of a display memory dividing a power supply according to a second embodiment.",
"FIG.",
"6 is a schematic view of an address array of the display memory and the array of pixels on a display screen according to a third embodiment.",
"FIG.",
"7 is a view of the configuration for accessing a display memory in units of lines according to the third embodiment.",
"FIG.",
"8 is a view of the configuration of principal parts of a display memory able to write data for every bit according to a fourth embodiment.",
"FIG.",
"9 is a view of the schematic circuit configuration on a CPU side of a driver circuit according to a fifth embodiment.",
"FIGS.",
"10A to 10 F are timing charts of an operation for writing data in units of lines of the driver circuit according to the fifth embodiment.",
"FIGS.",
"11A to 11 F are timing charts of an operation for reading data in units of lines of the driver circuit according to the fifth embodiment.",
"FIG.",
"12 is a view of the schematic circuit configuration at the time of the writing for every pixel of the driver circuit according to a sixth embodiment.",
"FIG.",
"13 is a view of the configuration enabling writing of data into the display memory for every pixel in the driver circuit according to the sixth embodiment.",
"FIGS.",
"14A to 14 F are timing charts of an operation for writing data into the display memory for every pixel using a write flag signal according to the sixth embodiment.",
"FIG.",
"15 is a view of the schematic circuit configuration on a display screen side of the driver circuit according to a seventh embodiment.",
"FIG.",
"16 is a view of the configuration of principal parts of a display according to an eighth embodiment.",
"FIGS.",
"17A to 17 F are timing charts of RGB time division of image data in a display according to the eighth embodiment.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to a display memory for storing pixel data to be supplied to pixels of a display, a driver circuit having a display memory and driving pixels arrayed in a matrix of the display by signals corresponding to the image data, a display using the driver circuit, and a portable information device.",
"BACKGROUND ART Liquid crystal displays are being widely used as display systems of mobile phones and PDAs (Personal Digital Assistants) and other portable information devices by making use of their light weight, thinness, low power consumption, and other features.",
"Further, due to the spread of mobile phones and the Internet, the displays of portable information devices are being required to be further enlarged in size, offer color, and otherwise be improved in quality and are being strongly required to be ultra-low in power consumption for realizing long hours of usage.",
"In liquid crystal drivers, therefore, it has become important to realize lower power consumption while handling larger screens and color.",
"In conventional liquid crystal drivers, the power consumption of the logic circuits inside the LSI has been lowered by a variety of methods, but if dealing with the enlargement of size of screens or colorization and other improvements in image quality, the number of drive devices increases, so the power consumption rises accordingly.",
"In order to realize lower power consumption, the method of building a display memory (also referred to as a “frame memory”) into a liquid crystal driver has been employed.",
"This eliminates the need for a controller memory for transfer of display data, slashes the number of parts, and realizes a reduction of the power consumption.",
"Further, a new drive system may be employed to reduce the power consumption.",
"Concerning this subject, for example, Japanese Unexamined Patent Publication (Kokai) No.",
"7-64514 discloses a liquid crystal driver having a built-in general purpose memory realizing high speed and lower power and a liquid crystal display using that driver.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"2000-293144 discloses a liquid crystal display device using a liquid crystal driver with a built-in memory generating graphics with a low power consumption and at a high speed and able to reduce the load of the CPU.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"7-281634 discloses a liquid crystal display using a liquid crystal driver with a built-in memory achieving lower power consumption and realizing high speed graphic drawing access.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"7-230265 realizes a liquid crystal drive device improving the means of supply of power and having a built-in memory with a low power consumption and a large capacity.",
"Further, Japanese Unexamined Patent Publication (Kokai) No.",
"7-175445 discloses a technique achieving lower power consumption and higher speed graphic drawing without lowering the operating efficiency of the system by building into the liquid crystal driver a display memory accessible by a general purpose memory interface.",
"In the layout of an LSI of a liquid crystal driver having a built-in conventional display memory, however, the interface has terminals at one side of the general purpose memory cells, so general purpose interface signal interconnects must be detoured around them.",
"Power is taken for the amount of those interconnects.",
"Further, a conventional display memory uses data buses, address buses, and control signal buses for display and graphics drawing and requires bus arbitration.",
"Due to this, if the number of accesses for display is large, the time for the drawing is reduced.",
"Further, in the conventional system, the memory is accessed from the CPU for every group of pixels.",
"Therefore, for example, when desiring to store one screen's worth of data from the CPU into the memory, (one screens's worth of number of pixels)/(number of pixels in group of pixels) of write operations to the memory are required, so the number of times of operation of the memory was large.",
"The operating power consumption of the memory is proportional to the number of times of read/write operations, therefore consequently an increase of the power consumption is induced.",
"Further, when transferring display data from the memory to the liquid crystal panel, one horizontal line on the display screen's worth of the display data was simultaneously output, but the data was read from the memory for this purpose not in amounts of one horizontal line at one time, but in amounts of an output data line of the liquid crystal driver.",
"For example, when desiring to display one screen's worth of data stored in a memory on an LCD display screen, (one screen's worth of number of pixels)/(group of pixels) of read operations of the memory become necessary, so there is the disadvantage that power of the amount of the number of times of access is consumed.",
"Further, in the conventional system, the operation has to be performed at the high frequency of the memory.",
"No margin can be given to the access time of the CPU.",
"Therefore, there is a disadvantage that this is not suited display of a moving picture requiring quick switching of the screen.",
"Further, when using a conventional memory, the images of the memory array and the pixel array of the liquid crystal are not the same, so it is necessary to calculate where a pixel is in the memory at the time of drawing.",
"Further, a conventional display memory rewrites all data to be written at one time when writing data.",
"Accordingly, when there is a data which is not desired to be changed in the data to be written at one time, a so-called read-modify-write system which reads out the data in advance before rewriting the data, modifies the bits to be rewritten while masking the data not desired to be rewritten, and then writes the data into the memory is employed.",
"For this reason, there were the disadvantages that the number of operation was large and power was consumed.",
"Further, conventionally, when outputting image data stored in a display memory to a digital/analog converter (DAC), since RGB data corresponding to the three primary colors of the color cannot be output in a time division manner, the outputs of the display memory were directly connected to DACs in one-to-one correspondence.",
"In this way, conventionally, since a DAC was necessary for every RGB data, the number of DACs was large and an increase of the power consumption was induced.",
"In order to reduce the power consumption of the DACs, the settling time must be adjusted.",
"Since the operating speeds of the DACs and the display memory are different, they must be separately controlled.",
"Depending on the characteristics of the DACs, the phases of the input signals must be adjusted.",
"Conventionally, however, when outputting the data of the display memory to the DACs, the timing for outputting the RGB data is fixed.",
"The phase of the data cannot be freely changed to match with the characteristics of the DACs, so this necessity could not be coped with.",
"Further, in order to lower the power consumption of a liquid crystal display, there is the method of lowering the power supply voltage.",
"When the operating power supply voltage becomes smaller than 3.0V, however, malfunctions occur.",
"Further, for a method of supplying power considering power conservation, there is a partial display mode used in a standby screen of mobile phones, but in this partial display mode, although nothing is displayed on the screen, leakage current of the memory cells still flows, so there is the disadvantage of consumption of power.",
"DISCLOSURE OF THE INVENTION An object of the present invention is to provide a display memory able to reduce the power consumption, able to draw graphics at a high speed, and free from the need for memory mapping, a driver circuit provided with this display memory, a display using that driver circuit, and a portable information device.",
"To attain the above object, a first aspect of the present invention is a display memory for storing pixel data to be supplied to pixels of a display, comprising at least one pair of bit lines; at least one column of memory cells each having a first storage node and a second storage node able to hold states of a complementary first level and second level; a first read circuit for reading the stored data of the first storage node output to one bit line of the pair of bit lines; and a second read circuit for reading the stored data of the second storage node output to the other bit line of the pair of bit lines.",
"Further, the second read circuit inverts and outputs the level of the stored data of the second storage node output to the other bit line.",
"Provision is further made of a write circuit for outputting the data of the first level and second level to the first and second storage nodes of the memory cells to each the pair of bit lines and writing the data into the memory cells.",
"Further, the display memory comprises a controlling means for controlling the operation of the display memory, a write port including at least one the write circuit, a first read port including at least one the first read circuit, and a second read port including at least one the second read circuit, wherein the first read port supplies the data stored in the memory cell to the display, the second read port reads the data from the memory cell and outputs the same to the controlling means, and the write port writes the data from the controlling means into the memory cell.",
"Further, in a first level period of a clock signal of the display memory, the first read port performs a first access for outputting the data read via the first read circuit to the display, and in a second level period of the clock signal of the display memory, the second read port and the write port perform a second access for outputting the data read via the second read circuit to the controlling means and inputting the write data to be written into the memory cell from the controlling means.",
"Further, the display memory comprises a bit selecting means for selecting the memory cell into which the data is to be written and a write control signal for controlling the writing of the data into the memory cell into which the data is to be written, and the write circuit is controlled by the bit selecting means and the write control signal and outputs the data of the first level and second level at the first and second storage nodes of the memory cell selected by the bit selecting means to each of the pair of bit lines of the memory cell to be written.",
"Further, the display memory has a drive use power supply voltage source for the display memory and a switching device for selectively connecting a power supply voltage supply end of at least one memory cell and the drive use power supply voltage source.",
"Further, signal terminals for the first access are arrayed at one side part of the display memory, signal terminals for the second access are arrayed in the other side part different from that one side part, and the first access use first interface and the second access use second interface are connected to the first access use signal terminals and the second access use signal terminals of the display memory while sandwiching the display memory therebetween.",
"Preferably, the first interface has a first line latch for storing one line's worth of image data in a horizontal direction of pixels arrayed in the matrix, and, via the first line latch, the write port outputs the one line's worth of data to the selected bit line and the second read port outputs the one line's worth of data from the display memory to the controlling means.",
"Preferably, the second interface has a second line latch for storing one line's worth of image data in the horizontal direction of pixels arrayed in a matrix, and the first read port outputs the one line's worth of data from the display memory to the display via the second line latch.",
"Further, in the display, a plurality of pixel cells are arrayed in a matrix, in the display memory, a plurality of memory cells are arrayed in a matrix corresponding to the matrix array of the plurality of pixel cells, in each memory cell of the display memory, the pixel data for driving the corresponding pixel cell of the matrix of the display is stored by the write port, and the first read port latches the image data in the second line latch in units of lines and supplies the same to the pixels of the corresponding line of the display.",
"A second aspect of the present invention is a driver circuit for driving pixels arrayed in a matrix of a display by signals corresponding to image data stored in a display memory, wherein the display memory comprises at least one pair of bit lines; at least one column of memory cells each having a first storage node and a second storage node able to hold states of a complementary first level and second level; a first read circuit for reading the stored data of the first storage node output to one bit line of the pair of bit lines; and a second read circuit for reading the stored data of the second storage node output to the other bit line of the pair of bit lines.",
"Further, in the driver circuit, the first interface has a first line latch for storing one line's worth of image data in a horizontal direction of the pixels arrayed in a matrix, and, via the first line latch, the write port outputs the one line's worth of data to the selected bit line and the second read port outputs the one line's worth of data from the display memory to the controlling means.",
"Further, the first line latch stores write control data for designating the pixel data to be written into the display memory for every pixel among the pixel data latched by the first line latch, and the write port writes the pixel data latched at the first line latch designated by the write control data into the display memory.",
"A third aspect of the present invention is a driver circuit for driving pixels arrayed in a matrix of a display by signals corresponding to pixel data supplied from a controlling means and stored in the display memory, comprising a line latch for storing one line's worth of pixel data in a horizontal direction of the pixels arrayed in a matrix and a driving means for writing the data supplied from the controlling means into the display memory via the line latch in units of the one line's worth of the image data, reading the image data from the display memory, and outputting the same to the controlling means.",
"Concretely, the driving means stores the image data in the line latch up to the amount of one line, then writes the same into the display memory at one time.",
"Further, the driving means outputs one line's worth of the image data in the horizontal direction of the pixels arrayed in a matrix at one time from the display memory to the line latch.",
"Further, the driving means stores each pixel data in one line's worth of pixel data of the pixels arrayed in a matrix held in the line latch in the display memory as pixel data for driving a corresponding pixel in pixels of a corresponding line among the pixels arrayed in a matrix.",
"Further, the line latch stores for every pixel write control data for designating the pixel data to be written into the display memory in the pixel data held in the line latch, and the driving means writes the pixel data held in the line latch designated by the write control data into the display memory.",
"A fourth aspect of the present invention is a driver circuit for driving pixels arrayed in a matrix of a display by signals corresponding to pixel data supplied from a controlling means and stored in the display memory, comprising a line latch for storing one line's worth of pixel data in a horizontal direction of the pixels arrayed in a matrix and an outputting means for reading the image data from the display memory via the line latch in units of the one line's worth of the image data and outputting the same to the corresponding pixels of the display.",
"Preferably, the outputting means performs a first access for outputting the image data stored in the display memory to the pixels in a first level period of a clock signal of the display memory, and the controlling means performs a second access for reading the image data stored in the display memory and writing the data to be written into the display memory in a second level period of the clock signal of the display memory.",
"Further, provision is further made of a selection circuit for sequentially selecting R, G, B data included in the image data held in the line latch and converting the image data to time divided signals and digital/analog converting means for converting digital signals to analog signals, the selection circuit outputs the time divided signals obtained by time division of the R, G, B data included in the image data to the digital/analog converting means, and the digital/analog converting means convert the time divided signals to the analog signals and supply the same to the display.",
"Further, the selection circuit selects the R, G, B data included in the pixel data held in the line latch asynchronously to the clock signal of the display memory and converts them to time divided signals.",
"A display according to a fifth aspect of the present invention comprises a display screen wherein pixels are arrayed in a matrix; a scanning circuit for scanning the pixel matrix by each row and supplying voltage to a selected row; a driver circuit for outputting signals corresponding to image data to the pixels; and a display memory for storing the image data, wherein the display memory has at least one pair of bit lines, at least one column of memory cells each having a first storage node and a second storage node able to hold states of a complementary first level and second level, a first read circuit for reading the stored data of the first storage node output to one bit line of the pair of bit lines, and a second read circuit for reading the stored data of the second storage node output to the other bit line of the pair of bit lines.",
"A display according to a sixth aspect of the present invention comprises a display screen wherein pixels are arrayed in a matrix; a scanning circuit for scanning the pixel matrix by each one row and supplying a voltage to a selected row; a driver circuit for outputting signals corresponding to image data to the pixels; and a display memory for storing the image data, wherein the driver circuit has a line latch for storing one line's worth of image data in a horizontal direction of the pixels arrayed in a matrix and a driving means for writing the data supplied from the controlling means into the display memory or reading the image data from the display memory via the line latch in units of the one line's worth of the image data and outputting the same to the controlling means.",
"A display according to a seventh aspect of the present invention comprises a display screen wherein pixels are arrayed in a matrix; a scanning circuit for scanning the pixel matrix by each row and supplying a voltage to a selected row; a driver circuit for outputting signals corresponding to the image data supplied from the controlling means to the pixels; and a display memory for storing the image data, wherein the driver circuit has a line latch for storing one line's worth of image data in a horizontal direction of pixels arrayed in the matrix state and an outputting means for reading the image data from the display memory via the line latch in units of the one line's worth of image data and supplying the same to corresponding pixels of the display.",
"A portable information device according to a seventh aspect of the present invention comprises a display wherein a plurality of pixel cells are arrayed in a matrix and a display memory for storing pixel data to be supplied to pixel cells of the display, wherein the display memory has a controlling means for controlling the operation of the display memory, a plurality of memory cells, each having a first storage node and a second storage node able to hold states of a complementary first level and second level, arrayed in a matrix corresponding to the matrix array of the plurality of pixel cells, a first read port for reading the stored data of the first storage node of each memory cell, a second read port for reading the stored data of the second storage node of each memory cell, a write port for writing pixel data for driving corresponding pixel cells of the matrix of the display into the memory cells, a first line latch for storing one line's worth of pixel data in the horizontal direction of the pixel cells arrayed in a matrix, and a second line latch for storing one line's worth of image data in the horizontal direction of the pixel cells arrayed in a matrix; the write port outputs the one line's worth of data to a plurality of the memory cells via the first line latch; the first read port latches the image data in the second line latch in units of lines and outputs the same to corresponding pixel cells of the display; and the second read port outputs the one line's worth of data to the controlling means via the first line latch.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a view of the overall configuration of a display according to the present invention.",
"FIG.",
"2 is a circuit diagram of a concrete example of the configuration of a memory cell of a display memory according to a first embodiment.",
"FIG.",
"3 is a view of the configuration of principal parts of a driver circuit according to the first embodiment.",
"FIGS.",
"4A to 4F are timing charts of the operation of the display memory according to the first embodiment of the present invention.",
"FIG.",
"5 is a view of the configuration of a display memory dividing a power supply according to a second embodiment.",
"FIG.",
"6 is a schematic view of an address array of the display memory and the array of pixels on a display screen according to a third embodiment.",
"FIG.",
"7 is a view of the configuration for accessing a display memory in units of lines according to the third embodiment.",
"FIG.",
"8 is a view of the configuration of principal parts of a display memory able to write data for every bit according to a fourth embodiment.",
"FIG.",
"9 is a view of the schematic circuit configuration on a CPU side of a driver circuit according to a fifth embodiment.",
"FIGS.",
"10A to 10F are timing charts of an operation for writing data in units of lines of the driver circuit according to the fifth embodiment.",
"FIGS.",
"11A to 11F are timing charts of an operation for reading data in units of lines of the driver circuit according to the fifth embodiment.",
"FIG.",
"12 is a view of the schematic circuit configuration at the time of the writing for every pixel of the driver circuit according to a sixth embodiment.",
"FIG.",
"13 is a view of the configuration enabling writing of data into the display memory for every pixel in the driver circuit according to the sixth embodiment.",
"FIGS.",
"14A to 14F are timing charts of an operation for writing data into the display memory for every pixel using a write flag signal according to the sixth embodiment.",
"FIG.",
"15 is a view of the schematic circuit configuration on a display screen side of the driver circuit according to a seventh embodiment.",
"FIG.",
"16 is a view of the configuration of principal parts of a display according to an eighth embodiment.",
"FIGS.",
"17A to 17F are timing charts of RGB time division of image data in a display according to the eighth embodiment.",
"BEST MODE FOR WORKING THE INVENTION Below, embodiments of a display memory, a driver circuit, and a display using the driver circuit according to the present invention will be explained with reference to the attached drawings.",
"First Embodiment FIG.",
"1 is an overall view of the configuration of a first embodiment of a display 1 according to the present invention.",
"Here, the explanation will be given by taking as an example a liquid crystal driver and a liquid crystal display using the liquid crystal driver circuit.",
"In the liquid crystal display 1 shown in FIG.",
"1, a processor (CPU) 2 for controlling the operation of the entire device, a liquid crystal driver 3, a display screen 4 (liquid crystal panel 4 in the case of a liquid crystal display) for displaying an image, and a scanning circuit 5 for selecting a row of pixels, to which addresses are given in a horizontal direction of the liquid crystal panel 4, and supplying voltage to pixels to turn them on are included.",
"The liquid crystal driver 3 has a display memory 7, a CPU side interface (CPU I/F) 6 for receiving the data for every pixel from the CPU 2 and writing the same into the display memory 7 or reading the pixel data stored in the display memory 7, and a panel side interface (LCD I/F) 8 for receiving pixel data including red (R), green (G), and blue (B) colors output by the display memory 7 and outputting the same to the liquid crystal panel 4 to display the same.",
"The CPU side interface (CPU I/F) 6 has a data latch 9 for storing the pixel data from the CPU 2 and a selector circuit 10.The panel side interface (LCI I/F) 8 includes a data latch 11 for buffering the output of the memory, a selector circuit 12, and a digital/analog converter (DAC) 13 for converting the image data to be displayed from digital signals to analog signals and outputting the same to the pixels of the liquid crystal panel 4.In order to display an image on the liquid crystal panel 4, the data for every pixel is transferred from the CPU 2 and stored up to the amount of one line in the horizontal direction of the liquid crystal panel 4 by the data latch 9 of the CPU I/F 6, then the one line's worth of data are simultaneously transferred to the display memory 7.From the display memory 7, one line's worth of pixel data in the horizontal direction of the liquid crystal panel 4 are simultaneously output and latched by the data latch 11 of the LCD I/F 8, then voltages in accordance with the pixel data are simultaneously supplied to the liquid crystal panel 4.By this, the pixel data is displayed on the screen.",
"In the present embodiment, the display memory 7 is configured by for example a single port SRAM.",
"FIG.",
"2 is a circuit diagram of a concrete example of the configuration of a memory cell of a display memory according to the present embodiment.",
"As shown in FIG.",
"2, the display memory 7 has a memory cell 21, a sense amplifier 22 as a first read circuit, a sense amplifier 23 as a second read circuit, a write circuit 24, a pair of bit lines (BL) 25a and 25b, and a word line (WL) 26.In FIG.",
"2, the memory cell 21 of the display memory 7 has two inverters 29a and 29b having inputs and outputs connected to each other and NMOS transistors 27a and 27b as access transistors.",
"A first storage node 28a is configured by a connection point of the output of the inverter 29a and the input of the inverter 29b, while a second storage node 28b is configured by a connection point of the input of the inverter 29a and the output of the inverter 29b.",
"The bit line 25a is connected via the NMOS transistor 27a to the first storage node 28a, while the bit line 25b is connected via the NMOS transistor 27b to the second storage node 28b.",
"Gates of the NMOS transistors 27a and 27b of the memory cell 21 are connected to a common word line 26.When outputting data to the liquid crystal panel 4, the image data is read from the memory 7 by using the sense amplifier 22.The sense amplifier 23 is used when the CPU 2 reads the data from the memory 7.The CPU 2 writes the data into the memory 7 by using the write circuit 24.RC1 and RC2 indicate control signals (sense amplifier control) of the sense amplifiers 22 and 23, while RD1 and RD2 indicate output data (read data) of the sense amplifiers 22 and 23.WC and WD indicate a control signal (write control) of the write circuit 24 and write data to the memory cell 21.The write circuit 24 has first drivers 24a and 24b connected in series and operating upon receipt of the low level and active control signal WC.",
"The display memory 7 of the present embodiment is for example a custom ARAM built into the liquid crystal driver 3.As shown in FIG.",
"2, as the components of the memory cell 21, the read sense amplifier 22 at the time of the display and the sense amplifier 23 for the CPU 2 to read the data from the memory cell are connected to both bit lines 25a and 25b.",
"The sense amplifiers 22 and 23 can independently control the read operation.",
"The sense amplifier 23 and the write circuit 24 can simultaneously operate.",
"That is, it is possible to read data while writing data.",
"Next, the operation of the display memory 7 will be explained.",
"The pair of CMOS inverters 29a and 29b, for example, are supplied with a drive use power supply voltage of VDD=3.3V.",
"The pair of CMOS inverters 29a and 29b form a bi-stable flip-flop circuit.",
"Among the bi-stable states, for example, the state where the node 28a is at a high level and the node 28b is at a low level is defined as meaning data “1” is stored, while conversely the state where the node 28a is at a low level and the node 28b is at a high level is defined as meaning that data “0” is stored.",
"When reading the data stored in the memory cell 21, first the scanning circuit 5 scans the memory cell matrix, a word line designated by a not illustrated row address decoder, for example, the word line 26 is selected, a voltage is supplied, and the NMOS transistors 27a and 27b become conductive in state.",
"When reading the data for every bit, a not illustrated column address decoder is used to designate a memory cell to be further read from, for example, the memory cell 21.At this time, the read control signal RC1 or RC2 becomes the high level, and the sense amplifier 22 or the sense amplifier 23 is turned on.",
"When reading data for every line or for every plurality of memory cells, a not illustrated means is used for example to designate a memory cell line including the memory cell 21 and to be read from or a plurality of memory cells.",
"Since the NMOS transistors 27a and 27b have become conductive in state, the states of the nodes 28a and 28b are transmitted to the sense amplifiers 22 and 23 connected to the bit lines 25a and 25b.",
"When outputting the data stored in the memory to the liquid crystal panel, the read control signal RC1 becomes the high level, the sense amplifier 22 turns on, and the present state of the memory cell 21, that is, “1” or “0”, stored at the node 28a is extracted from the sense amplifier 22.When reading the data stored in the memory from the CPU 2, the read control signal RC2 becomes the high level, the sense amplifier 23 turns on, and the value “0” or “1” which is a complementary to the node 28a stored at the node 28b is inverted at the sense amplifier 23 and data having the same value as that at the node 28a is extracted.",
"When writing the data from the CPU 2 into the memory cell 21, a memory cell or a plurality of memory cells are selected as described above, a word voltage is supplied, and the NMOS transistors 27a and 27b are made conductive in state.",
"The write control signal WC of the selected memory cell becomes the low level, and the write circuit 24 turns on.",
"As shown in FIG.",
"2, the write circuit 24 has a first write driver 24a and a second write driver 24b, the write data WD input to the write circuit 24 is first inverted at the second write driver 24b and stored in the storage node 28b via the now on NMOS transistor 27b.",
"The inverted output of the second write driver 24b is input to the first write driver 24a and further inverted and stored in the storage node 28a via the now on NMOS transistor 27a.",
"For example, when the value of the write data WD is “1”, it becomes “0” by the output of the second write driver 24b and is stored at the storage node 28b.",
"The output “0” of the second write driver 24b is input to the first write driver 24a, then “1” is output and stored at the storage node 28a.",
"When the value of the write data WD is “0”, similarly, “0” is stored at the storage node 28a, and “1” is stored at the storage node 28b.",
"FIG.",
"3 shows principal parts of the liquid crystal driver 3 having the above built-in display memory 7.In FIG.",
"3, the same reference numerals are used for the same components as those of FIG.",
"1.In FIG.",
"3, an interface circuit (CPU I/F) 6 on the CPU side includes a data latch 9, selector 10, etc.",
"Reference numeral 7 indicates the display memory of the present embodiment, while 8 indicates the interface circuit for the liquid crystal panel display.",
"The display use interface 8 includes circuits such as a data latch 11, a selector 12, and a DAC 13.Reference numerals 34 and 35 are a data bus for transferring the image data output by the memory 7 to the liquid crystal panel and a data bus for the CPU 2 to transfer the data to the memory 7.The liquid crystal driver 3 shown in FIG.",
"3 operates as follows.",
"When writing the pixel data into the display memory 7, the CPU 2 sends the image data to be displayed to the display memory 7 for every pixel.",
"The pixel data sent for every pixel is first stored in the data latch 9.The data stored in the data latch 9 up to a predetermined number of bits is output to the selector 10, selected there, and written into the display memory 7 through the data bus 35.Alternatively, when the CPU 2 reads the pixel data stored in the display memory 7, the pixel data stored in the display memory 7 passes through the data bus 35 in units of a predetermined number of bits, and is held at the data latch 9 via the selector 10, then the data held at that data latch 9 is read to the CPU 2 for every pixel.",
"When reading the pixel data stored in the display memory 7 and displaying it on the liquid crystal panel, the pixel data stored in the display memory 7 passes through the data bus 34 in units of a predetermined number of bits and is held in the data latch 11.Then, the data held in the data latch 11 is output to the selector 12, and R, G, B portions of each pixel data are sequentially selected by the selector 12 by a predetermined method, output to the digital/analog converters (DACs) 13, and further output to the pixels of the liquid crystal panel.",
"In the present embodiment, the data bus 34 holds the number of bits of data required for one line in the horizontal direction of the liquid crystal panel.",
"One line's worth of data can be calculated by one line's worth of the number of pixels×colors (number of bits).",
"Concretely, where one line's worth of the number of pixels is 176 pixels and the colors are comprised of 18 bits (6 bits for each of R, G, B), it becomes an output data bus for 3168 bits.",
"The number of bits of the data bus 35 is one line's worth of the number of bits of data in the same way as the data bus 34.When the number of pixels is 176 and the colors are comprised of 18 bits, the result is 3168 bits.",
"As shown in FIG.",
"3 and as described above, the display memory 7 has two read ports and one write port, assigns one read port and one write port for the access from the CPU 2, assigns the other read port for the liquid crystal panel 4, and assigns the pixel data to the display.",
"The read and write access from the CPU 2 to the display memory can be simultaneously carried out since the read access from the display memory to the liquid crystal panel is independently controlled.",
"Further, the read and write access with respect to the display memory 7 of the CPU 2 and the read access from the display memory 7 to the liquid crystal panel 4 are assigned to the high level period and the low level period of the clock signal for controlling the operation of the display memory 7.The access from the CPU 2 and the read operation to the liquid crystal panel 4 do not interfere with each other, but are carried out in parallel.",
"FIGS.",
"4A to 4F are timing charts of the above operation.",
"FIG.",
"4A shows an address signal DRA of the read access when displaying an image.",
"The address signal DRA is generated once for every display of a row.",
"FIG.",
"4B shows an address signal CAA for the CPU 2 to access the display memory 7.FIG.",
"4C shows a clock signal MCLK of the display memory 7.A high level period of the clock signal MCLK is the period for the CPU 2 to access the display memory 7.In this period, the CPU 2 reads pixel data from the display memory 7, or the CPU 2 writes image data into the display memory 7.A low level period of the clock signal MCLK is used for the read period for the display.",
"In this period, the image data stored in the display memory 7 is read and output to the pixels of the liquid crystal panel.",
"FIG.",
"4D shows a signal DR showing the read period for display.",
"The read operation from the display memory is carried out in the period where the clock signal MCLK of the display memory 7 is at the low level.",
"FIG.",
"4E shows a signal CR indicating the period for the CPU 2 to read data from the display memory 7.The CPU 2 reads data from the display memory in the period where the clock signal MCLK of the display memory 7 is at the high level.",
"FIG.",
"4F shows a signal CW indicating the period for the CPU 2 to write data into the display memory 7.The CPU 2 writes data into the display memory in the period where the clock signal MCLK of the display memory 7 is at the high level.",
"According to the present embodiment, in a custom display memory built into a liquid crystal driver, each memory cell is equipped with two read sense amplifiers for the CPU and display on the two ends of the bit line and is provided with a write driver for the CPU, whereby it becomes possible to independently control the access for display and the read access from the CPU.",
"By this, two systems of read ports and one system of write ports can be equipped.",
"Therefore, if assigning them to the CPU and the liquid crystal panel display and further assigning the access of the CPU and the access for display to the high level period and the low level period of the system clock, the access from the CPU and the operation of reading for display can be simultaneously carried out in parallel and will not overlap.",
"Namely, the operation for display and drawing and the reading of the data can be independently carried out.",
"By this, even if the number of times of access for display increases, the time for the drawing and reading will not be reduced and the CPU will not be made to wait for display.",
"Further, in the display memory of the present embodiment, terminals are equipped at the facing sides of the display memory, and two interfaces are arranged sandwiching the display memory therebetween.",
"One of them is used as the interface for the CPU side, and the other is used as the interface for the liquid crystal panel side.",
"The two can be directly connected to the display memory.",
"By this, there is no detouring of the signal lines, the amount of the interconnects can be reduced in comparison with the conventional general purpose interface, and the power consumption can be reduced by the amount of the interconnects.",
"Further, in comparison with the case of using a usual dual port SRAM, the single port SRAM of the present embodiment can greatly reduce the cell size.",
"Second Embodiment In the second embodiment, an example where the power consumption is further reduced by dividing the power supply of the memory and independently providing power to different image data regions of the memory will be explained.",
"The display memory in the second embodiment has the configuration of the display memory of the first embodiment.",
"Further, in the second embodiment, the display memory is divided into a plurality of regions, and ON/OFF state of the power is controlled for every separated region or operation mode.",
"FIG.",
"5 is a circuit diagram of the configuration of a display memory dividing the power supply.",
"In FIG.",
"5, the same reference numerals are used for part of the components the same as those of FIG.",
"2.In FIG.",
"5, 51a, 51b, and 51c indicate memory cells of the display memory 7 according to the first embodiment shown in FIG.",
"2, 52a and 52b indicate a pair of bit lines (BL), 53a, 53b and 53c indicate word lines (WL), 54a, 54b, and 54c indicate N wells, and 55a, 55b, and 55c indicate P wells.",
"In the memory cell 51a, PMOS transistors P1 and P2 are formed at the N well 54a, and NMOS transistors N1, N2, 27a, and 27b are formed at the P well 55a.",
"The NMOS transistor N1 and the PMOS transistor P1 form a CMOS inverter circuit 29a, while the NMOS transistor N2 and the PMOS transistor P2 form a CMOS inverter circuit 29b.",
"Inputs and outputs are cross-connected to each other so that this pair of CMOS inverters 29a and 29b form a flip-flop, whereby a bi-stable flip-flop circuit is obtained.",
"When supplying a drive voltage VDD to this pair of CMOS inverters 29a and 29b by a drive power supply line 56a, the above bi-stable flip-flop circuit holds two complementary stable states at the nodes 28a and 28b.",
"The nodes 28a and 28b become storage nodes able to store data.",
"For example, the state where the node 28a is at the high level and the node 28b is at the low level is defined as meaning that the data “1” is stored, while conversely the state where the node 28a is at the low level and the node 28b is at the high level is defined as meaning that the information “0” is stored.",
"When reading this data, first, a word line voltage is supplied to the word line designated by a not illustrated row address decoder, for example, the word line 53a, to set the NMOS transistors 27a and 27b in the conductive state.",
"When reading data for every bit, a not illustrated column address decoder is used to designate the memory cells to be read, for example, memory cells 51a, 51b, and 51c.",
"Along with the designation of the word line, the memory cell 51a will be selected.",
"When reading data for every line or for every plurality of memory cells, for example, a memory cell line including the memory cell 51a or a plurality of memory cells is designated.",
"Since the NMOS transistors 27a and 27b become conductive in state, the states of the nodes 28a and 28b are transmitted to a not illustrated read sense amplifier connected to the pair of bit lines 52a and 52b.",
"When outputting the data stored in the memory to the liquid crystal panel, a not illustrated display use sense amplifier is used to extract the present state of the memory cell 51a.",
"Further, when reading the data stored in the memory from the CPU 2, a not illustrated CPU 2 sense amplifier is used to extract the present state (data) of the memory cell 21.Further, when writing data from the CPU 2 into the memory cell 51a, the line of the memory cell or a plurality of memory cells or one memory cell is selected as described above and the NMOS transistors 27a and 27b are set to the conductive state.",
"Then, the write data input to the not illustrated write driver is stored at the two storage nodes 28a and 28b via the NMOS transistors 27a and 27b.",
"Namely, when the value of the write data is “1”, the storage node 28a is set to the high level and the storage node 28b is set to the low level, while when the value of the data is “0”, the storage node 28a is set to the low level and the storage node 28b is set to the high level.",
"The memory cells 51b and 51c have exactly the same configurations as that of the memory cell 51a and operate in the same way as 51a.",
"Therefore, in the memory cells 51b and 51c, the same reference numerals as those for the memory cell 51a are used for components other than the power supply.",
"Further, in the present embodiment, as shown in FIG.",
"5, PMOS transistors Tr1, Tr2, and Tr3 acting as power supply switches are connected to the drive power supply lines 56a, 56b, and 56c of the memory cells 51a, 51b and 51c and control the ON/OFF states of the power supply to the memory cells 51a, 51b, and 51c.",
"The N wells 54a, 54b, and 54c to which the drive power supply lines 56a, 56b, and 56c of the memory cells 51a, 51b, and 51c are connected are separated from each other.",
"Further, the drive power supply lines 56a, 56b, and 56c are connected to the drive power supply lines 56a, 56b, and 56c of the PMOS transistors of the memory cells 51a, 51b, and 51c via the transistors Tr1, Tr2, and Tr3 for turning the power supply ON/OFF, therefore the supplies of power to the memory cells 51a, 51b, and 51c are separated from each other.",
"In FIG.",
"5, VDD controllers VCTR1, VCTR2, and VCTR3 control the ON/OFF states of the transistors Tr1, Tr2, and Tr3 and thereby control the ON/OFF states of the power supplies of the memory cells 51a, 51b, and 51c.",
"This control is set by the operation modes of the VDD controllers VCTR1, VCTR2, and VCTR3.Here, an example of three cells is shown, but the same also applies for the case of the division for more than three cells.",
"Further, one power supply switch transistor is provided in each memory cell here, but there is nothing stopping control of power supplies of memory cells of predetermined regions of the memory together in accordance with actual conditions.",
"According to the display memory of the second embodiment, by dividing the power supply for every predetermined region of the memory and independently controlling the on/off states of the power supplies, the leakage current of memory cells of the unused regions can be reduced.",
"Further, by separating N wells of memory cells, the supply of power to the unused regions of memory cells can be cut to reduce the power consumption.",
"Third Embodiment The display memory according to the third embodiment has a similar basic configuration to that of the display memory of the first embodiment.",
"Note, in the third embodiment, the address array of the display memory corresponds to the pixel array of the liquid crystal panel so the image of the image data stored in the display memory becomes the same as the screen of the liquid crystal panel.",
"Further, the read or write access with respect to the display memory is carried out in units of one row's worth of the pixel data on the screen.",
"FIG.",
"6 is a schematic view of the address array of the display memory and the array of pixels of the liquid crystal panel according to the third embodiment.",
"In FIG.",
"6, the address array of the memory and the pixel matrix of the liquid crystal panel are expressed by an array having lines 1n0 to 1nN and pixels px0 to pxN as suffixes.",
"The arrays of addresses of the memory and pixels of the liquid crystal panel become the same in image.",
"Namely, addresses of the memory are distributed according to the array of pixels of the liquid crystal panel.",
"For example, the number of memory cells connected to one word line of the memory and the number of memory cells connected to a pair of bit lines are determined according to the number of pixels of one row of the liquid crystal screen, the number of pixels of one column, and the number of bits of the colors of the pixels.",
"By the array of addresses of the memory and the array of pixels of the liquid crystal panel becoming the same, the data of pixels to be accessed can be designated among the data stored in the memory having the lines 1n0 to 1nN and pixels px0 to pxN as suffixes.",
"The CPU 2 designates the line address and pixel address and reads and writes data.",
"When displaying data on a liquid crystal panel, it operates to designating the line address and read one line's worth of data together.",
"Next, a read or write operation in units of rows of pixel data will be concretely explained.",
"FIG.",
"7 shows the configuration for accessing the display memory for every line.",
"In FIG.",
"7, 71 indicates a plurality of display use sense amplifiers, 72 indicates one line's worth of memory cells of the liquid crystal panel, 73 indicates a plurality of write drivers for the CPU, and 74 indicates a plurality of sense amplifiers for the CPU.",
"One line's worth of memory cells 72 of the liquid crystal panel becomes the unit of the transfer data when reading and writing data.",
"Data is read and written by this amount of data.",
"Display use sense amplifiers 71 are provided in a number of the amount of one row's worth of pixels of the liquid crystal panel.",
"When reading data stored in the display memory and outputting the same to the liquid crystal panel, these sense amplifiers all operate at one time.",
"The CPU use write drivers 73 are provided in the same number as the display use sense amplifiers 71.When the CPU 2 reads data stored in the display memory, these write drivers 73 also all simultaneously operate.",
"The CPU use sense amplifiers 74 are provided in the same number as the display use sense amplifiers 71 or the CPU use write drivers 73.When the CPU 2 writes data into the display memory, these sense amplifiers all simultaneously operate.",
"Note that at the time of writing, the write drivers can simultaneously write data into required portions (bits or predetermined plurality of bits) according to the write control signal for every bit explained later.",
"In the present embodiment, by employing simple mapping able to handle the pixel array of the liquid crystal panel and the memory address array by the same suffixes, the calculation for linking the addresses and the pixels of the liquid crystal panel becomes unnecessary and liquid crystal panels having a variety of numbers of pixels can be easily handled.",
"Further, the number of times of reading of the memory for one line's worth of the display may be one time.",
"Further, the display memory has a circuit enabling access from the CPU 2 in units of rows and access to the pixel information in that as well.",
"Namely, the operation of the memory is based on access for one line's worth of data.",
"By this, the number of times of operation of the memory can be reduced and low power consumption can be realized.",
"Fourth Embodiment In the conventional display memory, when writing predetermined bits, a read-modify-write operation is necessary.",
"Namely, in the conventional display memory, the data is read out in advance before rewriting the data, the bits to be rewritten are modified while masking the data which are not desired to be rewritten, and then the data is written into the memory.",
"In the third embodiment, an explanation will be given of a display memory providing a column decoder designating a memory cell in the bit direction and a write signal for controlling the write operation on the above display memory and enabling selection of any one memory cell and writing of any bits.",
"The display memory in the present embodiment has the basic configuration of the display memory of the first embodiment.",
"FIG.",
"8 is a view of principal parts of a display memory according to the present embodiment.",
"In FIG.",
"8, the same reference numerals are used for part of the components the same as those of FIG.",
"2.In FIG.",
"8, 81a and 81b indicate memory cells, 82 indicates the row decoder of the memory, and 83a and 83b indicate write drivers of the memory cells 81a and 81b.",
"Further, 84a and 84b indicate column decoders, 85 indicates a read row address latch, 86 indicates a pixel address latch, and 87 indicates a write data latch.",
"Reference numerals 88a and 88b and reference numerals 88c and 88d indicate pairs of bit lines of the memory cells 81a and 81b, and 89 indicates a word line common to the memory cells 81a and 81b.",
"In FIG.",
"8, the memory cell 81a has two inverters 29a and 29b having inputs and outputs connected to each other and has NMOS transistors 27a and 27b as access transistors.",
"A first storage node 28a is configured by the connection point of the output of the inverter 29a and the input of the inverter 29b, while a second storage node 28b is configured by the connection point of the input of the inverter 29a and the output of the inverter 29b.",
"The bit line 88a is connected via the NMOS transistor 27a to the first storage node 28a, while the bit line 88b is connected via the NMOS transistor 27b to the second storage node 28b.",
"Gates of the NMOS transistors 27a and 27b of the memory cell 81a are connected to the common word line 89.The write circuit 83a has first drivers 24a and 24b connected in series and operating by a control signal comprised of the low level, active output of the column decoder 84a.",
"The row address decoder 82 outputs the word line-voltage to the common word line of a predetermined memory cell row based on the row address data of the read row address latch 85 and sets the NMOS transistors 27a and 27b to the conductive state.",
"Based on the column address data of the pixel address latch 86, the output of the column address decoder 84a is inverted and input to the write drivers 24a and 24b of the memory cell column to be written in the bit direction to actuate them.",
"The write signal WRT is input to the column decoder circuits 84a and 84b.",
"The column decoders 84a and 84b operate only in the case where the write signal WRT is at the high level.",
"Next, the operation of a memory having the above configuration will be explained.",
"When supplying the drive voltage VDD to the pair of CMOS inverters 29a and 29b, the CMOS inverters 29a and 29b forming a bi-stable flip-flop circuit hold two complementary stable states at the nodes 28a and 28b, whereby the nodes 28a and 28b can store data.",
"For example, the state where the node 28a is at the high level and the node 28b is at the low level is defined as meaning the data “1” is stored, while conversely the state where the node 28a is at the low level and the node 28b is at the high level is defined as meaning the data “0” is stored.",
"Since the NMOS transistors 27a and 27b have become conductive in state, the nodes 28a and 28b are connected to the write driver 83a via the pair of bit lines 88a and 88b and data can be written.",
"For example, when writing data into the memory cell 81a from the CPU 2, based on the row address data of the read row address latch 85, the row address decoder 82 selects for example the word line 89, supplies voltage to the word line 89, and thus sets the NMOS transistors 27a and 27b to the conductive state.",
"Next, based on the column address data of the pixel address latch 86, the column address decoder 84a designates the memory cell to be written in the bit direction.",
"For example, assume that the memory cell 81a is designated.",
"Along with the designation of the word line, the memory cell 81a will be selected.",
"In the fourth embodiment, the write signal WRT for controlling the write operation to a memory cell is input to the column decoder circuits 84a and 84b.",
"Only when the write signal WRT is at the high level is the writing into the memory cell designated by the column decoders 84a and 84b possible.",
"For example, as described above, when the memory cell 81a is selected and the write signal WRT is at the high level, the output of the column decoder device 84a becomes the low level and enables the operation of the write driver 83a.",
"Accordingly, the data held in the write data latch 87 can be written into the memory cell 81a designated by the row decoder 82 and the column decoder 84.As shown in FIG.",
"8, the write driver 84a has a first write driver 24a and a second write driver 24b.",
"The data held in the write data latch 87 are input to the write driver 84a one after another.",
"The data of each bit thereof is first inverted at the second write driver 24b and stored at the storage node 28b via the now on NMOS transistor 27b.",
"The inverted output of the second write driver 24b is input to the first write driver 24a and further inverted and stored at the storage node 28a via the now on NMOS transistor 27a.",
"For example, when the value of the write data is “1”, it becomes “0” by the output of the second write driver 24b and is stored at the storage node 28b.",
"The output “0” of the second write driver 24b is input to the first write driver 24a, whereby “1” is output and stored at the storage node 28a.",
"When the value of the write data is “0”, similarly, “0” is stored at the storage node 28a, and “1” is stored at the storage node 28b.",
"On the other hand, when the write signal WRT is at the low level, the output of the decoder device 84a designating the memory cell 81a becomes the high level, and the write driver 83a of the memory cell 81a becomes unable to operate.",
"Accordingly, the data held in the write data latch 87 cannot be written into the memory cell 81a designated by the row decoder 82 and the column decoder 84.The memory cell 81b operates in the same way.",
"The display memory of the fourth embodiment has a write control signal (write signal) for every bit.",
"The CPU 2 can write any one bit into the display memory based on this control signal.",
"When comparing this with the conventional display memory, similar effects are realized by just a write operation without performing a read operation in advance.",
"According to the fourth embodiment, by using a write system not requiring a read-modify-write operation, the number of times of operation of the memory can be reduced.",
"Due to this, the power consumption of the memory can be reduced.",
"Fifth Embodiment As already explained, in the display memory of the present invention, terminals are arranged on facing sides of the memory while sandwiching the memory therebetween, therefore one terminal can be arranged for the CPU, and another terminal can be arranged for the liquid crystal panel.",
"The liquid crystal driver of the present invention has a configuration wherein the CPU use interface and the liquid crystal panel use interface sandwich the display memory and are arranged at the two ends of the display memory.",
"It has a CPU use interface between the display memory and the CPU 2 and has a liquid crystal panel use interface between the display memory and the liquid crystal panel.",
"The fifth embodiment relates to the data transfer between the CPU use interface and the display memory.",
"FIG.",
"9 is a view of the schematic circuit configuration of the part on the CPU side of the liquid crystal driver according to the fifth embodiment.",
"In FIG.",
"9, 91 indicates a line latch circuit, 92 indicates a selector circuit, 93 indicates a data bus, and 94 indicates a display memory.",
"The image data is sent from the CPU 2 or the logic circuit for every pixel.",
"The pixel data sent for every pixel is first stored in a data latch 91.When one line of the liquid crystal panel's worth of data is stored in the data latch 91, that data is output to the selector 92, selected there, and written into the display memory 94 via the data bus 93.Alternatively, when the CPU 2 reads the pixel data stored in the display memory 94, the pixel data stored in the display memory 94 is held in the data latch 91 in units of one line's worth of the data through the data bus 94 and via the selector 92, then the data held in the data latch 91 is read to the CPU 2 for every pixel.",
"The data of the display memory 94 is read to the liquid crystal panel side and displayed.",
"The bit width of the line latch 91 is the same as the bit width of one line's worth of the image data in the horizontal direction of the display screen.",
"For example, when the size of the liquid crystal panel is 176 pixels×240 rows, the data of each of the three colors of R, G, B is expressed by 6 bits, and display of 260,000 colors is possible, the required capacity of the memory becomes 176×3×6×240=760,320 bits and the data capacity and bit width of the line latch 91 become 176×3×6×1=3168 bits.",
"The data bus 93 has the same bit width.",
"FIGS.",
"10A to 10F show timing charts of the write operation by units of lines according to the circuit configuration of FIG.",
"9.FIG.",
"10A shows 1 pixel's worth of the image data DAT sent from the CPU side; and FIGS.",
"10B and 10C show addresses ADD-X and ADD-Y in the X-direction (column direction) and in the Y-direction (row direction) in the display memory 94.FIG.",
"10D shows a write command XLATW from the CPU 2 to the line latch 91; FIG.",
"10E shows a write command XRAMW from the line latch 91 to the display memory 94; and FIG.",
"10F shows latch data LDAT.",
"Note that it is also possible to read out the stored data of the line latch 91 to the CPU side.",
"One line's worth of the image data is input from the CPU side while designating the X-address for every pixel.",
"At this time, “L” is input as the write command to the line latch 91, and the image data of pixels are sequentially stored at locations corresponding to X-addresses in the line latch 91.After one line's worth of the image data is stored in the line latch 91, when the Y-addresses are designated and the write command XRAMW to the display memory 94 is set to “L”, the one line's worth of the image data stored in the line latch 91 are written at the locations designated by the Y-addresses of the display memory 94.Here, the read command from the line latch 91 to the display memory 94 is made XRAMR.",
"FIGS.",
"11A to 11F show timing charts of the read operation of units of lines according to the circuit configuration of FIG.",
"9.FIGS.",
"11A and 11B show addresses ADD-X and ADD-Y in the X-direction (column direction) and in the Y-direction (row direction) in the display memory 94.FIG.",
"11C shows a read command XLATR from the line latch 91; FIG.",
"11D shows a read command XRAMR from the line latch 91 to the display memory 94; FIG.",
"11E shows latch data LDAT; and FIG.",
"11F shows read one pixel's worth of the image data DAT.",
"When the CPU side designates the Y-addresses of the locations of the display memory 94 from which the data are desired to be read out and sets the read command XRAMR to “L”, the data at the locations designated by the Y-addresses in the display memory 94 are read out and one line's worth of the data is stored in the line latch 91.After one line's worth of the data is stored in the line latch 91, the read command XLATR from the line latch 91 is set to “L” and the X-address is designated for every pixel, to thereby read out the data stored in the line latch 91.In this way, the read and write access with respect to the memory can be carried out in units of one line.",
"By providing one line's worth of the line latch between the display memory and the CPU 2, operations of reading and writing with respect to the display memory are simultaneously carried out for the amount of one line.",
"By this, the number of times of access to the display memory is reduced.",
"The operating power consumption of the display memory is proportional to the number of times of access, so a lower power consumption can be realized.",
"Sixth Embodiment In the liquid crystal driver according to the sixth embodiment, based on the configuration of the fifth embodiment, the array of the pixels on the liquid crystal panel and the array of addresses of the display memory and the addresses of the data in the line latch are brought into one-to-one correspondence.",
"Further, the data can be written from the line latch into the display memory for every pixel.",
"This is similar to the display memory explained in the third embodiment in the point that the array of pixels on the liquid crystal panel and the array of addresses of the display memory are in a one-to-one correspondence in the liquid crystal driver of the sixth embodiment.",
"Namely, a display memory having X-directional and Y-directional addresses corresponding to X-(column), Y-(row) coordinates on the liquid crystal panel is provided, and the X-, Y-coordinates on the display panel and the X-directional and Y-directional addresses of the display memory are set into one-to-one correspondence.",
"Next, an explanation will be given of the write operation for every pixel from the line latch to the display memory in the liquid crystal driver of the present embodiment by using FIG.",
"12 and FIG.",
"13 while referring to the timing charts of FIG.",
"10.FIG.",
"12 shows the operation of writing data for every pixel.",
"In FIG.",
"12, 121 indicates a data bus of the image data sent from a CPU 2 or the logic circuit (one pixel's worth of the number of bits of data), 122 indicates a line latch, 123 indicates a data bus for reading the data to the display memory from the line latch 122 or writing the data (one line's worth of the number of bits of data), 124 indicates a display memory, and 125 indicates a data bus for sending the data to the liquid crystal panel side for displaying the data of the display memory.",
"The display memory 24 has X-directional and Y-directional addresses corresponding to the X-, Y-coordinates on the not illustrated liquid crystal panel.",
"The sizes in the X-direction and Y-direction are data sizes in the X-direction and Y-direction of one screen.",
"The line latch 122 stores one line's worth of the data from the not illustrated CPU 2.The X-directional positions of this line latch 122 and X-directional addresses in the memory 125 and the X-coordinate on the screen are in one-to-one correspondence.",
"Next, the operation of writing the image data at addresses (05H, 03H) of the display memory 124 will be explained as an example.",
"First, when writing data by designating the image data and X-address (05H) from the CPU side (that is, XLATW=“L” in FIG.",
"10), the image data is stored at the location indicated by the address 05H on the line latch 122.After the image data is simultaneously written into the line latch 122, if the Y-address (03H) is designated as the write command XRAMW=“L”, the color data of 1 pixel is written at the address positions of (05H, 03H) in the memory.",
"Next, using FIG.",
"13, the technique for realizing an operation of writing data into the display memory 124 for every pixel described above will be explained.",
"In FIG.",
"13, 131 indicates part of the display memory, and 132 is the line latch.",
"In the line latch 132, 133 is the storage region occupied by one pixel, and 134 is a write flag provided for every pixel.",
"As shown in FIG.",
"13, at the line latch 132, a write flag for writing data from the line latch 132 into the display memory 131 is provided for the address of each pixel.",
"The write flag is set (that is, WRITE FLAG=1) for only a pixel for which data is written from the CPU side to the line latch 132.When writing data into the display memory 131, data is written for only pixels where the write flag is “1”, therefore it is possible to write data for only the desired pixels and there is no effect on the surrounding pixel data.",
"Further, it is also possible to rewrite the data for any plurality of pixels on the same line by using these write flags.",
"After writing the data from the line latch 132 into the display memory 131, the write flags are all reset to “0”.",
"FIGS.",
"14A to 14F are timing charts of the above operation.",
"FIG.",
"14A shows a latch write signal LCWRQ; FIG.",
"14B shows a line write signal LNWRQ; and FIG.",
"14C shows a write address signal WADR, a clock signal CK, a write flag signal WF, and a word line signal WL.",
"As shown in FIGS.",
"14A to 14F, when writing data for a pixel of the line latch 132 indicated by the write address signal WADR, the latch write signal LCWRQ for the pixel becomes the high level.",
"That is, LCWRQ becomes equal to “1”.",
"Then, the write flag signal WF of the pixel is set, that is, becomes the high level (WF=“1”).",
"The line write signal LNWRQ is set and becomes the high level for the pixel of the memory 131 corresponding to the pixel where the write flag WF=“1”.",
"Namely, LNWRQ becomes equal to “1”.",
"A voltage is supplied to the word line WL designated by the write address signal WADR of the display memory 131, writing to a pixel of the memory related to this word line WL is enabled, and then the writing starts.",
"Namely, when writing data into the display memory 131, the data is written into only a pixel corresponding to a pixel where the write flag WF=“1” of the line latch 132 of the display memory 131 (LNWRQ=“1”).",
"It is also possible to rewrite any plurality of pixels on the same line by using the write flags.",
"After writing the data from the line latch 132 into the display memory 131 (Write End), the write flag WF is reset to “0”.",
"Conventionally, the read/write operation with respect to the display memory is carried out for every group of pixels, therefore, when desiring to write data for a certain single pixel in the display memory from the CPU 2, if trying to write one pixel's worth of data as it is, the plurality of pixels around that will be rewritten.",
"Therefore, the read-modify-write sequence of reading a group of pixels once, then rewriting only the data of pixels desired to be rewritten outside the memory, and again storing the rewritten group of pixels in the memory has been performed.",
"By imparting the write flags WF to the line latch as in the sixth embodiment, it is possible to rewrite data for only pixels desired to be written.",
"By imparting the write flags WF to the line latch for every pixel, it is possible to write the desired pixel data without any effect upon the pixel data around the pixels to be written.",
"Therefore, according to the sixth embodiment, there is the advantage that the read-modify-write sequence which has been conventionally required becomes unnecessary.",
"Further, it is not necessary to generate memory addresses corresponding to X-, Y-coordinates on the screen outside the display memory.",
"Image data can be written in units of pixels at the locations of the memory corresponding to the screen by just designating the X-, Y-coordinates on the screen as X-, Y-addresses from the CPU side.",
"Further, when writing data for a plurality of pixels existing on the same line, the line latch and the display memory need only be accessed one time.",
"Seventh Embodiment As already explained, in the display memory of the present invention, terminals are arranged at the facing sides of the memory while sandwiching the memory therebetween, therefore one terminal can be arranged for the CPU, and another terminal can be arranged for the liquid crystal panel.",
"The liquid crystal display of the present invention is configured with the CPU use interface and the liquid crystal panel use interface sandwiching the display memory therebetween and arranged at the two ends of the display memory.",
"It has the CPU use interface between the display memory and the CPU 2 and has the liquid crystal panel use interface between the display memory and the liquid crystal panel.",
"The seventh embodiment relates to the data transfer from the display memory to the liquid crystal panel use interface.",
"FIG.",
"15 is a view of the circuit configuration of the part on the panel side of the liquid crystal display according to the seventh embodiment.",
"In FIG.",
"15, 141 indicates a display memory, 142 indicates a data latch circuit, 143 indicates a selector circuit, and 144 indicates a digital/analog converter (DAC).",
"Reference numeral 145 indicates a data bus for the liquid crystal panel.",
"Pixel data is read out to a not illustrated liquid crystal panel from the display memory 141 through the data bus 145 for the liquid crystal panel.",
"The line latch 142 can store one line's worth of the data in the horizontal direction on the screen.",
"The bit width is the same as one line's worth of the bit width.",
"For example, when the size of the liquid crystal panel is 176 pixels×240 rows, the data of each of the three colors of R, G, B is expressed by 6 bits, and display of 260,000 colors is possible, the required capacity of the memory becomes 176×3×6×240=760,320 bits and the data capacity and bit width of the line latch 142 become 176×3×6×1=3168 bits.",
"When reading out the pixel data stored in the display memory 141 and displayed it on the liquid crystal panel, data is held in the data latch 142 through the data bus 145 in units of one line's worth of the pixel data in the horizontal direction of the not illustrated liquid crystal panel.",
"Then, the data held in the data latch 142 is output to the selector 143.The selector 143 sequentially selects the R, G, B portions of each pixel data by a predetermined system, outputs them to the DACs 144, and further outputs them to the pixels of the liquid crystal panel.",
"Due to this, the pixel data is displayed on the screen.",
"In this way, the line latch 142 performs a series of operations for fetching one line's worth of the data in the horizontal direction on the liquid crystal screen from the display memory 145 in a constant cycle and outputting the same to the DACs 144.Further, the operation of writing one line's worth of the data held in the display memory 145 into the line latch 142 is carried out in synchronization with a clock of the display memory.",
"After holding one line's worth of the data in the line latch 142, the memory 145 can be freed up, so the time after that can be used for the access time of the CPU 2.As a result, a moving picture display etc.",
"requiring quick switching of the screen can also be handled.",
"As described above, in the liquid crystal driver having the built-in display memory, in order to drive one line in the horizontal direction on the liquid crystal panel screen at a time, a latch circuit for holding the data of simultaneously operating DACs is necessary.",
"By providing a latch circuit having a capacity required for holding one line's worth of the data in the horizontal direction on the liquid crystal panel screen between the display memory and the DACs, it becomes possible to read and write one line's worth of data in the horizontal direction on the liquid crystal panel screen at one time, the number of times of access to the memory is reduced, and thus a lower power consumption can be achieved.",
"Eighth Embodiment The configuration of the liquid crystal display according to the eighth embodiment is substantially the same as that of the seventh embodiment.",
"The difference thereof resides in that a selector circuit able to output data in a time division manner for three colors of red, green, and blue (RGB time division) when outputting data held in the data latch to the digital/analog converters (DACs) (hereinafter, referred to as a RGB selector) is included.",
"FIG.",
"16 is a circuit diagram of the configuration of the principal parts of a liquid crystal display according to the eighth embodiment.",
"In FIG.",
"16, 150 indicates a liquid crystal panel, 151 indicates an RGB selector circuit, 152 indicates a line latch circuit, 153 indicates a data bus for the image data sent from the display memory, 154 indicates a data bus for the image data output from the line latch 152, 155 indicates a display memory, 156 indicates the data bus for the image data output from the selector circuit 151, 157 indicates a digital/analog converter (DAC), 158 indicates a selector circuit for converting the image data having red, green, and blue colors divided by the RGB selector 151 to the parallel data of R, G, B, and 159 indicates a pixel cell expressed by red, green, and blue colors.",
"The liquid crystal display having the above configuration operates as follows.",
"The image data sent from the display memory 155 is output to the line latch 152 and held there in units of lines.",
"The data held in the line latch 152 is output to the DACs 157 in synchronization with the horizontal synchronization signal (Hsync).",
"At that time, the R, G, B components of the image data are switched by the RGB selector 151 asynchronously with respect to the clock of the memory, time divided, and then output to the DACs 157.By this, the number of the output terminals of the selector 151 and DACs 157 becomes one-third of the bit width of the line latch 152.The R, G, B data in the time-divided image data output from the DACs 157 are separated by the selector circuit 158 to become the parallel data of R, G, and B which are in turn output to the pixel cells 159 for display.",
"For example, as explained above, when the size of the liquid crystal panel 150 is 176 pixels×240 rows, each of the data of the three colors of R, G, B is represented by 6 bits, and the display of 260,000 colors is possible, the RGB selector 151 has input terminals for 3168 bits or the same as the bit width of the line latch 152 and, for one DAC 157, switches R, G, B data each consisting of 6 bits by time division and outputs the same.",
"Accordingly, the selector 151 has output terminals for 1056 bits.",
"The data held in the line latch 152 is output to the DACs 157 in synchronization with the horizontal synchronization signal (Hsync).",
"At that time, the R, G, B components of the color image data are switched at the RGB selector 151, time divided, and output.",
"Conventionally, when outputting the data of a memory to DACs, the data was not output by the time division of the RGB data, but the outputs of the memory were directly connected to the DACs by one-to-one correspondence.",
"According to the eighth embodiment, by outputting the image data by time division by RGB, in comparison with the case where the outputs of the line latch 152 are directly connected to the DACs 157 by one-to-one correspondence, the number of DACs 157 can be decreased to one-third.",
"Further, when outputting the data held in the line latch 152 to the digital/analog converters (DAC) 157, the switching of RGB of the image data of color is controlled asynchronously with respect to the clock of the memory.",
"FIGS.",
"17A to 17F show timing charts of the RGB time division of the output data of the line latch 152.FIG.",
"17A shows a clock signal CLK of the memory; FIG.",
"17B shows output data D152 (3168 bits) of the line latch 152; FIG.",
"17C shows red (R) data; FIG.",
"17D shows green (G) data; FIG.",
"17E shows blue (B) data; and FIG.",
"17F shows RGB data D151 (1056 bits) output by the RGB selector circuit 151.The R, G, B data output from the line latch 152 are converted to the time divided signals asynchronously with the clock by the RGB selector circuit 151 and output from the same terminals of the RGB selector circuit 151.The 3168 bits of data output from the line latch 152 become 1056 bits at the output terminals of the RGB selector circuit 151.Conventionally, in order to reduce the power consumption of the DACs, it was necessary to adjust the settling time.",
"Since the operating speed is different between the DACs and the memory, they must be separately controlled.",
"When outputting the data of the display memory to the DACs, however, the timing of outputting the RGB data is fixed, so the phase of the data could not be changed freely to match with the characteristics of the DACs.",
"According to the eighth embodiment, by enabling the asynchronous control of the switching of RGB of the data output to the DACs with respect to the clock of the memory, adjustment matching with the settling time of the DACs can be carried out, so the read system is not disturbed even if an interruption occurs.",
"Further, the timing can be adjusted matching with the settling time of the DACs, so the power consumption can be reduced.",
"The DACs and memory can be separately controlled, and different operating speeds can be coped with.",
"Further, the phase of the input signal can be easily adjusted.",
"By providing the RGB selector able to output the data to be output to the DACs by time division by RGB, in comparison with the case where the outputs of the line latch are directly connected to the DACs in one-to-one correspondence, the number of DACs is greatly decreased (two-thirds) and thus the power consumption can be greatly reduced.",
"Next, an explanation will be given of an example of a preferred configuration of the liquid crystal driver according to the embodiment explained above.",
"The present liquid crystal driver is for example a one-chip driver IC having a built-in single port or dual port display memory (frame memory), oscillator, timing generator, liquid crystal tone display reference voltage source, and interface circuit with the CPU.",
"Concretely, it is designed so as to have a built-in dual-port memory of 176(H)×3×6 (RGB)×240(V)=760,320 bits and to be compatible for liquid crystal panels having different numbers of pixels such as 120×160 dots, 132×176 dots, 144×176 dots, and 176×240 dots by setting.",
"In the applied liquid crystal panel, for example, the diagonal length is about 2.2 inch, the driver in the horizontal direction includes a TFT selector and the driver IC with the built-in memory of the present invention, the driver in the vertical direction becomes the TFT driver, and the chip is mounted by the COF method or COG method.",
"As the inversion system, an IH/IV (VCOM inversion) system is employed.",
"The logic system terminals of the present liquid crystal driver IC include CPU interface chip selection, read, write, data bus, address bus, reset, main clock, horizontal synchronization, vertical synchronization, serial data, and other terminals and further includes terminals for liquid crystal panel control.",
"Assume that by setting a mode register of the present liquid crystal driver, it is possible to change among the asynchronous mode, synchronous mode, color mode, screen mode, alternation mode, refresh rate, standby mode, etc.",
"Explaining this in detail, in the asynchronous mode, the timing of scanning of the TFT panel and the timing of rewriting the display memory by the CPU may be asynchronous.",
"The display memory is a dual port memory, and the CPU is not allowed to wait.",
"When the scans of the display memory and the TFT panel are synchronous and the contents of the built-in display memory are output to the DACs in parallel for each of the R, G, B colors for every row by the clock of the internal/external oscillator (self refresh), the data of the blue color is output in the first ⅓ period of one cycle of the clock signal of the shift register of the vertical driver, the data of the green color is output in the middle ⅓ period, and the data of the red color is output in the last ⅓ period.",
"The CPU interface of the asynchronous mode becomes a parallel interface.",
"When not using a parallel interface, the same function as that of an 8-bit parallel interface is achieved by using a serial interface.",
"Note that a serial interface is used only for writing and cannot perform reading.",
"In the synchronous mode, the image data are continuously sent in synchronization with the image use clock, the horizontal synchronization signal, and the vertical synchronization signal.",
"The TFT panel is scanned by using the horizontal and/or vertical synchronization signal, so all timings are synchronous also with the scanning of the TFT panel.",
"In the synchronous mode, normally, the image data is directly written into the line buffer immediately before the DACs.",
"The display memory holds the information before switching to the synchronous mode.",
"In the synchronous mode, the image data is transferred without break, therefore a buffer for transferring the data to the DACs and a buffer for sequentially receiving the data exist.",
"The RGB data are input with the width of 18 bits to line buffers alternating by the cycle of the horizontal synchronization signal (Hsync).",
"When output, the R data is first sent to the DACs with the width of 6 bits in the first ⅓ period of the horizontal synchronization signal Hsync, next the G data is sent to the DACs with the width of 6 bits in the middle ⅓ period of the horizontal synchronization signal Hsync, then the B data is sent to the DACs with the width of 6 bits in the last ⅓ period of the horizontal synchronization signal Hsync.",
"In the synchronous mode, there also exists the so-called “capture” system of handling image data where image data is fetched once into the display memory.",
"The RGFB parallel bus interface of the synchronous mode will be explained next.",
"The image data is latched at the rising edge of the image signal clock synchronized with the image signal by default, but this can be changed from the CPU.",
"The polarity of the horizontal synchronization signal is negative (can be changed from CPU) by default.",
"One cycle is formed by a vertical blanking period+video signal period.",
"The image signal is latched by the image clock.",
"For the CPU interface of the synchronous mode, only a serial interface can be used in the synchronous mode.",
"The serial interface is used only for writing and cannot perform reading.",
"In the serial interface, the operation is similar to that of a parallel 8-bit bus mode.",
"By setting the mode register of the liquid crystal driver, various color modes can be set.",
"In the full color mode, the built-in 6-bit DACs are used to convert 6 bits of RGB to 64 stages of voltage for output.",
"In the reduced color mode (8-color mode), the ground or output amplifier use high voltage power supply level VCC is output according to the page indicated by a special effect register, that is, for the most significant bit (MSB) among 6 bits of the RGB when the page is 1, for the second bit from the most significant bit when the page is 2, or for the least significant bit (LSB) when the page is 6.At this time, the supply of the power to the built-in 6-bit DACs is stopped.",
"The screen mode will be explained next.",
"In the full screen mode, the entire screen is displayed by the color mode designated by the status register.",
"In the partial screen mode, only the portion designated by the status register is displayed by the color mode designated by the status register.",
"When a portion other than this is scanned, white is displayed by the designated color mode.",
"Next, the standby mode will be explained.",
"In a transition period of the standby mode, the value of the standby mode of the mode register is referred to for each one phase for every field cycle.",
"When the awake mode is entered again during a transition from the awake mode to the asleep mode according to this value, feedback is given while maintaining the sequence.",
"After turning on the power or after a hardware reset, the liquid crystal driver IC enters the asleep mode.",
"In the awake mode, from the asleep state, the sequence of: Start oscillation of built-in oscillator Activate DC/DC converter Reset panel Rapidly charge coupling capacitor of common voltage Display white on entire screen is executed, then the awake (normal) mode is entered.",
"In the asleep mode, from the awake state, the sequence of: Display white on entire screen Rapidly discharge coupling capacitor of common voltage Reset panel Stop DC/DC converter Start oscillation of built-in oscillator is executed, then the asleep mode is entered.",
"The display memory access mode will be explained next.",
"According to the contents of the display memory access mode register, eight types of sequential memory accesses are possible such as portrait, landscape, normal, mirror, normal, and upset.",
"Special functions of the liquid crystal driver will be explained next.",
"In the image fetching function, the content of the frame memory for a moving picture signal is held for the period where the capture flag of the frame memory access register is “0”.",
"When the capture flag becomes “1”, one frame after the next vertical synchronization signal is fetched into the frame memory.",
"When the capture flag changes from “1” to “0”, after the next vertical synchronization signal, the content of the frame memory is held.",
"In the common voltage initial charging function, the DC cut capacitor of the output terminal of the common voltage can be rapidly charged and discharged.",
"Facing the DC cut capacitor of the output terminal of the common voltage, a DC offset terminal is connected and sag occurs.",
"In order to keep the sag small in the display mode as well, the DC offset terminal is made a high resistance and a long time is taken for the charging and discharging of the DC offset to and from the capacitor.",
"At the time of turning on/off the power supply, however, if the DC offset is not rapidly charged or discharged, the display quality is lowered in the period of transition from the initial state to normal state.",
"Particularly, at the time of discharge, an after image is displayed if the DC offset still remains even after the power is cut.",
"For this reason, rapid charging and discharging become necessary.",
"In the reset function, the hardware is reset by a reset signal from a reset pin connected to the CPU.",
"The register/frame memory is not reset.",
"The software is reset by a command from the CPU.",
"The contents of the display memory/some registers are held.",
"In the contrast control function, since a display using much black consumes a large power, the contrast is lowered and black display is avoided (definition of contrast is luminance of white/luminance of black, so lowering of contrast in this case means raising luminance of black while keeping luminance of white as it is).",
"In the case of 6-bit RGB data, 00H→charge and discharge panel by 6V amplitude→display black→large power consumption.",
"20H→charge and discharge panel by 3V amplitude→display gray.",
"3FH→charge panel by 0.4V amplitude→display white.",
"Therefore, divide 6 bits by 2 (discard least significant 1 bit) and add 20H, 00H→20H→charge and discharge panel by 3V amplitude→display black, 20H→30H→charge and discharge panel by 1.5V amplitude→display gray, 3FH→3FH→charge and discharge panel by 0.4V amplitude→display white.",
"A reduction of contrast is realized by making 32,000 colors.",
"In the scroll function, the panel end memory pointer is controlled so as to change the data to be transferred from the frame memory to the panel so that it appears to roll on the display.",
"It is possible to control the roll starting row, roll row width, and roll speed/direction by a dedicated register.",
"In the negative-positive inversion function, when two points on the screen are designated by the dedicated register, the inside of a rectangle having the two points as diagonals inverts between negative and positive.",
"The panel end memory pointer is monitored, and the output of the display memory is inverted then sent to the DACs in the period where the pointer is in a designated range.",
"In the blinking function, when two points on the screen are designated by the dedicated register, the inside of a rectangle having the two points as diagonals blinks.",
"The panel end memory pointer is monitored, and a logical AND of the output of the display memory and the output of a blinking cycle counter is sent to the DAsC in the period where the pointer is in a designated range.",
"In the built-in DC/DC converter control function, the CPU can control the switch for setting usage/sealing of the built-in DC/DC converter and the ON/OFF switches of the channels of the DC/DC converter.",
"In the built-in LED driver control function, the CPU can set the switch for setting usage/sealing of the built-in LED driver and the current sink capability adjustment (8 stages) of the LED driver.",
"The liquid crystal driver is provided with a large number of registers and pointers to realize the above specifications.",
"The present invention is not limited to the embodiments explained above.",
"Various modifications are possible in a range not out of the gist of the present invention.",
"In the first embodiment, the first access for outputting data from the display memory to the pixels was carried out in the low level period of the clock signal of the display memory, while the second access for an external controlling means to read data from the display memory and write data into the display memory was carried out in the high level period of the clock signal of the display memory, but it is also possible to perform the first access in the high level period of the clock signal and perform the second access in the low level period of the clock signal.",
"Further, in the second embodiment, one power supply switch transistor is provided for every memory cell, but it is also possible to control the power supplies of memory cells of predetermined regions of the memory all together in accordance with actual conditions.",
"As explained above, according to the present invention, by imparting two systems of read ports and one system of write ports to the two sides of the display memory, the cell size can be greatly reduced in comparison with the case of using an ordinary dual port memory, the interconnect resources can be reduced, and the power for the amount of the interconnects can be reduced.",
"Further, by assigning the display use access and the CPU use access to the memory to the high level period and low level period of the clock signal of the memory, the waiting time of the CPU for display can be reduced.",
"By dividing the power supply to supply the drive power supply voltage to the memory and by cutting the supply of power to regions of the memory cells which are not used, the power consumption can be reduced.",
"Further, by the system of writing for every bit or for every pixel not requiring a read-modify-write sequence, the number of times of operation of the memory can be reduced.",
"Since data can be written into the memory for any single pixel by a single access, the read-modify-write sequence becomes unnecessary.",
"Rewriting in units of pixels also consumes less power in comparison with the conventional case.",
"By enabling simple mapping of the driver circuit and memory array, calculation for linking addresses and pixels of the display screen becomes unnecessary.",
"Further, dealing with driver circuits for a variety of numbers of pixels becomes easy.",
"It is possible to link the screen, memory mapping, and line latch and write data to the memory for any single pixel, possible to write data for any plurality of pixels on the same line by one access to the memory, and possible to designate X, Y-coordinates on the display screen as the address from the CPU side.",
"By imparting a line latch between the processor and the display memory and operating it by one read operation per row display, the number of times of operation of the memory is reduced.",
"By this, the power consumption of the memory can be reduced.",
"In a display memory built into a driver circuit, by providing a line latch having a capacity required for holding one line's worth of data in the horizontal direction on the LCD panel screen between the display memory and the DACs and providing a bit width the same as one line's worth of the bit width in the line latch, it becomes possible to read and write one line's worth of the data in any horizontal direction on the screen at one time.",
"By reducing the number of times of access to the memory, the power consumption can be reduced.",
"By reading and writing one line's worth of data held in the memory at one time in synchronization with the clock of the memory, the period after holding one line's worth of the data can be used for the access time of the CPU, therefore it is possible to deal with even display of a moving picture requiring quick switching of the screen.",
"By the RGB selector selection circuit able to output data to be output to the DACs by time division by RGB, in comparison with the case where the outputs of the line latch are directly connected to the DACs by one-to-one correspondence, the number of DACs can be decreased to one-third and the power consumption can be reduced.",
"By enabling the control of the switching of RGB of the data to be output to the DACs asynchronously with respect to the clock of the memory, the DACs and the memory can be separately controlled and different operating speeds can be coped with.",
"Further, even if the interruption occurs, the read system is not disturbed and the phase of the input signal can be easily adjusted.",
"By adjusting the timing matched with the settling time of the DACs, the power consumption can be reduced.",
"INDUSTRIAL APPLICABILITY According to the display memory, driver circuit, and display of the present invention, the power consumption can be reduced, graphics can be generated at a high speed, and there is no need for memory mapping, therefore they can be applied to the display system of a mobile phone, PDA, or other portable information device (portable information apparatus)."
]
] | Patent_10432937 |
[
[
"Phase transfer of nanoparticles",
"The invention relates to phase transfers of nanoparticles and to a catalysis using said nanoparticles.",
"The aim of the invention is to facilitate a transfer of nanoparticles from an organic solution to an inorganic, especially, aqueous solution.",
"To this end, a generically describable substance class, for example the commercially available 4-dimethylaminopyridine (DMAP), which is for example dissolved in water, is added to the organic solution in sufficient amounts.",
"This measure has the effect that the nanoparticles are readily transferred in a one-step process from the organic phase (in each case in the top section) to the inorganic phase (in each case in the lower section) in the sample container."
],
[
"1-86.",
"(canceled) 87.A method of transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with the organic phase, said method using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic component Z, and concatenating the component X and Y, wherein the hydrophobic component Y comprises a weakly basic group, the hydrophillic component X comprises a strong basic, tertiary amino group and the groups are in conjugation via the organic component Z.",
"88.A method of transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with the organic phase, said method using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic component Z, and concatenating the components X and Y, the hydrophobic component Y comprising a thiol group and the hydrophillic component X comprising a carboxylic group.",
"89.A method of transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with the organic phase, said method using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic component Z, and concatenating the component X and Y, said transfer phase catalyst being mercaptopropyletrimethoxysilane.",
"90.The method of claim 87, wherein said phase transfer catalyst is 4-dimethylaminopyridine (DMAP).",
"91.The method according to claim 88, wherein said phase transfer catalyst is mercaptoundecanoic acid (MUA).",
"92.The method according to claim 87, wherein the ratio of the number of surface atoms of a colloid particle to the number of phase transfer catalyst molecules bound thereto is in a range from 0.1 to 10.93.The method according to claim 88, wherein the ratio of the number of surface atoms of a colloid particle to the number of phase transfer catalyst molecules bound thereto is in a range from 0.1 to 10.94.The method according to claim 89, wherein the ratio of the number of surface atoms of a colloid particle to the number of phase transfer catalyst molecules bound thereto is in a range from 0.1 to 10.95.The method according to claim 92, wherein said ratio is about 1.96.The method according to claim 93, wherein said ratio is about 1.97.The method according to claim 94, wherein said ratio is about 1.98.The method according to claim 87, wherein said colloid particles are at least one member of the group consisting of metal colloids, metal oxide colloids, and metal alloy colloids.",
"99.The method according to claim 88, wherein said colloid particles are at least one member of the group consisting of metal colloids, metal oxide colloids, and metal alloy colloids.",
"100.The method according to claim 89, wherein said colloid particles are at least one member of the group consisting of metal colloids, metal oxide colloids, and metal alloy colloids.",
"101.The method according to claim 98, wherein said metal is at least one member of the group consisting of gold, silver, iridium, platinum, palladium, nickel, iron, rhodium and ruthenium.",
"102.The method according to claim 99, wherein said metal is at least one member of the group consisting of gold, silver, iridium, platinum, palladium, nickel, iron, rhodium and ruthenium.",
"103.The method according to claim 100, wherein said metal is at least one member of the group consisting of gold, silver, iridium, platinum, palladium, nickel, iron, rhodium and ruthenium.",
"104.The method according to claim 98, wherein said metal oxide is at least one member of the group consisting of iron oxide, zinc oxide, titanium dioxide and tin oxide.",
"105.The method according to claim 99, wherein said metal oxide is at least one member of the group consisting of iron oxide, zinc oxide, titanium dioxide and tin oxide.",
"106.The method according to claim 100, wherein said metal oxide is at least one member of the group consisting of iron oxide, zinc oxide, titanium dioxide and tin oxide.",
"107.The method according to claim 87, wherein said colloid particles are semiconductor nanoparticles.",
"108.The method according to claim 88, wherein said colloid particles are semiconductor nanoparticles.",
"109.The method according to claim 89, wherein said colloid particles are semiconductor nanoparticles.",
"110.The method according to claim 87, wherein said colloid particles are inorganic nanoparticles which comprise rare earth elements.",
"111.The method according to claim 88, wherein said colloid particles are inorganic nanoparticles which comprise rare earth elements.",
"112.The method according to claim 89, wherein said colloid particles are inorganic nanoparticles which comprise rare earth elements.",
"113.The method according to claim 87, wherein said aqueous phase comprises water soluble components.",
"114.The method according to claim 88, wherein said aqueous phase comprises water soluble components.",
"115.The method according to claim 89, wherein said aqueous phase comprises water soluble components.",
"116.The method according to claim 1 13, wherein said aqueous phase comprises alcohols.",
"117.The method according to claim 1 14, wherein said aqueous phase comprises alcohols.",
"118.The method according to claim 115, wherein said aqueous phase comprises alcohols.",
"119.The method according to claim 87, wherein after said phase transfer has completed, said aqueous or alcoholic phase is separated from said organic phase.",
"120.The method according to claim 88, wherein after said phase transfer has completed, said aqueous or alcoholic phase is separated from said organic phase.",
"121.The method according to claim 89, wherein after said phase transfer has completed, said aqueous or alcoholic phase is separated from said organic phase.",
"122.An aqueous or alcoholic phase obtainable by the method according to claim 87.123.An aqueous or alcoholic phase obtainable by the method according to claim 88.124.An aqueous or alcoholic phase obtainable by the method according to claim 89.125.A method for selectively coating surfaces of macroscopic bodies, comprising the step of using an aqueous or alcoholic phase obtainable by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y, one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"126.A method for selectively coating carrier particles, comprising the step of using an aqueous or alcoholic phase obtainable by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"127.A method for marking biologically derived molecules, comprising the step of using an aqueous or alcoholic phase obtainable by transferring inorganic colloid particles, from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"128.A paint, comprising a dye solution having an aqueous or alcoholic phase produced by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"129.A printing ink, comprising a dye solution having an aqueous or alcoholic phase produced by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y one condition being present from the group consisting of: (1) said hydrophobic component y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"130.A varnish, comprising a dye solution having an aqueous or alcoholic phase produced by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components x and y one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"131.An article, having a coating formed of one of a varnish, a paint and an ink produced by using an aqueous or alcoholic phase obtainable by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"132.A method of homogeneous, heterogeneous, or mixed-phase catalysis, comprising the step of utilizing an aqueous or alcoholic phase obtainable by transferring inorganic colloid particles from an organic phase into an aqueous or alcoholic phase, immiscible with said organic phase, by using a phase transfer catalyst which comprises a hydrophobic component Y, a hydrophillic component X and an organic molecule Z, and concatenating said components X and Y one condition being present from the group consisting of: (1) said hydrophobic component Y comprising a weakly basic group, said hydrophillic component X comprising a strongly basic, tertiary amino group, and said groups being in conjugation via said organic component Z, (2) said hydrophobic component Y comprising a thiol group and said hydrophillic component X comprising a carboxylic group, and (3) said phase transfer catalyst being mercaptopropyletrimethoxysilane.",
"133.The method of claim 125, further comprising the step of removing the solvent from said aqueous or alcoholic phase so as to obtain one of a powder and a slurry comprising a powder.",
"134.The method of claim 126, further comprising the step of removing the solvent from said aqueous or alcoholic phase so as to obtain one of a powder and a slurry comprising a powder.",
"135.The method of claim 127, further comprising the step of removing the solvent from said aqueous or alcoholic phase so as to obtain one of a powder and a slurry comprising a powder.",
"136.The method according to claim 133, further including washing the powder or slurry with a solvent for removing said phase transfer catalyst.",
"137.The method according to claim 134, further including washing the powder or slurry with a solvent for removing said phase transfer catalyst.",
"138.The method according to claim 135, further including washing the powder or slurry with a solvent for removing said phase transfer catalyst.",
"139.The paint according to claim 128, wherein the aqueous or alcoholic phase has a solvent removed so that one of a powder and a slurry comprising a powder is obtainable.",
"140.A printing ink according to claim 129, wherein the aqueous or alcoholic phase has a solvent removed so that one of a powder and a slurry comprising a powder is obtainable.",
"141.A varnish according to claim 130, wherein the aqueous or alcoholic phase has a solvent removed so that one of a powder and a slurry comprising a powder is obtainable.",
"142.The method according to claim 87, wherein said colloid particles are comprised of gold colloids.",
"143.The method of claim 87, wherein said phase transfer catalyst is DMAP.",
"144.Aqueous phase obtainable by the method according to claim 142.145.Aqueous phase obtained by the method according to claim 143."
],
[
"PRIOR ART The present invention relates to phase transfers of colloids, in particular of nanoparticles, and both homogeneous and heterogeneous catalysis using nanoparticles.",
"The widespread use of colloids and in particular very small colloids, namely nanoparticles, in the fields of biotechnology, nanotechnology, colloid and surface science, in catalysis, electronics, solid state physics and materials science at present occupies a central position in current research and development.",
"In the following, the term “colloid” is used synonymously with the term “nanoparticle”, since the latter are merely the particular case of very small colloids.",
"The present invention can be applied at least to colloids in the size range from 1000 nanometers to 0.1 nanometers.",
"However, the respective, specific uses of the colloids often require specific sizes, for example relatively small sizes, if the particles are to be sprayed as colloid through narrow nozzles.",
"Synthetic organic preparative methods can be used in a known manner for producing nanoparticulate materials of desired morphology, size and shape in relatively high concentrations which appear suitable for many applications and for transport of the nanoparticles.",
"However, a large proportion of the applications of these particles requires them to be present in aqueous medium, i.e.",
"in aqueous solution or in solutions which are miscible with water, e.g.",
"alcohols.",
"A direct synthesis in water leads, however, only to low concentrations of the nanoparticles, since they precipitate at relatively high concentrations.",
"In particular, such a direct synthesis suffers from problems due, firstly, to the occurrence of ionic interactions.",
"These problems are usually overcome by means of low reactant concentrations, e.g.",
"5×10−4 M, cf.",
"J. Turkevich, P. C. Stevensen, J. Hillier, Diskuss.",
"Faraday Society.",
"1951, SS.",
"Secondly, it can be difficult for the stabilizers required for the synthesis to be removed later.",
"In contrast, such particles can be prepared in organic solvents at relatively high concentrations up to 1M in respect of the starting material with predictable size and shape.",
"This is disclosed, for example, in: M. Green, P. Obrian, Chem.",
"Commun.",
"1999, 2235, or in: M. P. Pileni, New J. Chem.",
"1998, 22, 693.These particles also display an improved monodispersity compared to those which have been synthesized in aqueous solutions.",
"However, they are not miscible with water, which restricts their uses.",
"Dissolved nanoparticles are, however, necessary for many applications, since in coagulated form they lose not only their ability to be readily applied to substrates but also many of their advantageous chemical and physical properties.",
"A high concentration of the nanoparticles in the solution is specifically preferred for many reasons, depending on the field of application.",
"A general advantage of a high concentration is that the solution having a high concentration of nanoparticles has only a low weight for transport compared to a solution having a low concentration.",
"The abovementioned relationship suggests the desirability of synthesizing colloids or nanoparticles in an organic solvent and subsequently transferring them into an aqueous or similarly useable solution.",
"In this case, a phase transfer takes place between the organic starting solution into an essentially inorganic target solution, in particular an aqueous target solution.",
"Such a phase transfer process has been disclosed in both directions in Liu, H., Toshima, N.: “Transferring Colloidal Metal Particles from an Organic To an Aqueous Medium and vice versa by Ligand Coordination”, Journal of the Chemical Society, Chemical Communications, Number 16, 1992, pp.",
"1095 to 1096.This publication teaches coating the nanoparticles covalently with sodium diphenylphosphinobenzenesulfonate (DPPS), a water-soluble phosphine ligand.",
"Such covalent bonding is, for the purposes of the present invention, designated as “irreversible” since it can be broken again only with difficulty.",
"However, this covalent bonding permanently alters the chemistry of the particle surface as a result of the strong bond between ligand and nanoparticle.",
"Furthermore, the possible applications for such DPPS-coated nanoparticles are limited, since the DPPS molecules are undesirable as an outer shell for many applications.",
"However, the DPPS ligands can be removed again from the nanoparticle surface only with great difficulty.",
"In addition, the maximum concentration of nanoparticles in the target solution which is obtainable in good yield in the single-step phase transfer is relatively low, since a good transfer yield can only be achieved at a fixed DPPS concentration in the aqueous solution which is optimal for the “Liu process”.",
"These are disadvantages which cannot be accepted, or are difficult to accept, for many applications for the nanoparticles.",
"It would therefore be particularly desirable to be able to obtain concentrations of the nanoparticles in aqueous solution which are of similar magnitude to the concentrations in the synthesis of the nanoparticles in organic solution, if possible without being restricted in terms of the future use of the nanoparticles.",
"ADVANTAGES OF THE INVENTION A first main aspect disclosed by the present invention is the use of a substance for transferring inorganic colloids, in particular nanoparticles, from an organic starting solution into a target solution, where the target solution is either an inorganic, in particular aqueous, solution or an inorganic solution comprising water-soluble compounds, in particular alcohols, in a concentration of from 0% to 100%, where the substance comprises: a.)",
"at least one constituent Y (12) which can bind chemically to the surface of the colloids, b.)",
"at least one constituent X (16) which chemically changes the surface properties of the colloids so that phase transfer of the colloids from the organic solution into the inorganic solution is effected, and c.) at least one spacer constituent Z (14) which is able to link at least the constituent Y (12) and the constituent X (16) and has a molecular size which is sufficiently large for the constituents Y (12) and X (16) to be able to display their chemical actions based on their respective chemical affinities.",
"This use is characterized in that the substance used for the phase transfer is selected so as to be simultaneously functionalized for the future use of the nanoparticles.",
"The target solution can also be a solution comprising water-soluble compounds, in particular alcohols, in a concentration of from 0% to 100%.",
"The basic idea of the present invention is thus to select the phase transfer reactant, i.e.",
"the “substance” in the abovementioned sense, both for the phase transfer and also with a view to the future use of the nanoparticles and to configure it according to the desired chemical functions of its constituents X, Y and Z.",
"There is thus a generic class of substances for the reactant, which class is hereinafter abbreviated as “MM” and comprises the abovementioned constituents.",
"The addition of the reactant substance results in the nanoparticles readily going over from the organic phase into the inorganic phase in a single-step process.",
"This produces an aqueous solution of nanoparticles whose concentration depends on the amount of water added.",
"The transfer can be accelerated by introduction of energy, for example in the form of stirring or shaking of the mixture.",
"When the amount of water is equal to that of the organic solvent, the concentrations in the water are, as desired, as high as those in the organic solvent before the phase transfer.",
"Advantageously, a simple separation of the inorganic phase from the organic phase can subsequently be carried out after a sufficient delay time, since the usually denser inorganic phase can easily be separated from the organic phase.",
"The subordinate claims provide advantageous embodiments of and improvements to the respective subject matter of the invention.",
"To enable the constituent Y of the reactant to be detached readily from the nanoparticles after phase transfer has occurred, it is proposed that a reversible bond between the nanoparticle surface and the constituent Y of the phase transfer substance coupled thereto be used.",
"For the present purposes, a “reversible” bond means essentially a type of bond which is mainly due to van der Waals forces.",
"At least a strong covalent or an ionic bond to the nanoparticle should thus be ruled out here so as not to endanger the ready removability of the shell molecules from the nanoparticle surface 10.In particular, it is proposed for this purpose that the commercially available 4-dimethylaminopyridine, hereinafter referred to as DMAP for short, e.g.",
"in aqueous solution, be added in a sufficient amount to the organic solution.",
"As a result of its ring shape, the pyridine simultaneously represents the spacer constituent Z and the coupling constituent Y.",
"The nitrogen atom in the ring binds to the nanoparticle surface.",
"This is in most cases a relatively weak type of bond which is nevertheless sufficient to stabilize the nanoparticles in aqueous solution.",
"The addition of DMAP simply in a minimum amount without having to pay attention to keeping below a maximum amount in relation to the amount of starting solution is an advantage compared to the abovementioned prior art of Liu et al.",
"The DMAP shell molecules around the nanoparticles can easily be washed off again after phase transfer has occurred, for example by means of toluene, should this be necessary for any reason.",
"Such a reason can be, for example, the creation of very large free nanoparticle surface areas which are to be available in active form for catalytic purposes, or when an electric current is to flow with very little resistance through a layer of metal nanoparticles.",
"Here, the shell molecules would otherwise increase the resistance, since the metal nanoparticles do not touch.",
"Should, on the other hand, a very stable bond be wanted between shell molecules and nanoparticle surface after phase transfer has occurred, as is desirable for many applications, the substance is, according to the invention, selected so that an effectively irreversible type of bond is obtained.",
"This can be achieved, for example, by means of a covalent bond between nanoparticle surface and the coupling constiuent Y of the MM.",
"A preferred substance for this purpose is mercaptoundecanoic acid (MUA).",
"As can be derived directly from the above, the preparative methods of the prior art for nanoparticles or colloids which are based on an organic synthesis can be supplemented quite generally by the phase transfer process in accordance with the first main aspect of the invention which follows the synthesis so as to achieve many advantages.",
"The abovementioned MM substances have the following generic chemical formula: X----Z-----Y This generic formula comprises an organic spacer Z, a hydrophilic constituent X bound thereto and a constituent Y which is likewise bound to Z and can bind to the surface of the colloid or nanoparticle.",
"Examples of such substances are 4-dimethylaminopyridine and the abovementioned concentrated 11-mercaptoundecanoic acid (MUA) solution in toluene.",
"The constituent X is selected so that it chemically changes the surface properties of the colloids or nanoparticles so that phase transfer in the abovementioned sense can occur.",
"It can advantageously be or contain a functional group.",
"In particular, the constituent X which points away from the nanoparticle surface can be deliberately chosen so as to have a reactivity which makes it particularly suitable for the further use of the nanoparticles.",
"For example, it can serve to couple to biologically derived molecules, e.g.",
"particular proteins, so as to be transported with the protein as carrier to cancer cells.",
"After attachment, cancer cells can then be combated by targeted effects by exploiting physical or chemical prpoperties of the nanoparticles in a targeted fashion.",
"An example which may be mentioned is increasing the temperature of the nanoparticles in a targeted manner by irradiation, so that the nanoparticles then transfer their energy via heat conduction to the cancer cells which can then be made harmless in this way.",
"The spacer constituent or constituents Z has to be able to link at least the constituent Y and the constituent X (16), and it has to have a molecular size which is sufficiently large for the constituents Y and X to be able to display their chemical actions based on their respective chemical affinities.",
"It is possible for a multiatom compound, for instance a cyclic compound, to be able to perform the functions of two of the three constituents, or even of all three constituents.",
"In this way, the most recent advances in the prior art, namely the ability to synthesize virtually all relevant materials as nanoparticles in an organic phase, can now be directly utilized in an economically advantageous fashion, since they can be kept in aqueous solution in very high concentration using MM as stabilizer.",
"The concentration can also be increased by, for example, distilling off part of the water, or adding the MM as a solution in only relatively little water.",
"The constituent X preferably has hydrophilic properties with an affinity to water which is sufficiently great to effect phase transfer.",
"In this way, inorganic colloids, in particular nanoparticles, which have diameters in the range from 0.1 nm to 1000 nm or larger in the case of colloids and have been obtained by reactions of inorganic salts or a mixture of inorganic salts in nonaqueous solvents can be transferred into aqueous or alcoholic solutions by a sufficient amount of substances designated as MM being absorbed onto them.",
"The use of a thiol as constituent Y and a carboxylic acid as constituent X in the substance used according to the invention is advantageous.",
"If the constituent Y is selected so that it forms a covalent bond to the colloid surface, for instance as in the case of the use of MUA with an aliphatic compound as MM, a stable, permanent bond which is desirable for many applications, for example in the production of paints, inks, etc., is obtained.",
"If the constituent Y forms a noncovalent bond to the nanoparticle surface, this has the advantage that the surface of the nanoparticles is modified only temporarily and not permanently.",
"This can be achieved, for example, by use of DMAP having a conjugated bond.",
"Washing enables the nanoparticle surface to be exposed again in unmodified form after phase transfer has occurred.",
"Thus, for example, adjacent nanoparticles can conduct electric current when metallic nanoparticles are used.",
"Furthermore, a washed surface of the nanoparticles can be exploited in an advantageous fashion, for example in catalysis to increase its efficiency.",
"The chemical action of this particular measure according to the invention is produced by application of MM to the surface of nanoparticles, e.g.",
"metal or noble metal nanoparticles, gold, silver, iridium, platinum, palladium, without modifying it by means of a covalent bond as is the case in the prior art, for example with formation of a shell of gold sulfide around a nanoparticle.",
"An example of a molecule used according to the invention comprises essentially a hydrophilic part which readily couples to water, a further part which couples to the nanoparticle and a spacer located between them.",
"The abovementioned DMAP is an example of such a molecule.",
"In its broadest form, the phase transfer process of the invention for colloids and in particular nanoparticles is as follows: A process for transferring colloids, in particular nanoparticles, from an organic starting solution into a target solution, where the target solution is either an inorganic, in particular aqueous, solution or a solution comprising water-soluble compounds, in particular alcohols, in a concentration of from 0% to 100% is disclosed and claimed.",
"It is characterized by the steps: a.)",
"addition of a sufficient amount of a preselected substance as claimed in any of the preceding claims to the organic starting solution, b.)",
"allowing the nanoparticles to go over from the organic phase to the inorganic phase, c.) separation of the inorganic phase from the organic phase.",
"In this way, high nanoparticle concentrations are obtained in the target solution, with the concentration being dependent on the amount of target solution which is originally present or is added to the starting solution.",
"If an MM substance used according to the invention, i.e., for example DMAP or MUA, is added as an aqueous solution or a solution comprising water-soluble compounds, in particular alcohols, in a concentration of from 0% to 100% at a predetermined concentration in the solution, the process can be matched to the respective production requirements.",
"In particular, aqueous solutions having very high concentrations of nanoparticles can be produced.",
"When the amount of the MM substance added is sufficiently large to form a monolayer around a nanoparticle in the solution, a high stability of the nanoparticles is obtained, particularly in the case of MUA as MM substance.",
"The ratio of the number of surface atoms of a nanoparticle to the number of the MM molecules bound thereto is preferably in a range from 0.1 to 10, more preferably about 1.It is possible to transfer metallic colloids, in particular metallic nanoparticles, and colloids or nanoparticles of alloys in this way.",
"The transfer of nanoparticles of gold, silver, iridium, platinum, palladium, nickel, iron, rhodium, ruthenium or metal oxides, in particular iron oxide, zinc oxide, titanium dioxide, tin oxide, in each case results in desirable effects, for example colorants having long-term stability or coatings having other desired physical or chemical properties, e.g.",
"electrical, magnetic or other properties.",
"Semiconductor nanoparticles and inorganic nanoparticles containing rare earth elements can also be transferred.",
"The additional step: Separation of the colloids, in particular nanoparticles, from the solvent of the target solution to give a powder or slurry comprising particles, in particular nanoparticles, makes it possible to obtain a further state of the nanoparticles which may be a preferred alternative to the liquid form of the target solution for various types of further processing.",
"MM is advantageously added in an amount which is sufficiently large to form a monolayer around a nanoparticle in the solution.",
"This layer thus contains the number of MM molecules required to cover the surface of the nanoparticle.",
"However, a larger amount of MM is not harmful for the purposes of the invention.",
"If gold nanoparticles are used, the ratio of the number of surface atoms of a nanoparticle to the number of MM molecules bound thereto is preferably in a range from 0.1 to 10, more preferably about 1.High concentrations of noble metal nanoparticles: according to the invention, noble metal colloids can be used as solutions in water and in a form which can be specifically matched to the future use of the nanoparticles.",
"This reduces transport costs, since the achievable concentrations can be increased by a factor of from 106 to 109 compared to present-day concentrations in water.",
"As a result, the transport weight of a solution containing the nanoparticles is reduced by the same factor while maintaining the chemical activity.",
"However, the term “high” concentration is also used to refer to concentrations which are less than 106 times the concentrations in water which are obtainable at present, as are, for example, marketed commercially at the present time.",
"In this way, water-based, colorfast colorants having long-term stability can be produced.",
"Lipsticks, motor vehicle paints and even printing inks can profit considerably from these properties.",
"This is because such inks can be printed when produced according to the invention because the colorants produced according to the invention no longer block very fine nozzles of a printing machine, e.g.",
"an inkjet printer, as a result of the small size of the color-imparting nanoparticles.",
"A paint or surface coating according to the invention is much finer in terms of its surface and interior structure compared to paints having larger micron-sized pigment particles of the prior art.",
"As a result, a paint is suitable for the first time for many fields of application, since the paint layer no longer flakes off as easily because of its homogeneous structure.",
"At least the following uses of the target solution or powder or slurry are encompassed by the present invention: For the selective coating of surfaces of macroscopic bodies, or as paint/ink, in particular as printing ink, or surface coating, or for attachment to biologically derived molecules, in particular as biomarker; for the production of structures which are applied to a support body and have predetermined magnetic properties; for the production of structures which are applied to a support body and have predetermined electrical properties; in sol/gel processing; or the use of a target solution to coat support particles with at least one layer of colloids, in particular nanoparticles, with there being sufficient affinity between support particles and colloid or nanoparticles for this purpose.",
"This process step can be one of mixing a support solution containing the support particles with the target solution.",
"Alternatively, the support particles can be mixed in another form into the target solution.",
"The additional step can be carried out to remove residues of the MM substance by washing with a suitable (organic) solvent.",
"For many uses of the aqueous solutions of the invention, for example for surface coatings in the motor vehicle sector, an aqueous solution can be firstly applied as such or in admixture with another component to small support particles (beads), for example by spraying by the inkjet process according to the prior art, to be added in a later step as component for a surface coating to one or more other components, and can be applied in a customary manner together with this and homogeneously distributed therein to the article to be coated.",
"Spraying onto beads is known per se from the prior art, for example in pearl-effect or metallic surface coatings.",
"The abovementioned support particles (beads) are, owing to the ready handleability, an independent subject matter of the claims when provided with nanoparticles from aqueous, high-concentration solution.",
"Their size is in principle dependent on the prior art paint application process chosen in the particular case.",
"Such support particles can then be used industrially in accordance with the desired function of the nanoparticle properties.",
"Nanoparticles having a functionalized shell can be particularly advantageously applied to particularly small support bodies having a size of 0.02 micron or larger.",
"Depending on the desired further use of the system comprising support bodies and nanoparticles, it is then wished to leave the shell of nanoparticles thereon (in which case MUA, for example, should be used) or to be able to remove them relatively easily (in which case DMAP is appropriate), for example by washing with toluene.",
"Particularly useful nanoparticles of this type are noble metal, in particular gold (Au), nanoparticles having a DMAP shell when they are applied, for example, to polyelectrolyte-coated spheres of polystyrene, polymethyl methacrylate (PMMA) or silicon oxide, etc., and are then treated with a solution comprising hydroxylamine hydrochloride and hydrogen tetrachloroaurate (electroless plating) to remove the reversibly bound MM substances, e.g.",
"DMAP or residues thereof, to form a coherent metal shell or gold shell on the cores of the spheres.",
"Such spheres/surfaces can then be advantageously employed in a wide range of industrial or medical applications, for instance in the field of photonics, cancer therapy, pharmacy and catalysis, as mentioned above.",
"According to the present invention, spheres having a high loading of nanoparticles can be produced in a single absorption step, giving a high level of homogenous shells.",
"Here too, considerable advantages are achieved by means of the invention, in each case specifically according to the application.",
"A further independent subject matter of the invention comprises colorant liquids into which the nanoparticles have been introduced from aqueous solution according to the invention.",
"Furthermore, the invention advantageously provides a primary color set of aqueous solutions for producing mixed colors, using gold nanoparticles for producing the primary color red, silver nanoparticles for producing the primary color yellow and iridium nanoparticles for producing the primary color blue.",
"Using appropriate amounts of the primary colors, it is therefore possible to produce all mixed colors in a customary fashion, e.g.",
"the mixed color green by mixing aqueous solutions or beads containing iridium nanoparticles (blue) and silver nanoparticles (yellow).",
"When metallic nanoparticles are transferred in the process of the invention, this results, owing to the wide range of process end products, in highly concentrated nanoparticle solutions containing the respective desired metals.",
"When, for example, an iron solution and a platinum solution are prepared, a contribution can be made to producing sprayable liquids for producing magnetizable or permanent magnetic or electrically active structures, for instance conductor tracks, sprayed or otherwise applied onto a substrate, by means of which, for example, very finely structured data storage media can be “printed”.",
"These can then be read or written on in a known manner by means of an induction head of appropriately small size.",
"According to a second main aspect of the present invention, the inventive principle can be applied directly to carrying out a catalytic reaction, either a homogeneous or heterogeneous catalysis which can be applied in the customary fields, e.g.",
"polymer production, and is thus of immense importance for many chemical processes carried out industrially and processes occurring in everyday life: According to this aspect, noble metal nanoparticles, for example, which may be added, for example, as an organic solution to an organic reaction mixture act as catalyst.",
"They disperse uniformly (homogeneously) in the reaction liquid and are not bound to support molecules such as Ceolite, as a result of which they display a catalytic efficiency which is many times higher than that in heterogeneous catalysis with appropriate binding to a support such as Ceolite, carbon, etc.",
"It is then possible to carry out the fundamental reaction, where the catalytic action is advantageously brought to bear because now many times the surface area, e.g.",
"200 m2 per gram for 6 nanometer nanoparticles, is catalytically active compared to heterogeneous catalysts in which a major part of the catalyst has to remain chemically inactive because it remains hidden in the interior of the support molecule.",
"After the reaction is complete, the catalyst should advantageously be able to be removed again from the reaction mixture.",
"This is achieved by adding water and a substance according to the invention.",
"The nanoparticles then go back, as described above, from the organic phase into the water and thus can advantageously be recovered completely.",
"At the same time, the reaction mixture is advantageously freed of the catalyst.",
"The application of nanoparticles to a substrate suitable for a heterogeneously catalyzed process, e.g.",
"Ceolite or carbon, using an aqueous solution having a high concentration of nanoparticles, e.g.",
"gold or platinum nanoparticles, which may have been obtained according to the invention can be improved according to the invention by the nanoparticles being able to be applied to most hydrophilic support materials in a simpler manner, for example without evaporation of the nanoparticle solution, when the aqueous solution of the invention is added.",
"This makes catalysts used industrially simpler to produce and cheaper.",
"The phase transfer process of the invention is applicable to all nanoparticles which are disclosed in the associated patent application PCT/DE-00/03130.In this way, the present process can be combined in an advantageous manner with the disclosure of the patent application mentioned, resulting in the advantages known to a person skilled in the art.",
"DRAWINGS Examples illustrating the invention are depicted in the drawings and are described in more detail in the following description.",
"In the figures: FIG.",
"1 schematically shows a nanoparticle with coupled-on MM molecules having, by way of example, the DMAP structure; FIG.",
"2 shows a photograph of the gold nanoparticles in a 2-phase mixture before (at right) and after (at left) transfer within 2 mL Eppendorf tubes; FIG.",
"3 shows a transmission electron micrograph of the gold nanoparticles from FIG.",
"1 after they have been transferred into water; FIG.",
"4 shows photographs of 5 different nanoparticle samples in pairs (A,B), (B,C), .",
".",
".",
"in each case before and after phase transfer using MUA as MM, FIG.",
"5 shows UV/VIS spectra of gold nanoparticles in toluene (continuous line) and after transfer (broken line), FIG.",
"6 shows transmission electron micrographs of gold nanoparticles after their preparation in toluene (A) and 1 month after transfer, FIG.",
"7 shows transmission electron micrographs of palladium nanoparticles in toluene (A) and after transfer into water (B), effected by means of DMAP as M, FIG.",
"8 shows EDAX spectra of palladium nanoparticles as synthesized (top) and after phase transfer into water (bottom), FIG.",
"9 shows a sketch of a mechanism (scheme 1) for the phase transfer of gold and palladium nanoparticles from toluene into water by addition of DMAP, R=C8H17, FIG.",
"10 shows photographs which illustrate the phase transfer as a function of time; gold nanoparticles from toluene into 0.1M DMAP solution.",
"DESCRIPTION OF THE EXAMPLES FIG.",
"1 schematically shows in the center the surface 10 of a gold nanoparticle.",
"The surface is drawn as a line having a smooth contour which is shown by way of example and purely schematically as an octagonal shape.",
"The surface is shown in idealized form as a smooth surface, but is made up of individual gold atoms.",
"Eight DMAP molecules are shown schematically coupled onto the surface 10; of these, only one (the uppermost) is shown in greater detail in the interest of simplicity.",
"Depending on the size of the phase-transferred nanoparticles, a considerably greater number of shell molecules are naturally arranged in the plane of the drawing.",
"The description below applies likewise to the other DMAP shell molecules which are bound to the nanoparticles (including those located outside the plane of the drawing): The endocyclic nitrogen (N) atom 12 of the pyridine ring binds, as constituent Y of the DMAP molecule used according to the invention, to the nanoparticle surface 10.The pyridine ring 14 itself acts as spacer Z to the hydrophilic dimethylamine 16 depicted at the top, which forms the constituent X of the MM molecule.",
"The chemical action is brought about by the application of the DMAP molecules to the surface of nanoparticles, so that at least a large part of the surface of the nanoparticle is occupied by such molecules in the form of a monolayer with gaps which are not too large.",
"If no gaps are present or a surplus of DMAP molecules is present, this does not impair the desired action.",
"In this example, a readily removable layer is therefore formed around each individual nanoparticle without the surface of the particle being modified, as is the case in the prior art, for instance in the formation of a shell of gold sulfide around a nanoparticle.",
"FIG.",
"2 shows a photograph of the gold nanoparticles in a 2-phase mixture before (at right) and after (at left) transfer in 2 mL Eppendorf tubes.",
"The upper phase is toluene, and the lower phase is water.",
"It can clearly be seen from the Figure that no agglomeration of nanoparticles has taken place, neither in the organic phase nor in the inorganic phase.",
"FIG.",
"3 shows a transmission electron micrograph of the gold nanoparticles of FIG.",
"2 after they have been transferred into water.",
"Here too, it can clearly be seen that neither agglomeration nor a change in the size, shape or morphology of the nanoparticles has taken place during phase transfer.",
"Even 6 months after phase transfer, the transferred particles display no signs of decomposition or aggregation.",
"It can therefore be assumed that they are stable for an indefinite time.",
"Advantageously, this process requires neither precipitate formation nor solvent replacement, and the particles transferred by means of the process are not stabilized by covalently bound ligands.",
"With a view to subsequent uses, this is an important difference from thiol-stabilized particles.",
"The availability of such concentrated, aqueous solutions of nanoparticles opens up new possibilities for cyto-labeling, heterogeneous and homogeneous catalysis, solid state physics and for applications in the field of colloidal crystals.",
"In addition, it is in principle possible to carry out coating of surfaces with the purpose of changing the surface so that it takes on certain, desired properties of the nanoparticles or properties of elements which are present in the nanoparticles.",
"Further applications exist in the field of homogeneous or heterogeneous catalysis, as are described below.",
"Since the nanoparticles are readily available industrially due to their presence as fine dispersions in aqueous solution or in, for example, alcohol-enriched solution, they can also be applied to, for example, small support particles, viz.",
"beads.",
"Suitable support particles have a size in the range from about twice the size of the nanoparticles used (in the case of a homogeneous size distribution), i.e.",
"from about one nanometer, through to the macroscopic range, i.e.",
"up to several millimeters in size.",
"Here there are possible industrial applications in the field of colloidal crystal applications and photonics, in the field of optical waveguides for telecommunications for producing desired, optical lattice properties.",
"The steps for preparing the phase-transferred nanoparticles are indicated below: All chemical starting materials necessary for the examples described below were used as they are commercially available from SIGMA-Aldrich.",
"The water used was passed through a MILLIPORE purification system before use; after this treatment it has a resistance of greater than 18 M Ω cm.",
"UV/VIS samples are placed in fused silica cells (Hellmar, SUPRASIL, path length −1.000 cm) and measured using a double-beam spectrophotometer (CARY 4E, Varian).",
"A solvent spectrum is subtracted from all spectra.",
"Samples which require sedimentation are centrifuged in 2 ml Eppendorf tubes which can be stood upright (3K30, SIGMA Laboratory Centrifuges).",
"Samples for TEM measurement (Philips CM70, acceleration voltage=120 kV) are applied to copper grids provided with a standard carbon layer and dried before examination.",
"The particle size distribution is determined by means of ultracentrifugation using absorption optics (Beckmann Optima XL-I) or by TEM analysis.",
"Samples for TEM analysis are prepared by placing a drop of this solution on a carbon-coated copper grid and drying it in air.",
"The particles are synthesized by methods published in: Hayat, M.A.",
"(Academic PressInc., San Diego, USA, 1989), or in Goia, D.V.",
"& Matijevic, E. Tailoring the particle size of monodispersed colloidal gold.",
"Colloids Surf.",
"A 146, 139-152 (1999), or in Green, M. & O'Brien, P. Recent advances in the preparation of semiconductors as isolated nanometric particles: new routes to quantum dots.",
"Chem.",
"Commun., 2235-2241 (1999).",
"In case of doubt, the disclosures of these sources can be consulted.",
"1) One milliliter of an aqueous 4-dimethylaminopyridine solution (DMAP) are then, according to the invention, added to a one milliliter aliquot of nanoparticle mixtures, for example comprising gold, silver, iridium, platinum, palladium, rhodium or ruthenium, synthesized in toluene and stabilized by the tetraalkylammonium salt method.",
"Larger volumes of nanoparticles in their reaction solution can likewise be transferred successfully into one milliliter of water, which makes subsequent recycling of the ammonium salt possible.",
"Direct phase transfer through the organic/aqueous boundary is complete within a period of from one hour to three hours without any further action being necessary.",
"More rapid phase transfer can be achieved by, for example, the use of centrifugation, shaking, stirring, i.e.",
"introduction of energy.",
"This gives high concentrations of the nanoparticles which can be diluted by a factor of about 1000 for subsequent use (analysis, photography).",
"2) 100 microliters of a concentrated 11-mercaptoundecanoic acid (MUA)/toluene solution are added to a 1 milliliter aliquot of the nanoparticle mixture as has been described above, i.e., for example, comprising gold, silver, platinum, iridium, etc.",
"and synthesized in toluene and stabilized by the tetraalkylammonium salt method or synthesized by the Wilcoxon-AOT method as disclosed in U.S. Pat.",
"No.",
"5,147,841, with no prior purification being necessary.",
"The adsorption of the phase transfer catalyst can be observed with the naked eye as a red shift in the color of the solution, followed by development of turbidity produced by particle agglomeration and precipitation.",
"Coated particles can either be carefully centrifuged from the organic solution or can be left to sediment overnight.",
"Washing the precipitate with 2 aliquots of the starting solution, followed by one aliquot of methanol removes all by-products of the reaction and the excess of phase transfer catalyst.",
"This is followed by washing with methanol.",
"Careful shaking of the precipitate in basic water results in a stable, clear solution of the nanoparticles.",
"Since the particles are added as solid to the water, solutions of any concentration can be produced.",
"A phase transfer carried out using MUA results in a precipitate which is stable for a long period when stored as, for example, powder or slurry.",
"This applies particularly when MUA molecules envelop the nanoparticles so completely that the particles do not come into contact with one another.",
"No agglomeration or clumping together is therefore possible.",
"3) 10 microliters of mercaptopropyltrimethoxysilane are added to a 1 milliliter aliquot of the gold nanoparticle mixture as can be prepared by the method described above, synthesized in toluene or synthesized by the Wilcoxon-AOT process, U.S. Pat.",
"No.",
"5,147,841, without prior purification.",
"The adsorption of the phase transfer catalyst can be observed with the naked eye as a red shift in the color of the solution followed by development of turbidity caused by particle agglomeration and precipitation.",
"Coated particles can either be centrifuged carefully from the organic solution or can be sedimented overnight.",
"Washing of the precipitate with two aliquots of the initial solution followed by one aliquot of methanol removes all by-products of the reaction and excess phase transfer catalyst.",
"The phase transfer catalyst is added to the organic nanoparticle solution at a metal concentration of from 1×10−6 to 100% by weight, in particular 1 mg per ml of metal in a concentration of from 1×10−6 to 100% by weight, in particular equal volumes of a 0.01M aqueous solution.",
"This results in complete phase transfer from organic solution to water.",
"As can be seen from the description above, the phase transfer of the nanoparticles from organic solution into aqueous solution as disclosed according to the invention can be achieved by means of the two examples above.",
"It is also possible to employ other substances if they have the required binding properties, cf.",
"above description for FIG.",
"1.The phase transfer process of the invention can be used advantageously for metallic nanoparticles such as gold, silver, iridium, platinum, palladium, nickel, iron, metal oxide, in particular iron oxide, zinc oxide, titanium dioxide and tin oxide nanoparticles and also for rhodium and ruthenium nanoparticles.",
"In this way, colorants which are present in aqueous solution and are stable for a prolonged time can be obtained in an advantageous fashion.",
"These can thus also be employed for applications in which use of such nanoparticles in organic solution is normally ruled out for environmental reasons, or on health grounds or for other reasons.",
"The process as described above can likewise be used for marking materials in electron microscopy.",
"Semiconductor nanoparticles can likewise be transferred.",
"The following applications and advantages are made possible: Biological applications, in particular bio-labeling, production of lighting effects by means of fluorescence, and other applications which are disclosed in the abovementioned patent application which has been submitted simultaneously, all applications related to environmental protection which profit from the solubility of the nanoparticles in water instead of organics, regardless of the type of nanoparticles.",
"Furthermore, any inorganic, in particular aqueous, solution as is obtained by the process of the invention can be used and economically exploited in many ways.",
"It can be used, in particular, as paint, paint component, printing ink or surface coating composition or as constituent of a surface coating.",
"Advantages can be gained from the particular fineness of the nanoparticles and their narrow size distribution in cases in which a printing ink containing nanoparticles has to be passed through the fine nozzles of an inkjet printer.",
"According to the invention, such nozzles then do not become blocked.",
"When a paint/ink containing such nanoparticles or a similar surface coating is applied to a substrate other than by passage through a nozzle, then the narrow size distribution and small size of the nanoparticles results in a paint layer or surface coating which is less brittle than when color-imparting particles having particle sizes in the micron range are used.",
"Complete phase transfer of the metallic nanoparticles is achieved, cf.",
"FIG.",
"2.This is also shown in further examples in which complete phase transfer according to the invention occurs.",
"Such results are shown in FIG.",
"4 ff.",
"Here, the following experimental set-up was employed: Synthesis of the nanoparticles: 30 milliliters of an aqueous solution of a metal chloride (HAuCl4 or Na2PdCl4, 30 milliliters) were added to a 25 mM solution of tetraoctylammonium bromide in toluene (80 ml).",
"The transfer of the metal salt into the toluene phase could be clearly recognized visually within a few seconds.",
"A 0.4 M solution of freshly prepared NaBH4 (25 ml) was added to the stirred mixture, resulting in immediate commencement of the reduction.",
"After 30 minutes, the two phases were separated and the toluene phase was washed with 0.1 M H2SO4, 0.1 M NaOH and three times with H2O and finally dried over anhydrous NaSO4.Earlier high-resolution TEM analyses showed that nanoparticles prepared in a similar manner are crystalline and have a capped octahedral morphology.",
"Phase transfer: 1 ml of an aqueous 0.1 M solution of DMAP was added to aliquots (1 ml) of the nanoparticle mixture.",
"This DMAP concentration was found to be sufficient to effect complete and spontaneous phase transfer of the nanoparticles.",
"It should be noted that it was also possible to transfer nanoparticles from larger volumes of the reaction solution (up to 0.5 l) into water (1 ml) and to recover the tetraalkylammonium salt.",
"The direct phase transfer through the organic/aqueous interface had proceeded to completion within one hour, without stirring or shaking being necessary.",
"Furthermore, it is also possible to add the DMAP directly to the toluene solution so as to precipitate the particles; these can then be resuspended in water.",
"Phase transfer was also successful using similar particles synthesized in chloroform but not when using particles whose synthesis had been carried out using other organic stabilizers, e.g.",
"sodium 5,14-diethyl-8,11-dioxo-7,12-dioxaoctadecane-2-sulfonate (Na AOT) or dodecyldimethylammonium bromide.",
"All reagents were purchased from Sigma-Aldrich and used without further pretreatment.",
"The UV/Vis spectra were recorded on a UV/Vis spectrophotometer from Cary (Model 4E), resolution: 0.2 nanometers.",
"The zeta potentials of the nanoparticles were determined using a Zetasizer 4 from Malvern, with the mean of five steady-state measurements being taken.",
"The mobilities were converted into electrophoretic potentials by means of the Smoluchowski relationship.",
"The TEM examinations were carried out using a CM12 microscope from Philips at 120 kV.",
"The particle size distribution was calculated from the sedimentation velocities determined by analytical ultracentrifugation at 20° C. The measurements were carried out using an Optima XL-1 ultracentrifuge from Beckman-Coulter which was equipped with absorption optics for detection.",
"“Home-made” double sector middle pieces of titanium having a diameter of 12 millimeters were used.",
"In the analytical ultracentrifugation, a dilute sample of the nanoparticles is subjected to a constant centrifugal force.",
"At the beginning of an experiment, a scan at a fixed wavelength over the radius of the cell gives a constant absorption value, which indicates a constant distribution of the colloids over the volume of the cell.",
"During the experiment, the time-dependent sedimentation of the particles can be followed by sequential radial scans of the local colloid concentration.",
"The fractionation of the particles during the experiment allows the distribution of the sedimentation coefficients to be calculated from a series of radial scans carried out at various times.",
"In this way, both the density of the solvent and the viscosity of the solution and also the size distribution and density of particles can be determined, even when their size is in the Angström range.",
"FIG.",
"4 shows five diluted nanoparticle solutions in pairs from left to right, in each case before and after phase transfer.",
"The particles can be seen by the dark color in the sample container.",
"All primary colors can be produced, but of course cannot be seen in such a black and white depiction.",
"Phase transfers were in each case carried out from reaction mixtures (toluene) using MUA into water as mentioned above.",
"Examples A, B showed the transfer of the silver nanoparticles, C, D showed the transfer of gold nanoparticles, E, F showed the transfer of platinum nanoparticles, G, H showed the transfer of further gold nanoparticles which have been produced in a different way, and I, J showed the transfer of palladium nanoparticles.",
"UV/Vis spectra of the dissolved gold nanoparticles were recorded before and after phase transfer, since particle aggregation, reversible or irreversible, flocculation or coagulation and also changes in the dielectric constant of the environment of the nanoparticles can be observed in a known manner in the optical spectra.",
"In toluene, the maximum of the surface plasmon band was at a wavelength of 518 nanometers, as shown in FIG.",
"5.After phase transfer, the band had undergone a blue shift of 6 nanometers to 512 nanometers.",
"This shift could be based on the combined action of the change in the index of refraction of the medium from 1.47 to 1.33 and the replacement of the adsorbed molecules during transfer.",
"Any form of particle aggregation would lead to a red shift and a broadening of the plasmon absorption band.",
"The UV/Vis spectra obtained thus clearly showed that the DMAP-induced phase transfer gives gold nanoparticles which are well dispersed in an aqueous solution.",
"No UV/Vis experiments were carried out on the palladium nanoparticles, since they do not have a strong surface plasmon absorption band.",
"FIG.",
"6 shows results of transmission electron microscopy (TEM).",
"TEM indicated no visible differences in the morphologies of the gold and palladium nanoparticles after phase transfer.",
"This can be seen from the micrographs, where A in the upper region shows toluene-synthesized gold nanoparticles and the micrograph B shows the sample one month after transfer into water effected by addition of DMAP.",
"Analysis of the micrographs of the gold nanoparticle samples in both the toluene solution and the aqueous solution indicated a mean nanoparticle diameter of 5.5 nanometers with a standard deviation (SD) of 0.7.These figures are based on 153 particles, and an SD of 0.8 was obtained from a count of 115 particles.",
"The palladium nanoparticles gave a mean diameter of 4.5 nanometers and an SD of 0.9 for 145 counted particles, and a diameter of 4.8 nanometers and an SD of 1.2 for 122 particles.",
"This is shown in FIG.",
"7, where palladium nanoparticles synthesized in toluene are shown in the upper region A and the region B shows the same sample after transfer into water effected by addition of DMAP.",
"Energy-dispersive X-ray fluorescence analysis (energy dispersive analysis of X-rays, EDAX) indicates that no bromide ions are present on the particles which have gone over into the aqueous phase and have been dried on a TEM grid.",
"It should also be stated that the bromide ions are the counterions of the tetraalkylammonium ions.",
"However, traces of the organic salt can still be adsorbed on the particle surface.",
"The EDAX spectra of palladium nanoparticles are shown in FIG.",
"8: “as synthesized” at the top and after phase transfer into water at the bottom.",
"Since the information on the morphology of the nanoparticles which is obtained by TEM relates to the dry state, measurements were also carried out by means of analytical ultracentrifugation (AU) in order to obtain the size distributions of the nanoparticles in solution.",
"AU analysis of the gold nanoparticle samples indicates mean diameters of 5.1 nm (SD=1.1) in toluene and 5.2 nm (SD=1.1) in water.",
"AU analysis of the palladium nanoparticles indicated diameters of 2.8 nm (SD=1.5) and 3.1 nm (SD=1.6), respectively.",
"These results agree well with the values determined by TEM and once again confirm that no significant aggregation of the nanoparticles has taken place as a result of the transfer.",
"The stability of the DMAP-stabilized particles was examined as a function of salt concentration and pH.",
"All samples (Au.",
"Pd.",
"pH 10.5) were stable for a period of at least 6 months in 3M NaCl solution (for gold=514 nm).",
"Determination of the zeta potential of the DMAP-stabilized nanoparticles in aqueous solution (pH 10.5) by means of microelectrophoresis gave an average value of +25 mV (from five measurements on samples from three different transfer experiments).",
"This indicates a positively charged particle surface.",
"The particles are stable as colloids (with a zeta potential of about 35 mV) in the pH range from 7 to 12, even though the proportion of flocculated particles increased (as could be observed visually; see background information) when the pH was decreased from 10.5 to 3.0 by stepwise addition of dilute acid (1 mM HCl, pH 3).",
"This observation agrees with the postulated mechanism of phase transfer (see Scheme 1), since the decrease in the pH should lead to a greater proportion of the endocyclic nitrogen atoms being protonated and thus no longer being able to bind to the surface of the nanoparticles in order to stabilize them.",
"As a consequence, regions of the nanoparticle surface were “deprotected”, which would lead to reversible aggregation.",
"A decrease in the degree of particle flocculation (which can be recognized by the blue shift in the peak of the plasmon absorption band) was achieved by addition of a dilute base (1 mM NaOH), which again increased the pH to its original value (pH 10.5).",
"The separation of the aggregated particles did not occur immediately, but could be detected only after a few days; however, the phenomenon was always repeatable.",
"As would be expected on the basis of simple acid-base equilibria, increasing the pH of the solution to values above pH 13 by addition of a dilute base led to aggregation of the particles since the proportion of charged DMAP molecules decreases (pH=9.6).",
"To obtain a better understanding of the mechanism of the spontaneous transfer of the metallic nanoparticles from the organic phase into the aqueous phase, the effectiveness of various compounds was tested.",
"These were in each case added as 0.1M aqueous solutions to aliquots of the solution of the gold nanoparticles.",
"Pyridine and 4-aminopyridine led to immediate aggregation of the particles suspended in toluene, which could be recognized by a color change from red to blue and subsequent precipitation formation.",
"Only in the container in which DMAP was present did transfer of the nanoparticles into the aqueous phase take place; in the case of the other samples, the precipitate formed collected at the interface between toluene and water.",
"These results indicate that a tertiary (strongly basic) amino group conjugated to an electron-donating (weakly basic) group is necessary to bring about phase transfer.",
"To demonstrate that strong covalent bonds have not formed between the stabilizing molecules (DMAP) and the surfaces of the metallic nanoparticles, the aqueous phase was washed a number of times with toluene.",
"The DMAP content of the aqueous phase decreased continuously with increasing number of extractions, until the particles finally formed aggregates.",
"This effect indicates that DMAP is detached from the particle surfaces.",
"Covalently bound molecules cannot be removed by simple washing with a solvent.",
"A possible mechanism of the spontaneous phase transfer of the nanoparticles in the presence of DMAP molecules is shown in Scheme 1 in FIG.",
"9: the addition of an aqueous DMAP solution to the dispersion of the nanoparticles in toluene leads to partition of the DMAP between the aqueous and toluene phases of the mixture (this was confirmed by thin layer chromatography of the organic phase) and to physisorption of the DMAP on the surface of the nanoparticles.",
"Simple calculations of acid-base equilibria show that 98% of the DMAP molecules are present as free base in a 0.1 M aqueous solution.",
"We assume that the DMAP molecules form labile donor-acceptor complexes with the atoms of the metal surface via the endocyclic nitrogen atoms, as has been described above for planar gold substrates; a charge on the surface is then necessary for transfer into the aqueous phase, and this can be achieved by partially protonating the exocyclic nitrogen atoms which point away from the surface of the nanoparticles.",
"FIG.",
"10 depicts photographs which show the progress of phase transfer over time: gold nanoparticles from toluene into 0.1M DMAP solution; at top left, immediately after initiation of phase transfer, at right beside it one minute later, with the commencement of migration of the nanoparticles being able to be seen clearly.",
"The photograph at bottom left is after 10 minutes, with migration having progressed further, and that at bottom right is after one hour, with phase transfer being virtually complete.",
"The method described here is a general method of transferring gold and palladium nanoparticles with high efficiency from an organic solvent (in this case toluene) into water.",
"This method offers at least three advantages: The first is that it replaces the hydrosol synthesis methods which require high dilutions and time-consuming dialysis purification processes.",
"Secondly, syntheses in organic solvents give high concentrations of nanoparticles having a monodispersity which is significantly better than that of particles formed in water; the method described here makes it possible for researchers whose experiments are based on aqueous solutions to obtain such particles.",
"Thirdly, since the transfer of the nanoparticles from the organic phase occurs without precipitate formation, it is possible to recover the expensive ammonium salts.",
"Furthermore, the water-dispersible metallic nanoparticles can be isolated as solids, which is important when highly concentrated solutions of the particles are required, e.g.",
"in applications in the field of colloidal crystals.",
"The expected strong affinity of the DMAP-stabilized particles to negatively charged substrates as are customarily used in heterogeneous catalysis is likewise deserving of further examination.",
"According to a further, very wide-ranging aspect of the present invention, the inorganic, in particular aqueous solution as has been obtained by the above-described phase transfer process can advantageously also be used quite generally to recover nanoparticles used as homogeneous catalyst in a liquid after the catalyzed reaction.",
"For example, gold nanoparticles are introduced as catalyst into a reaction mixture, after which the chemical reaction which is to be catalyzed by the gold nanoparticles is carried out.",
"The catalyst, i.e.",
"in this case the gold nanoparticles, can once again be removed according to the invention from the reaction mixture: This is achieved by addition of one of the substances having the abovementioned, generic formula, i.e., for example, by means of 4-dimethylaminopyridine, preferably in solution in water.",
"As described above, this substance brings about the migration of the catalyst into the water.",
"The water which now contains the nanoparticles can then be taken from the reaction zone.",
"This gives the advantage that it is possible to achieve a catalytic efficiency of the catalyst which is many times as high as when the catalyst is, for instance, bound to Ceolite or carbon molecules, as is the case in the prior art.",
"This step change in efficiency is brought about by a large increase in the catalytically active surface area for a constant number of catalyst molecules, because the catalyst particles are dispersed homogeneously in the reaction mixture and virtually all of their surface is available for the catalytic reaction.",
"For this reason, much less catalyst is required for the same effect, which drastically reduces the costs, especially since noble metal nanoparticles are very expensive.",
"It may also be remarked that in catalysis processes of the prior art in which, for insatnce, Ceolite is used as a catalyst support, a high proportion of potentially active catalyst surface is lost as a result of a substantial part of the catalyst remaining inactive in the interior of the support molecule because it does not have an exposed surface which can act catalytically.",
"A further advantage of the homogeneous catalysis process presented here is that the catalyst can be recovered to an extent of almost 100% after the reaction is complete, by exploiting the abovementioned inventive principle.",
"In addition, this recycling of the catalyst can be carried out in a simple manner.",
"This, too, contributes to a considerable reduction in costs.",
"A process variant according to the invention provides a transfer process for nanoparticles from inorganic, in particular aqueous solution into organic solution.",
"In such a case, for example to obtain pure nanoparticles in the organic solution, the nanoparticles can, as described above, be selectively separated off by transferring them into water, as has been proposed in the first main aspect of the invention.",
"The organic solvent “contaminated” with the by-products is then present in isolated form and can be disposed of or passed to another use.",
"This is followed by the two steps described below, namely removal of the water and addition of fresh, uncontaminated organic solvent to the nanoparticles.",
"This variant comprises essentially the two steps: a.)",
"removal of part of the inorganic solvent to obtain the nanoparticles with a reduced content of inorganic solvent, b.)",
"addition of organic solvent to the nanoparticles.",
"These essential process steps can be used, for example, for purified reuse of the nanoparticles in fresh organic solution after such nanoparticles have been prepared in organic solutions and, in the process, undesirable by-products have also been formed in addition to the nanoparticles.",
"In a further embodiment of the teachings of the present invention disclosed here, the following 3 different types of catalysis, which are known from the prior art, can be improved by employing the inventive step of phase transfer as part of the catalytic process, The 3 types are as follows: 1.homogenous catalysis, 2.heterogeneous catalysis, and 3.mixed-phase catalysis.",
"As regards homogeneous catalysis: The term homogeneous catalysis is used to describe a catalytic reaction in which the catalyst, in this case the colloids or nanoparticles, is dispersed in the reaction solution.",
"This has the advantage that the entire surface of the nanoparticles is available to the starting material in the reaction solution.",
"The reaction can then take place on or in the vicinity of the particle surface.",
"The reaction products are therefore present in the same solution as the nanoparticles.",
"This is a disadvantage of homogeneous catalysis carried out in the manner of the prior art, because nanoparticles cannot be removed from the solution in a simple fashion using known separation techniques, e.g.",
"filtration.",
"The present invention provides two alternatives: a) the nanoparticles present in an organic solvent can be used as homogeneous catalysts in the organic solvents.",
"After the reaction has taken place, the nanoparticles can then be removed from the solution using the phase transfer process of the invention.",
"b) nanoparticles present in an organic solvent can firstly be transferred into water, with or without a certain alcohol content, using the phase transfer process of the invention.",
"The nanoparticles can then be used for homogeneous catalysis in such a water-soluble solvent.",
"The particles transferred into the aqueous solution also have improved properties compared to particles produced in water; for example, they require no cocatalyst in the solution.",
"Regarding heterogeneous catalysis: As described above, the main disadvantage of homogeneous catalysis is the fact that it is difficult to separate the catalyst from the reaction mixture (in order to recycle or purify the product).",
"However, if the catalyst is immobilized on a larger object having a dimension greater than 100 nanometers, then the separation is considerably simpler.",
"Supports known from the prior art have particle sizes in the micron range.",
"The present invention can improve heterogeneous catalysis in the following way: Nanoparticles synthesized in organic solvents can be transferred into water using the phase transfer process of the invention.",
"Addition of a support compound which is chosen so that it has an affinity for the phase-transferred nanoparticles to the solution in which the nanoparticles are present results in the particles being bound to the support compound.",
"The support compound can then be washed and/or made ready in a normal manner for use in the catalytic reaction, as is known from the prior art.",
"After the catalytic reaction is complete, the nanoparticles adhering to the supports can then be recovered from the reaction mixture using known techniques for recycling or separation.",
"Regarding mixed-phases catalysis: Alternatively, the catalytic reaction can also take place when different phases are mixed, with the nanoparticles being present as catalyst in one of these.",
"Here, “phase” is 1 or more of: solids, gas or hydrophilic or hydrophobic liquids.",
"If 2 of these phases are mixed, they separate in time.",
"Nanoparticles can then be used as catalyst as described above, with the phase transfer process of the invention being able to be used in 3 different ways: 1.for separating off the catalyst after the reaction, for example into a mixture of hydrophobic and hydrophilic liquid, or 2.using phase transfer to form a solid-state catalyst which is then used, for example, in a solid/gas mixture, or 3.to form water-soluble nanoparticles which can then react at the interface to another phase, for example at the interface between hydrophilic liquid and solid or at the interface between hydrophobic and hydrophilic liquid in an appropriate mixture.",
"Mixed forms of all 3 phases, viz.",
"solid, liquid and gas, are also possible in a corresponding way.",
"Although the present invention has been described above for a preferred example, it is not restricted thereto but can be modified in a variety of ways.",
"In particular, the quantitative data provided are provided only by way of example and do not restrict the scope of the invention as defined in the claims.",
"For example, the MM component used in the phase transfer process of the invention can be added not in aqueous solution (with or without a proportion of alcohol) but also in isolated, undissolved form.",
"The addition can be carried out either directly to the starting solution, for instance as a third phase in powder or slurry form, or after addition of a predetermined amount of target solution."
]
] | Patent_10432942 |
[
[
"Ultrasonic reflux system for one-step purification of carbon nanostructures",
"Reflux systems and methods for purifying carbon nanostructures using same are provided.",
"The reflux system includes a solvent flask, an extraction tube connected to the solvent flask by a siphon tube and a vapor tube each extending between the extraction tube and the solvent flask, and an energy application disposed around the bottom portion of the extraction tube.",
"The reflux systems can be used in a one-step method of purifying carbon nanostructures that includes placing a soot sample that contains the carbon nanostructures and amorphous carbon in a filter and disposing the filter in the extraction tube."
],
[
"1-31.",
"(canceled) 32.A reflux system comprising: a solvent supply device; an extraction tube connected to the solvent supply device, wherein the extraction tube has a top portion and a bottom portion; a siphon tube extending from the bottom portion of the extraction tube, and connected to the solvent source; and an energy applicator disposed around the bottom portion of the extraction tube.",
"33.The reflux system according to claim 32, wherein the solvent supply device is a solvent flask, and the reflux system further comprises a vapor tube connected between the solvent flask and the extraction tube.",
"34.The reflux system according to claim 33, further comprising a condenser connected to the top portion of the extraction tube.",
"35.The reflux system according to claim 33, further comprising a supply tube connected to the extraction tube through which material can be introduced into the extraction tube.",
"36.The reflux system according to claim 32, wherein the energy applicator is an ultrasonic vibrator.",
"37.A reflux system comprising: a solvent source including a solvent flask and a vapor tube connected to the solvent flask; an extraction tube having a top portion and a bottom portion, wherein the extraction tube is connected to the vapor tube allowing the extraction tube to be in communication with the solvent flask; a condenser connected to the top portion of the extraction tube, wherein the condenser is in communication with the vapor tube; a siphon tube extending from the bottom portion of the extraction tube, and connected to the solvent flask; and a supply tube connected to the extraction tube through which material can be introduced into the extraction tube.",
"38.The reflux system according to claim 37, further comprising an energy applicator disposed around the bottom portion of the extraction tube.",
"39.The reflux system according to claim 38, wherein the energy applicator is an ultrasonic vibrator.",
"40.A one-step method of purifying carbon nanotubes, comprising: placing a soot sample that contains the carbon nanotubes together with amorphous carbon in a filter and disposing the filter in a lower portion of an extraction tube; introducing an oxidizing agent into the extraction tube to oxidize the amorphous carbon; introducing a solvent into the extraction tube so as to contact the filter, collect in the lower portion of the extraction tube, and dissolve the oxidized amorphous carbon from the soot sample; and removing the solvent from the extraction tube allowing the carbon nanotubes to remain in the filter, wherein the method of purifying carbon nanotubes is carried out at ambient temperature.",
"41.The method according to claim 40, wherein the soot sample includes metal catalyst particles, and the method further comprises introducing acid into the extraction tube allowing the acid to remove the metal catalyst particles from the soot sample.",
"42.The method according to claim 41, wherein the step of introducing an oxidizing agent includes introducing oxidizing gas, the step of introducing acid into the extraction tube includes introducing acid vapor, and further wherein the acid vapor is simultaneously introduced with the oxidizing gas.",
"43.The method according to claim 41, wherein the step of introducing solvent includes introducing solvent vapor to the extraction tube and condensing the solvent vapor, and wherein the step of introducing acid into the extraction tube includes introducing acid vapor along with the solvent vapor.",
"44.The method according to claim 40, further comprising applying energy to the soot sample so as to disperse agglomerations.",
"45.The method according to claim 44, wherein the energy is ultrasonic vibration.",
"46.The method according to claim 45, wherein the step of applying energy is performed simultaneously with the step of introducing an oxidizing agent and simultaneously with the step of introducing solvent.",
"47.The method according to claim 40, wherein the solvent has a dipole greater than or equal to about 1.48.A one-step method of purifying carbon nanostructures, comprising: placing a soot sample that contains the carbon nanostructures in combination with amorphous carbon in a filter and disposing the filter in a lower portion of an extraction tube; introducing solvent into the extraction tube so as to contact the filter, collect in the lower portion of the extraction tube, and dissolve one of the amorphous carbon and the carbon nanostructures from the soot sample; applying energy to the soot sample in the extraction tube so as to disperse agglomerations; and removing the solvent, and the one of the amorphous carbon and carbon nanostructures dissolved therein, from the extraction tube so that the other one of the amorphous carbon and the carbon nanostructures remains in the filter.",
"49.The method according to claim 48, wherein the step of applying energy includes applying ultrasonic vibration.",
"50.The method according to claim 48, further comprising carrying out the method of purifying carbon nanostructures at ambient temperature.",
"51.The method according to claim 48, further comprising introducing an oxidizing agent into the extraction tube to oxidize the amorphous carbon.",
"52.The method according to claim 51, wherein the step of introducing solvent includes introducing a solvent having a dipole greater than or equal to about 1 so that the carbon nanostructures remain in the filter, whereas the oxidized amorphous carbon is dissolved in the solvent.",
"53.The method according to claim 51, further comprising introducing acid into the extraction tube to remove metallic particles from the soot sample.",
"54.The method according to claim 53, wherein the step of introducing an oxidizing agent includes introducing an oxidizing gas, wherein the step of introducing acid into the extraction tube includes introducing acid vapor, and wherein the acid vapor is introduced simultaneously with the oxidizing gas.",
"55.The method according to claim 53, wherein the step of introducing solvent to the extraction tube includes introducing solvent vapor into the extraction tube and condensing the solvent vapor, and wherein the step of introducing acid into the extraction tube includes introducing acid vapor along with the solvent vapor.",
"56.The method according to claim 48, wherein the step of introducing solvent includes introducing a solvent having a dipole less than about 1, so that the carbon nanostructures are dispersed in the solvent, whereas the amorphous carbon remains in the filter.",
"57.The method according to claim 56, wherein the step of introducing solvent includes introducing solvent vapor with an inert gas, and then condensing the solvent vapor.",
"58.A one-step method of purifying carbon fullerenes, comprising: placing a soot sample that contains the carbon fullerenes together with amorphous carbon in a filter and disposing the filter in a lower portion of an extraction tube; introducing a solvent into the extraction tube so as to contact the filter, collect in the lower portion of the extraction tube, and form a solution with the fullerenes from the soot sample, wherein the solvent has a dipole moment less than about 1; and removing the solvent containing the fullerenes from the extraction tube so that the amorphous carbon remains in the filter, wherein the above steps are carried out at ambient temperature.",
"59.The method according to claim 58, further comprising applying ultrasonic energy to the soot sample so as to disperse agglomerations.",
"60.The method according to claim 59, wherein the step of applying energy is performed simultaneously with the step of introducing solvent.",
"61.The method according to claim 58, wherein the step of introducing solvent includes evaporating the solvent from a flask, causing the solvent to travel along an evaporation tube to a condenser, and condensing the evaporated solvent in the condenser so that the solvent is introduced to the extraction tube, and wherein the step of removing the solvent includes returning the solvent to the flask.",
"62.The method according to claim 61, wherein the step of introducing solvent includes using an inert gas to assist in causing the evaporated solvent to travel along an evaporation tube, and further comprising maintaining an atmosphere, in the extraction tube, without oxidizing agents."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to a reflux systems, and methods, for purifying carbon nanostructures.",
"More particularly, the present invention relates to improved apparatusses and systems and methods of using same to purify carbon nanostructures, including single wall nanotubes (SWNTs), multi-wall nanotubes (MWNTs), fullerenes, endohedral metallofullerenes, carbon nanofibers, and other carbon-containing nano-materials.",
"The reflux systems and methods are particularly useful for purifying SWNTs.",
"One known method of purifying carbon nanostructures includes baking a soot sample at 750° C. in air for about thirty minutes.",
"See “Purification of nanotubes” by Ebbesen et al, Nature, vol.",
"367, 10 February 1994, p. 519.However, Ebbesen's method is directed to the purification of MWNTs; such high heat in this process tends to damage, or even destroy, SWNTs.",
"Other known methods of purifying carbon nanostructures involve multiple steps carried out in multiple apparatuses.",
"See “Purification Procedure for Single-Walled Nanotubes” by K. Tohji et al., J. Phys.",
"Chem.",
"B, vol.",
"101, 1997, p. 1974-1978, for example.",
"That is, soot produced by arc-discharge includes many byproducts such as metal particles, fullerenes, buckyonions, and a large amount of amorphous carbon together with the desired SWNTs.",
"Thus, heretofore, many steps carried out in multiple apparatuses have been necessary for purifying SWNTs.",
"The steps typically include, for example, hydrothermally initiated dynamic extraction (HIDE), sonication, filtration, drying, washing, heat treatment, and acid treatment.",
"But many of the processes are performed in different apparatuses, thereby necessitating removal of the soot sample from one apparatus and placing it in another apparatus.",
"Still other known methods include microfiltration, and some even use ultrasound to assist in the filtration.",
"See “Purification of single-wall carbon nanotubes by ultrasonically assisted filtration” by Konstantin B. Shelimov et al., Chem.",
"Phys.",
"Lett., vol.",
"282, 1998, p. 429-434, for example.",
"In such methods, however, multiple steps are still necessary, and the yield remains low.",
"That is, the soot is first suspended in toluene and filtered to extract soluble fullerenes.",
"Then, the toluene-insoluble fraction is re-suspended in methanol and filtered with assistance of an ultrasonic horn inserted into the filtration funnel.",
"Finally, a separate acid wash is performed to remove metal particles.",
"Therefore, because of the many steps and apparatuses necessary, these methods have been implemented mainly for diluted and relatively pure raw materials such as those synthesized by laser ablation; they are inefficient for large quantities of low-purity raw materials.",
"Lastly, a dilute nitric acid reflux technique has been performed to purify SWNTs.",
"See “A Simple and Complete Purification of Single-Walled Carbon Nanotube Materials”, by Anne C. Dillon et al., Advanced Materials 1999, vol.",
"11, no.",
"16, p. 1354-1358.But this process still requires three steps—including an oxidation step in which the carbon is heated to 550° C.—carried out in different apparatuses.",
"Therefore, this process suffers the same drawbacks as like processes discussed above.",
"Namely, the different steps require transference of the soot, the heating step damages or destroys SWNTs, and the method is effective only for high-purity soot.",
"Because the related art purification methods include multiple steps, performed in multiple apparatuses, these methods are time consuming and labor intensive.",
"Additionally, there is risk that some of the sample is lost, contaminated, or destroyed in transit from one apparatus to another.",
"Further, because of the large amount of amorphous carbon in the soot samples, and the heating steps, these methods have only been able to achieve a low yield (about 5 wt %) of 95% pure SWNTs."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to improved reflux systems and methods for purifying carbon nanostructures.",
"For example, the present invention can avoid using heat, especially high heat, to purify carbon nanostructures because such high heat tends to damage the carbon nanostructures.",
"In fact, high heat tends to destroy SWNTs altogether, whereas it merely tends to burn off the outer layers of MWNTs.",
"The present invention can provide methods and apparatuses that are useful for purifying large quantities of low-purity raw materials, such as those synthesized by arc-discharge.",
"The present invention can also purify such materials in a highly efficient manner which yields a high percentage of the desired carbon nanostructures.",
"Still further, the present invention can provide apparatuses and methods that are simple and less complex in design and construction by which various forms of carbon nanostructures can be purified.",
"That is, the present apparatus and method can be used to purify carbon nanotubes, extract fullerenes, or both, from a given soot sample.",
"In order to avoid using heat to purify carbon nanostructures, the present invention is carried out at ambient, or room temperature according to an embodiment.",
"When purifying carbon nanotubes, an oxidizing gas is introduced into the soot sample in order to oxidize the amorphous carbon therein, and a solvent is used to remove the oxidized amorphous carbon.",
"When purifying fullerenes, the amorphous carbon is not oxidized but, instead, a solvent is used to remove the fullerenes from the soot sample.",
"In any case, because the carbon nanostructures are purified at ambient temperature, they are not damaged by high heat.",
"Further, the use of little, or no, heat leads to an increased yield of carbon nanostructures, especially SWNTs, because the carbon nanostructures are not destroyed in the purification process.",
"In order to avoid transferring the soot sample between apparatuses, thereby reducing the time required for purification as well as reducing the risk of contaminating or damaging a sample, the methods of the present invention can be performed in a single apparatus.",
"That is, the soot sample and products separated therefrom can remain in one apparatus until the desired structures are purified.",
"Further, because the present invention does not require soot transference, it is less labor intensive and, therefore, less costly.",
"In order to increase the yield of the desired carbon nanostructure specially SWNTs—from low-purity raw materials, the present method and apparatus use a one-step process in an embodiment.",
"In the one-step process, amorphous carbon is oxidized, oxidized amorphous carbon is removed, and metallic particles are removed, in a short period of time because these processes are carried out by the same apparatus.",
"Additionally, the processes can be performed simultaneously thereby further increasing the speed of the process.",
"Moreover, energy—such as ultrasonic vibrations, or microwaves, for exampl—can be used to assist in dispersing agglomerations thereby making more of the soot sample available to the other processes and, hence, make the process more efficiently attain a higher yield.",
"The ultrasonic energy is applied with the soot remaining in the same apparatus, and may be applied at the same time as the other processes, thereby reducing the time necessary to purify the sample.",
"Because the time for purification is reduced, a relatively large, low-purity, sample efficiently can be purified.",
"A reflux system including a solvent flask, an extraction tube connected to the solvent flask by a siphon tube and a vapor tube each extending between the extraction tube and the solvent flask, and an energy applicator disposed around the bottom portion of the extraction tube is provided pursuant to an embodiment of the present invention.",
"Further, a condenser is connected to the top portion of the extraction tube.",
"A supply tube is connected to the extraction tube, whereby material can be introduced into the extraction tube.",
"The reflux system is used in a one-step method, of purifying carbon nanostructures, including placing a soot sample—containing the carbon nanostructures and amorphous carbon—in a filter and disposing the filter in the extraction tube.",
"Solvent is then introduced into the extraction tube so as to collect in the lower portion thereof, and remove one of the amorphous carbon and the carbon nanostructures from the soot.",
"Further, the energy applicator is used to apply ultrasonic vibrations to the soot so as to disperse agglomerations therein.",
"The solvent, and the one of the amorphous carbon and carbon nanostructures dissolved therein, is then removed from the extraction tube so that the other one of the amorphous carbon and the carbon nanostructures remains in the filter.",
"Further, the method is performed at ambient temperature, an oxidizing gas is introduced into the extraction tube to oxidize the amorphous carbon, and acid is introduced into the extraction tube to remove metallic particles from the soot.",
"Additional features and advantages of the present invention are described in, and will be apparent from, the following Detailed Description of the Invention and the figures."
],
[
"CROSS REFERENCES TO RELATED APPLICATIONS The present application claims priority to Japanese Patent Document No.",
"2000-375043 filed on Dec. 8, 2000, the disclosure of which is incorporated herein by reference.",
"BACKGROUND OF THE INVENTION The present invention relates to a reflux systems, and methods, for purifying carbon nanostructures.",
"More particularly, the present invention relates to improved apparatusses and systems and methods of using same to purify carbon nanostructures, including single wall nanotubes (SWNTs), multi-wall nanotubes (MWNTs), fullerenes, endohedral metallofullerenes, carbon nanofibers, and other carbon-containing nano-materials.",
"The reflux systems and methods are particularly useful for purifying SWNTs.",
"One known method of purifying carbon nanostructures includes baking a soot sample at 750° C. in air for about thirty minutes.",
"See “Purification of nanotubes” by Ebbesen et al, Nature, vol.",
"367, 10 February 1994, p. 519.However, Ebbesen's method is directed to the purification of MWNTs; such high heat in this process tends to damage, or even destroy, SWNTs.",
"Other known methods of purifying carbon nanostructures involve multiple steps carried out in multiple apparatuses.",
"See “Purification Procedure for Single-Walled Nanotubes” by K. Tohji et al., J. Phys.",
"Chem.",
"B, vol.",
"101, 1997, p. 1974-1978, for example.",
"That is, soot produced by arc-discharge includes many byproducts such as metal particles, fullerenes, buckyonions, and a large amount of amorphous carbon together with the desired SWNTs.",
"Thus, heretofore, many steps carried out in multiple apparatuses have been necessary for purifying SWNTs.",
"The steps typically include, for example, hydrothermally initiated dynamic extraction (HIDE), sonication, filtration, drying, washing, heat treatment, and acid treatment.",
"But many of the processes are performed in different apparatuses, thereby necessitating removal of the soot sample from one apparatus and placing it in another apparatus.",
"Still other known methods include microfiltration, and some even use ultrasound to assist in the filtration.",
"See “Purification of single-wall carbon nanotubes by ultrasonically assisted filtration” by Konstantin B. Shelimov et al., Chem.",
"Phys.",
"Lett., vol.",
"282, 1998, p. 429-434, for example.",
"In such methods, however, multiple steps are still necessary, and the yield remains low.",
"That is, the soot is first suspended in toluene and filtered to extract soluble fullerenes.",
"Then, the toluene-insoluble fraction is re-suspended in methanol and filtered with assistance of an ultrasonic horn inserted into the filtration funnel.",
"Finally, a separate acid wash is performed to remove metal particles.",
"Therefore, because of the many steps and apparatuses necessary, these methods have been implemented mainly for diluted and relatively pure raw materials such as those synthesized by laser ablation; they are inefficient for large quantities of low-purity raw materials.",
"Lastly, a dilute nitric acid reflux technique has been performed to purify SWNTs.",
"See “A Simple and Complete Purification of Single-Walled Carbon Nanotube Materials”, by Anne C. Dillon et al., Advanced Materials 1999, vol.",
"11, no.",
"16, p. 1354-1358.But this process still requires three steps—including an oxidation step in which the carbon is heated to 550° C.—carried out in different apparatuses.",
"Therefore, this process suffers the same drawbacks as like processes discussed above.",
"Namely, the different steps require transference of the soot, the heating step damages or destroys SWNTs, and the method is effective only for high-purity soot.",
"Because the related art purification methods include multiple steps, performed in multiple apparatuses, these methods are time consuming and labor intensive.",
"Additionally, there is risk that some of the sample is lost, contaminated, or destroyed in transit from one apparatus to another.",
"Further, because of the large amount of amorphous carbon in the soot samples, and the heating steps, these methods have only been able to achieve a low yield (about 5 wt %) of 95% pure SWNTs.",
"SUMMARY OF THE INVENTION The present invention relates to improved reflux systems and methods for purifying carbon nanostructures.",
"For example, the present invention can avoid using heat, especially high heat, to purify carbon nanostructures because such high heat tends to damage the carbon nanostructures.",
"In fact, high heat tends to destroy SWNTs altogether, whereas it merely tends to burn off the outer layers of MWNTs.",
"The present invention can provide methods and apparatuses that are useful for purifying large quantities of low-purity raw materials, such as those synthesized by arc-discharge.",
"The present invention can also purify such materials in a highly efficient manner which yields a high percentage of the desired carbon nanostructures.",
"Still further, the present invention can provide apparatuses and methods that are simple and less complex in design and construction by which various forms of carbon nanostructures can be purified.",
"That is, the present apparatus and method can be used to purify carbon nanotubes, extract fullerenes, or both, from a given soot sample.",
"In order to avoid using heat to purify carbon nanostructures, the present invention is carried out at ambient, or room temperature according to an embodiment.",
"When purifying carbon nanotubes, an oxidizing gas is introduced into the soot sample in order to oxidize the amorphous carbon therein, and a solvent is used to remove the oxidized amorphous carbon.",
"When purifying fullerenes, the amorphous carbon is not oxidized but, instead, a solvent is used to remove the fullerenes from the soot sample.",
"In any case, because the carbon nanostructures are purified at ambient temperature, they are not damaged by high heat.",
"Further, the use of little, or no, heat leads to an increased yield of carbon nanostructures, especially SWNTs, because the carbon nanostructures are not destroyed in the purification process.",
"In order to avoid transferring the soot sample between apparatuses, thereby reducing the time required for purification as well as reducing the risk of contaminating or damaging a sample, the methods of the present invention can be performed in a single apparatus.",
"That is, the soot sample and products separated therefrom can remain in one apparatus until the desired structures are purified.",
"Further, because the present invention does not require soot transference, it is less labor intensive and, therefore, less costly.",
"In order to increase the yield of the desired carbon nanostructure specially SWNTs—from low-purity raw materials, the present method and apparatus use a one-step process in an embodiment.",
"In the one-step process, amorphous carbon is oxidized, oxidized amorphous carbon is removed, and metallic particles are removed, in a short period of time because these processes are carried out by the same apparatus.",
"Additionally, the processes can be performed simultaneously thereby further increasing the speed of the process.",
"Moreover, energy—such as ultrasonic vibrations, or microwaves, for exampl—can be used to assist in dispersing agglomerations thereby making more of the soot sample available to the other processes and, hence, make the process more efficiently attain a higher yield.",
"The ultrasonic energy is applied with the soot remaining in the same apparatus, and may be applied at the same time as the other processes, thereby reducing the time necessary to purify the sample.",
"Because the time for purification is reduced, a relatively large, low-purity, sample efficiently can be purified.",
"A reflux system including a solvent flask, an extraction tube connected to the solvent flask by a siphon tube and a vapor tube each extending between the extraction tube and the solvent flask, and an energy applicator disposed around the bottom portion of the extraction tube is provided pursuant to an embodiment of the present invention.",
"Further, a condenser is connected to the top portion of the extraction tube.",
"A supply tube is connected to the extraction tube, whereby material can be introduced into the extraction tube.",
"The reflux system is used in a one-step method, of purifying carbon nanostructures, including placing a soot sample—containing the carbon nanostructures and amorphous carbon—in a filter and disposing the filter in the extraction tube.",
"Solvent is then introduced into the extraction tube so as to collect in the lower portion thereof, and remove one of the amorphous carbon and the carbon nanostructures from the soot.",
"Further, the energy applicator is used to apply ultrasonic vibrations to the soot so as to disperse agglomerations therein.",
"The solvent, and the one of the amorphous carbon and carbon nanostructures dissolved therein, is then removed from the extraction tube so that the other one of the amorphous carbon and the carbon nanostructures remains in the filter.",
"Further, the method is performed at ambient temperature, an oxidizing gas is introduced into the extraction tube to oxidize the amorphous carbon, and acid is introduced into the extraction tube to remove metallic particles from the soot.",
"Additional features and advantages of the present invention are described in, and will be apparent from, the following Detailed Description of the Invention and the figures.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1 is a schematic, partial cross sectional, view showing a reflux system according to an embodiment of the present invention.",
"DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to reflux systems and methods for purifying carbon nanostructures.",
"The reflux system of the present invention in an embodiment allows carbon nanostructures to be purified in one step by filtration, extraction, or both, carried out at ambient temperature.",
"That is, soot containing the desired carbon nanostructures as well as unwanted byproducts is put into a filter, is placed into the reflux system and, through various processes performed in the reflux system, the desired carbon nanostructures are removed from the reflux system.",
"Therefore, neither the soot, nor any intermediate products, need be removed from the reflux system until the purification process is complete; the entire purification process takes place within the reflux system and takes place at ambient temperature.",
"The reflux system includes an extractor 1, a condenser 20, and an energy applicator 30.The extractor 1 includes a solvent flask 2, a thermal mantle 4, and an extraction tube 7.The solvent flask 2 sits in the thermal mantle 4 so as to be heated thereby.",
"The thermal mantle 4 is configured so that it can produce a variable amount of heat for evaporating various solvents held within the solvent flask 2.Additionally, the solvent flask 2 has a flask inlet 3 through which solvent, and gases, can be introduced into the flask 2.A vapor tube 5 and a siphon tube 11 are connected between the solvent flask 2 and the extraction tube 7, so that the solvent flask 2 and extraction tube 7 are in communication with one another.",
"The extraction tube 7 includes a top portion 7′ and a bottom portion 7″.",
"A stopper 8 is disposed in the extraction-tube top portion 7′ so as to form a vapor chamber 9 in the extraction tube 7.The vapor tube 5 is connected to the extraction tube 7 so as to be in communication with the vapor chamber 9, whereas the siphon tube 11 is connected to the bottom portion 7″ of the extraction tube 7.Spacers 12 are disposed between the vapor tube 5 and the siphon tube 11, as well as between the siphon tube 11 and the extraction tube 7.Additionally, a supply tube 13 is connected to the bottom portion 7″ of the extraction tube 7.The supply tube 13 allows material, in particular gases used during a filtration process, to be introduced into the extraction tube 7.Spacers 12 are also disposed between the supply tube 13 and the extraction tube 7.The extraction tube 7 is sized and configured to hold a filter 10 therein.",
"The filter 10 initially holds the sample to be purified and, after the purification process, holds the undissolved portion of the sample.",
"The condenser 20 is connected to the upper portion 7′ of the extraction tube 7 so as to receive vapors from the vapor chamber 9.More particularly, the condenser 20 includes a condenser tube 21 having a condenser-tube inlet 22 and a condenser-tube gas outlet 23.The condenser-tube inlet 22 is connected to the stopper 8 so as to communicate with the vapor chamber 9.The condenser-tube gas outlet 23 allows some gases to escape from the top of the condenser tube 21.Further, the condenser tube 21 includes a cooling-fluid jacket 24 having a cooling-fluid inlet 25 and a cooling-fluid outlet 26.The energy applicator 30 is disposed about the bottom portion 7″ of the extraction tube 7 so as to apply energy to a sample disposed in filter 10.The energy applicator 30 can be, for example, an ultrasonic vibrator, or a microwave applicator.",
"The energy applicator 30 assists in dispersing agglomerations in the sample disposed in filter 10 so that the sample is more easily, and thoroughly, purified.",
"That is, the energy applicator 30 allows the apparatus to achieve a higher purity, higher yield, of desired product from the sample.",
"A general purification process, using the above-described reflux system, will now be described according to an embodiment of the present invention.",
"First, a sample to be purified is placed in the filter 10 which, in turn, is disposed within the extraction tube 7.A solvent, for removing the soluble portion of the sample, is disposed in the solvent flask 2 wherein it is heated so as to evaporate.",
"The evaporated solvent enters evaporation tube 5, which is insulated by vapor-tube insulation 6 so as to maintain the solvent in its evaporated state as it travels through the evaporation tube 5.The evaporated solvent then travels through the evaporation tube 5, along the direction of arrow A, so as to enter the vapor chamber 9.In order to assist in driving the evaporated solvent through the evaporation tube 5, gas may be pumped through the flask inlet 3.After driving the evaporated solvent to the evaporation chamber 9 and, subsequently, to the condenser tube 21, the gas is allowed to exit through the condenser-tube gas outlet 23.Vapor from vapor chamber 9 enters the condenser-tube inlet 22 and passes up through the condenser tube 21, wherein it is condensed.",
"The condensate then falls back through the condenser-tube inlet 22 and down onto the filter 10 disposed in the extraction tube 7.The condensate collects in the extraction tube 7 and enters the filter 10 so as to react with the soluble portion of the sample contained therein.",
"When the solvent level in the extraction tube 7 rises above the highest portion of the siphon tube 11, the solvent then flows through the siphon tube 11, in the direction of arrow B, back down into the solvent flask 2 carrying the soluble portion of the sample with it.",
"Because the siphon tube 11 is connected to the bottom portion 7″ of the extraction tube 7, substantially all of the solvent—including soluble portions of the sample dissolved therein—are removed from the extraction tube 7.The evaporation process is again carried out as necessary, so that the soluble portion of the sample is collected in the solvent flask 2.That is, the temperature of the thermal mantle is selected so that only the solvent, not the soluble portion of the sample, is evaporated from the solvent flask 2.In order to assist with separating the desired portion of the sample from the impurities, gases or other materials may be introduced into the extraction tube 7 through supply tube 13.Generally, gase—such as oxidizing gases, acid vapor and the like—will be introduced and, therefore, the supply tube 13 is connected to the bottom portion 7″ of the extraction tube 7 so that the gasses flow up through the filter 10 and through the sample contained therein.",
"Further, any unused portion of the gases introduced through the supply tube 13 are allowed to exit through the condenser-tube gas outlet 23.Although the supply tube 13 preferably is connected to the bottom portion 7″, it can be connected anywhere along the extraction tube 7, especially if liquids are to be introduced therethrough.",
"To further assist with separating the desired portion of the sample from the impurities, the energy applicator 30 may be used to apply energy to the sample contained in filter 10.For example, the energy applicator 30 may be an ultrasonic vibrator which assists purification by dispersing agglomerated portions of the sample through agitation.",
"The energy applicator may be used continuously or intermittently throughout the purification process.",
"When the desired portion of the sample is that which is soluble, it is collected in the solvent flask 2 together with solvent.",
"In such a case, the solvent flask can be disconnected from the extraction tube, the solvent evaporated, and the desired portion of the sample easily is collected.",
"Further, the undissolved portions of the sample, which may be either wanted or unwanted, are then collected in the filter 10.When the desired portion of the sample is that which has not been dissolved, such is retained in the filter 10, and easily is removed.",
"Next, a purification process for obtaining carbon nanotubes, and in particular SWNTs, will be described.",
"In order to carry out a one-step purification of SWNTs, the reflux system of the present invention according to an embodiment combines the functions of ultrasound agitation, low temperature oxidation, and instant filtration.",
"First, a soot sample to be purified is placed in the filter 10 which, in turn, is disposed within the extraction tube 7.The soot sample contains the desired carbon nanostructures—SWNTs in this example—along with one or more of the following: amorphous carbon; metal catalyst particles; fullerenes; and other carbon nanoparticles.",
"A solvent, for removing oxidized amorphous carbon from the sample, is disposed in the solvent flask 2 wherein it is heated so as to evaporate.",
"In this example, a solvent having a dipole moment larger than one is used to assist in dispersing agglomerations in the soot and so as to easily dissolve and loosen oxidized amorphous carbon.",
"Preferably, the dipole moment of the solvent is in the range of from greater than or equal to about 1, to about 4.Examples of solvent which may be used include water (H2O), DMSO, dimethylformamide (DMF), THF, the like and suitable combinations thereof.",
"The evaporated solvent enters evaporation tube 5, and then travels through the evaporation tube 5, along the direction of arrow A, so as to enter the vapor chamber 9.In order to assist in driving the evaporated solvent through the evaporation tube 5, gas may be pumped through the flask inlet 3.For example, the gas pumped through the flask inlet 3 may be air or oxygen.",
"After driving the evaporated solvent to the evaporation chamber 9 and, subsequently, to the condenser tube 21, the gas is allowed to exit through the condenser-tube gas outlet 23, although some gas may remain in the extraction tube 7.In either case, when oxygen is used, it assists in oxidizing amorphous carbon.",
"Solvent vapor from vapor chamber 9 enters the condenser-tube inlet 22 and passes up through the condenser tube 21, wherein it is condensed.",
"The solvent condensate then falls back through the condenser-tube inlet 22 and down onto the filter 10 disposed in the extraction tube 7.In order to oxidize the amorphous carbon portion of the sample, an oxidizing agent—such as oxidizing gases, for example, oxygen (O2) or ozone (O3), or oxidizing liquids, for example, H2O2—is introduced into the extraction tube 7 through supply tube 13.The gasses flow up through the filter 10 and through the sample contained therein to oxidize the amorphous carbon.",
"The oxidizing agent may be continuously or intermittently introduced to the extraction tube.",
"The oxidized amorphous carbon is then carried with the solvent through the siphon tube 11 and into the solvent flask 2, as described below.",
"Any unused portion of the oxidizing gasses, which were introduced through the supply tube 13, are allowed to exit through the condenser-tube gas outlet 23.Because oxidizing gases are introduced into the extraction tube 7, and to the sample in filter 10, heat is not necessary to oxidize the amorphous carbon.",
"That is, the purification process of the present invention can be carried out at low temperatures such as, for example, ambient or room temperature.",
"By carrying out the purification process at ambient temperature, the SWNTs and other carbon nanostructures are not damaged, or destroyed, as they are at high temperatures.",
"Further, although oxidizing gas has been disclosed, an oxidizing liquid such as H2O2 may be used.",
"However, oxidizing gas is preferred because the oxidizing liquid takes up more volume in the extraction tube and, therefore, there is less volume available for the solvent.",
"In order to remove the metal catalyst portions of the sample, acid vapor is introduced into the extraction tube 7 through the supply tube 13.The acid vapor may be introduced along with the oxidizing gasses, or may be introduced either before or after the oxidizing gasses.",
"As the acid vapor enters the extraction tube 7 and, thus, the soot sample in filter 10, it reacts with the metal particles in the sample thereby forming metal salts.",
"The type of acid used depends on the solvent used.",
"Acid may be contained in the solvent and, thus, may be disposed in the solvent flask 2.That is, if only the acid and the solvent can co-evaporate, they may be disposed in the solvent flask 2, evaporated, and condensed together.",
"Introducing the acid and solvent together is preferable, as long as the acid does not have a tendency to react with, or decompose in, the solvent vapor which may be hot.",
"In still another embodiment, the acid may be introduced as vapor through the flask inlet 3 and, thereby, also may be used to assist in driving solvent vapor through the vapor tube 5.Each of the above three manners of introducing acid to the extraction tube may be used either separately, or in combination with one or more of the other manners of introducing acid to the extraction tube.",
"Further, the acid may be continuously, or intermittently, introduced.",
"To further assist with separating the desired portion of the sample from the impurities, the energy applicator 30 may be used to apply energy to the sample contained in filter 10.For example, the energy applicator 30 may be an ultrasonic vibrator which assists purification by dispersing agglomerated portions of the sample, which agglomerations include amorphous carbon, metal catalyst particles, and the desired SWNTs.",
"For example, ultrasonic vibration of about 100 W to about 1000 W, preferably about 350 W to about 500 W, can be applied to the soot sample.",
"By dispersing the agglomerations, the solvent, and acid vapor, readily can react with more of the sample and, thus, a higher purity can be achieved.",
"That is, because the agglomerations are dispersed into smaller particles, a greater surface area is available for the solvent, oxidizing agent, and acid.",
"The energy applicator 30 may be operated continuously, or intermittently, throughout the purification process.",
"The solvent condensate, received from the condenser, collects in the extraction tube 7 and enters the filter 10 so as to dissolve the oxidized amorphous carbon portion of the sample.",
"The solvent also washes out of the sample any fullerenes that are present.",
"When the solvent level in the extraction tube 7 rises above the highest portion of the siphon tube 11, the solvent then flows through the siphon tube 11, in the direction of arrow B, back down into the solvent flask 2 carrying the oxidized amorphous carbon, and metal salt, portions of the sample with it.",
"Because the siphon tube 11 is connected to the bottom portion 7″ of the extraction tube 7, substantially all of the solvent—including the oxidized amorphous carbon, and metal salt, portions of the sample contained therein—are removed from the extraction tube 7.The evaporation process is again carried out as necessary, so that the oxidized amorphous portion of the sample is collected in the solvent flask 2.That is, the temperature of the thermal mantle is selected so that only the solvent and acid are evaporated from the solvent flask 2, leaving the amorphous carbon, metal salts, and fullerenes in the solvent flask 2.What is left in the solvent flask 2, however, depends on what was included in the soot sample first placed in filter 10.That is, if no fullerenes were present in the original soot sample, then none will be present in the solvent flask 2.Similarly, if there were no metal catalyst particles in the original soot sample, then there will be no metal salts in the solvent flask 2.But if there were fullerenes in the original soot sample, they are collected in the solvent flask 2 and easily may be extracted therefrom.",
"That is, the apparatus can purify a sample containing both carbon nanotubes and fullerenes, and can do so such that both structures are purified at the same time.",
"When purifying carbon nanotubes and fullerenes at the same time, it is preferable to first use a solvent with a dipole less than about 1, before introducing an oxidizing agent to the sample, to increase the yield of fullerenes which may be damaged by the oxidizing agent.",
"In order to retain the desired SWNTs in the filter 10, a filter having a pore size of less than about 1 μm is used.",
"Such pore size allows fullerenes, but not nanotubes, to pass therethrough.",
"Additionally, the filter may be made of any material that will withstand attack from the acid introduced to remove the metal catalyst particles.",
"For example, the filter may be made of Teflon, or paper fiber which is stable in an acid environment.",
"Further, preferably, the filter 10 is one which encloses, or envelopes, the soot sample so that no carbon nanotubes are washed out when the solvent is removed from the extraction tube 7.Thus, in the above one-step purification process, the desired SWNTs are filtered and left in the filter 10, whereas any fullerenes are extracted and are present in the solvent flask 2.The process is a one-step process in that the soot sample, and/or intermediate products therefrom, do not need to be removed from one apparatus until purification of the desired carbon nanostructures contained in sample is complete.",
"The above described method, for purifying SWNTs, may also be used to purify MWNTs, or any other carbon nanotubes or nano-fibers.",
"All that is necessary to purify these other structures is to have them in the original soot sample which is placed in the filter 10.That is, if the original soot sample contains MWNTs, such structures will be collected in the filter 10, whereas fullerenes, amorphous carbon, and metal salts will be collected in solvent flask 2.Similarly, if the original soot sample contains other carbon nanotubes, or nano-fibers, these structures will be purified and collected in the filter 10.However, at present, the filter 10 does not distinguish between SWNTs, MWNTs, other nanotubes, or other nano-fibers.",
"Therefore, any of such structures which are present in the original soot sample will be collected in the filter 10.In one example of the above-described process for purifying SWNTs according to an embodiment of the present invention, water was used for the solvent, and HNO3 was used as the acid.",
"The acid was mixed with the water in the solvent flask 2 before heating it.",
"The water and HNO3 were then evaporated together, and condensed together.",
"Oxygen gas was continuously introduced through flask inlet 3 at about 50 ml/min to assist in driving the solvent and acid vapor through the vapor tube 5.Also, a flow of oxygen gas containing about 2% of ozone was introduced to the extraction tube 7 through supply tube 13 at about 50 ml/min.",
"Thus, the oxidizing agent for this example includes oxygen and ozone gasses, wherein the content of ozone was limited to about 2% of the gas introduced through supply tube 13 because if the concentration of ozone is too high, it may destroy the SWNTs.",
"The energy applicator was an ultrasonic vibrator operated at 350 W, and was operated continuously throughout the purification process.",
"All of the previously described conditions—heating and vapor condensation of both H2O and HNO3 together, introduction of gasses through both flask inlet 3 and supply tube 13, and ultrasonic vibration—were carried out simultaneously.",
"For a 10 g soot sample, produced by an arc-discharge operation, containing at least SWNTs, amorphous carbon, metal catalyst particles, and a trace amount of fullerenes, the above process was carried out under the previously described conditions for about 3 to about 4 hours, and resulted in a 95 wt % yield of SWNTs having a purity of 95%.",
"This yield, at such a high purity of SWNTs, is believed to be greater than has been achieved as compared to known processes, thus exemplifying the advantages of the present invention.",
"Although specific process parameters have been given here, they are not intended to be limiting to the scope of the present invention.",
"For example, these parameters may be varied in accordance with the guidance given throughout the specification.",
"The present invention in an embodiment is also applicable to the extraction of fullerenes.",
"That is, the apparatus and method of the present invention in an embodiment may be used to purify an original soot sample mainly containing fullerenes as the desired product.",
"In such a case, the above-described apparatus is used in the above-described manner, except that: no oxidizing gasses are introduced; no acid vapor is introduced; an inert gas may be used to drive the solvent vapor through the vapor tube 5; the extraction tube has an inert gas environment; and a solvent having a dipole less than about 1 is used pursuant to an embodiment of the present invention.",
"Such solvents include, for example, CS2, toluene, benzene the like, and suitable combinations thereof.",
"By using a solvent with a dipole less than about 1, the solvent readily extracts the fullerenes from the sample while leaving the amorphous carbon and metallic particles in the filter.",
"Further, because the amorphous carbon is not oxidized, and because the metal catalyst particles are not reacted with acid, such products are contained in the filter 10 along with any carbon nanotubes that were present in the original soot sample.",
"Thus, only the solvent and fullerenes are collected in the solvent flask 2 thereby making it easy to collect the desired fullerenes.",
"It is contemplated that numerous modifications may be made to the reflux system and purification method of the present invention without departing from the spirit and scope of the invention as defined in the claims.",
"For example, although the reflux system was described as being used to purify carbon nanostructures, it can be used in the same manner as a traditional SOXLET extractor to purify, or extract, any desired substance from a given sample.",
"Because the process is carried out at ambient temperature, with little or no heating of the soot sample, SWNTs are not damaged or destroyed thereby producing an increased yield of SWNTs.",
"Additionally, because the process in an embodiment is carried out in one apparatus—i.e., it is a one-step process—it can be done quickly, at a reduced cost, with reduced risk of contaminating or damaging the sample.",
"Further, the apparatus systems and method of the present invention are capable of efficiently purifying large amounts of low-purity soot to a high degree with a high yield of the desired carbon nanostructures.",
"Moreover, the present invention in an embodiment can be used easily to purify carbon nanotubes, fullerenes, or other suitable substances.",
"It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art.",
"Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its intended advantages.",
"It is therefore intended that such changes and modifications be covered by the appended claims.",
"Description of Reference Numerals 1 extractor 2 solvent flask 3 flask inlet 4 thermal mantle 5 vapor tube 6 vapor-tube insulation 7 extraction tube 7′ top portion of extraction tube 7″ bottom portion of extraction tube 8 stopper 9 vapor chamber 10 filter 11 siphon tube 12 spacers 13 supply tube 20 condenser 21 condenser tube 22 condenser-tube inlet 23 condenser-tube gas outlet 24 cooling-fluid jacket 25 cooling-fluid inlet 26 cooling-fluid outlet 30 energy applicator"
]
] | Patent_10433125 |
[
[
"Method and system to automatically qualifying a party to participate in a network-based commerce transaction",
"A system to facilitate network-based commerce includes receiving information relating to an item to be transacted via a network-based commerce system from a first party.",
"Criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system is received from the first party.",
"An automatic determination is made as to whether the second party satisfies the at least one criterion and if so, then the second party is automatically qualified to transact for the item via the network-based commerce system."
],
[
"1.A method to facilitate network-based commerce, the method including: receiving, from a first party, item information relating to an item to be transacted via a network-based commerce system; receiving, from the first party, criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system; automatically determining whether the second party satisfies the at least one criterion; and if so, then automatically qualifying the second party to transact for the item via the network-based commerce system.",
"2.The method of claim 1, wherein if the second party fails to satisfy the at least one criterion, automatically disqualifying the second party from transacting for the item via the network-based commerce system.",
"3.The method of claim 1, wherein the criterion information specifies a plurality of criterion.",
"4.The method of claim 3, wherein the criterion information specifies a relationship among the plurality of criterion so as to define a criterion function.",
"5.The method of claim 1, wherein the at least one criterion comprises an identification criterion identifying the second party, and wherein the determining includes determining whether the second party satisfies the identification criterion.",
"6.The method of claim 1, including receiving a request from the second party to transact the item.",
"7.The method of claim 1, wherein the at least one criterion comprises a geographic criterion specifying a geographic location associated with the second party.",
"8.The method of claim 1, wherein the at least one criterion comprises an age criterion.",
"9.The method of claim 1, wherein the at least one criterion comprises a reputation criterion relating to a reputation of the second party within the network-based commerce system.",
"10.The method of claim 9, wherein the reputation on the second party is at least partially determined by feedback information provided to the network-based commerce system by a third party.",
"11.The method of claim 10, wherein the feedback information indicates a score on a feedback scale.",
"12.The method of claim 10, wherein the feedback information is positive or negative.",
"13.The method of claim 1, wherein the at least one criterion comprises an activity criterion relating to previous transaction activity of the second party within the network-based commerce system.",
"14.The method of claim 13, wherein the activity criterion identifies a specific previous transaction activity that, if performed by the second party, disqualifies the second party from being authorized to transact for the item via the network-based commerce system.",
"15.The method of claim 13, wherein the activity criterion specifies a threshold for the previous transaction activity that, if exceeded by the second party, disqualifies the second party from being authorized to transact for the item via the network-based commerce system.",
"16.The method of claim 13, wherein the previous transaction activity includes a retraction of a bid offer.",
"17.The method of claim 1, wherein the at least one criterion comprises a transaction criterion relating to transaction activity pertaining to a transaction for the item.",
"18.The method of claim 17, wherein the transaction criterion includes submitting a purchase offer price for the item that exceeds a threshold value.",
"19.The method of claim 1, wherein the criterion information is associated with the item information and pertains only to the item.",
"20.The method of claim 1, wherein the criterion information is associated with the first party, and pertains to a plurality of items to be transacted by the first party via the network-based commerce system.",
"21.The method of claim 1, wherein the item information identifies and describes at least one of a product and a service.",
"22.The method of claim 1, wherein the determining includes accessing second party information concerning the second party stored by the network-based commerce system.",
"23.The method claim 22, wherein the second party information comprises profile information profiling the second party.",
"24.The method of claim 23, wherein the profile information is submitted by the second party.",
"25.The method of claim 23, wherein the profile information is automatically generated by the network-based commerce system.",
"26.The method of claim 22, wherein the second party information includes activity information collected by the network-based commerce system, the activity information to include transaction activity of the second party while utilizing the network-based commerce system.",
"27.The method of claim 1, wherein the network-based commerce system is an auction system, and the transaction for the item includes an auction process.",
"28.The method of claim 27, wherein the qualification of the second party includes enabling the second party to submit a bid for the item within the auction process.",
"29.The method of claim 1, wherein the network-based commerce system is a fixed-price transaction system, and the transaction for the item includes a fixed-price transaction process.",
"30.The method of claim 29, wherein the qualification of the second party to transact for the item includes enabling the second party to accept an offer for the item within the fixed-priced transaction process.",
"31.The method of claim 1, wherein the qualification of the second party includes, at the commerce system, recording the second party as qualified to transact with respect to the item.",
"32.The method of claim 2, wherein the qualification of the second party includes, at the commerce system, enabling the second party to establish a transaction with respect to the item, and wherein the disqualification of the second party includes, at the commerce system, preventing the second party from establishing a transaction with respect to the item.",
"33.The method of claim 1, wherein the qualification of the second party includes, at the commerce system, making the item information relating to the item available to the second party, and wherein the disqualification of the second party includes, at the commerce system, hiding in the information relating to the item from the second party.",
"34.A network-based commerce system, the system including: a parser to receive, from a first party, item information relating to an item to be transacted via a network-based commerce system and criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system; and a qualification module automatically to determine whether the second party satisfies the at least one criterion, and if so, then the qualification module automatically to qualify the second party to transact for the item via the network-based commerce system.",
"35.The system of claim 34, wherein the qualification module is automatically to disqualify the second party from transacting for the item via the network-based commerce system if the second party fails to satisfy the at least one criterion.",
"36.The system of claim 34, wherein the criterion information specifies a plurality of criterion.",
"37.The system of claim 36, wherein the criterion information specifies a relationship among the plurality of criterion so as to define a criterion function.",
"38.The system of claim 34, wherein the qualification module is to utilize the at least one criterion as an identification criterion to identify the second party, and wherein the qualification module is to determine based on whether the second party satisfies the identification criterion.",
"39.The system of claim 34, including a transaction module to receive a request from the second party to transact the item.",
"40.The system of claim 34, wherein the at least one criterion comprises a geographic criterion specifying a geographic location associated with the second party.",
"41.The system of claim 34, wherein the at least one criterion comprises an age criterion.",
"42.The system of claim 34, wherein the at least one criterion comprises a reputation criterion relating to a reputation of the second party within the network-based commerce system.",
"43.The system of claim 42, wherein the qualification module is at least partially to determine the reputation of the second party based on feedback information provided to the network-based commerce system by a third party.",
"44.The system of claim 43, wherein the feedback information indicates a score on a feedback scale.",
"45.The system of claim 43, wherein the feedback information is positive or negative.",
"46.The system of claim 34, wherein the at least one criterion comprises an activity criterion relating to previous transaction activity of the second party within the network-based commerce system.",
"47.The system of claim 46, wherein the activity criterion identifies a specific previous transaction activity that, if performed by the second party, disqualifies the second party from being authorized to transact for the item via the network-based commerce system.",
"48.The system of claim 46, wherein the activity criterion specifies a threshold for the previous transaction activity that, if exceeded by the second party, disqualifies the second party from being authorized to transact for the item via the network-based commerce system.",
"49.The system of claim 46, wherein the previous transaction activity includes a retraction of a bid offer.",
"50.The system of claim 34, wherein the at least one criterion comprises a transaction criterion relating to transaction activity pertaining to a transaction for the item.",
"51.The system of claim 50, wherein the transaction criterion includes submitting a purchase offer price for the item that exceeds a threshold value.",
"52.The system of claim 34, wherein the criterion information is associated with the item information and pertains only to the item.",
"53.The system of claim 34, wherein the criterion information is associated with the first party, and pertains to a plurality of items to be transacted by the first party via the network-based commerce system.",
"54.The system of claim 34, wherein the item information identifies and describes at least one of a product and a service.",
"55.The system of claim 34, wherein the qualification module is automatically to determine whether the second party satisfies the at least one criterion by accessing second party information concerning the second party stored by the network-based commerce system.",
"56.The system claim 55, wherein the second party information comprises profile information profiling the second party.",
"57.The system of claim 56, wherein the profile information is submitted by the second party.",
"58.The system of claim 56, wherein the network-based commerce system automatically generates the profile information.",
"59.The system of claim 55, wherein the network-based commerce system collects the second party information that includes activity information, the activity information recording activity of the second party while utilizing the network-based commerce system.",
"60.The system of claim 34, wherein the network-based commerce system is an auction system, and the transaction for the item includes an auction process.",
"61.The system of claim 60, wherein the qualification module to automatically qualify is to enable the second party to submit a bid for the item within the auction process.",
"62.The system of claim 34, wherein the network-based commerce system is a fixed-price transaction system, and the transaction for the item includes a fixed-price transaction process.",
"63.The system of claim 52, wherein the qualification module to automatically qualify is to enable the second party to accept an offer for the item within the fixed-priced transaction process.",
"64.The system of claim 34, wherein the qualification module to automatically qualify is to record the second party as qualified to transact with respect to the item.",
"65.The system of claim 35, wherein the qualification module is to qualify the second party by enabling the second party to establish a transaction with respect to the item, and wherein the qualification module is to disqualify the second party by preventing a transaction by the second party with respect to the item.",
"66.The system of claim 34, wherein the qualification module is to qualify the second party by making the item information that relates to the item available to the second party, and wherein the qualification module is to disqualify the second party by hiding information that relates to the item from the second party.",
"67.A machine readable medium storing a set of instructions that, when executed by the machine, cause the machine to: receive, from a first party, item information that relates to an item to be transacted via a network-based commerce system; receive, from the first party, criterion information to specify at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system; automatically determine whether the second party satisfies the at least one criterion; and if so, then automatically qualify the second party to transact for the item via the network-based commerce system.",
"68.A system to facilitate network-based commerce, the system including: a first means for receiving, from a first party, item information relating to an item to be transacted via a network-based commerce system and criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system; and a second means automatically for determining whether the second party satisfies the at least one criterion, and if so, for automatically qualifying the second party to transact for the item via the network-based commerce system."
],
[
"<SOH> ART BACKGROUND <EOH>More and more Internet users are realizing the ease and convenience of buying and selling online by way of person-to-person online trading (or transaction processing) pioneered by eBay Inc. As a result, collectors, hobbyists, small dealers, unique item seekers, bargain hunters, and other consumers are able to buy and sell millions of items at various online shopping sites.",
"The success of the online shopping sites, such as the Internet-based shopping facilities, depends upon their ability to provide enjoyable shopping experiences and easy-to-use and reliable environments in which buyers and sellers can conduct business efficiently.",
"The online shopping sites can offer their services by facilitating auctions or by allowing sellers to offer their offerings for fixed prices.",
"The current Internet-based shopping facilities have been presented with public relations risks due to excessive bid retraction and cancellation activities.",
"For example, the reputation of eBay Inc. as a safe trading place was threatened because of the excessive bid retraction and cancellation activities during the recent auction of the Titanic deck chair and other high profile listings.",
"It is estimated that as many as eighty percent of the bids made on the Internet-based shopping facilities are bogus.",
"Network-based commerce has of course found broad application beyond person-to-person trading, and is extensively used to perform business-to-business (B2B) trading.",
"Within the B2B environment, a party (e.g., potential buyer) may engage in a transaction activity or have a profile that is undesirable from the perspective of a further party (e.g., a potential seller).",
"In the light of the foregoing, there is a need to enhance the trust and confidence within online transaction facilities.",
"Particularly, it would be valuable and useful to provide a party to an online transaction with a degree of confidence that a further party is sincere and qualified to engage in a transaction process."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of the present invention, there is provided a method to facilitate network-based commerce.",
"Item information relating to an item to be transacted via a network-based commerce system is received from a first party.",
"Criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system is received from the first party.",
"An automatic determination is made as to whether the second party satisfies the at least one criterion and if so, then the second party is automatically qualified to transact for the item via the network-based commerce system.",
"According to a further aspect of the present invention, there is provided a method to facilitate network-based shopping.",
"A communication between a network-based auction facility and a seller is facilitated whereby the seller authorizes a bidder to bid on an offering offered for sale by the seller is disclosed.",
"Furthermore, the method comprises automatically recording the bidder as authorized to bid on the offering responsive to the communication.",
"Other features of the present invention will be apparent from the accompanying drawings and from the detailed description that follows."
],
[
"RELATED APPLICATIONS This application is a national application based on International Application WO 02/44860, filed Nov. 30, 2001, which claimed the benefit of the filing date of U.S. utility patent application Ser.",
"No.",
"09/881,911, filed Jun.",
"15, 2001, and the benefit under 35 U.S.C.",
"§ 119(e) of U.S. provisional application No.",
"60/250,637, filed Nov. 30, 2000.FIELD OF INVENTION The present invention relates to the field of network-based and electronic commerce.",
"Specifically, the present invention relates to the qualification of a party to participate within a network-based commerce transaction facilitated by a network-based commerce facility such as, for example, an Internet-based shopping or auction facility.",
"ART BACKGROUND More and more Internet users are realizing the ease and convenience of buying and selling online by way of person-to-person online trading (or transaction processing) pioneered by eBay Inc. As a result, collectors, hobbyists, small dealers, unique item seekers, bargain hunters, and other consumers are able to buy and sell millions of items at various online shopping sites.",
"The success of the online shopping sites, such as the Internet-based shopping facilities, depends upon their ability to provide enjoyable shopping experiences and easy-to-use and reliable environments in which buyers and sellers can conduct business efficiently.",
"The online shopping sites can offer their services by facilitating auctions or by allowing sellers to offer their offerings for fixed prices.",
"The current Internet-based shopping facilities have been presented with public relations risks due to excessive bid retraction and cancellation activities.",
"For example, the reputation of eBay Inc. as a safe trading place was threatened because of the excessive bid retraction and cancellation activities during the recent auction of the Titanic deck chair and other high profile listings.",
"It is estimated that as many as eighty percent of the bids made on the Internet-based shopping facilities are bogus.",
"Network-based commerce has of course found broad application beyond person-to-person trading, and is extensively used to perform business-to-business (B2B) trading.",
"Within the B2B environment, a party (e.g., potential buyer) may engage in a transaction activity or have a profile that is undesirable from the perspective of a further party (e.g., a potential seller).",
"In the light of the foregoing, there is a need to enhance the trust and confidence within online transaction facilities.",
"Particularly, it would be valuable and useful to provide a party to an online transaction with a degree of confidence that a further party is sincere and qualified to engage in a transaction process.",
"SUMMARY OF THE INVENTION According to one aspect of the present invention, there is provided a method to facilitate network-based commerce.",
"Item information relating to an item to be transacted via a network-based commerce system is received from a first party.",
"Criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for the item via the network-based commerce system is received from the first party.",
"An automatic determination is made as to whether the second party satisfies the at least one criterion and if so, then the second party is automatically qualified to transact for the item via the network-based commerce system.",
"According to a further aspect of the present invention, there is provided a method to facilitate network-based shopping.",
"A communication between a network-based auction facility and a seller is facilitated whereby the seller authorizes a bidder to bid on an offering offered for sale by the seller is disclosed.",
"Furthermore, the method comprises automatically recording the bidder as authorized to bid on the offering responsive to the communication.",
"Other features of the present invention will be apparent from the accompanying drawings and from the detailed description that follows.",
"BRIEF DESCRIPTION OF THE DRAWINGS The present invention is illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements, and in which: FIG.",
"1 is block diagram illustrating an exemplary network-based commerce facility in the form of an Internet-based auction facility.",
"FIG.",
"2 illustrates the web home page for an exemplary Internet-based auction facility.",
"FIG.",
"3 illustrates an exemplary pre-approve bidders main web page for an exemplary Internet-based auction facility.",
"FIG.",
"4 illustrates an exemplary pre-approve bidders logon web page for an exemplary Internet-based auctions facility.",
"FIG.",
"5 illustrates an exemplary pre-approve bidders form web page for an exemplary Internet-based auctions facility.",
"FIG.",
"6A and 6B illustrate an exemplary view item web page for an exemplary Internet-based auction facility.",
"FIG.",
"6C illustrates an exemplary error message.",
"FIG.",
"7 is a database diagram illustrating an exemplary database for the Internet-based auction facility.",
"FIG.",
"8 is a diagrammatic representation of an exemplary embodiment of the bidder feedback profile summary table.",
"FIG.",
"9 is a diagrammatic representation of one exemplary embodiment of the bidder feedback profile details table.",
"FIG.",
"10 illustrates the flow chart of one embodiment of the method for seller authorized bidding through an Internet-based auction facility.",
"FIG.",
"11 is a block diagram illustrating an exemplary commerce system that may at least partially perform an automatic qualification, or disqualification, of a second party to transact for an item.",
"FIG.",
"12 is a flow chart illustrating a method, according to an exemplary embodiment of the present invention, whereby a first party (e.g., a seller user) may define and specify criteria to be satisfied by a second party (e.g., a buyer user) in order for the second party to be automatically qualified to transact for a specific item, or for a number of items (e.g., all items offered for sale by the first party).",
"FIG.",
"13 is a diagrammatic representation of an item and criterion information input user interface, according to one exemplary embodiment of the present invention.",
"FIG.",
"14 is a diagrammatic representation of an exemplary items table.",
"FIG.",
"15 is a diagrammatic representation of an exemplary criteria table.",
"FIG.",
"16 is a flow chart illustrating a first exemplary method whereby a commerce system may automatically qualify a second party to transact, or disqualify a second party from transacting, with respect to a particular item, or group of items.",
"FIG.",
"17 is a flow chart illustrating a second exemplary method whereby a commerce system may automatically qualify a second party to transact, or disqualify a second party from transacting, with respect to a particular item, or group of items.",
"FIG.",
"18 provides diagrammatic representations of exemplary profile tables.",
"FIG.",
"19 shows a diagrammatic representation of machine in the exemplary form of a computer system within which a set of instructions, for causing the machine to perform any one of the methodologies discussed above, may be executed.",
"DETAILED DESCRIPTION A method and system to qualify a party to transact with respect to an item via a network-based commerce system are described.",
"In one embodiment, this qualification is manually performed by implementing seller-authorized transacting privileges.",
"In one embodiment, and the present invention proposes a method and system whereby a first party (e.g., a seller) can authorize transacting privileges (e.g., buying privileges) for a second party (e.g., a buyer) to transact for an item message dated with the first party.",
"The transacting privileges can include, for example, the authorization to bid on an auction listing of the first party and/or the authorization to offer to buy a fixed price listing of the first party.",
"A listing may, for example, relate to an item.",
"In this description, the terms listing and item and offering are used interchangeably.",
"Unauthorized bidders are disabled (or barred) from transacting for an item.",
"An advantage of one embodiment of the present invention is that a first party (e.g., a seller) does not manually have to monitor or participate in transacting activities because only the parties manually pre-approved by the first party, or automatically qualified by the commerce system utilizing predefined criteria specified by the first party, are allowed to transact (e.g., bid on or offer to buy) for an item associated with the first party.",
"The shoppers (or potential buyers) also benefit from this embodiment of the present invention because a healthier trading environment is created because only the pre-approved (or qualified) candidates are allowed to compete for a particular item.",
"The community of users of a commerce system benefits in general because the commerce system is perceived to facilitate worthwhile transacting because only the serious partys are allowed to transact for an item (e.g., bid on and offer to buy the listings).",
"Seller authorized buying privileges can be requested by any seller with privileges to list on the commerce system (e.g., a shopping facility) and for any listing, although the sellers with high profile or high value listings would have more incentive to do so.",
"Further, sellers wishing to restrict a pool of potential buyers would also find certain embodiments of the present invention useful.",
"Of course, the invention may also find application where a buyer is soliciting offers (e.g., request for quotes) and wishes to restrict a pool of potential suppliers.",
"In the at least part of ensuing description, a method and system to qualify a party to transact with respect to an item utilizing a commerce system in the exemplary form of network-based auction facility are described.",
"It will be appreciated that the method and system are also applicable to a commerce system in the form of a network-based fixed-price facility or a commerce system that provides a multitude of transaction processes (e.g., any number of types of auction transaction processes and any number of fixed-price transaction processes).",
"In various embodiments of the present invention, a first party is empowered to use different mechanisms to qualify parties to transact for a particular item.",
"For example, considering an auction facility as an example of a commerce system, a seller may view a potential bidder's bidding history and profile to determine whether or not to pre-approve the bidder to bid on a listing.",
"It is understood that if the vetting process is too strict, the conversion rate on the item will be affected.",
"To mitigate, in one embodiment, the commerce system educates the sellers to use proper vetting mechanism to choose their bidders.",
"Also, in one embodiment, the seller may remove the pre-approval restriction anytime during an auction.",
"For example, the seller may wish to take the risk to open the listing to all potential bidders if the pre-approval restriction produces no bids or the bids amounts are low.",
"In one embodiment, the seller may add and remove the pre-approval restriction multiple times during the auction.",
"In one embodiment, the seller may remove a bidder from the pre-approved list after the seller has added the bidder to the pre-approved list.",
"In one embodiment, the seller may add bidders to and remove bidders from the pre-approved list multiple times during the auction of the listing.",
"In one embodiment, the seller may request the pre-approval of bidders for his/her listing without specifying any bidders initially, and then add the bidders to the pre-approved list from time to time.",
"In one embodiment, the seller has the choice to apply the pre-approve bidders list from a prior or current listing to all on-going listings with the auction facility and/or any future listings.",
"In one embodiment, the seller may pre-approve the bidders individually or in a bulk.",
"In one embodiment, the seller may view the list of the pre-approved bidders and their respective Usernames/email addresses by logging on to the auction facility web site and providing the listing identification number.",
"In one embodiment, the parties within a specific geographical region (e.g.",
"the United States of America) may be automatically pre-approved to bid on an item.",
"In one embodiment, only the predetermined currencies can be used to bid on a listing.",
"Further, while the exemplary embodiment described herein describe sellers as qualifying potential buyers, it will be appreciated that the invention may be utilized by buyers to qualify potential sellers.",
"In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention.",
"It will be evident, however, to one skilled in the art that the present invention may be practiced without these specific details.",
"For the purposes of the present specification, the terms “items” shall be deemed to include products, goods and services.",
"The term “party” shall be deemed to include any party (human or automated) that is capable of transacting for an item or utilizing commerce system.",
"The term “party” shall accordingly include buyers, sellers, shoppers, customers, bidders etc.",
"Qualification/Disqualification of a Party to Transact-Auction Facility Exemplary Embodiment FIG.",
"1 is block diagram illustrating an exemplary commerce system in the form of an Internet-based auction facility 10.While an exemplary embodiment of the present invention is described within the context of an auction facility 10, it will be appreciated by those skilled in the art that the invention will find application in many different types of network-based commerce systems.",
"The auction facility 10 includes an authorization module 40 and a communications module 42.The authorization module 40 includes CGI servers 18 (or application servers) that provide an intelligent interface to the back-end of the auction facility 10, a database engine server 22 and a database 23.The communications module 42 includes one or more of a number of types of front-end servers, namely page servers 12 (or Web servers) that deliver web pages (e.g., markup language documents), picture servers 14 that dynamically deliver images to be displayed within Web pages, listing servers 16, CGI servers 18, and search servers 20 that handle search requests to the facility 10.E-mail servers 21 provide, inter alia, automated e-mail communications to the users of the auction facility 10.The back-end servers include a database engine server 22, a search index server 24 and a credit card database server 26, each of which maintains and facilitates access to a respective database.",
"The Internet-based auction facility 10 may be accessed by a client program 30, such as a browser (e.g., the Internet Explorer distributed by Microsoft Corp. of Redmond, Wash.) that executes on a client machine 32 and accesses the auction facility 10 via a network such as, for example, the Internet 34.The sellers and the buyers (or bidders) access the auction facility 10 through the client machines 32.Other examples of networks that a client may utilize to access the auction facility 10 include a wide area network (WAN), a local area network (LAN), a wireless network (e.g., a cellular network), the Plain Old Telephone Service (POTS) network or as the Public Switched Telephone Network (PSTN).",
"FIG.",
"2 illustrates an exemplary web home page 100 that may be generated by the Internet-based auction facility 10.The home page 100 includes a “seller services” link 102, which provides access to the seller services page.",
"The seller services page, in turn, includes a buying and selling tools link, which provides access to the buying and selling tools page.",
"The buying and selling tools page, in turn, includes a pre-approve bidders link, which provides access to the pre-approve bidders logon web page.",
"FIG.",
"3 illustrates an exemplary pre-approve bidders logon web page 200 that may be generated by the Internet-based auction facility 10.The pre-approve logon page 200 prompts the seller to provide a proper username 202 and password 204.When the seller provides the proper username 202 and password 204, the logon page 200 provides access to the pre-approve bidders main web page.",
"FIG.",
"4 illustrates an exemplary pre-approve bidders main web page 300 that may be generated by the Internet-based auction facility 10.The pre-approve bidders main page 300 displays currently active auction listings 302 and past auction listings 304 for the particular seller.",
"The pre-approve bidders main page 300 includes an “edit” link 306, which allows the seller to edit the pre-approve bidders list for the corresponding listing 302.The editing can include adding bidders to or subtracting bidders from the pre-approve bidders list.",
"The pre-approve bidders main page 300 also includes a “deactivate” link 308, which allows the seller to deactivate the pre-approve bidders list such that potential bidders need not seek the seller's authorization to bid on the listing.",
"If the seller removes the pre-approval restriction during an auction, the auction facility 10 requests the seller to inform the pre-approved bidders that the listing is now available to all potential bidders.",
"In one embodiment, the seller can inform the pre-approved bidders of the removal of the pre-approval restriction through email.",
"The pre-approve bidders main page 300 also includes an “add a new item” link 310, which provides access to the pre-approve bidders form page.",
"FIG.",
"5 illustrates an exemplary pre-approve bidders form web page 400 generated by the Internet-based auction facility 10.The pre-approve bidders form page 400 prompts the seller to provide an item number 402.The item number 402 can be provided by the auction facility 10 and corresponds to the item for which the seller wishes to pre-approve the bidders.",
"The form page 400 also prompts the seller to add or remove the identifiers 404 for the bidders whom the seller wishes to authorize to bid on the particular item.",
"The identifier 404 can include the bidder Username.",
"The bidder identifiers 404 that are added to the form page 400 are stored in an authorized bidders table described below with reference to FIG.",
"7.The view item web page described below with references to FIG.",
"6A and 6B is updated to include the information submitted through the form page 400.In one embodiment, if the seller's username 302 does not match with the item number 402, the auction facility 10 prompts an error message asking the seller to recheck the item number 402.In one embodiment, if the Username for the bidder does not match with a Username in the bidder table in the database 23, the auction facility 10 prompts an error message indicating that the bidder is not registered, suspended, terminated or merged.",
"FIGS.",
"6A and 6B illustrate an exemplary view item web page 500 generated by the Internet-based auction facility 10.If the seller has requested pre-approval restriction for the item, the auction facility 10 flashes an error message 520 when the unauthorized bidders attempt to bid on the item.",
"An exemplary error message 520 is illustrated in FIG.",
"6C.",
"The error message 520 advises the unauthorized bidder to contact the seller to seek the pre-approval to bid.",
"The error message 520 can appear in the bidder box area 510.In one embodiment, if the potential bidder is on the pre-approve bidders list to bid on this item, the auction facility 10 prompts him/her with a message to continue with the bidding process.",
"The view item page 500 includes an “about me” page link 502, which provides access to an about me web page.",
"The unauthorized bidders may visit the about me page for more details regarding the seller, including the seller's vetting process/guidelines.",
"In one embodiment, the “about me” page link 502 is added in the item description area 504.In one embodiment, when the seller removes the pre-approval restriction, the restricted message is removed from the bid box area.",
"In one embodiment, the seller can request pre-approval restriction after the auction has begun for the remaining time on the auction.",
"In such a case, in one embodiment, the seller can manually cancel the bids made prior to the implementation of the pre-approval restriction.",
"Database Structure The auction facility 10 provides the seller with information regarding the potential bidder such that the seller can make an informed determination regarding whether to pre-approve the bidder.",
"The information may include the bidder's bidding history and feedback profile.",
"The information is included in the database 23.In one embodiment, the seller provides the auction facility 10 with the bidder contact information to obtain information regarding the bidder.",
"The bidder contact information can include the bidder Username or email address.",
"The seller may obtain the bidder contact information directly from the bidder or from the auction facility 10.The auction facility 10 matches the contact information provided by the seller with the contact information stored in the database 23 to provide the seller with user information.",
"FIG.",
"7 is a database diagram illustrating an exemplary database 23, maintained by and accessed via the database engine server 22, which at least partially implements and supports the auction facility 10.The database 23 may, in one embodiment, be implemented as a relational database, and includes a number of tables having entries, or records, that are linked by indices and keys.",
"In an alternative embodiment, the database 23 may be implemented as a collection of objects in an object-oriented database.",
"The database 23 includes a bidder (or party) table 602, which contains a listing of the registered bidders of the auction facility 10.The bidder table 602 can also be referred to as the user table because each user may operate as both a bidder and a seller within the auction facility 10.The bidder table includes a link to a bidding history table 604 for each registered bidder.",
"Each bidding history table 604 is populated with the particular bidder's bidding history records.",
"Each bidding history record may include, inter alia, the title of a listing that was/is being auctioned via the auction facility 10, the bidder's bidding amount, and bid retraction information.",
"The bid retraction information indicates whether the bidder retracted his/her bid on a particular item.",
"Two other tables are also shown linked to the bidder table 602, namely a bidder feedback profile summary table 606 and a bidder feedback profile details table 608.The database 23 also includes an authorized bidders table 610 for each item for which the seller has requested the pre-approval of the bidders.",
"The authorized bidders table 610 includes a list of bidder identifiers 404 that are authorized to bid on the particular item.",
"The bidder identifier 404 can include the bidder Username.",
"FIG.",
"8 is a diagrammatic representation of an exemplary embodiment of the bidder feedback profile summary table 606.The summary table 606 stores a summary of the feedback information regarding the bidders.",
"Sellers and bidders that have experienced a particular bidder's behavior during the past auctions provide the feedback information (or comments) regarding to the bidder.",
"The summary table 606 includes a bidder identifier column 702 that stores, for each bidder, a bidder identifier providing a pointer to the bidder table 602.The total score column 704 stores the total number of feedback comments (e.g., negative, positive and neutral) received for each bidder.",
"The total negative column 706 stores the total number of negative feedback comments received for each bidder, and the total positive column 708 similarly stores the total number of positive feedback comments received for each bidder.",
"The number of retractions column 710 stores the total number of threads that each bidder has retracted from auctions.",
"The summary table 606 provides a summary of the impressions of the users of the auction facility 10 regarding a particular bidder.",
"Each bidder of the summary table 606 is linked to a bidder feedback profile details table 608.It is contemplated that other embodiments of the summary table 606 can include additional information, such as whether the bidder has a credit card on file with the auction facility 10 and whether the bidder is agreeable to use of an online payment service (e.g., Billpoint).",
"FIG.",
"9 is a diagrammatic representation of one embodiment of the bidder feedback profile details table 608.The details table 608 is populated with entries reflecting the details of each feedback comment or opinion submitted by users to the auction facility 10 regarding a particular bidder.",
"Typically, the users submitting the comments include sellers on whose auction listings the bidder has bid in the past.",
"In one exemplary embodiment, the users are only permitted to provide feedback pertaining to a transaction upon conclusion of that transaction.",
"The feedback details table 608 includes the item number column 802 that identifies the items for which the comments were submitted.",
"The comment column 804 stores the actual texts of the feedbacks, comments, or opinions.",
"The type column 806 stores the indications as to whether the comments are positive, negative or neutral.",
"The date column 808 stores the dates on which the feedbacks, comments or opinions were received.",
"The response column 810 stores the texts of the responses submitted by the bidder in response to the comments texts stored in column 804.Similarly, the rebuttal column 812 stores the texts of the rebuttals to such responses.",
"The commentator column 814 stores the identifiers of the users that submitted the original comments stored in column 804.It is appreciated that further dates and other descriptive information may also populate the details table 608.The tables 602, 604, 606 and 608 include information that can provide the seller with valuable insights when evaluating a potential bidder.",
"In one embodiment, the information contained in the tables 602, 604, 606 and 608 is easily accessible to the sellers.",
"The seller can provide the bidder's identifier such as the Username or email address to access the information stored in the tables 602, 604, 606 and 608.It is contemplated that the databases of alternate embodiments can include additional tables that provide additional bidders related information.",
"FIG.",
"10 illustrates the flow chart of one embodiment of a method 900 for seller authorized bidding through an Internet-based auction facility 10.It will be appreciated by those skilled in the art that with certain modifications the method 900 is applicable in many different types of network-based, and network-based, commerce systems.",
"At block 902, a seller registered with the auction facility 10 logs on to a website that provides access to the auction facility 10.If the seller were already logged on, then he/she need not logon again to use request the pre-approval restriction.",
"The suspended, merged, or terminated seller who cannot use any other feature on the auction facility 10 is prohibited from using the seller authorized bidding feature.",
"At block 904, the seller identifies the item for which he wishes to add the pre-approval restriction.",
"At block 906, an alert text appears on the item web page to alert the potential bidders to get a pre-approval from the seller to bid on the item.",
"The item web page or another web page linked to the item web page provides the potential bidder with the seller contact information and vetting process information.",
"At block 908, the potential bidder contacts the seller and requests permission to bid on the item.",
"In one embodiment, the bidder must be registered with and logged on to the auction facility 10.The bidder provides the seller with a bidder identifier, such as the Username or email address.",
"At block 910, the seller uses the bidder identifier to retrieve and view the bidder's bidding history and profile information.",
"At block 912, the seller determines whether to add the bidder to the pre-approve bidders list (i.e., whether to qualifying the bidder to transact (e.g., bid) for the item).",
"At block 914, if the determination is positive, the seller adds in the bidder identifier identifying the potential bidder to the pre-approve bidders list.",
"The bidder identifier is then added to an appropriate authorization table within the database 23.At block 916, if the determination is negative, the bidder identifier is not added to the authorization table.",
"In one embodiment, the potential bidder is informed through email that the seller has rejected his/her request for pre-approval.",
"At block 918, the seller may edit the pre-approve bidder list.",
"The editing can include the addition of the potential bidder to the list that was rejected at block 912.The editing can also include the removal of a bidder from the list.",
"When a bidder attempts to bid on an item, the authorization module 40 checks whether the bidder identifier is included in the item authorization table.",
"If the bidder identifier is included in the item authorization table, the bidder is allowed (or enabled) to bid on the item.",
"If the bidder identifier is not included in the item authorization table, the bidder receives an error message.",
"Automatic Qualification/Disqualification of a Party to Transact The embodiment of the present invention described above implemented a partially manual qualification/disqualification process where a first party (e.g., a seller) manually qualified or disqualified a second party (e.g., a potential buyer) to transact for an item via a network-based commerce system (e.g., of the auction facility 10).",
"Thereafter, the qualification of the second party was performed by having the first party submit identification criteria (e.g., a username or e-mail address), identifying the second party, to the network-based commerce system.",
"The network-based commerce system then automatically qualifies the second party to transact for an item when the second satisfies the identification criterion (e.g., when the identity of the second party is confirmed through an appropriate login process).",
"A further embodiment of the present invention is described below wherein the automatic qualification of the second party to transact for an item within a commerce system may be performed utilizing a broad scope of criteria.",
"In this way, a first party (e.g., seller) can potentially have the commerce system automatic qualify or disqualify a second party from transacting with respect to a specific item, or with respect to a number of items, associated with the first party without requiring that the first party manually to approve or disapprove the second party.",
"FIG.",
"11 is a block diagram illustrating an exemplary commerce system 1100 that may at least partially perform the automatic qualification, or disqualification, of a second party to transact for an item.",
"The commerce system 110 may implement any one or more of a number of transaction processes, such as auction, fixed price, reverse auction, declining price auction, or bulk-purchase processes.",
"The commerce system 1100 illustrated in FIG.",
"11 includes an application server 1102 (e.g., the CGI server 18 illustrated in FIG.",
"1) that communicates with a web server 1104 (e.g., a page server 112 as illustrated in FIG.",
"1) and a database engine server 1106 (e.g., the database engine server 22 illustrated in FIG.",
"1).",
"The application server 1102 hosts a number of application modules that perform functions related to the operation of the commerce system 1100.For example, the application server 1102 is shown to include a qualification module 1108 that operates, in a manner described below, to qualify parties to transact with respect to an item via the commerce system 110.The application server 1102 also incorporates a transaction module 1110 that operates to implement a transaction process (e.g., an auction or fixed price transaction process) via which an item may be transacted between two or more parties.",
"Data required by the various modules of the application server 1102 is requested by, and communicated to, the application server 1102 from the database engine server 1106.To this end, the database engine server 1106 may host a number of queries, or stored procedures, that operate to retrieve requested data from a database 1112.For example, the database engine server 1106 is shown to host item records queries 1114, criteria queries 1116 and profile queries 1118.The utilization of these queries will be described in further detail below.",
"The application server 1102 also communicates data to, and receives data from, a web server 1104.The web server 1104 is responsible, in one embodiment, for the generation and transmission of user interface information (e.g., a markup language document such as an HTML document) that may be utilized by a client application (e.g., a browser) executing on a computing device (e.g., a personal computer, Personal Digital Assistant (PDA), mobile telephone, etc.)",
"to generate a user interface for the display of data to, and the receipt of data from, a user of the commerce system 110.To this end, the web server 1104 is shown to include a page build module 1120 to construct user interface information and a parser 1122 utilized to deconstruct data transmissions received via a communications network (e.g., the Internet 34).",
"Having now provided an architectural description of an exemplary commerce system 1100, a description of the operation of the exemplary commerce system 1100 will be provided below with reference to a number of flow charts and user interface diagrams.",
"FIG.",
"12 is a flow chart illustrating a method 1200, according to an exemplary embodiment of the present invention, whereby a first party (e.g., a seller user of the commerce system 1100) may define and specify criteria to be satisfied by a second party (e.g., a buyer user of the commerce system 1100) in order for the second party automatically to be qualified to transact for a specific item, or for a number of items (e.g., all items offered for sale by the first party).",
"It will accordingly be appreciated that the specified criteria may be associated with a specific item, or associated with a specific party (e.g., the first party).",
"The method 1200 commences at block 1202 at the commerce system 1100, with the generation and transmission to a first party of data specifying (or relating to) an item and criterion information input user interface.",
"At block 1204, the item and criterion information input user interface is generated and displayed to the first party (e.g., a selling user) on a computing device of the first party.",
"FIG.",
"13 is a diagrammatic representation of an item and criterion information input user interface 1300, according to one exemplary embodiment of the present invention.",
"The input user interface 1300 is shown to include an item information portion 1302 and a criteria information portion 1304.The item information portion 1302 is shown to include a number of input fields into which the first user may input item information, such as name, price, transaction preference, item description and transaction condition (or parameter) information.",
"Similarly, the criteria information portion 1304 includes a number of input fields into which the first party may optionally input criteria that must be satisfied by a second party in order to qualify the second party to transact with respect to the item described in the item information portion 1302, and according to the transaction preferences and transaction conditions described in the item information portion 1302.It will be appreciated that the criteria options that are presented in the criteria information portion 1304 may be dependent upon the type of transaction being facilitated by the commerce system 1100, the nature of the item to which the transaction pertains, user preferences and any number of variables.",
"It will further be appreciated that criteria information types (e.g., the non-exhaustive list of exemplary criteria options described below) may also be used for the purpose of manual qualification of party to participate in a network-based commerce transaction.",
"For example, each of the criteria options described below may be retrieved from the bidders bidding history or profile information thereby enabling the seller to make a determination whether the bidder may be added to a preapproved bidders list.",
"FIG.",
"13 provides a non-exhaustive list of exemplary criteria options that may be presented to the first party.",
"A geographic criterion (or constraint) to be satisfied by a qualified second party may specify a geographic location (e.g., continent, country, state, city, town, zip code) in which a second party must decide to qualify to transact.",
"A geographic constraint may also be expressed as a distance from a predetermined location (e.g., a maximum distance from the hometown of the first party).",
"An age criteria (or constraint) may restrict a qualified second party to exceeding a predetermined minimum age threshold, to being below a predetermined maximum age threshold, or to be within a particular age range (e.g., 20-34 years old).",
"A reputation criterion (or constraint) may require that a qualified second party have a minimum predetermined reputation within the commerce system 1100.For example, the commerce system 1100 may implement a reputation system whereby the reputation of a particular user is expressed according to a particular scale or as a score.",
"Any number of factors may contribute towards the establishment and definition of a reputation of a user, such as feedback from parties with whom the relevant user has interacted utilizing the commerce system 1100 (e.g., a number of negative of positive feedback comments), a history of transaction activity by the relevant user with respect to the commerce system 1100, and a history of violations of rules established by the commerce system 1100.A reputation measure may also be established by other factors, such as the amount of time that a particular user has been an active or registered user of the commerce system 1100, the age of the user, a financial standing of the user, etc.",
"A prior activity criterion (or constraint) may require that a qualified second party not have undertaken, or engaged in (or alternatively have positively undertaken or engaged in) a specified prior transaction activity.",
"For example, the prior activity criterion may dictate that a qualified second party not have previously retracted a bid within an auction transaction process facilitated by the commerce system 1100, or that the qualified second party not have retracted more than a predetermined maximum threshold number of bids within one or more auction transaction processes, optionally within a predetermined time.",
"On the other hand, the prior activity criterion may require that a qualified second party have made a payment to a further party with which the second party transacted within a predetermined time period, or have delivered a purchased item within a predetermined time period or in a predetermined condition, in order for the second party to be qualified.",
"A time/date criterion (or constraint) may limit the time/date during which a second party is qualified to transact with respect to an item, or may act as a supplement criterion to the define a further criterion.",
"For example, in the time/date criterion may be utilized to identify a time interval within which the prior activity specified by the prior activity criterion must have occurred in order to qualify or disqualified the second party (e.g., may specify a time interval within which a predetermined number of bid retractions must have occurred in order to disqualify the second party).",
"A financial criterion (or constraint) may require that a qualified second party, for example, have a credit rating above a predetermined minimum value, or not have previously been declared bankrupt.",
"A financial criterion may also require that a qualified second party have a history of making payment within a predetermined maximum time period, or have a predetermined amount of funds (or credit resource) within an account with the commerce system 1100, or with a financial institution associated with or accessible by the commerce system 1100.The financial criterion may also require that to the second party have a credit card on record with the commerce system 1100, or agreed to use a particular payment service (e.g., Billpoint or PayPal).",
"A language criterion (or constraint) may require that a qualified second party indicate a predetermined language preference, or have previously transacted via the commerce system 1100 in a particular language, in order to qualify.",
"Similarly, a currency activity criterion (or constraint) may require that a qualified second party have previously transacted in a predetermined currency.",
"Finally, the exemplary criterion information may also allow the first party to implement a manual override for a fully automatic approval process, whereby manual approval by the first party of a second party is required in order to finally qualify the second party to transact, even if the second party succeeds in satisfying the criterion associated with a particular item.",
"The input user interface 1300 may optionally also allow the first party to specify relationships between one or more criterion so as to facilitate the formulation of a “qualification formula” or a complex qualification policy that is expressed in terms of multiple criterion.",
"For example, the input user interface 1300 may facilitate specification of an AND or OR operation between two or more criterion.",
"In this matter, the first party may, for example, specify qualification formula that requires that a qualified second party reside within a predetermined geographic area and not have received more than a predetermined number of negative feedback comments.",
"Returning now to the method 1200 illustrated in FIG.",
"12, having generated and displayed the input user interface at block 1204, at block 1206 the first party (e.g., a selling user) inputs items and criterion information into the item and criterion information input user interface.",
"At block 1208, the inputted item and criterion information is transmitted from the first party to the commerce system 1100.At block 1210, the commerce system 1100 generates an item record that is written into an items table within the database 1112 and a criteria record that is written into a criteria table, also maintained within the database 1112.Specifically, upon receipt of the transmitted item and criterion information at a web server 1104, the parser 1122 extracts the item and criterion information, which is then communicated to the database engine server 1106.The database engine server 1106 proceeds to build the appropriate records and write them into the appropriate tables within the database 1112.FIG.",
"14 is a diagrammatic representation of an exemplary items table 1400, and indicates the various fields that may be populated for each record within this table 1400.FIG.",
"15 is a diagrammatic representation of an exemplary criteria table 1500.Each record within the criteria table 1500 is shown to include a criteria identifier 1502 that may operate as a primary key for the table 1500, and be utilized to associate a particular criterion record with one or more item records within the items table 1400 or with one or more party (e.g., user) records of within a party table 602.A record within the criteria table 1500 may also include an Approved Second Party entry 1504 that identifies the parties (e.g., users) for which records exist within the party table 602 and that have been manually approved to transact by the first party.",
"In an alternative embodiment, as discussed above, a Preapproved Parties table may be maintained separate of the criteria table 1500.An entry within the Approved Second Party field 1504 of a particular record within the criteria table 1500 may also be linked to a record within an authorized party table 610.Having received item information relating to and describing an item, and criterion information specifying at least one criterion to be satisfied by a second party in order for the second party to be qualified to transact for an item, according to one embodiment of the present invention, a criteria enforcement process is implemented by the commerce system 1100.In one embodiment, the criteria enforcement process involves automatically determining whether a second party satisfies at least one criterion specified by the criterion information and, if so, then automatically qualifying the second party to transact for an item, or group of items, via the commerce system 110.As described above, through the item and criterion information input user interface 1300, the commerce system 1100 allows a first party to specify one or more criterion to be satisfied by a second party to qualify to transact.",
"As also described above, the first user, when specifying multiple criteria, can define a qualification formula or function utilizing the multiple criteria.",
"For example, the first party has the option of specifying AND and OR relationships between individual criterion so as to construct a customized qualification formula (or function).",
"In one embodiment, the criteria enforcement process is performed by the qualification module 1108 of the application server 1102.The qualification module 1108 makes an assessment as to whether a second party satisfies one or more criterion specified by a first party utilizing input received from the second party via a user interface and communicated to the web server 1104, or utilizing data regarding the second party extracted from the database 1112 by the database engine server 1106.FIG.",
"16 is a flow chart illustrating a first exemplary method 1600 whereby a commerce system 1100 may automatically qualify a second party to transact, or disqualify a second party from transacting, with respect to a particular item, or group of items.",
"The method 1600 commences at block 1602 with the generation and transmission of navigation user interface data from the commerce system 1100 to a second user (e.g., to a personal computer or mobile device operated by the second user).",
"Specifically, the page build module 1120 of the web server 1104 may, in one embodiment, construct a markup language document that instructs the generation of a suitable navigation user interface to the second party.",
"The navigation user interface may facilitate navigation of a large number of items offered for transacting by the commerce system 1100 according to any one or more of a number of transaction processes.",
"For example, the navigation user interface may allow the second party to perform key word searches of item descriptions contained in the items table 1400.The navigation user interface may also allow the second party to locate items of interest by browsing established categories supported by the commerce system 1100.For example, with reference to FIG.",
"14, it will be noted that the items table 1400 includes a category field whereby a particular item may be conveniently categorized according to a category scheme supported by the commerce system 1100.At block 1604, a navigation user interface is generated and displayed to the second party.",
"For example, where the navigation user interface data comprises a markup language document, a browser operating on a personal computer of the second user may utilize the user interface data to generate and display the navigation user interface.",
"At block 1606, the second party (e.g., a buyer user), inputs navigation information into the navigation user interface with the purpose of locating one or more item records that are of interest.",
"As mentioned above, the second party may, for example, provide search key words, or specify a certain item category, with a view to locating item records of interest.",
"At block 1608, the navigation information inputted into the navigation user interface, as well as an identifier identifying the second party, is transmitted to the commerce system 1100.For example, the identifier identifying the second party may be a session ID established during an online session between the second party and the commerce system 1100, or an identifier extracted from a cookie stored on a computing device operated by the second party.",
"Further, the identifier for the second party may be a user ID entered by the second party into the navigation user interface (or a preceding logon interface) At block 1610, the navigation information is received by the commerce system 1100, and specifically by the parser 1122 of the web server 1104 that operates to extract the navigation information from a network transmission.",
"The parser 1122 then communicates the navigation information (e.g., a search term or a category identifier) to an appropriate item records query 1114 that locates item records within the items table 1400 according to the navigation information.",
"The located item records are then communicated from the item records query 1114 to the qualification module 1108.At block 1612, in one embodiment, identifiers for the located item records are communicated from the item records query 1114 to a criteria query 116 that, at block 1612, accesses, searches and locates criteria records within the criteria table 1500 that are associated with the located items.",
"As noted above with reference to FIG.",
"16, a criteria identifier 1502 may map to one or more records within the items table 1400.A criteria record within the criteria table 1500 may also be associated with a particular party, for which a record exists within the party table 602.In this case, an appropriate criteria query 1116 may operate to identify a first party associated with a specific located item record (e.g., a seller), and then perform a query against the party table 602 to identify one or more criteria identifiers 1502 associated with the relevant first party.",
"Having then located a criteria identifier associated with the first party, the criteria query 116 may then access the criteria table 1500 to locate and retrieve an appropriate criteria record.",
"It will be appreciated that where multiple located item records exist, different criteria records may be associated with each of these item records, either directly or indirectly through a first party (e.g., a seller).",
"Accordingly, a criteria record may in this way be associated with each of the located item records.",
"At decision box 1614, for each located item record, a determination is made as to whether a criterion, or multiple criteria, specified within a criteria record associated with the item record is satisfied.",
"It will of course be appreciated that this determination is dependent upon the criterion specified by the appropriate criteria record, and optionally also by the nature of the criteria formula (or function) that may be expressed in terms of such multiple criterion.",
"Examples of criterion that may be specified are described above with reference to the input user interface 1300 illustrated in FIG.",
"13 and the criteria table 1500, illustrated in FIG.",
"15.In order to assess whether a single criterion, multiple criteria, or even a criteria formula is satisfied by the second party, it will be appreciated that information regarding the second party is required.",
"In one exemplary embodiment, the determination made at decision block 1614 is made by the qualification module 1108 of the application server 1102, utilizing criteria records located by one or more criteria queries 1116, and profile data concerning the second party extracted by one or more profile queries 1118 from profile tables maintained within the database 1112.The identifier for the second party, transmitted to the commerce system 1100 at block 1608 is parsed by the parser 1122 of the web server 1104, and utilized at decision block 1614 to identify profile records within profile tables for the second party.",
"FIG.",
"18 provides diagrammatic representations of two such exemplary profile tables that may be maintained within the database 1112, namely a master profile table 1800 and an activity profile table 1802.The exemplary master profile table 1800 is populated with records for each party that has registered to utilize the commerce system 1100.Each record contains personal information regarding the appropriate party that may be voluntary submitted by the relevant party, or gleaned from external sources.",
"For example, a record within the master profile table 1800 may indicate the address, language preference, currency preference, age and credit rating of an appropriate party.",
"The address, language preference, currency preference and age information may be gleaned from the relevant party as part of a registration process for utilization of the commerce system 1100.The credit rating information may, as illustrated, be obtained from a third party credit bureau.",
"The activity profile table 1802 may similarly contain a record for each party that utilizes the commerce system 1100.However, this profile table 1802 may contain information reflecting behavioral and transactional characteristics of the relevant party, as observed or tracked by the commerce system 1100 over a period of time.",
"Both positive and negative characteristics or activities of a particular party may be recorded within the profile table 1802.For example, the exemplary activity profile table 1802 is show to maintain an indication of a number of bids retracted by a particular party within the context of auction process transactions.",
"A record within the activity profile table 1802 may also indicate a number of payment failures associated with the relevant party, a number of delivery failures associated with the relevant party, a total of number of complaints received against, or issued by, the relevant party, and a total number of violations of rules of the commerce system 1100 attributable to the relevant party.",
"It will of course be appreciated that a wide range of other activities and characteristics of a particular party may be tracked within one or more tables similar to the activity profile table 1802.Of course, the activities or characteristics tracked within a profile table 1802 may be such so as to support the various criteria options that may be presented to a first party within a criterion information portion 1304 of an item and criterion information input user interface 1300.In the exemplary embodiment, the qualification module 1108, having collected one or more criteria records and the appropriate profile information regarding the second user, is able to make a determination as to whether the second party satisfies one or more criterion associated with a specific item record.",
"If the criteria expressed by a criteria record associated with a particular item record are determined to have been satisfied at decision block 1614, at block 1616, the relevant item record is added to a set of navigation results.",
"The set of navigation results includes, in one embodiment, only those item records for which the second party is identified as being a qualified party.",
"At decision block 1618, a determination is made as to whether there are further item records for which associated criteria must be assessed.",
"If so, the method 1600 loops back to decision block 1614.Similarly, if the criteria associated with a particular located item record are determined at decision block 1614 not to be satisfied, the method 1600 progresses directly from decision block 1614 to decision block 1618.Once it has been determined at decision block 1618 that no further item records require consideration, the method 1600 progresses to block 1620.At block 1620, the commerce system 1100, and specifically the page build module 1120 of the web server 1104, generates and transmits navigation result user interface data to the user.",
"The navigation result user interface data includes an identifier for each of the located item records that were included within the navigation results at block 1616.The page build module 1120 receives the navigation results from the qualification module 1108.In addition to communicating the navigation results to the page build module 1120, the qualification module 1108 also operates to enable the second party to transact for items identified in the navigation results.",
"In one embodiment, this enablement is achieved by including an identifier for the second party within the Approved Second Party field 1504 of the appropriate criteria table 1500.In an alternative embodiment, the items table 1400 may include an Approved Second Party field (not shown) in which an indication of second parties that have been approved, manually or automatically, is stored.",
"At block 1628, an application executing on a computing device (e.g., a browser executing on a personal computer) generates and displays a navigation result user interface, identifying the navigation results.",
"At block 1624, the second party selects an item from the navigation results to transact.",
"From block 1624, a transaction process, facilitated in one embodiment by a transaction module 1110 of the application server 1102, is commenced.",
"Further details regarding an exemplary transaction process are described below with reference to FIG.",
"17.In summary, it will be appreciated that the exemplary criterion enforcement process implemented by the method 1600 operates to enable a qualified second party to transact with respect to an item by only presenting details regarding the item to the second party once the second party has been qualified.",
"In other words, the qualification process acts as a filter so that the second party is only presented with the details for items for which the second party has been automatically (or manually) pre-qualified.",
"This exemplary embodiment has the advantage of not frustrating the second party by allowing the commencement of a transaction process with respect to an item for which the second party may not qualify to transact.",
"FIG.",
"17 is a flow chart illustrating a second exemplary enforcement process that may be implemented by a method 1700.Blocks 1702-1710 correspond substantially to the operations performed at blocks 1602-1610 described above with reference to FIG.",
"16.Moving on to block 1712, a set of navigation results is constructed to include all item records located at block 1710 utilizing the navigation information (e.g., a search term or a category identifier).",
"The method 1700 thus differs from the method 1600 in that the navigation results include all record items located by a search and not only item records for which a particular second party qualifies.",
"At block 1714, the page build module 1120 of the web page server 1104 builds navigation results user interface data utilizing the navigation results as received directly from an item records query 1114 and transmits this navigation result user interface data to a computing device operated by a second party for display.",
"At block 1716, an appropriate application executing on a computing device operated by the second party (e.g., a browser being executed on a personal computer) operates to generate and display a navigation result user interface that includes identifiers for each item record included within the navigation results.",
"At block 1718, the second party selects one or more items from the navigation results for which the second party wishes to commence a transaction process utilizing the commerce system 1100.For example, the second party may select a Uniform Resource Locator (URL) associated with a particular item to perform the selection of the item.",
"At block 1720, information identifying the selected item is transmitted from the computing device operated by the second user to the commerce system 1100.For example, an HTTP PUT request may be dispatched from the computing device responsive to user selection of a URL associated with the selected item.",
"At block 1722, the parser 122 of the web server 1104 receives the data transmission initiated at block 1720, and parses the transmission to locate an identifier identifying the selected item.",
"The parser 1122 then communicates the extracted identifier to an appropriate item record query 1114 of the database engine server that performs access, search and location operations with respect to the criteria table 1500 to identify a criteria record, specifying one or more criterion, associated with the selected item.",
"It will of course be appreciated that this lookup operation may involve initially performing a lookup on an items table to obtain a relevant criteria ID with which to perform a lookup on the criteria table 1500.Again, a criteria record may be associated directly with an items record, or may be associated indirectly with an item record through being associated directly with a party record for a party (e.g., the first party acting in the capacity of a seller) associated with an appropriate items record.",
"At decision block 1724, the qualification module 1108 of the application server 1102 operates to make a determination regarding whether a single criterion, or criteria, specified by the located criteria record is satisfied.",
"As described above with reference to FIG.",
"16, this determination at block 1724 may, in one embodiment, involve the qualification module 1108 invoking one or more profile queries 1106 against profile tables stored within the database 1112 in order to retrieve profile information (e.g., from a master profile table 1800 and an activity profile table 1802) pertaining to the second party.",
"The second party may be identified in any one of a number of ways (e.g., utilizing a session identifier initiated following a logon process, or utilizing identify information stored within a cookie on a computing device of the second party).",
"Following a positive determination by the qualification module 1108, at block 1726, the second party is qualified and enabled to transact for the relevant item.",
"As described above, the enablement of the second user may involve storing appropriate identifier information within a relevant record within the criteria table 1500, the items table 1400 and/or the party table 602.As a result of the qualification and enablement at block 1726, the transaction module 1110 is invoked to generate and communicate transaction information pertaining to the relevant item to the page build module 1120, which then generates and transmits transaction user interface data to a computing device of the second user.",
"At block 1730, an application executing on the client computing device (e.g., a browser executing on a personal computer) operates to generate and display a transaction user interface.",
"The transaction module 1110 may operate to facilitate one or more transaction processes (e.g., a regular auction process and/or a fixed price process) via which the item may be transacted.",
"Accordingly, the transaction user interface data, and the transaction user interface itself will reflect information concerning the one or more transaction processes supported by the transaction module 1110.At block 1732, the second party then inputs appropriate transaction data.",
"For example, within an auction process, the transaction data may include a bid specifying a price and other information specific to the auction type.",
"In the case of a fixed price process, the transaction data may be acceptance of an offer to purchase an item at a fixed price, or an offer to purchase an item at a particular fixed price.",
"At block 1734, the transaction data is transmitted by the computing device of the second party to the commerce system 1100 for processing by the transaction module 1110 within the context of an appropriate transaction process.",
"Returning to decision block 1724, following a negative determination at block 1726, the second party is disqualified and disabled from transacting for the particular item, and a decline user interface data is generated and transmitted to the second party at block 1738.At block 1740 an application executing on a client machine operated by the second party generates and displays a decline user interface, advising the second party that the second party has been disqualified from transacting for the item.",
"In this case, the decline user interface may optionally provide one or more reasons to the second party for the disqualification by the qualification module 1108.For example, the second party may be advised that he or she has been disqualified as a result of an excessive number of retracted bids within a predetermined time period (e.g., one month) preceding a current date.",
"In summary, the exemplary criteria enforcement process implemented by the method 1700 is advantageous in that the qualification assessment, performed at block 1724, is only performed for a selected item in which a second party indicates express interest.",
"This is different from the exemplary criteria enforcement process of method 1600 where the qualification process is performed with respect to each and every item located by a search.",
"Accordingly, the criterion enforcement process of method 1700 is advantageous in that it may be computationally less demanding of the commerce system 1100.It will be appreciated that the exemplary enforcement criteria enforcement processes described above as being implemented by methods 1600 and 1700 are merely two examples of multiple ways in which a criteria enforcement policy may be implemented.",
"For example, a qualification assessment may be made at any stage during a particular navigation or transaction process.",
"Furthermore, a criterion expressed within a criteria record may be of such a nature that a determination as to whether the criterion is satisfied may only be made once the transaction process has progressed to a certain stage.",
"For example, the criterion may comprise a transaction criterion that relates to transaction activity pertaining to the transaction of the specific item.",
"In this case, the transaction criterion may specify that a second party becomes disqualified from transacting for the item when the second party submits a bid price offer that is in excess of a threshold value that clearly exceeds any reasonable offer price for the relevant item.",
"Such an excessive bid offer price may be indicative of the fact that the bid offer lacks sincerity, and is in fact a hoax.",
"Further, the transaction criteria may operate automatically to disqualify the second party from transacting further with respect to a particular item when the second party performs a particular transaction activity.",
"For example, were the second party to retract a bid for a particular item, this transaction activity may disqualify the second party from attempting to again transact for the specific item (e.g., submit a further bid) for the relevant item.",
"The above exemplary embodiments of the present invention have been described with reference to transactions pertaining to an item.",
"It will be understood that an item covers both a product (or goods) and a service (or services).",
"FIG.",
"19 shows a diagrammatic representation of machine in the exemplary form of a computer system 1900 within which a set of instructions, for causing the machine to perform any one of the methodologies discussed above, may be executed.",
"In alternative embodiments, the machine may comprise a network router, a network switch, a network bridge, Personal Digital Assistant (PDA), a cellular telephone, a web appliance or any machine capable of executing a sequence of instructions that specify actions to be taken by that machine.",
"Within the context of the above exemplary embodiments, the machine may a server machine on which any of the described servers may be hosted.",
"The machine may also comprise a computing device utilized by a party to access and interact with the commerce system 1100.The computer system 1900 includes a processor 1902, a main memory 1904 and a static memory 1906, which communicate with each other via a bus 1908.The computer system 1900 may further include a video display unit 1910 (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)).",
"The computer system 1900 also includes an alphanumeric input device 1912 (e.g., a keyboard), a cursor control device 1914 (e.g., a mouse), a disk drive unit 1916, a signal generation device 1918 (e.g., a speaker) and a network interface device 1920.The disk drive unit 1916 includes a machine-readable medium 1922 on which is stored a set of instructions (i.e., software) 1924 embodying any one, or all, of the methodologies described above.",
"The software 1924 is also shown to reside, completely or at least partially, within the main memory 1904 and/or within the processor 1902.The software 1924 may further be transmitted or received via the network interface device 1920.For the purposes of this specification, the term “machine-readable medium” shall be taken to include any medium that is capable of storing, carrying or encoding a sequence of instructions for execution by the machine and that cause the machine to perform any one of the methodologies of the present invention.",
"The term “machine-readable medium” shall accordingly be taken to included, but not be limited to, solid-state memories, optical and magnetic disks, and carrier wave signals.",
"Thus, a method and system to qualify a party to transact within a network-based commerce facility have been described.",
"Although the present invention has been described with reference to specific exemplary embodiments, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the invention.",
"Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense."
]
] | Patent_10433173 |
[
[
"Devices and methods for controlled delivery from a drug delivery device",
"The invention features a plug for use with a drug delivery device, wherein the plug defines an expansion control channel, which accommodates thermal expansion of a formulation in a reservoir of a drug delivery device, and an exit channel.",
"In one embodiment, the plug comprises an inner plug member and an outer plug member, which members define an expansion control channel to facilitate release of entrapped air and to accommodate thermal expansion of formulation from the sealed drug reservoir.",
"The plug further defines an exit channel, and may optionally further comprise a frit positioned within the flow pathway just prior to the delivery outlet, or both."
],
[
"1.A plug defining an inlet, an expansion control channel, and an outlet, wherein during use the inlet, expansion control channel and outlet define a flow path from a reservoir of a drug delivery device, through the plug, and out of the drug delivery device.",
"2.The plug of claim 1, wherein the plug comprises: an inner plug member; and an outer plug member.",
"3.The plug of claim 2, wherein the outer plug member is adapted to receive the inner plug member.",
"4.The plug of claim 2, wherein the inner plug member is slidable within the expansion control channel.",
"5.The plug of claim 2, wherein the expansion control channel is defined by the outer plug member.",
"6.The plug of claim 2, wherein the expansion control channel is defined by an inner wall of the drug delivery device.",
"7.The plug of claim 4, wherein during use the inner plug member is slidable to a first position to accommodate thermal expansion of a formulation in the drug delivery device reservoir.",
"8.The plug of claim 4, wherein the inner plug member is slidable to a second position to provide for fluid communication between the expansion control channel and the outlet.",
"9.The plug of claim 8, wherein the plug further defines an exit channel positioned between the expansion control channel and the outlet.",
"10.The plug of claim 2, wherein at least a portion of the expansion control channel is defined by an outer wall of the inner plug member and an inner wall of the outer plug member.",
"11.The plug of claim 2, wherein the plug further comprises a frit positioned in the flow path prior to the outlet.",
"12.The plug of claim 1, wherein the plug further defines an exit channel positioned in the flow path between the expansion control channel and the outlet.",
"13.The plug of claim 12, wherein the exit channel is at least partially filled with an drug formulation-immiscible fluid.",
"14.The plug of claim 12, wherein the exit channel is defined by a groove in an outer wall of the outer plug member and an inner wall of a reservoir following insertion of the plug into a reservoir of a drug delivery device.",
"15.The plug of claim 12, wherein the plug further comprises a frit positioned in the flow path between the exit channel and the outlet.",
"16.The plug of claim 2, wherein the expansion control channel comprises a first channel extending longitudinally through the inner plug member, a second channel extending laterally through the inner plug member, and a third helical channel extending in a spiral fashion along or through the inner plug member.",
"17.The plug of claim 16, wherein the third helical channel is defined by mating surfaces of an outer wall of the inner plug member and an inner wall of the outer plug member.",
"18.A plug comprising, an inner plug member comprising a first end and a second end and an inner plug member body; and an outer plug member adapted to receive the inner plug member; wherein the plug defines an expansion control channel, the expansion control channel extending from a plug inlet, through the inner plug member body, and to an outlet defined by the outer plug member, and wherein the inlet, expansion control channel, and outlet define a flow path through the plug.",
"19.The plug of claim 18, wherein the expansion control channel is defined by the outer plug member, and the inner plug member is slidable within the expansion control channel.",
"20.The plug of claim 19, wherein the inner plug member is slidable to a first position to accommodate thermal expansion of a formulation in the drug delivery device reservoir.",
"21.The plug of claim 19, wherein the inner plug member is slidable to a second position to provide for fluid communication between the expansion control channel and the outlet.",
"22.The plug of claim 18, wherein the plug further comprises a frit positioned in the flow path prior to the outlet.",
"23.The plug of claim 18, wherein the plug further defines an exit channel positioned in the flow path between the expansion control channel and the outlet.",
"24.The plug of claim 23, wherein the exit channel is defined by a groove in an outer wall of the outer plug member and an inner wall of a reservoir following insertion of the plug into a reservoir of a drug delivery device, and wherein a passage in a wall of the outer plug member provides for fluid communication between the expansion control channel and the exit channel.",
"25.The plug of claim 23, wherein the exit channel comprises a flowable material immiscible with the formulation to be delivered through the flow path.",
"26.The plug of claim 23, wherein the plug further comprises a frit positioned in the flow path between the exit channel and the outlet.",
"27.The plug of claim 18, wherein the expansion control channel comprises a first channel extending longitudinally through the inner plug member, a second channel extending laterally through the inner plug member, and a third helical channel extending in a spiral fashion along or through the inner plug member.",
"28.The plug of claim 27, wherein the third helical channel is defined by mating surfaces of an outer wall of the inner plug member and an inner wall of the outer plug member.",
"29.A drug delivery device comprising: a reservoir body defining a reservoir for retaining a formulation comprising a drug; and a plug according to claim 1; wherein the plug is seated within the reservoir to provide for a flow pathway from the reservoir and out the outlet of the plug.",
"30.The drug delivery device of claim 29, wherein the drug delivery device is implantable.",
"31.The drug delivery device of claim 29, wherein the drug is selected from the group consisting of peptides, polypeptides, nucleic acids, and hormones.",
"32.The drug delivery device of claim 29, wherein the drug delivery device is operably attached to a catheter for delivery of the formulation from the reservoir to a delivery site.",
"33.A method of delivering a drug to a delivery site, the method comprising: implanting at least a portion of drug delivery device in a subject, the drug delivery device comprising a plug according to claim 1 and a reservoir body defining a reservoir for retaining a formulation comprising a drug, wherein the plug is seated within the reservoir to provide for a flow pathway from the reservoir and out the outlet of the plug; delivering the formulation from the reservoir and to a delivery site in a subject.",
"34.The method of claim 33, wherein the drug delivery device further comprises a catheter operably attached for delivery of the formulation from the reservoir and to the delivery site, wherein at least a distal end of the catheter comprising a drug delivery outlet is implanted in the subject.",
"35.The method of claim 33, wherein the drug is selected from the group consisting of peptides, polypeptides, nucleic acids, and hormones."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Implantable drug delivery devices provide an attractive therapeutic tool for treatment of a variety of conditions and diseases, especially where therapy requires a prolonged period of therapy.",
"Implantable drug delivery devices avoid the inconvenience and discomfort that can be associated with administration of multiple doses of an agent, and further provides for enhanced therapeutic benefits due to, for example, avoidance of bolus doses (e.g., in contrast to parenteral injection) and improved patient compliance.",
"Devices that provide for precisely controlled drug delivery are of particular interest, as such devices can provide for delivery of drug at doses and rates that are both predictable and reliable (e.g., not affected by the environment in which the device is implanted).",
"Various implantable drug delivery devices have been developed, and are based upon various different mechanisms to accomplish movement of drug from a reservoir of the device to a treatment site in the subject.",
"In general, these delivery technologies can be based upon, for example, diffusive, erodible, or convective mechanisms.",
"Implantable drug delivery devices based upon convective systems are of particular interest in the field, generally due to advantageous features such as the ability to refill the device, and the compatibility of the device for use with a catheter to effect local delivery of drug to a treatment site.",
"Exemplary convective systems include, but are not limited to, electromechanical pumps, osmotic pumps, electroosmotic pumps, electrochemical pumps, hydrolytic systems, piezoelectric pumps, elastomeric pumps, vapor pressure pumps, and electrolytic pumps.",
"The development of implantable devices, particularly for controlled delivery of drug, has posed several challenges in the drug delivery field.",
"One such challenge is the ability to provide for precisely controlled delivery of drug even from the moment of start-up, e.g.",
"the period just after implanting the drug delivery device.",
"For example, variations in environmental temperature during storage and following implantation can cause expansion and contraction of the formulation, which in turn can affect the amount of formulation delivered following implantation.",
"Expansion of the formulation following implantation can result in “extra dosing,” an uncontrolled release of a small amount of formulation, which can be particularly problematic where highly concentrated drug formulations are used.",
"Environmental temperature shifts can also cause expansion and contraction of any air that may be trapped in the reservoir or between the reservoir and the outlet, which can also adversely affect the ability to provide for controlled delivery of drug formulation at start-up.",
"In addition, the formulation flow can “split” during start-up, a phenomenon in which the formulation is not a substantially continuous stream of fluid, but rather is composed of one or more discrete leading volumes separated by air or gas voids (e.g., “burst(s)”) which precede the main formulation stream.",
"As a result, at start-up the formulation may be delivered in a manner that is not precisely controlled.",
"One approach to solving these problems involves making the channel through which the formulation flows out of the reservoir small enough to regulate flow even when the formulation is in an expanded state.",
"However, a channel small enough to regulate flow is generally unacceptably long (and, for example, unacceptably delaying delivery at start-up) or provides a volume insufficient to accommodate thermal expansion, which can result in leakage of formulation from the reservoir.",
"Accurately filling a reservoir of a drug delivery device during manufacture in a manner that allows for capacity for the formulation to expand (e.g., due to variations in environment temperature during storage and following implantation) without loss of contents from the reservoir has proven extremely difficult.",
"As is evident from the above, there is a need for a device that can be used with drug delivery devices, particularly with convective drug delivery devices, that avoids the problems associated with drug delivery at start-up.",
"The present invention addresses this problem."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention features a plug for use with a drug delivery device, wherein the plug defines an expansion control channel, which accommodates thermal expansion of a formulation in a reservoir of a drug delivery device, and an exit channel.",
"In one embodiment, the plug comprises an inner plug member and an outer plug member, which members define an expansion control channel to facilitate release of entrapped air and to accommodate thermal expansion of formulation from the sealed drug reservoir.",
"The plug further defines an exit channel, and may optionally further comprise a frit positioned within the flow pathway just prior to the delivery outlet, or both.",
"In one aspect the invention features a plug comprised of an inner plug member and outer plug member adapted to receive the inner plug member, wherein the plug defines an inlet, an expansion control channel, and an outlet.",
"The inlet, expansion control channel and outlet of the plug define a flow path through and out of the plug.",
"In one embodiment, at least a portion of the expansion control channel defines a passageway through the inner plug member body.",
"In another embodiment, the inner plug member is slidable within the expansion control channel.",
"In related embodiments, the expansion control channel is defined by the outer plug member so that the slidable inner plug member is received within the outer plug member.",
"In a related alternative embodiment, the expansion control channel is defined by adjacent ends of the inner plug member and the outer plug member and an inner wall of the reservoir body of the drug delivery device.",
"In another aspect, the invention features a drug delivery device comprising a plug of the invention.",
"In one embodiment, the drug delivery device is implantable.",
"In still another aspect the invention features methods for delivery of a drug using a drug delivery device comprising a plug of the invention.",
"In one embodiment, the drug delivery device is implantable.",
"A primary object of the invention is to provide for controlled delivery of drug while avoiding problems associated with conventional devices such as formulation leakage (e.g., due to thermal expansion of formulation during storage), delivery of a burst or bolus of formulation at start-up, and the like.",
"One advantage of the invention is that that the invention allows for accurate filling of a reservoir while maintaining an outflow track which serves to dampen the effects of thermal expansion of the fluid in the reservoir.",
"One important advantage of the invention is that the plug helps to prevent “extra dosing” or the initial, uncontrolled release of drug (e.g., “burst”) that can result from thermal expansion due to short-term changes in environmental temperatures.",
"Another advantage is that the invention minimizes or avoids entrapment of air during assembly, thereby minimizing or avoiding the problems such entrapped air can pose (e.g., due to the differences in the expansion rate of air compared to the expansion rate of formulation in the reservoir).",
"Another advantage of the invention is that it provides for precise control of start-up time for the drug delivery device.",
"Another advantage of the invention is that it provides for an extended outflow track while maintaining a more streamlined and volume efficient reservoir and delivery system size.",
"Still another advantage of the invention is that the plug allows for a smaller size reservoir to be filled and to withstand temperature variations, while still allowing for visual or other inspection to ensure a proper fill before completion of reservoir closure.",
"Another advantage of the invention is that it contains the drug formulation within the delivery device until the desired time for delivery, e.g., the plug inhibits leakage of formulation out of the reservoir during storage, shipping, etc.",
"These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the methodology and compositions as more fully set forth below."
],
[
"FIELD OF THE INVENTION The invention relates to devices and methods to facilitate controlled drug delivery.",
"BACKGROUND OF THE INVENTION Implantable drug delivery devices provide an attractive therapeutic tool for treatment of a variety of conditions and diseases, especially where therapy requires a prolonged period of therapy.",
"Implantable drug delivery devices avoid the inconvenience and discomfort that can be associated with administration of multiple doses of an agent, and further provides for enhanced therapeutic benefits due to, for example, avoidance of bolus doses (e.g., in contrast to parenteral injection) and improved patient compliance.",
"Devices that provide for precisely controlled drug delivery are of particular interest, as such devices can provide for delivery of drug at doses and rates that are both predictable and reliable (e.g., not affected by the environment in which the device is implanted).",
"Various implantable drug delivery devices have been developed, and are based upon various different mechanisms to accomplish movement of drug from a reservoir of the device to a treatment site in the subject.",
"In general, these delivery technologies can be based upon, for example, diffusive, erodible, or convective mechanisms.",
"Implantable drug delivery devices based upon convective systems are of particular interest in the field, generally due to advantageous features such as the ability to refill the device, and the compatibility of the device for use with a catheter to effect local delivery of drug to a treatment site.",
"Exemplary convective systems include, but are not limited to, electromechanical pumps, osmotic pumps, electroosmotic pumps, electrochemical pumps, hydrolytic systems, piezoelectric pumps, elastomeric pumps, vapor pressure pumps, and electrolytic pumps.",
"The development of implantable devices, particularly for controlled delivery of drug, has posed several challenges in the drug delivery field.",
"One such challenge is the ability to provide for precisely controlled delivery of drug even from the moment of start-up, e.g.",
"the period just after implanting the drug delivery device.",
"For example, variations in environmental temperature during storage and following implantation can cause expansion and contraction of the formulation, which in turn can affect the amount of formulation delivered following implantation.",
"Expansion of the formulation following implantation can result in “extra dosing,” an uncontrolled release of a small amount of formulation, which can be particularly problematic where highly concentrated drug formulations are used.",
"Environmental temperature shifts can also cause expansion and contraction of any air that may be trapped in the reservoir or between the reservoir and the outlet, which can also adversely affect the ability to provide for controlled delivery of drug formulation at start-up.",
"In addition, the formulation flow can “split” during start-up, a phenomenon in which the formulation is not a substantially continuous stream of fluid, but rather is composed of one or more discrete leading volumes separated by air or gas voids (e.g., “burst(s)”) which precede the main formulation stream.",
"As a result, at start-up the formulation may be delivered in a manner that is not precisely controlled.",
"One approach to solving these problems involves making the channel through which the formulation flows out of the reservoir small enough to regulate flow even when the formulation is in an expanded state.",
"However, a channel small enough to regulate flow is generally unacceptably long (and, for example, unacceptably delaying delivery at start-up) or provides a volume insufficient to accommodate thermal expansion, which can result in leakage of formulation from the reservoir.",
"Accurately filling a reservoir of a drug delivery device during manufacture in a manner that allows for capacity for the formulation to expand (e.g., due to variations in environment temperature during storage and following implantation) without loss of contents from the reservoir has proven extremely difficult.",
"As is evident from the above, there is a need for a device that can be used with drug delivery devices, particularly with convective drug delivery devices, that avoids the problems associated with drug delivery at start-up.",
"The present invention addresses this problem.",
"SUMMARY OF THE INVENTION The invention features a plug for use with a drug delivery device, wherein the plug defines an expansion control channel, which accommodates thermal expansion of a formulation in a reservoir of a drug delivery device, and an exit channel.",
"In one embodiment, the plug comprises an inner plug member and an outer plug member, which members define an expansion control channel to facilitate release of entrapped air and to accommodate thermal expansion of formulation from the sealed drug reservoir.",
"The plug further defines an exit channel, and may optionally further comprise a frit positioned within the flow pathway just prior to the delivery outlet, or both.",
"In one aspect the invention features a plug comprised of an inner plug member and outer plug member adapted to receive the inner plug member, wherein the plug defines an inlet, an expansion control channel, and an outlet.",
"The inlet, expansion control channel and outlet of the plug define a flow path through and out of the plug.",
"In one embodiment, at least a portion of the expansion control channel defines a passageway through the inner plug member body.",
"In another embodiment, the inner plug member is slidable within the expansion control channel.",
"In related embodiments, the expansion control channel is defined by the outer plug member so that the slidable inner plug member is received within the outer plug member.",
"In a related alternative embodiment, the expansion control channel is defined by adjacent ends of the inner plug member and the outer plug member and an inner wall of the reservoir body of the drug delivery device.",
"In another aspect, the invention features a drug delivery device comprising a plug of the invention.",
"In one embodiment, the drug delivery device is implantable.",
"In still another aspect the invention features methods for delivery of a drug using a drug delivery device comprising a plug of the invention.",
"In one embodiment, the drug delivery device is implantable.",
"A primary object of the invention is to provide for controlled delivery of drug while avoiding problems associated with conventional devices such as formulation leakage (e.g., due to thermal expansion of formulation during storage), delivery of a burst or bolus of formulation at start-up, and the like.",
"One advantage of the invention is that that the invention allows for accurate filling of a reservoir while maintaining an outflow track which serves to dampen the effects of thermal expansion of the fluid in the reservoir.",
"One important advantage of the invention is that the plug helps to prevent “extra dosing” or the initial, uncontrolled release of drug (e.g., “burst”) that can result from thermal expansion due to short-term changes in environmental temperatures.",
"Another advantage is that the invention minimizes or avoids entrapment of air during assembly, thereby minimizing or avoiding the problems such entrapped air can pose (e.g., due to the differences in the expansion rate of air compared to the expansion rate of formulation in the reservoir).",
"Another advantage of the invention is that it provides for precise control of start-up time for the drug delivery device.",
"Another advantage of the invention is that it provides for an extended outflow track while maintaining a more streamlined and volume efficient reservoir and delivery system size.",
"Still another advantage of the invention is that the plug allows for a smaller size reservoir to be filled and to withstand temperature variations, while still allowing for visual or other inspection to ensure a proper fill before completion of reservoir closure.",
"Another advantage of the invention is that it contains the drug formulation within the delivery device until the desired time for delivery, e.g., the plug inhibits leakage of formulation out of the reservoir during storage, shipping, etc.",
"These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the methodology and compositions as more fully set forth below.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a schematic of a convective delivery system.",
"FIG.",
"2 is a cut-away view of a plug of the invention inserted for use in a reservoir of a drug delivery device.",
"FIG.",
"3 is a cut-away view of a plug of the invention assembled for use with a drug delivery device.",
"FIG.",
"4 is a cut-away view of a plug of the invention comprising a frit positioned within the formulation flow path and prior to the outflow channel.",
"FIG.",
"5 is a cut-away view of a plug of the invention comprising a frit positioned with the flow path of the exit channel.",
"FIGS.",
"6-10 are cut-away views of the plug of the invention and a reservoir of a drug delivery device during insertion of the plug into the reservoir for use.",
"FIGS.",
"11 and 12 are cut-away views of an exemplary plug of the invention in which the inner plug member is slidable within an expansion control channel defined by the outer plug member.",
"FIGS.",
"13 and 14 are cut-away views of an exemplary plug of the invention in which the inner plug member is slidable within an expansion control channel defined by the outer plug member.",
"FIGS.",
"15 and 16 are cut-away views of an exemplary plug of the invention in which the inner plug member is slidable within an expansion control channel defined by the reservoir body.",
"FIGS.",
"17 and 18 are cut-away views of an exemplary plug of the invention in which the inner plug member is slidable within an expansion control channel defined by the reservoir body, where the inner plug member includes and O-ring.",
"DESCRIPTION OF THE PREFERRED EMBODIMENTS Before the present invention is described, it is to be understood that this invention is not limited to the specific devices, materials, formulations, or exemplary embodiments described as such may, of course, vary.",
"It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.",
"It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise.",
"Thus, for example, reference to “a drug delivery device” includes a plurality of such devices and reference to “the assembly method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.",
"Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.",
"Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.",
"All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the compositions and methodologies which are described in the publications which might be used in connection with the presently described invention.",
"The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application.",
"Nothing herein is to be construed as an admission that the invention is not entitled to antedate such a disclosure by virtue of prior invention.",
"Definitions The term “drug” as used herein is meant to encompass any substance suitable for delivery to a treatment site of a subject, which substances can include pharmaceutically active drugs, as well as biocompatible substances that do not exhibit a pharmaceutical activity in and of themselves, but that provide for a desired effect at a treatment site, e.g.",
"to flush or irrigate a treatment site (e.g., saline).",
"The term “therapeutically effective amount” is meant an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent, effective to facilitate a desired therapeutic effect.",
"The precise desired therapeutic effect will vary according to the condition to be treated, the formulation to be administered, and a variety of other factors that are appreciated by those of ordinary skill in the art.",
"“Delivery site” as used herein is generally meant to refer to an area of the body to which drug is delivered for either local therapy or entry into the systemic circulation.",
"Exemplary delivery sites include, but are not necessarily limited to, subcutaneous, intravenous, intra-arterial, intramuscular, intra-adipose tissue, and intra-lymphatic sites.",
"The term “implantation site” is used to refer to a site within the body of a subject at which a drug delivery device is introduced and positioned.",
"“Drug delivery device” as used herein is meant to any device, generally an implantable device, suitable for delivering a drug of choice, and in general suitable for use with the present invention.",
"“Drug delivery device” thus encompasses any implantable device with any mechanism of action compatible with the claimed invention including, but not necessarily limited to, these convective systems (e.g., osmotic pumps, electromechanical pumps, electroosmotic pumps, electrochemical pumps, hydrolytic systems, piezoelectric pumps, elastomeric systems, vapor pressure pumps, and electrolytic pumps.",
"“Patterned” or “temporal” as used in the context of drug delivery is meant delivery of drug in a pattern, generally a substantially regular pattern, over a pre-selected period of time (e.g., other than a period associated with, for example a bolus injection).",
"“Patterned” or “temporal” drug delivery is meant to encompass delivery of drug at an increasing, decreasing, substantially constant, or pulsatile, rate or range of rates (e.g., amount of drug per unit time, or volume of drug formulation for a unit time), and further encompasses delivery that is continuous or substantially continuous, or chronic.",
"The term “controlled drug delivery device” is meant to encompass any device wherein the release (e.g., rate, timing of release) of a drug or other desired substance contained therein is controlled by or determined by the device itself and not the environment of use.",
"The term “subject” is meant any subject, generally a mammal (e.g., human, canine, feline, equine, bovine, etc.",
"), to which delivery is desired.",
"Overview of the Invention The invention features devices and methods to provide for delivery of an agent from a drug delivery device while avoiding the problems associated with, for example, expansion and contraction of the formulation within the reservoir of the drug delivery device without allowing significant amounts or substantially any air into the sealed drug reservoir.",
"Another requirement of this system was to provide an exit pathway from the device so as to allow regulation of flow of formulation through the channel, particularly at very low flow rates and in very low volumes.",
"In general, the invention must provide a pathway small enough to accommodate regulated delivery of the formulation from a drug delivery device reservoir, but that at the same time is not unacceptably long or of insufficient volume to accommodate thermal expansion.",
"The present invention accomplishes these goals by providing a plug comprising inner and outer plug members, and defining a first channel of sufficient volume for thermal expansion (referred to herein as an expansion control channel) and a second, smaller channel (referred to herein as the exit channel) that provides for regulated delivery of the formulation from the reservoir without wicking of the formulation out of the reservoir or allowing seepage of fluids surrounding the drug delivery device into the plug and into the drug reservoir.",
"The invention will now be described in more detail.",
"Expansion Control Channel In all embodiments, at least a portion of the plug defines an expansion control channel.",
"The expansion control channel is of a volume sufficient to accommodate thermal expansion of a formulation to be delivered from a reservoir of a drug delivery device, and during delivery of formulation communicates with an exit channel to allow for flow of formulation from the reservoir and out an outlet of the device.",
"In considering the dimensions of the expansion control channel, one can assume a convective delivery system 100 (see schematic in FIG.",
"1) having a drug reservoir 65 and an orifice 37 (depicted in FIG.",
"1 as a linear path extending from reservoir 65).",
"Reservoir 65 has a volume Vd with volume Vo as additional volume that may impact thermal expansion, Vt then the total volume that participates in thermal expansion can be expressed: Vt=Vd+Vo (1) Assume that delivery system 100 is filled with formulation to the mark A to extend into the linear orifice volume Vl.",
"Mark A is reached at storage temperatures TS and on implantation the volume expansion of the liquid reaches to the mark B at body temperature TB.",
"The rise of the volume expansion chamber Vlx can then be calculated as follows.",
"The volume expansion coefficient of the liquid formulation in the system is “a” as per the equation: 1/Vt(ΔV/ΔT)=a (2) Assuming that a is constant over the temperature range, it then follows that the rise of the volume expansion chamber Vlx can be calculated by the equation: Vlx=aVt(TB−TS) (3) Thus a volume expansion chamber can be designed according to the invention by taking into account, for example, the values a (the volume expansion coefficient of the formulation to be delivered), Vl (the volume of the orifice, which varies with the length of the pathway through such orifice), (TB−TS) (the difference between the body (implantation) temperature TB and the storage temperature TS), the orifice diameter, and the length (B−A) along the orifice.",
"For example, for an organic drug formulation, ao=1.1×10−3/° C., and for an aqueous formulation aa=3×10−4/° C. According to equation (3), Vlx for organic and water-based formulations, with a drug reservoir of V=0.200 cm3 and ΔT (or (TB−TS))=17° C., are: Vlx=a(0.2)(17)=a(3.4) Vlx=ao(3.4)=(1.1×10−3)(3.4)=3.7 μl for an organic liquid formulation, and Vlx=aa(3.4)=(3.1×10−4)(3.4)=1.0 μl for an aqueous formulation, and The system can also be designed to take into account the coefficient of linear expansion of the material through which the formulation flows.",
"Values for the thermal expansion of formulations and components of such formulations are readily available and/or can be readily determined by methods well known in the art.",
"The expansion control channel can be provided by the plug of the invention in a variety of ways.",
"For example, the expansion control channel can extend from one end of the inner plug member (a first end that will be in contact with the formulation of the drug reservoir during use), and through nearly all or a portion of the length of the inner plug member body to communicate with the exit channel of the plug.",
"In one exemplary embodiment, the expansion control channel comprises a longitudinal channel which passes through the inner plug member body, a lateral passage, and a helical section.",
"The longitudinal channel, which defines an inlet at a first end of the inner plug member (e.g., the end that is in contact with formulation in a reservoir during use), extends through the body of the inner plug member until it extends a desired distance toward the inner plug member second end.",
"This longitudinal section can be positioned in the center of the inner plug member body, or may be off-center, as desired.",
"The lateral section of the expansion control channel extends from the longitudinal section and to the helical section of the expansion control channel to provide a fluid passage between the longitudinal section to the outer wall.",
"The helical section of the expansion control channel provides for fluid communication from the lateral section to the exit channel defined at least in part by the outer plug member (described below) or, where used in combination with a frit, to an outlet (described below).",
"The helical section of the expansion control channel is defined at least in part by the inner plug member body.",
"In one embodiment, the helical section of the expansion control channel is defined by the mating surfaces of the inner plug member outer wall and the outer plug member inner wall (e.g., by a groove in the inner plug member outer wall and an inner wall of the outer plug member).",
"Each of these sections of the expansion control channel can be of varying dimensions, as will be readily appreciated by the ordinarily skilled artisan upon reading the instant disclosure.",
"In another embodiment, the expansion control channel is defined by the outer plug member body and an end of the inner plug member which is to be proximal to the reservoir during use.",
"In this embodiment, the inner plug member is slidably received within the outer plug member body.",
"Expansion of the formulation against the inner plug member proximal end causes the inner plug member to slide within the outer plug member, thereby providing an expansion control channel within the proximal end of the plug.",
"The inner plug is prevented from moving out the distal end of the drug delivery device by the outer plug member, which is stably seated in the device.",
"In another embodiment, the inner plug member is slidable along the walls of the reservoir body of the drug delivery device, with the outer plug member being stably seated within the drug delivery device distal end.",
"Expansion of the formulation against the inner plug member causes the inner plug member to slide within the reservoir body.",
"outer plug member, thereby providing an expansion control channel within the proximal end of the plug.",
"The outer plug member, which is again stably seated within the delivery device reservoir, prevents the inner plug from moving out the distal end of the drug delivery device.",
"The expansion control channel volume can be increased by varying the geometry of the channel.",
"For example, the expansion control channel can be made longer by making all or a portion of the channel a spiral winding through or along the side of the inner plug member.",
"Inner Plug Member The inner plug member of the plug of the invention is received by and at least a portion seated within the outer plug member.",
"The inner plug member, generally either alone or in combination with the outer plug member, defines an expansion control channel, the conduit through which formulation will initially flow during use with a drug delivery device.",
"The expansion control channel is of a size (diameter and length) that provides for control of air entrapment during assembly (e.g., insertion of the plug into a drug delivery device to provide for a sealed drug reservoir).",
"In addition, the expansion control channel is of a size that accommodates thermal expansion of air and/or formulation so as to prevent or substantially diminish release of drug from the reservoir prior to start-up.",
"The size of the inner member can be minimized by defining the expansion control channel as a groove along an outside wall of the inner plug member or along an inside wall of the outer plug member, so that the complete expansion control channel is defined by the mating surfaces of the inner member outer wall and an inner wall of the outer plug member.",
"In one embodiment, a first portion of the expansion control channel extends through the length of the inner plug member body, extends laterally through the inner plug member body, and then is defined as a helical groove that spirals along and down the outside wall of the inner plug member body such that the helical groove defines a complete expansion control channel when the inner member is positioned within the outer plug member.",
"Suitable materials are hard plastics, metals, metal alloys, ceramics, polymers, and the like, where the materials provide for well-defined dimensions of the exit channel that can be maintained.",
"It is desirable to have materials of low surface energy such that they prevent wicking of liquids through the channels.",
"Materials such as metals can also be surface coated to reduce surface energy.",
"Outer Plug Member In general, the outer plug member is the plug portion that defines the distal end of the plug, and further defines the outlet end of the drug delivery device when in use, e.g., the outer plug member provides at least a portion of the distal end of the drug delivery device, the end through which formulation is delivered to a delivery site or into a catheter operatively attached to the drug delivery device (e.g., by a press-fit, threaded, snap-fit, or adhesive attachment element and the like, and/or by welding, bonding, molding, and the like).",
"In one embodiment, the outer plug member is adapted to receive the inner plug member, which inner plug member may be either stably seated or slidable within the outer plug member.",
"In another embodiment, the inner plug member is slidable within the reservoir body, and is prevented from being pushed out the drug delivery device by the outer plug member, i.e., the inner plug member slides through the reservoir until it contacts a proximal end of the outer plug member.",
"In one embodiment, the outer plug member defines, either alone or in combination with an inner wall of the drug delivery device, an exit channel through which the formulation flows out of the device during use.",
"The exit channel is of a size that provides for regulated flow of formulation from the reservoir and the inner plug member channel during drug delivery, prevents wicking of formulation from the reservoir and out of the device, and prevents backflow of formulation or environmental fluids into the plug and reservoir during use.",
"Backflow can have undesirable effects including contamination of the interior of the delivery device, and can result in dilution, destabilization, or other undesirable effect upon the formulation in the reservoir.",
"Parameters for the dimensions of an exit flow pathway that provides for controlled flow of a formulation through the pathway and with regulation of back-diffusion are described in, for example, U.S. Pat.",
"Nos.",
"5,985,305 and 5,728,396.Briefly, the length, interior cross-sectional shape and area of the exit channel 41 are selected so that the average linear velocity of the released formulation is higher than that of the linear inward flux of materials in the environment of use due to diffusion or osmosis, so as to attenuate or moderate back-diffusion.",
"The release rate of formulation (and thus of drug) can be modified by modifying the outlet pathway geometry, which are related as discussed below.",
"The convective flow of active agent out of outlet 45 is set by the delivery rate of the system and the concentration of drug A in the formulation 75 of reservoir 65.The relationship of these variables can be represented by the following formula: Qca=(Q)(Ca) (4) where Qca is the convective transport of drug A in mg/day; Q is the overall convective transport of the agent and its diluents in cm3/day; and Ca is the concentration of a drug in the formulation within reservoir Qca 65 in mg/cm3.The diffusive flow of drug through the material in the exit channel 41 is a function of drug concentration, cross-sectional configuration of the exit channel 41, drug diffusivity, and length of exit channel 41, and can be represented as follows: Qda=Dπr2ΔCa/L (5) where Qda is the diffusive transport of drug A in mg/day; D is the diffusivity through the material in exit channel 41 in cm2/day, r is the effective inner radius of the flow path in cm, ΔCa is the difference between the concentration of drug A in the reservoir and in the body outside of the outlet 45 in mg/cm3; and L is the length of the flow path in cm.",
"In general, the concentration of drug in the reservoir is much greater than the concentration of drug in the body outside of the outlet such that the difference, ΔCa, can be approximated by the concentration of agent within the reservoir, Ca.",
"Qda=Dπr2Ca/L (6) In general, the diffusive flux of drug is kept at less than 10% of the convective flow of drug.",
"This is represented as follows: Qda/Qca=Dπr2Ca/QCaL=Dπr2/QL≦0.1 (7) For a delivery system with a purging rate of 1.5 μl/day, with a reservoir volume of 150 μl, where the system is to deliver the formulation over 100 days and has an orifice length of 1.1 cm and orifice diameter of 10 mil, assuming a diffusion coefficient of D=2×10−6 cm2/sec, the ratio of (Qda/Qca)=5.2×10−3 and satisfies equation (7).",
"For this orifice size the volume Vl=5 μl and satisfies the criteria Vlx<V1 projected in FIG.",
"1.Exemplary ranges for Vlx are then from 0.1 to 0.9 times Vl.",
"The exit channel generally extends through a side wall of the outer plug member body and is in fluid communication with the expansion control channel.",
"The exit channel can extend nearly all or a portion of the length of the outer plug member body before providing an outflow channel for release of formulation out of the plug and device.",
"An exit channel of desired length can be provided without requiring a concomitant increase in the dimensions of the outer plug member by varying the shape of the exit channel.",
"For example, the exit channel can be provided as a spiral extending along all or a portion of the outer plug member body.",
"In one embodiment, the exit channel is provided as a groove along the outer wall of the outer plug member, with the complete exit channel provided by the mating surfaces of an inner wall of the drug delivery device (e.g., an inner wall of the drug reservoir) and an outer wall of the outer plug member following assembly of the plug for use with the device.",
"In this embodiment, the diameter of the outer plug member may be minimized for use with smaller, implantable devices or microdevices.",
"In another embodiment, the exit channel is provided by a combination of a channel through the outer plug member body that communicates with the expansion channel, and groove within the inner wall of the drug delivery device.",
"In another embodiment in which the outer plug member is adapted to receive a slidable inner plug member, the exit channel can be defined by the mating surfaces of the outer plug member and the inner plug member, e.g., the exit channel can be defined by an outer wall of the inner plug member and an inner wall of the outer plug member.",
"Materials suitable for the outer plug member are the same as those suitable for use for the inner plug member.",
"The materials used for the inner plug member and the outer plug member can be the same or different.",
"Where the outer plug member is adapted to slidably receive the inner plug member, the inner wall of the outer plug member may be coated or other wise treated to facilitate sliding of the inner plug member.",
"Additional Plug Components or Elements The plug of the invention can comprise other components that can serve to further enhance controlled delivery of drug through the plug.",
"For example, in one embodiment, the plug comprises a frit positioned within the flow pathway so as to provide a void volume.",
"The frit is generally positioned within the outer member and just prior to an outflow channel of the plug.",
"In general, the frit comprises a porous material that is positioned within the path through which the formulation must pass, and which provides a void volume sufficient to trap liquid and allow air to escape.",
"In use, the frit “captures” the leading edge of formulation flow (the “burst”) at start-up, and traps this formulation volume until the main body of formulation joins the initial formulation burst.",
"Where the plug comprises a frit, the outer member plug may optionally define an exit channel as described above, e.g., where a frit is positioned with in the flow pathway, the exit may not be required to provide for the desired level of controlled delivery.",
"It will be readily appreciated that the materials suitable for use in the frit will vary according to a variety of factors including, but not necessarily limited to, the formulation to be passed through the frit (e.g., the formulation pH, hydrophobicity, hydrophilicity, wetting characteristics, and the like).",
"In general, the frit material is selected so that it is capable of wetting and wicking the formulation, and is both compatible and chemically resistant to the formulation.",
"Suitable frit materials include, but are not necessarily limited to, metals, glass, polymers, and cellulose.",
"Likewise, the dimensions of the frit and, thus the void volume provided by the frit, will vary according to a variety of factors that will be readily appreciated by the ordinarily skilled artisan.",
"The frit materials are typically made with materials of high surface energy, such that they wet easily.",
"In general, the frit materials are porous elements with porosities in the range of 30% to 90%, preferably 50% to 90%, with open pore structure.",
"The internal volume of the porous element is in the range of 0.1 to 0.5 times Vlx as defined in FIG.",
"1.In general, the volume provided by the frit is selected so that it can handle either the thermal expansion volume of the formulation (Vlx), and may also be designed to handles any volume of fluid which may break away from the main stream of fluid exiting the pump, e.g., handling a small volume (e.g., 1-2 μl) of fluid which breaks from the fluid front and wick forward toward the plug outlet.",
"Drug Delivery Devices for Use with the Invention Any of a variety of drug delivery devices are compatible for use with the invention.",
"In general, drug delivery devices suitable for use with the plug of the invention are those comprising a drug reservoir that is designed to contain a flowable drug formulation, and which further comprises a portion that can receive the plug to provide for sealing of the drug reservoir by the plug, e.g., the plug can be positioned in the drug delivery device so as to provide for a flow pathway from the drug reservoir and out of the device.",
"The drug delivery device is generally capable of carrying a drug formulation in such quantities and concentration as therapeutically required, and of providing sufficient protection to the formulation from attack by body processes for the duration of implantation and delivery.",
"The exterior is thus preferably made of a material that has properties to diminish the risk of leakage, cracking, breakage, or distortion so as to prevent expelling of its contents in an uncontrolled manner under stresses it would be subjected to during use, e.g., due to physical forces exerted upon the drug release device as a result of movement by the subject or physical forces associated with pressure generated within the reservoir associated with drug delivery.",
"The drug reservoir or other means for holding or containing the drug must also be of such material as to avoid unintended reactions with the active agent formulation, and is preferably biocompatible (e.g., where the device is implanted, it is substantially non-reactive with respect to a subject's body or body fluids).",
"Suitable materials for the reservoir body or drug holding means of the drug delivery devices of the invention are well known in the art.",
"For example, the reservoir material may comprise a non-reactive polymer or a biocompatible metal or alloy.",
"Suitable polymers include, but are not necessarily limited to, acrylonitrile polymers such as acrylonitrile-butadiene-styrene polymer, and the like; halogenated polymers such as polytetrafluoroethylene, polyurethane, polychlorotrifluoroethylene, copolymer tetrafluoroethylene and hexafluoropropylene; polyethylene vinylacetate (EVA), polyimide; polysulfone; polycarbonate; polyethylene; polypropylene; polyvinylchloride-acrylic copolymer; polycarbonate-acrylonitrile-butadiene-styrene; polystyrene; cellulosic polymers; and the like.",
"Further exemplary polymers are described in The Handbook of Common Polymers, Scott and Roff, CRC Press, Cleveland Rubber Co., Cleveland, Ohio.",
"Metallic materials suitable for use in the reservoir body include stainless steel, titanium, platinum, tantalum, gold and their alloys; gold-plated ferrous alloys; platinum-plated titanium, stainless steel, tantalum, gold and their alloys as well as other ferrous alloys; cobalt-chromium alloys; and titanium nitride-coated stainless steel, titanium, platinum, tantalum, gold, and their alloys.",
"Laminates of the above materials can also be used in the reservoir body.",
"Where the drug formulation is stored in a reservoir comprising metal or a metal alloy, particularly titanium or a titanium alloy having greater than 60%, often greater than 85% titanium is preferred for the most size-critical applications, for high payload capability and for long duration applications and for those applications where the formulation is sensitive to body chemistry at the implantation site or where the body is sensitive to the formulation.",
"Most preferably, the drug delivery devices are designed for storage with drug at room temperature or higher.",
"Drug delivery devices suitable for use with the invention can be based on any of a variety of modes of operation.",
"In general, the plug of the invention can be used with any drug delivery device comprising a reservoir for containing a flowable drug formulation, and from which the formulation is to be delivered to a treatment site in the subject.",
"The plug of the invention finds particular use with a convective device comprising a drug reservoir.",
"Exemplary devices include, but are not necessarily limited to, electromechanical pumps, osmotic pumps, electroosmotic pumps, electrochemical pumps, hydrolytic systems, piezoelectric pumps, vapor pressure pumps, and electrolytic pumps.",
"Drug release devices based upon a mechanical or electromechanical infusion pump, can also be suitable for use with the present invention.",
"Examples of mechanical or electromechanical infusion pumps include, but are not necessarily limited to, those described in, for example, U.S. Pat.",
"Nos.",
"4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like.",
"Exemplary osmotically-driven devices suitable for use in the invention include, but are not necessarily limited to, those described in U.S. Pat.",
"Nos.",
"3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and 5,985,305; in PCT Publication No.",
"WO 97/27840; and the like.",
"The plug of the invention is particularly useful where the drug delivery device is to provide for controlled delivery of drug, especially in very small amounts (e.g., 0.01 μg/hr to about 200 μg/hr) and/or low volumes (e.g., a volume rate of from about 0.01 μl/day to about 100 μl/day (i.e., from about 0.0004/to about 4 μl/hr), preferably from about 0.04 μl/day to about 10 μl/day, generally from about 0.2 μl/day to about 5 μl/day, typically from about 0.5 μl/day to about 1 μl/day).",
"In one embodiment, the volume/time delivery rate is substantially constant (e.g., delivery is generally at a rate ± about 5% to 10% of the cited volume over the cited time period.",
"In some embodiments it may be desirable to provide a drug delivery catheter with the drug delivery device, e.g., where the implantation site and the desired delivery site are not the same or adjacent.",
"The drug delivery catheter is generally a substantially hollow elongate member having a first end (or “proximal” end) associated with the drug delivery device, and a second end (or “distal” end) for delivery of the drug-comprising formulation to a desired delivery site.",
"Where a drug delivery catheter is used, a first end of the drug delivery catheter is associated with or attached to the drug delivery device so that the lumen of the drug delivery catheter is in communication with, via the channels defined by the plug of the invention, the drug reservoir in the drug delivery device, so that a formulation contained in a drug reservoir can move into the drug delivery catheter, and out a delivery outlet of the catheter which is positioned at the desired delivery site.",
"The body of the catheter defines a lumen, which lumen is to have a diameter compatible with providing leak-proof delivery of drug formulation from the drug delivery device.",
"Where the drug delivery device dispenses drug by convection (as in, e.g., osmotic drug delivery systems), the size of the catheter lumen leading from the reservoir of the drug release system can be designed as described by Theeuwes (1975) J. Pharm.",
"Sci.",
"64:1987-91 expressed in equation (7).",
"The body of the catheter can be of any of a variety of dimensions and geometries (e.g., curved, substantially straight, tapered, etc.",
"), that can be selected according to their suitability for the intended site for drug delivery.",
"The distal end of the drug delivery catheter can provide a distinct opening for delivery of drug, or as a series of openings.",
"Formulations for Delivery Any of a variety of formulations comprising any of a variety of drugs (active agents) can be delivered using a drug delivery device comprising a plug of the invention.",
"Classes of drugs suitable for delivery using an drug delivery device comprising a plug of the invention include, but are not necessarily limited to, pharmacologically active peptides, polypeptides, nucleic acid encoding a gene product of interest and such gene products (e.g., DNA, RNA, and other nucleic acid-based compounds), Compounds of interest include chemotherapeutic agents for neoplastic tissues, anti-inflammatory agents (e.g., for ischemic or inflamed tissues), hormones or hormone antagonists (e.g., for endocrine tissues), ion channel modifiers (e.g., for cardiovascular or other tissues), and neuroactive agents (e.g., for the central nervous system, including, but not necessarily limited to analgesics, including, but not limited to opioids, opioid derivatives, and the like).",
"Exemplary of pharmaceutical agents suitable for this invention are those described in The Pharmacological Basis of Therapeutics, Goodman and Gilman, McGraw-Hill, New York, N.Y., (1993) under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference.",
"Exemplary Embodiments Exemplary embodiments of the plug of the invention, as well as its use in connection with a drug delivery reservoir, will now be provided with reference to the drawings.",
"FIG.",
"2 shows a cut-away view of a plug 10 of the invention inserted for use within a reservoir 65 containing formulation 75, and defined by reservoir body 60 of drug delivery device 55.Plug 10 comprises inner plug member 20 and outer plug member 40.Inner plug member 20 is positioned within outer plug member 40.In this embodiment, expansion control channel 21 is composed of a longitudinally extending section, which extends from a first end of inner plug member 20 (which end defines inlet 35) and through the body of the inner plug member; a laterally extending section, which extends from the body of the inner plug member 20 and to an outer wall of the inner plug member 20, and a helical portion, which in this embodiment is defined by the mating surfaces of inner plug member 20 and outer plug member 40, specifically in this embodiment by the outer wall of inner plug member 20 (exemplified in FIG.",
"2 as a groove) and an inner wall of outer plug member 40.Plug 10 of FIG.",
"2 further defines exit channel 41, which in this embodiment is defined by the mating surfaces of an outer wall of outer plug member 40 and an inner wall of reservoir body 60.Exit channel 41 communicates with expansion control channel 21 via passage or “gate” 30 through a wall of outer plug member 40.Timing hole 28, represented by a dead-end passage at the end of inner plug member 20 adjacent reservoir 65 during use and positioned opposite gate 30, facilitates manufacture and assembly of the plug to ensure that expansion control channel 21 and exit channel 41 are in fluid communication.",
"In use, formulation 75 flows from reservoir 65 into inlet 35, through expansion channel 21, through exit channel 41, and out outlet 45, which in this embodiment is defined by an outer wall of outer plug member 40 and an inner wall of as illustrated by the flow pathway denoted by arrows 70, 71, 72, and 73.The seal between the mating surfaces of the plug 10 and the reservoir body 60 can be designed to withstand the maximum pressure of the formulation generated within the device.",
"In general, the pressure required to dislodge the plug 10 from the reservoir 65 is at least about ten times the pressure in reservoir 65.In one embodiment, a substantially drug-free, immiscible, flowable material is provided within exit channel 41.The material is immiscible with the formulation to be delivered from reservoir 65.The immiscible material can be, for example, a solid at room temperature or other storage temperature, and extrudable from the exit channel at body temperature such that the immiscible material is extruded out the exit channel and out the outlet following implantation of the device.",
"The immiscible material thus facilitates prevention of back diffusion into the plug and reservoir.",
"In another embodiment, exemplified in FIG.",
"3, exit channel 41 is defined by the mating surfaces of an outer wall of reservoir body 60 and an inner wall of outer plug member 40, where outer plug member 40 is adapted and sized to receive at least a portion of the reservoir body 60 and inner plug member 20 which is seated within reservoir body 60 (e.g., outer plug member 40 can fit over the end of reservoir body 60 in a cap configuration).",
"Expansion channel 21 is defined by at least a portion of inner plug member 20; in the embodiment exemplified in FIG.",
"3, expansion channel 21 is defined by the mating surfaces of an outer wall of inner plug member 20 and an inner wall of reservoir body 60.As illustrated by arrows 70, 71, and 72, in this embodiment, the formulation moves through a flow pathway that passes through inlet 35, through expansion channel 21, through gate 30, through exit channel 41 and out outlet 45.FIGS.",
"4 and 5 provide exemplary embodiments of the plug of the invention, where the plug comprises a frit positioned within the flow pathway.",
"In the embodiment of FIG.",
"4, frit 50 is seated within outer plug member 40 just prior to exit of the flow pathway outer outlet 45.Outer plug member 40 comprises an additional passage 47 which provides for fluid communication between exit channel 41 (which in this embodiment is provided in part by a groove in an outer wall of outer plug member 40) and frit 50.When plug 10 is positioned for use in a reservoir of a drug delivery device, formulation flows into inlet 35, through expansion control channel 21, through a first outer plug member wall passage or “gate” 30, through exit channel, 41, through a second outer plug member wall passage 47, into frit 50, and out outlet 45.In another embodiment, exemplified in FIG.",
"5, plug 10 comprises inner plug member 20, outer plug member 40, frit 50, and defines expansion control channel 21.The positioning of the frit in this embodiment avoids the need for an exit channel since the frit provides for regulated flow of formulation.",
"Thus, formulation flows into inlet 35, through expansion control channel 21, through inner plug member passage 26, and into frit 50.In another embodiment of the invention, illustrated in FIGS.",
"11-16, inner plug member 20 is slidably positioned within the expansion control channel.",
"Inner plug member 20 is positioned after fill and prior to delivery so that gate 30 is closed, thereby preventing fluid communication between expansion control channel 21 and exit channel 41.Inner plug member 20 is slidable a distance within expansion control channel 21 so as to make available a volume of expansion control channel 21 sufficient to accommodate thermal expansion of formulation 75 (represented by Vlx).",
"When delivery of formulation is desired, formulation pushes against the proximal end of inner plug member 20 so as to displace inner plug member 20 to create an inlet 35, and to displace inner plug member 20 a distance sufficient to slide the proximal end of inner plug member 20 past gate 30.Sliding of inner plug member 20 “opens” gate 30, providing for fluid communication between, and thus defining a flow pathway through, reservoir 65, expansion control channel 21, gate 30, exit channel 41, and outlet 45.In a preferred embodiment, exemplified in FIGS.",
"11 and 12, the walls of expansion channel 21 are defined by the body of outer plug member 40.In this embodiment, the walls are substantially smooth, allowing for smooth sliding of inner plug member 20 along the walls of expansion channel 21.As in the embodiment described above, sliding of inner plug member 20 provides for an expansion control channel 21 of a volume sufficient to accommodate thermal expansion of formulation 75 (represented by Vlx).",
"When delivery of formulation is desired, formulation pushes against the proximal end of inner plug member 20 so as to displace inner plug member 20 to create an inlet 35, and to displace inner plug member 20 a distance sufficient to slide the proximal end of inner plug member 20 past gate 30.Sliding of inner plug member 20 “opens” gate 30, providing for fluid communication between, and thus defining a flow pathway through, reservoir 65, expansion control channel 21, gate 30, exit channel 41, and outlet 45.Exit channel 41 is generally defined by the mating surfaces of an outer wall of outer plug member 40 and an inner wall of reservoir body 60.The exit channel is exemplified in FIGS.",
"11 and 12 as being defined by a groove on an outer wall of outer plug member 30 and an inner wall of reservoir body 60.Alternatively, the exit channel can be defined by an outer wall of the outer plug member and grooves along the inner wall of the reservoir body.",
"In a related embodiment, exemplified in FIGS.",
"13 and 14, exit channel 41 is defined by the mating surfaces of an outer wall of inner plug member 20 and an inner wall of expansion control channel 21.In this exemplary embodiment, outlet 45 is defined by the body of outer plug member 40.In another embodiment, exemplified in FIGS.",
"15 and 16, inner plug member 20 and outer plug member 40 are provided as separate components positioned within reservoir body 60.Outer plug member 40 is stably seated within reservoir body 65, while inner plug member is slidably positioned within expansion control channel 21 and at a position proximal to outer plug member 40 and adjacent formulation 75.As in the other embodiments described above, thermal expansion of formulation 75 forces movement of inner plug member 20 to provide a volume of expansion control channel 21 sufficient to accommodate thermal expansion (volume Vlx).",
"At start-up of delivery, formulation 75 is forced against the proximal end of inner plug member 20 until gate 30 is opened to bring expansion control channel 21 into fluid communication with exit channel 41.In this example, exit channel 41 is defined by the mating surfaces of an outer wall of inner plug member 20, an outer wall of outer plug member 40, and an inner wall of reservoir body 60.In the embodiment shown in FIGS.",
"17 and 18, the inner plug member 20 further comprises an O-ring 80 to help prevent fluid leakage of formulation 75 from reservoir 65 into expansion channel 21.Assembly and Use FIGS.",
"6-10 illustrate insertion of a plug of the invention for use with a drug delivery device.",
"Reservoir 65 is filled with formulation 75 and plug 10 inserted into reservoir 65 as shown by moving plug 10 in the direction of arrow 15.Air 5 entrapped during insertion of plug 10 moves through inlet 35 inner plug member 20, through expansion control channel 21, through exit channel 41 and out of an opening from between an inner wall of reservoir 60 and an outer wall of outer plug member 40.Flow of air 5 out of plug 10 and out of the drug delivery device (indicated in FIG.",
"8 by arrow 6) is followed by flow of formulation into inlet 35 and into expansion channel 21.As the formulation fluid level (indicated by arrow 76) rises, e.g., due to thermal expansion of formulation during storage, formulation moves through expansion channel 21, and can move into exit channel 41, but without movement of any or any substantial amount through outlet 45, as illustrated in FIGS.",
"9 and 10.Assembly of a drug delivery device such as that exemplified in FIG.",
"3 can be accomplished in a two-step plug assembly method.",
"Assembly begins with reservoir 65 being filled with formulation 75 so that, for example, at room temperature (filling temperature of about 22° C.) the formulation will substantially completely fill the reservoir and plug to the top following insertion of inner plug member 20.For assembly, inner plug member 20 is first inserted into reservoir 65, preferably in a manner that allows for movement of any entrapped air through expansion control channel 21 and out of the system.",
"Outer member plug 40 is then positioned over the assembled inner plug member 20 and reservoir body 60.Assembly of plugs exemplified in FIGS.",
"11-14 can be accomplished in a one-step process by insertion of the plug into the distal end of a reservoir of a drug delivery device.",
"The plug may be inserted prior to or after filling of the reservoir with formulation.",
"After assembly and filling of the reservoir, the inner plug member 20 is slidably positioned within expansion control channel 21 such that gate 30, which connects expansion control channel 21 with exit channel 41, is closed, e.g., is blocked by fluid communication with exit channel 41 by inner plug member 20.Thermal expansion or formulation 75 within the reservoir pushes against the proximal end of inner plug member 20.When the force of the formulation against inner plug member 20 is sufficient, inner plug member 20 slides through expansion control channel 21 to accommodate expanding formulation 75.Expansion control channel 21 is of a volume sufficient to accommodate thermal expansion without allowing for opening of gate 30 and thus without allowing for flow of formulation into exit channel 41.As illustrated in FIGS.",
"11 and 12, expansion control channel 21 can extend through a distal end of outer plug member 40 to allow for release of air that may be entrapped in the reservoir and to enable the inner plug member 20 to slide freely through the outer plug member 40 without a build up of pressure in expansion control channel 21.In the embodiments of FIGS.",
"13 and 14, the exit channel 45 can provide for release of air to relieve pressure during sliding of inner plug member 20.At start-up of delivery of formulation from the device, formulation is forced against the proximal end of inner plug member 20 (see, e.g., FIGS.",
"12, 14, and 16).",
"The force of formulation 75 against the inner plug member proximal end causes inner plug member 20 to slide through expansion control channel 21 until gate 30 is open.",
"Opening of gate 30 allows formulation 75 to flow into exit channel 41 and out outlet 45.EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.",
"Efforts have been made to ensure accuracy with respect to numbers used (e.g.",
"amounts, temperature, etc.)",
"but some experimental errors and deviations should be accounted for.",
"Example 1 A prototype plug as exemplified in FIG.",
"2 was manufactured.",
"The inner plug member was machined from titantium, and was approximately 2.2987 mm in diameter and 5.062 mm in length.",
"The longitudinal portion of the expansion control channel (inner diameter of 0.43 mm) was provided through the center of the inner plug member to a length of 4.689 mm.",
"The lateral portion of the expansion control channel of 0.254 mm diameter was provided to the outside of the inner plug member body.",
"The inner plug member outer wall defined grooves (0.400 mm pitch, 0.330 mm depth, 0.279 width) to further extend the expansion control channel.",
"The outer plug member was also machined from titanium.",
"The outer plug member had an external diameter of 0.1185 mm, and defined a chamber for receiving the inner plug member.",
"The receiving chamber had a depth of 0.200 mm and an inner diameter of 0.090 mm.",
"The gate (i.e., the passage from the inner wall of the outer plug member to the outer wall of the outer plug member) was provided as a hole of 0.0145 mm in diameter.",
"Grooves (0.0157 mm pitch, 0.0020 mm depth, and 0.0028 mm width) on the outer wall of the outer plug member were provided so as to define an exit channel when in use in a reservoir body.",
"The reservoir body into which the plug is to be inserted is approximately 3.150 mm in diameter, and approximately 43.18 mm in length.",
"The components were press fit together.",
"The plug was tested by filling the reservoir of the device, and placing the device with the plug in place in a 37° C. water bath to simulate implantation in the body.",
"Sample devices made with this plug showed no aberrant release of formulation upon placement into the water bath (i.e., release of formulation associated with thermal expansion of the formulation the device), indicating that the plug prevented the undesirable “burst” of drug that can be accompanied with implantation without the plug of the invention.",
"In contrast, devices without the plug of the invention released about 1.5 μl of formulation immediately upon implantation, which release is not consistent with the desired delivery pattern."
]
] | Patent_10433196 |
[
[
"Membrane molecule indicator compositions and methods",
"The invention provides membrane molecule indicators, including polypeptides, encoding nucleic acid molecules and cells containing such polypeptides and nucleic acid molecules.",
"The invention membrane molecule indicators are characterized in that fluorescence resonance energy transfer (FRET) between a donor fluorescent domain and an acceptor fluorescent domain indicates a property of the membrane molecule.",
"Also provided are methods of using the invention membrane molecule indicators to determine a property of a membrane molecule, and to identify compounds that modulates a property of a membrane molecule."
],
[
"1.A membrane molecule indicator, said indicator comprising: (a) at least one membrane molecule indicator domain; (b) a donor fluorescent domain; and (c) an acceptor fluorescent domain; wherein fluorescence resonance energy transfer (FRET) between said donor domain and said acceptor domain is indicative of a property of said membrane molecule, and wherein said donor fluorescent domain and said acceptor fluorescent domain are not attached to the membrane molecule.",
"2.The indicator of claim 1, which comprises one polypeptide.",
"3.The indicator of claim 1, which comprises two polypeptides.",
"4.The indicator of claim 1, further comprising a membrane anchoring domain.",
"5.The indicator of claim 1, further comprising a linker between said donor fluorescent domain and said acceptor fluorescent domain.",
"6.The indicator of claim 1, comprising two membrane molecule indicator domains.",
"7.The indicator of claim 6, wherein each of said two membrane molecule indicator domains associates with the same type of membrane molecule.",
"8.The indicator of claim 1, wherein the property indicated by said membrane molecule indicator is selected from the group consisting of localization abundance, conformation and post-translational modification state of said membrane molecule.",
"9.The indicator of claim 1, wherein said membrane molecule is a lipid.",
"10.The indicator of claim 9, wherein said lipid is phosphatidylinositol 4,5-bisphosphate (PIP2).",
"11.The indicator of claim 10, wherein said membrane molecule indicator domain is a pleckstrin homology (PH) domain.",
"12.The indicator of claim 11, wherein said PH domain is a PLCδ1 or PLCβ PH domain.",
"13.The indicator of claim 1, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"14.The indicator of claim 1, wherein said donor fluorescent domain is a CFP.",
"15.The indicator of claim 1, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"16.The indicator of claim 1, wherein said acceptor fluorescent domain is a YFP.",
"17.The indicator of claim 4, wherein said membrane anchoring domain comprises a CaaX motif.",
"18.A cell comprising the indicator of any of claims 1-17.19.A nucleic acid molecule which encodes a membrane molecule indicator, or a nucleic acid kit, the nucleic acid molecule components of which encode a membrane molecule indicator, said indicator comprising: (a) at least one membrane molecule indicator domain; (b) a donor fluorescent domain; and (c) an acceptor fluorescent domain; wherein fluorescence resonance energy transfer (FRET) between the donor domain and the acceptor domain is indicative of a property of said membrane molecule, and wherein said donor fluorescent domain and said acceptor fluorescent domain are not attached to the membrane molecule.",
"20.The nucleic acid molecule or kit of claim 19, wherein the encoding nucleotide sequences are operatively linked to one or more promoters of gene expression.",
"21.The nucleic acid molecule or kit of claim 19, wherein said indicator comprises one polypeptide.",
"22.The nucleic acid molecule or kit of claim 19, wherein said indicator comprises two polypeptides.",
"23.The nucleic acid molecule or kit of claim 19, wherein said indicator further comprises a membrane anchoring domain.",
"24.The nucleic acid molecule or kit of claim 19, wherein said indicator further comprises a linker between said donor fluorescent domain and said acceptor fluorescent domain.",
"25.The nucleic acid molecule or kit of claim 19, wherein said indicator comprises two membrane molecule indicator domains.",
"26.The nucleic acid molecule or kit of claim 25, wherein each of said two membrane molecule indicator domains associate with the same type of membrane molecule.",
"27.The nucleic acid molecule or kit of claim 19, wherein the property indicated by said membrane molecule indicator is selected from the group consisting of localization, abundance, conformation and post-translational modification state of said membrane molecule.",
"28.The nucleic acid molecule or kit of claim 19, wherein said membrane molecule is a lipid.",
"29.The nucleic acid molecule or kit of claim 28, wherein said lipid is PIP2.30.The nucleic acid molecule or kit of claim 29, wherein said membrane molecule indicator domain is a PH domain.",
"31.The nucleic acid molecule or kit of claim 30, wherein said PH domain is a PLCδ1 or PLCβ PH domain.",
"32.The nucleic acid molecule or kit of claim 19, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"33.The nucleic acid molecule or kit of claim 19, wherein said donor fluorescent domain is a CFP.",
"34.The nucleic acid molecule or kit of claim 19, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"35.The nucleic acid molecule or kit of claim 19, wherein said acceptor fluorescent domain is a YFP.",
"36.The nucleic acid molecule or kit of claim 23, wherein said membrane anchoring domain comprises a CaaX motif.",
"37.A cell expressing the nucleic acid molecule or nucleic acid molecule kit components of any of claims 19-36.38.A method of determining a proper of a membrane molecule in a cell, comprising: determining FRET between said donor fluorescent domain and said acceptor fluorescent domain in a cell comprising the membrane molecule indicator of any of claims 1-17, wherein FRET between said donor domain and said acceptor domain is indicative of a property of said membrane molecule, and wherein said donor fluorescent domain and said acceptor fluorescent domain are not attached to the membrane molecule.",
"39.The method of claim 38, wherein said cell recombinantly expresses a known or candidate signaling molecules 40.The method of claim 39, wherein said signaling molecule is a G-protein coupled receptor.",
"41.A method of identifying a compound that modulates a property of a membrane molecule, comprising: (a) determining in a cell, which comprises the membrane molecule indicator of any of claims 1-17 and further comprises said membrane molecule, FRET between said donor fluorescent domain and said acceptor fluorescent domain; (b) determining in a cell, which is in contact with one or more test compounds, wherein said cell comprises the membrane molecule indicator of any of claims 1-17 and further comprises said membrane molecule, FRET between said donor fluorescent domain and said acceptor fluorescent domain, wherein increased or decreased FRET in step (b) indicates that said test compound is a compound that modulates a property of said membrane molecule.",
"42.The method of claim 41, wherein said contacting is by administration of said test compound to the exterior of said cell.",
"43.The method of claim 41, wherein said contacting is by recombinant expression of said test compound in said cell.",
"44.The method of claim 41, wherein said compound that modulates a property of said membrane molecule is selected from the group consisting of a receptor agonist, antagonist, and inverse agonist.",
"45.The method of claim 41, wherein said cell recombinantly expresses a known or candidate signaling molecule.",
"46.The method of claim 45, wherein said known or candidate signaling molecule is a G-protein coupled receptor.",
"47.A phosphatidylinositol 4,5-bisphosphate (PIP2) indicator, said indicator comprising: (a) a first polypeptide comprising: (i) a pleckstrin homology (PH) domain; and (ii) a donor fluorescent domain (b) a second polypeptide comprising: (i) a pleckstrin homology (PH) domain; and (ii) an acceptor fluorescent domain; wherein fluorescence resonance energy transfer (FRET) between said donor domain and said acceptor domain indicates PIP2 levels.",
"48.The indicator of claim 47, wherein said PH domain is a PLCδ1 or PLCβ PH domain.",
"49.The indicator of claim 47, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"50.The indicator of claim 47, wherein said donor fluorescent domain is a CFP.",
"51.The indicator of claim 47, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"52.The indicator of claim 47, wherein said acceptor fluorescent domain is a YFP.",
"53.A cell comprising the indicator of claim 47.54.A nucleic acid kit, the nucleic acid molecule components of which encode a PIP2 indicator, said indicator comprising: (a) a first polypeptide comprising: (i) a PH domain; and (ii) a donor fluorescent domain (b) a second polypeptide comprising: (i) a PH domain; and (ii) an acceptor fluorescent domain; wherein fluorescence resonance energy transfer (FRET) between said donor domain and said acceptor domain indicates PIP2 levels.",
"55.The kit of claim 54, wherein said PH domain is a PLCδ1 or PLCβ PH domain.",
"56.The kit of claim 54, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"57.The kit of claim 54, wherein said donor fluorescent domain is a CFP.",
"58.The kit of claim 54, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"59.The kit of claim 54, wherein said acceptor fluorescent domain is a YFP.",
"60.A cell expressing the nucleic acid molecule components of the kit of claim 54.61.A method of indicating PIP2 levels in a cell, comprising: (a) providing a cell containing the PIP2 indicator of claim 47; and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain, wherein FRET between said donor domain and said acceptor domain indicates PIP2 levels in the cell.",
"62.A method of identifying a compound that modulates PIP2 levels in a cell, comprising: (a) contacting a cell containing the PIP2 indicator of claim 47 with one or more test compounds; and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET follow said contacting indicates that said test compound is a compound that modulates PIP2 levels in the cell.",
"63.The method of claim 61 or 62, wherein said PH domain is a PLCδ1 or PLCβ PH domain.",
"64.The method of claim 61 or 62, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"65.The method of claim 61 or 62, wherein said donor fluorescent domain is a CFP.",
"66.The method of claim 61 or 62, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.",
"67.The method of claim 61 or 62, wherein said acceptor fluorescent domain is a YFP.",
"68.The method of claim 62, wherein said contacting is by administration of said test compound to the exterior of said cell.",
"69.The method of claim 62, wherein said contacting is by recombinant expression of said test compound in said cell.",
"70.The method of claim 61 or 62, wherein said cell recombinantly expresses a G-protein coupled receptor."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates generally to the field of signal transduction and, more specifically, to compositions and methods for indicating properties of membrane molecules using fluorescence resonance energy transfer (FRET).",
"The transduction of signals from the outside to the inside of a cell underlies most cellular processes, including proliferation, differentiation, apoptosis, motility and invasion.",
"Therefore, there is considerable interest in developing improved methods of monitoring signal transduction in response to normal and abnormal stimuli.",
"Methods of monitoring signal transduction have numerous applications, such as in identifying or improving modulators of signal transduction pathways, which are candidate therapeutic drugs or therapeutic targets, and in detecting pathological alterations in cells.",
"Some of the earliest and most sensitive signals transduced in response to stimuli involve changes in properties of membrane molecules, including membrane lipids and polypeptides, such as changes in location, abundance, conformation or post-translational modification state.",
"Accordingly, there exists a need to develop compositions and methods suitable for indicating changes in properties of membrane molecule.",
"An early response to agonist stimulation of many tyrosine kinase and G-protein coupled receptors is the activation of the enzyme phospholipase C, which cleaves the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate second messengers that increase cytosolic free Ca 2+ concentration.",
"Although Ca 2+ indicators and methods have been described that allow monitoring of Ca 2+ concentration in single living cells with high spatial and temporal resolution, Ca 2+ fluxes, being more distal to receptor activation, may not as faithfully report receptor activation levels as changes in PIP2 levels.",
"In a recently developed method for detecting PIP2 dynamics in living cells, a pleckstrin homology (PH) domain tagged with a green fluorescent protein (GFP) has been used.",
"Detection of PIP2 hydrolysis was by in vivo visualization, such as by confocal imaging and post acquisition image analysis, of translocation of the fluorescence from the membrane to the cytosol.",
"However, this method suffers from several disadvantages.",
"First, it is hard to obtain quantitative data using confocal microscopy, since even minor focal drift and changes in cell morphology that often occur after stimulation render quantitative measurements unreliable.",
"Second, it is difficult to visualize translocation in very flat cells or in cellular subregions.",
"Third, at fast imaging rates, confocal imaging requires high excitation intensities that can cause severe cell damage in minutes.",
"Fourth, the imaging approach is not easily extended to cell populations.",
"Therefore, there exists a need to develop improved methods for detecting PIP2 dynamics in cells, and particulary methods amenable to high-throughput screening.",
"The present invention satisfies these needs and provides related advantages as well."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a membrane molecule indicator, the indicator containing: (a) at least one membrane molecule indicator domain; (b) a donor fluorescent domain; and (c) an acceptor fluorescent domain; wherein fluorescence resonance energy transfer (FRET) between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"Also provided is a nucleic acid molecule which encodes a membrane molecule indicator, or a nucleic acid kit, the nucleic acid molecule components of which encode a membrane molecule indicator, the indicator containing: (a) at least one membrane molecule indicator domain; (b) a donor fluorescent domain; and (c) an acceptor fluorescent domain; wherein FRET between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"The invention also provides a method of determining a property of a membrane molecule in a cell.",
"The method includes the steps of: (a) providing a cell containing a membrane molecule indicator; and (b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain, wherein FRET between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"Further provided is a method of identifying a compound that modulates a property of a membrane molecule.",
"The method includes the steps of: (a) contacting a cell containing a membrane molecule indicator with one or more test compounds, wherein the cell further comprises the membrane molecule; and (b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain following the contacting, wherein increased or decreased FRET following the contacting indicates that the test compound is a compound that modulates a property of the membrane molecule."
],
[
"BACKGROUND OF THE INVENTION The invention relates generally to the field of signal transduction and, more specifically, to compositions and methods for indicating properties of membrane molecules using fluorescence resonance energy transfer (FRET).",
"The transduction of signals from the outside to the inside of a cell underlies most cellular processes, including proliferation, differentiation, apoptosis, motility and invasion.",
"Therefore, there is considerable interest in developing improved methods of monitoring signal transduction in response to normal and abnormal stimuli.",
"Methods of monitoring signal transduction have numerous applications, such as in identifying or improving modulators of signal transduction pathways, which are candidate therapeutic drugs or therapeutic targets, and in detecting pathological alterations in cells.",
"Some of the earliest and most sensitive signals transduced in response to stimuli involve changes in properties of membrane molecules, including membrane lipids and polypeptides, such as changes in location, abundance, conformation or post-translational modification state.",
"Accordingly, there exists a need to develop compositions and methods suitable for indicating changes in properties of membrane molecule.",
"An early response to agonist stimulation of many tyrosine kinase and G-protein coupled receptors is the activation of the enzyme phospholipase C, which cleaves the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate second messengers that increase cytosolic free Ca2+ concentration.",
"Although Ca2+ indicators and methods have been described that allow monitoring of Ca2+ concentration in single living cells with high spatial and temporal resolution, Ca2+ fluxes, being more distal to receptor activation, may not as faithfully report receptor activation levels as changes in PIP2 levels.",
"In a recently developed method for detecting PIP2 dynamics in living cells, a pleckstrin homology (PH) domain tagged with a green fluorescent protein (GFP) has been used.",
"Detection of PIP2 hydrolysis was by in vivo visualization, such as by confocal imaging and post acquisition image analysis, of translocation of the fluorescence from the membrane to the cytosol.",
"However, this method suffers from several disadvantages.",
"First, it is hard to obtain quantitative data using confocal microscopy, since even minor focal drift and changes in cell morphology that often occur after stimulation render quantitative measurements unreliable.",
"Second, it is difficult to visualize translocation in very flat cells or in cellular subregions.",
"Third, at fast imaging rates, confocal imaging requires high excitation intensities that can cause severe cell damage in minutes.",
"Fourth, the imaging approach is not easily extended to cell populations.",
"Therefore, there exists a need to develop improved methods for detecting PIP2 dynamics in cells, and particulary methods amenable to high-throughput screening.",
"The present invention satisfies these needs and provides related advantages as well.",
"SUMMARY OF THE INVENTION The invention provides a membrane molecule indicator, the indicator containing: (a) at least one membrane molecule indicator domain; (b) a donor fluorescent domain; and (c) an acceptor fluorescent domain; wherein fluorescence resonance energy transfer (FRET) between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"Also provided is a nucleic acid molecule which encodes a membrane molecule indicator, or a nucleic acid kit, the nucleic acid molecule components of which encode a membrane molecule indicator, the indicator containing: (a) at least one membrane molecule indicator domain; (b) a donor fluorescent domain; and (c) an acceptor fluorescent domain; wherein FRET between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"The invention also provides a method of determining a property of a membrane molecule in a cell.",
"The method includes the steps of: (a) providing a cell containing a membrane molecule indicator; and (b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain, wherein FRET between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"Further provided is a method of identifying a compound that modulates a property of a membrane molecule.",
"The method includes the steps of: (a) contacting a cell containing a membrane molecule indicator with one or more test compounds, wherein the cell further comprises the membrane molecule; and (b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain following the contacting, wherein increased or decreased FRET following the contacting indicates that the test compound is a compound that modulates a property of the membrane molecule.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows four exemplary membrane molecule indicator compositions.",
"Solid bar: membrane anchoring domain.",
"Hatched and open boxes: fluorescent donor domain or fluorescent acceptor domain.",
"Thick semi-circle: MMID.",
"Thin semi-circle: linker.",
"Solid circle: membrane molecule.",
"Solid triangle: represents an altered property of membrane molecule.",
"(A-D): FRET is high due to association between membrane molecule indicator domain (MMID) and membrane molecule at the membrane.",
"(E-H): FRET is low due to dissociation between MMID and membrane molecule, as a result of an altered property of membrane molecule.",
"(I-L): FRET is low due to altered localization of membrane molecule.",
"FIG.",
"2 shows fluorescence resonance detection of PH domain translocation.",
"(A) Schematic representation of FRET occurring between CFP-PH and YFP-PH bound to the membrane.",
"Upon hydrolysis of PI(4,5)P2, PH domains translocate to the cytosol and FRET ceases.",
"(B) Emission signals of CFP and YFP, collected at 475 and 530 nm respectively, and the ratio of 530/475, recorded-from a single N1E-115 cell stimulated with bradykinin (BK, 1 μM).",
"Signals were low-pass filtered at 2 Hz and sampled at 3 Hz.",
"Scale bar for ratio signal shows percent deviation from baseline.",
"(C) Confocal detection of GFP-PH translocation, depicted on the same scale.",
"Images were collected once per 10 seconds, and the ratio of fluorescence intensities in membrane and cytosol (PM/Cyt) was deduced for each image by post-acquisition automated image analysis.",
"FIG.",
"3 shows characterization of fluorescence emission.",
"Cells expressing constructs as indicated were stimulated with 1 μM bradykinin and fluorescence emission was detected at the indicated wavelength.",
"FIG.",
"4 shows Fluorescence Recovery After Photobleaching (FRAP) to reveal dynamic movements of GFP-PH between cytosol and membrane.",
"Spots (approx.",
"1.3 μm full-width half maximum) were completely bleached in the basal membrane (or in the cytosol for B) with a 30 m-s pulse of 488 nm laser light, and recovery was monitored in line-scan mode in a confocal microscope.",
"(A) FRAP of membrane-delimited YFP-CAAX; (B) cytosolic PLCδ1PH(R40L)-GFP mutant that cannot bind PI(4,5)P2; (C) PLCδ1PH-GFP in a resting cell; (D) PLCδ1PH-GFP in a cell that has agonist-induced partial translocation of fluorescence.",
"Insets show confocal images for the distribution of these constructs, taken from distinct cells.",
"FIG.",
"5 shows PLC activation in single cells, neurites or in cell populations recorded by FRET.",
"(A) A single N1E-115 cell was stimulated repeatedly with neurokinin A (NKA) as indicated by the lines (dashes, 10 s pulses of 100 μM NKA from a puffer pipette; solid line, addition of 1 μM final concentration to the culture dish).",
"The response shows repeated PLC activation and partial desensitization.",
"PLC activation induced by subsequently added bradykinin (BK, 1 μM) was not desensitized by NKA pretreatment.",
"For calibration, maximal translocation was induced by adding 5 μM ionomycin+2 mM additional Ca2+.",
"(B) PLC activation in a single neurite of a neuroblastoma cell, differentiated by culturing in serum-free medium for 48 hours.",
"Area of measurement (2.5×9 μm) is indicated in the micrograph.",
"Excitation bandwidth was increased to 20 nm.",
"(C) FRET recording from a cluster of about 15 transfected cells demonstrates improved signal-to-noise ratio and averaged kinetics (note the same scale for B and C).",
"FIG.",
"6 shows that the PH domain of PLCδ1 reports changes in PI(4,5)P2 rather than in IP3 in N1E-115 cells.",
"(A) Cells expressing GFP-PH were loaded with both Fura-Red (20 FM) and caged IP3 (100 PM) by in-situ high frequency electroporation.",
"Shown is the response of a single cell, assayed simultaneously for GFP translocation and Ca2+ mobilization induced by flash photolysis of caged IP3.Arrows indicate photolysis of 1 μM, 10 μM and 90 μM.",
"For comparison, bradykinin (1 μM) was added afterwards.",
"Representative trace from 16 similar experiments.",
"(B) FRET response to bradykinin detected in a single cell, pretreated with 5 μM of phenyl arsine oxide for 10 minutes.",
"(C-D), time course of Ins(1,4,5)P3 and Ins(1,3,4)P3 formation in adrenal glomerulosa cells prelabeled with [3H]inositol, after stimulation with angiotensin II (Ang, 1 μM) in the presence of 2 mM Sr2+ or Ca2+.",
"(E) Angiotensin II-induced translocation as quantitated by analysis of serial confocal images of glomerulosa cells in the presence of Sr2+ or Ca2+.",
"Data points represent means±S.E.M., n=5.",
"(F) Bradykinin-induced translocation, with and without Sr2+, as detected by FRET in N1E-115 cells.",
"FIG.",
"7 shows heterogeneity of PLC activation responses to different GPCR agonists.",
"Single N1E-115 cells expressing CFP-PH and YFP-PH were stimulated with 1 μM bradykinin (BK), 1 μM neurokinin A (NKA), 50 μM thrombin-receptor activating peptide (TRP), 1 μM lysophosphatidate (LPA) or 10 μM histamine (HIS).",
"PLC activation as assayed by FRET, and intracellular Ca2+ recordings for these agonists detected ratiometrically using Yellow Cameleon 2.1 in separate experiments, are shown.",
"Changes in fluorescence ratio are expressed as percent of resting values.",
"Shown are representative examples of experiments performed at least 10 times.",
"FIG.",
"8 shows that PLC inactivation kinetics mirror receptor inactivation.",
"(A) FRET recording from a single N1E-115 cell stimulated with neurokinin A (NKA) and with 1 mM aluminum fluoride (AlF4−).",
"(B) Confocal micrographs of cells, taken 56 hours after transfection with PLCδ1PH-GFP (5 μg DNA/well) together with different amounts of constitutively active Gaq subunit (Gq*, 0.8 μg/well, and Gq* 1:10, 0.08 μg/well) or with constitutively active Gα12 at 0.8 μg/well (G12*).",
"(C) PLC activation detected by FRET in single neuroblastoma cells (left panel), expressing wild-type NKA receptors, stimulated with 10 second pulse from a puffer pipette with 100 μg NKA; and cells stimulated by prolonged addition of NKA (1 μM) to the medium, expressing either wild-type receptors (middle panel) or a mutant truncated at its C-terminus (right panel).",
"Recordings are all to the same scale.",
"(D) Kinetics of PLC activation by NKA in a N1E-115 cell transfected with the C terminally truncated NK2 receptors on an extended time scale.",
"FIG.",
"9 shows an exemplary membrane molecule indicator.",
"Oval: membrane molecule.",
"Trapezoid: MMID.",
"The donor and acceptor fluorescent domains are indicated.",
"Top: FRET is high due to association between MMID and the membrane molecule at the membrane and proximity of the donor and acceptor.",
"Bottom: FRET is low due to relocalization of membrane molecule and resulting separation of the donor and acceptor.",
"FIG.",
"10 shows an exemplary membrane molecule indicator.",
"Oval: membrane molecule.",
"Trapezoid: MMID.",
"The donor and acceptor fluorescent domains are indicated.",
"Top: FRET is low due to association between MMID and the membrane molecule at the membrane and separation of the donor and acceptor.",
"Bottom: FRET is high due to relocalization of membrane molecule and resulting proximity of the donor and acceptor.",
"DETAILED DESCRIPTION OF THE INVENTION The invention provides membrane molecule indicator compositions, including polypeptides, encoding nucleic acid molecules, and cells, as well as related methods for determining properties of a membrane molecule and for identifying modulatory compounds.",
"The membrane molecule indicator compositions of the invention are characterized by a membrane molecule indicator domain, a donor fluorescent domain and an acceptor fluorescent domain.",
"The donor fluorescent domain and acceptor fluorescent domain exhibit a characteristic fluorescence resonance energy transfer (FRET) when the membrane molecule indicator domain is associated with a membrane molecule at a membrane.",
"This characteristic FRET observed when the membrane molecule indicator domain and membrane molecule are associated at the membrane differs from FRET observed when the membrane molecule indicator domain dissociates from the membrane molecule, or when the membrane molecule is no longer localized to the membrane.",
"Therefore, FRET between the donor and acceptor fluorescent domains serves as an indicator of association at the membrane between the membrane molecule indicator domain and the membrane molecule, and thus serves as an indicator of a property of the membrane molecule.",
"In one embodiment, FRET is high when the membrane molecule indicator domain and membrane molecule are associated at the plasma membrane (e.g.",
"FIG.",
"1A-D and FIG.",
"9, top), and low when the membrane molecule indicator domain dissociates from the membrane molecule (e.g.",
"FIG.",
"1E-H), or when the membrane molecule relocalizes (e.g.",
"FIG.",
"1I-L and FIG.",
"9, bottom).",
"In another embodiment, FRET is low when the membrane molecule indicator domain and membrane molecule are associated at the plasma membrane (e.g.",
"FIG.",
"10, top), and high when the membrane molecule indicator domain dissociates from the membrane molecule, or when the membrane molecule relocalizes (e.g.",
"FIG.",
"10, bottom).",
"Properties of a membrane molecule that can affect its ability to associate at the membrane with an indicator domain include, for example, its localization, abundance, conformation and post-translational modifications.",
"These properties of membrane molecules are of considerable interest, as they often reflect changes that occur as a result of activation or inactivation of cellular signaling pathways that regulate fundamental cellular processes, including growth, differentiation, apoptosis, motility and invasion.",
"Therefore, the invention compositions and methods can be used to identify and determine the function of modulators of cellular signaling pathways, and thus have important therapeutic, diagnostic and research applications.",
"In one embodiment, the membrane molecule indicator compositions of the invention contain (or encode) a single polypeptide that contains a membrane molecule indicator domain, a membrane anchor, a donor fluorescent domain and an acceptor fluorescent domain (shown schematically in FIG.",
"1A).",
"In an alternative embodiment, the membrane molecule indicator compositions of the invention contain (or encode) two polypeptides, one containing a membrane molecule indicator domain, the other containing a membrane anchor domain, one of which further contains a donor fluorescent domain, the other of which further contains an acceptor fluorescent domain (shown schematically in FIG.",
"1B).",
"In another embodiment, the membrane molecule indicator compositions of the invention contain (or encode) a single polypeptide that contains two membrane molecule indicator domains, a donor fluorescent domain and an acceptor fluorescent domain (shown schematically in FIG.",
"1C).",
"In yet another embodiment, the membrane molecule indicator compositions of the invention contain (or encode) two polypeptides, each containing a membrane molecule indicator domain, one of which contains a donor fluorescent domain and the other of which contains an acceptor fluorescent domain (shown schematically in FIG.",
"1D).",
"In a further embodiment, the membrane molecule indicator compositions of the invention contain (or encode) one polypeptide, containing a central membrane molecule indicator domain, with a donor fluorescent domain and an acceptor fluorescent domain at the termini (shown schematically in FIGS.",
"9 and 10).",
"It will be appreciated by the skilled person that the membrane molecule indicators shown in FIGS.",
"1, 9 and 10 can be modified in a variety of ways, so long as the donor and fluorescent domains are operably positioned so as to exhibit a characteristic FRET when the membrane molecule indicator domain and membrane molecule are associated at the membrane, which differs from FRET observed when the membrane molecule indicator domain dissociates from the membrane molecule, or when the membrane molecule is no longer localized to the membrane.",
"For example, the relative locations of the donor fluorescent domain and acceptor fluorescent domain with respect to a membrane anchoring domain can be reversed in the compositions shown in FIGS.",
"1A and B.",
"The membrane molecule indicator compositions can also contain additional peptide or non-peptide domains, such as linker sequences between the donor fluorescent domain and acceptor fluorescent domain, or between a fluorescent domain and either the MMID or the membrane anchor.",
"Likewise, either the donor or acceptor fluorescent domains shown in FIGS.",
"9 and 10 can optionally contain membrane anchor domains.",
"When two MMIDs are present, the MMIDs can each associate with the same type of membrane molecule.",
"In such applications, the MMIDs can be identical, or different, so long as they associate with the same type of membrane molecule.",
"For other applications, it may be preferable that the MMIDs associate with different types of membrane molecules, which are commonly or differentially regulated.",
"Thus, such the membrane molecule indicator compositions can simultaneously, or alternatively, report the properties of two different membrane molecules.",
"As used herein, the term “membrane molecule” refers to a molecule that transiently, or permanently, resides at, partially or completely within, or across, a lipid bilayer of a cell.",
"A membrane molecule can thus be an integral membrane molecule, such as a lipid bilayer component or an integral membrane protein.",
"Alternatively, a membrane molecule can be a peripheral membrane molecule that directly associates with the lipid bilayer, or indirectly associates with the lipid bilayer by virtue of interaction with an integral membrane molecule.",
"A membrane molecule useful in the methods of the invention is a molecule that as a direct or indirect response to a normal or pathological stimulus, exhibits a change in a property that results in an increased or decreased association at the membrane between the membrane molecule and the particular membrane molecule indicator domain.",
"Exemplary properties of a membrane molecule that can change in response to a stimulus, and which can result in an increased or decreased association at the membrane between the membrane molecule and the MMID, include location (e.g.",
"translocation of the membrane molecule from its membrane location to a different cellular location, or vice versa), abundance (e.g.",
"local, or overall, increase or decrease in abundance of the membrane molecule at the membrane), conformation (e.g.",
"tertiary or quaternary structure, which can reflect activation state), and post-translational modification state (e.g.",
"acylation, biotinylation, mannosylation, farnesylation, formylation, geranyl-geranylation, hydroxylation, methylation, myristoylation, palmitoylation, phosphorylation, sulphation and the like).",
"Therefore, such properties of a membrane molecule, as indicated by its relative ability to associate with a membrane molecule indicator domain, reflect the presence and nature of the stimulus.",
"The appropriate property which changes in response to a stimulus, will depend on the nature of the membrane molecule and the stimulus.",
"As an example of a class of membrane molecules that exhibit changes in properties in response to stimuli, it is well known in the art that tyrosine kinase receptors often exhibit changes in location and abundance at the membrane (e.g.",
"by becoming internalized), conformation (e.g.",
"by adopting an activated tertiary conformation, dimerizing, or associating with effector molecules), and/or post-translational state (e.g.",
"by becoming tyrosine phosphorylated) in response to ligands.",
"Certain phospholipids exhibit changes in abundance (e.g.",
"by becoming hydrolyzed or produced) in response to agonist activation of receptors.",
"Other examples of membrane molecules and changes in their properties in response to stimuli, which can be detected using the methods and compositions of the invention, are known in the art and described further below.",
"As used herein, the term “membrane,” with respect to the location of a membrane molecule detected by the indicator compositions of the invention, refers to any lipid bilayer of a cell, including, but not limited to, the plasma membrane, Golgi membrane, endoplasmic reticulum (ER) membrane, mitochondrial membrane, endosomal membrane, peroxisomal membrane, lysosomal or vacuolar membrane, and nuclear membrane.",
"A membrane molecule can be of any nature, such as a lipid, protein, saccharide, or any combination thereof.",
"In one embodiment, the membrane molecule is a membrane lipid.",
"Exemplary membrane lipids include cholesterol, sphingolipids, polyisoprenoids, mono-, di- and triacylglycerols, acyl chains and their derivatives (e.g.",
"arachadonic acid and its metabolites, such as prostaglandins), and phospholipids (e.g.",
"phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, phosphaytidlyglycerols, lyso-derivatives thereof and phosphatidylinositols.",
"Exemplary phosphatidylinositols include PtdIns(4,5)P2 (also referred to as PIP2), PtdIns(3,4)P2, PtdIns(3,4,5) P3, PtdIns, PtdIns(3)P, PtdIns(4)P, as well as D-enantiomers (e.g.",
"D-Ins(1,4,5) P3), di-carboxy derivatives (e.g.",
"DiC8-PtdIns(4,5)P2) and glycerophosphoryl derivatives (e.g.",
"g-PtdIns(4,5)P2)of these molecules.",
"The structural and regulatory function of membrane lipids in normal and abnormal biological processes, as well as the changes in properties of lipids (e.g.",
"abundance, localization, conformation and post-translation modifications) that occur in response to normal and pathological stimuli, are well known in the art.",
"For example, a variety of sphingolipids have roles in signaling, such as sphingosine in inhibiting PKC, ceramide in modulating arachidonic acid (AA) release, and sphingosine-1-phosphate in mobilizing calcium (reviewed in Shayman, Kidney International 58:11-26 (2000).",
"As other examples of the role of membrane lipids in signaling, diacylglycerol (DAG) activates protein kinase C (PKC); phosphatidic acid (PA) activates certain kinases; and phosphatidyl choline serves as a substrate for phospholipase D to generate PA and then DAG, as well as a substrate for phospholipase A2 to generate AA, which is the precursor for eicosanoids and prostaglandins.",
"Phosphatidylinositols are particularly important signaling molecules.",
"For example, many cell surface receptors are coupled to phospholipase C activation.",
"PLC activation cleaves the phosphatidylinositol PIP2 to produce the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).",
"These second messengers increase intracellular Ca2+ concentration and activate the serine/threonine specific protein kinase C (PKC), respectively.",
"PIP2 also serves as a substrate for phosphatidyl inositol 3-kinase (PI3K), producing the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3).",
"PIP2 also is implicated in the regulation of the actin cytoskeleton, based on its ability to bind to and regulate the function of a number of actin severing, capping and bundling proteins.",
"Additionally, PIP2 modulates the activity of phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline.",
"PIP2 resides at the plasma membrane of resting cells.",
"Upon agonist stimulation of a receptor coupled to PLC, such as a tyrosine kinase receptor, or a G-protein coupled receptor (GPCR) that acts through a Gαq-containing effector G protein, PIP2 is hydrolyzed to yield soluble IP3 and membrane bound DAG.",
"PIP2 is then resynthesized and returns to the membrane.",
"Accordingly, the abundance of PIP2 at the plasma membrane reports the activation state of a PLC-coupled receptor, in that high abundance of PIP2 at the plasma membrane indicates the resting state, and low abundance indicates agonistic activity through the receptor.",
"In an alternative embodiment, a membrane molecule is a membrane protein.",
"Exemplary membrane proteins include integral membrane proteins such as cell surface receptors (e.g.",
"G-protein coupled receptors (GPCRs), tyrosine kinase receptors, integrins and the like) and ion channels; and proteins that shuttle between the membrane and cytosol in response to signaling (e.g.",
"Ras, Rac, Raf, Gα subunits, arrestins, Src and other effector proteins).",
"In certain embodiments, when specifically indicated, excluded from the scope of the invention is a membrane molecule that is a GPCR.",
"The structural and regulatory function of membrane proteins in normal and abnormal biological processes, as well as the changes in their properties (e.g.",
"abundance, localization, conformation and post-translation modifications) that occur in response to normal and pathological stimuli, are well known in the art.",
"As used herein, a “membrane molecule indicator domain” or “MMID” refers to a domain that associates with a membrane molecule with sufficient affinity and selectivity to report a property of the membrane molecule.",
"The choice of membrane molecule indicator domain will depend on the particular membrane molecule.",
"MMIDs for the membrane molecules described above are known in the art, or can be readily determined.",
"Suitable MMIDs include, for example, domains that mediate interaction with the membrane molecule that are present in its naturally occurring oligomeric partner(s), regulators and effectors, as well as functional variants of such domains.",
"Thus, for example, MMIDs that bind to membrane molecules can consist of SH2, SH3, PH, PTB, EH, PDZ, EVH1 and WW domains that bind the membrane molecule in vivo, as well as functional variants of such domains.",
"In certain embodiments, such as when the membrane molecule indicator is designed to indicate activation state of a GPCR, the MMID can comprise a G-protein subunit, such as a Gα, Gβ or Gγ subunit.",
"For example, high FRET between a Gα subunit linked to a donor fluorescent domain and a Gβ and/or Gγ subunit linked to an acceptor fluorescent domain (or vice versa) can indicate the inactive state of the GPCR, in which the trimeric G-protein complex is present at the membrane.",
"In contrast, low FRET can indicate activation of the GPCR and dissociation of the G-protein complex.",
"In other embodiments, when specifically indicated, excluded from the scope of the invention is an MMID which comprises a G-protein subunit.",
"MMIDs also include domains which do not normally interact with the membrane molecule in the cell, but are determined, by methods known in the art, to have sufficient affinity and selectivity to report a property of the membrane molecule.",
"Where the membrane molecule is a phosphatidylinositol, a suitable membrane molecule indicator domain is a phosphatidylinositol binding domain.",
"Phosphatidylinositol binding domains include, for example, “pleckstrin homology” or “PH” domains, “FYVE” domains, “C2” domains, “SH2” domains, PtdIns-binding domains of actin-binding proteins, PtdIns-binding domains of clathrin adaptor proteins, and START domains (reviewed in Bottomley et al., Bioc.",
"Biophys.",
"Acta 1436:165-183 (1998); Stenmark et al., J.",
"Cell Science 112:4175-4183 (1999); Janmey, Chem.",
"Biol.",
"2:61-65 (1995); and Ponting et al., TIBS 24:130-132 (1999)).",
"In one embodiment, the phosphatidylinositol indicator domain is a pleckstrin homology (PH) domain.",
"PH domains are generally around 120 amino acids long and share characteristic structural features that include two orthogonal β-sheets of three and four anti-parallel β-strands, which sandwich an α-helix at the C-terminus.",
"PH domains also contain clusters of lysine and arginine residues distal to the C-terminal α-helix that create a highly charged surface, and an almost invariant tryptophan residue near the C-terminus.",
"PH domains have been found in more than 100 different proteins, including mammalian, Drosophila, C. elegans and yeast proteins.",
"Many PH domain containing proteins are involved in intracellular signaling and cytoskeletal organization.",
"Examples of PH domain containing proteins include protein kinases (e.g.",
"Btk, β-ARK and Akt), all phospholipase C (PLC) isoforms (e.g.",
"PLCβ, γ and δ), insulin receptor substrates (IRS-1 and IRS-2), phosphoinositide 3-kinase (PI3 kinase) p110γ subunit, the guanine nucleotide release factor SOS, rasGAP, dynamin, CDC25, Tiam-1, Vav, guanine nucleotide exchange factors (e.g.",
"GRP-1, ARNO, cytohesin) and β-spectrin.",
"The sequences, ligands and relative binding affinities of a variety of PH domains are known in the art (see, for example, Bottomley et al., supra (1998)).",
"A preferred PH domain is a PH domain of a PLC, such as the PIP2-indicator PH domain of PLCδ1.The cloning and expression of the PH domain of PLCδ1, and its use in membrane molecule indicator polypeptides, is described in the Example, below.",
"An alternative PH domain of a PLC is the PH domain of PLCβ.",
"PLCβ is responsible for physically cleaving PIP2, and thus the PH domain therefrom can be used to determine tranlocation or disassociation from the membrane of the actual PIP2 lipids cleaved by PLCβ.",
"The PLCβ PH domain sequence is known in the art (e.g.",
"Rebecchi et al., Annu.",
"Rev.",
"Biophys.",
"Biomol.",
"Struct.",
"27:503-528 (1998)).",
"In another embodiment, the phosphatidylinositol indicator domain is an FYVE domain.",
"FYVE domains have been demonstrated to specifically bind to PtdIns(3)P. FYVE domains generally contain eight conserved cysteines, which coordinate two Zn2+ ions in a cross-braced topology, a conserved R(R/K)HHCRXCG (SEQ ID NO:1) motif surrounding the third and fourth cysteine residues, and several highly conserved hydrophobic residues (see, for example, Stenmark et al., supra (1999), and Gaullier et al., Chem.",
"Phys.",
"Lipids 98:87-94 (1999)).",
"FYVE domains have been found in mammalian, yeast and C. elegans proteins.",
"Exemplary FYVE domain containing proteins include EEA1, Fab1p, YOTB, Vac1p, Vps27p, Hrs, Smad anchor for receptor activation (SARA), Fgd1, and have also been described in a large number of proteins of unknown function whose sequences are available in public databases (Stenmark et al., supra (1999)).",
"In another embodiment, the phospholipid indicator domain is a C2 domain.",
"C2 domains are about 130 amino acids in length, and have been found in single or multiple copies in over 60 proteins.",
"C2 domains bind a variety of ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates and intracellular proteins.",
"C2 domains are found, for example, in synaptotagmin I-VIII, rabphilin, phosphatidylserine decarboxylase, protein kinase C, GAPs, perforin, PLC family members, BCR, ABR, PI3-kinase, cytosolic phospholipase A2, and have also been described in a large number of proteins of unknown function whose sequences are available in public databases (reviewed in Nalefski et al., Protein Science 5:2375-2390 (1996).",
"In yet another embodiment, the phospholipid indicator domain is an SH2 domain.",
"SH2 domains are well-characterized mediators of protein-protein interactions, but in addition certain SH2 domains bind phosphoinositides.",
"For example, the SH2 domains from p85α and c-Src have been shown to directly and selectively bind PtdIns (3,4,5) P3 (Bottomley et al., supra (1998)).",
"The sequences of a variety of SH2 domains are known in the art.",
"In a further embodiment, the phospholipid indicator domain is a lipid binding domain of an actin binding protein, such as the lipid binding domain of the actin monomer sequestering protein profilin; the actin filament severing proteins gelsolin, villin, severin, adseverin, destrin and cofilin; the protein gCap39, which blocks the ends of actin filaments; and the actin filament cross-linking protein α-actinin (reviewed in Janmey, supra (1995)).",
"The sequences of a variety of lipid binding domains of actin binding proteins are known in the art.",
"In another embodiment, the phospholipid indicator domain is a lipid binding domain of a clathrin adaptor protein, such as residues 5-80 of AP-2 (α-subunit), which specifically associates with PtdIns(3,4,5)P3, or residues 1-304 of AP-3, which specifically associates with pyrophosphate(PP)-InsP5 (reviewed in Bottomley et al., supra (1998)).",
"The sequences of a variety of lipid binding domains of clathrin adaptor proteins are known in the art.",
"Other membrane molecule indicator domains can be readily identified, for example, by database searching and by structural predictions based on sequence or structural homology to known membrane molecule indicator domains, as described above.",
"Association between a MMID and a membrane molecule can be determined by binding assays known in the art.",
"For example, association can be determined by co-immunoprecipitation assays, sedimentation assays, affinity chromatography, two-hybrid assays, gel-overlay assays, radiolabeled ligand binding assays, and the like.",
"Association between molecules can also be determined by surface plasmon resonance (SPR) on BIAcore, nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and mass spectroscopy.",
"Association between a MMID and lipid membrane molecule can conveniently be determined by adsorbing the MMID to a vesicle containing the lipid, and sedimenting the vesicle-bound protein by centrifugation.",
"Such methods are reviewed, for example, in Winzor, J. Mol.",
"Recognit.",
"13:279-298 (2000); and Bottomley et al., supra (1998).",
"The membrane molecule indicator domains described herein need not have the exact sequence of a domain found in a native sequence, so long as the domain retains the membrane molecule indicator function of the native sequence.",
"Thus, a membrane molecule indicator domain can be a variant sequence having one or several amino acid additions, deletions or substitutions compared with a native amino acid sequence.",
"Such modifications can be advantageous, for example, in enhancing the stability, expression level, or binding specificity of the domain, as well as for facilitating chimeric polypeptide construction.",
"The function of a variant MMID can be confirmed by the binding assays described above.",
"Modifications to the amino acid sequence of a MMID can be randomly generated, such as by random insertions, deletions or substitutions of nucleotides in a native MMID nucleic acid molecule.",
"Alternatively, modifications can be directed, such as by site-directed mutagenesis of a nucleic acid molecule encoding a native MMID.",
"The skilled person appreciates that extensive guidance in predicting which amino acid residues of a MMID can be modified, while retaining membrane molecule indicator ability, is provided by examining alignments between orthologs and other members of a particular MMID family.",
"It is well known in the art that evolutionarily conserved amino acid residues and motifs are more likely to be important for maintaining biological activity than less well-conserved residues and domains.",
"Thus, it would be expected that substituting a residue that is highly conserved among MMIDs within a family or across species with a non-conserved residue may be deleterious, whereas making the same substitution at a residue which varies widely would likely not have a significant effect on biological activity.",
"These guiding principles have been confirmed for a variety of MMID containing proteins by mutagenesis studies.",
"In general, a variant MMID will have at least 70% identity, more preferably at least 75% identity, including at least 80%, 85%, 90%, 95%, 98%, 99% or greater identity to the native domain to which the variant domain is most closely related.",
"Thus, as a non-limiting example, a PIP2 indicator domain can be a domain that has at least 70% identity, more preferably at least 75% identity, including at least 80%, 85%, 90%, 95%, 98% or greater identity to amino acids 1-174 of the human PLCδ-1 sequence (GenBank Accession No.",
"NM—006225).",
"As used herein, the term “membrane anchoring domain” refers to the portion of a membrane molecule indicator polypeptide that localizes the polypeptide to a particular membrane.",
"Membrane anchoring domains suitable for localizing polypeptides to membranes of interest are known in the art.",
"For example, a membrane anchoring domain suitable for localizing a polypeptide to the plasma membrane is the C-terminal sequence CaaX (where “a” is an aliphatic residue, and “X” is any residue, generally L).",
"An exemplary membrane anchoring domain suitable for localizing a polypeptide to the endoplasmic reticulum is the C-terminal sequence KDEL (SEQ ID NO:2), assuming a signal sequence present at the N-terminus.",
"Additionally, membrane anchoring domains can be small proteins, and portions of proteins, that confer appropriate localization to the membrane molecule indicator polypeptide when present in a chimera.",
"Optionally, the membrane anchoring domain can be a second membrane molecule indicator domain that associates with a different membrane molecule than the first membrane molecule indicator domain, and that is not co-regulated with the first membrane molecule.",
"For example, in order to determine membrane abundance of PIP2, an appropriate indicator composition can include a membrane molecule indicator domain that associates with PIP2 (e.g.",
"a PH domain) fused to a donor fluorescent domain, and a membrane molecule indicator domain that associates with a different membrane molecule that is not co-regulated with PIP2 fused to an acceptor domain, which thus serves to anchor the acceptor domain to the plasma membrane.",
"As used herein, the terms “donor fluorescent domain” and “acceptor fluorescent domain” refer to a pair of moieties selected so as to exhibit fluorescence resonance energy transfer (FRET) when the donor moiety is excited with appropriate electromagnetic radiation or becomes luminescent.",
"The donor fluorescent domain is excited by light of appropriate intensity within its excitation spectrum, and emits the absorbed energy as fluorescent light.",
"When the acceptor fluorescent domain is positioned to quench the donor fluorescent domain in the excited state, the fluorescence energy is transferred to the acceptor fluorescent domain, which can emit fluorescent light.",
"FRET can be manifested as a reduction in the intensity of the fluorescent signal emitted from the donor fluorescent domain, by reduction in the lifetime of the excited state of the donor fluorescent domain, or by emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor fluorescent domain.",
"When the association between the MMID and the corresponding membrane molecule changes, the donor and acceptor fluorescent domains physically separate (or come closer together), and FRET is decreased (or increased) accordingly (see FIG.",
"1).",
"One factor to be considered in choosing the fluorescent domain pair is the efficiency of fluorescence resonance energy transfer between them.",
"Preferably, the efficiency of FRET between the donor and acceptor moieties is at least 10%, more preferably at least 50% and even more preferably at least 80%.",
"The efficiency of FRET can easily be empirically tested using the methods described herein and known in the art.",
"The efficiency and detectability of FRET also depend on the separation distance and the orientation of the donor and acceptor fluorescent domains, as well as the choice of fluorescent domains.",
"Considerations for the choice of fluorescent domains are well known in the art, and described, for example, in U.S. Pat.",
"Nos.",
"5,998,204 and 5,981,200.For example, it is preferred that the emission spectrum of the donor fluorescent domain overlap as much as possible with the excitation spectrum of the acceptor fluorescent domain.",
"In addition, the excitation spectra of the donor and acceptor fluorescent domains should overlap as little as possible so that a wavelength region can be found at which the donor fluorescent domain can be excited selectively and efficiently without directly exciting the acceptor moiety.",
"Likewise, the emission spectra of the donor and acceptor fluorescent domains should have minimal overlap so that the two emissions can be distinguished.",
"Furthermore, it is desirable that the quantum yield of the donor fluorescent domain, the extinction coefficient of the acceptor fluorescent domain, and the quantum yield of the acceptor fluorescent domain be as large as possible.",
"For example, in a suitable pair of fluorescent domains, the donor fluorescent domain is excited by ultraviolet light (<400 nm) and emits blue light (<500 nm), while the acceptor fluorescent domain is efficiently excited by blue light (but not by ultraviolet light) and emits green light (>500 nm).",
"In an alternative pair of fluorescent domains, the donor fluorescent domain is excited by violet light (about 400-430 nm) and emits blue-green light (450-500 nm), while the acceptor fluorescent domain is efficiently excited by blue-green light (but not by violet light) and emits yellow-green light (about 520-530 nm).",
"Generally, the donor fluorescent domain and acceptor fluorescent domain will be fluorescent proteins, as described below.",
"Alternatively, the donor can contain a tag, such as an artificial tetracysteine-based peptide tag, to which a cell permeable fluorescent label, such as FLASH-EDT2, can bind (e.g.",
"Griffin et al., Science 281:269-272 (1998)).",
"Fluorescent proteins suitable for use as donor or acceptor fluorescent domains in the compositions and methods of the invention have been isolated from a number of species, including jellyfish (e.g.",
"Aequorea species) and coral (e.g.",
"Renilla species and Discosoma species).",
"In one embodiment, the donor and/or acceptor fluorescent domain is a “green fluorescent protein” or “GFP,” such as a native GFP from an Aequorea or Renilla species, an ortholog of a GFP from another genus, or a variant of a native GFP with optimized properties.",
"As used herein, the term “GFP variant” is intended to refer to polypeptides with at least about 70%, more preferably at least 75% identity, including at least 80%, 90%, 95% or greater identity to a native GFP, such as Aequorea victoria GFP.",
"A variety of GFP variants having useful excitation and emission spectra, have been engineered by modifying the amino acid sequence of a naturally occurring Aequorea or Renilla GFP (see, for example, U.S. Pat.",
"Nos.",
"5,625,048 and 5,998,204; Miyawaki et al., Nature 388:882-887 (1997); Delagrave et al., Biotechnology 13:151-154 (1995); Pollok et al., Trends in Cell Biol.",
"9:57-60 (1999)).",
"Additionally, a variety of enhanced GFPs (or EGFPs) with optimized codons for expression in human cells, are known in the art (e.g.",
"ECFP and EYFP).",
"GFP variants with optimized dimerization properties can also be prepared.",
"It is postulated that the weak dimerization observed between GFPs (e.g.",
"kD about 100 μM) allows donor and acceptor fluorescent domains present on separate polypeptide chains (e.g.",
"FIG.",
"1B or 1D) to associate at the membrane and exhibit FRET, even at low expression levels where based simply on polypeptide concentration at the membrane, FRET would not be expected.",
"The dimerization is suitably weak so that once dissociated from the membrane or from the membrane molecule, the donor and acceptor fluorescent domains separate so as to no longer exhibit FRET.",
"GFP variants with altered dimerization properties can be selected so as to optimize the differential in FRET between alternatives configurations.",
"For example, GFP variants with slightly higher, but still moderate, dimerization (e.g.",
"kD about 25 μM) are expected to provide for suitably high FRET at the membrane even at low polypeptide expression levels, while still separating once dissociated from the membrane or from the membrane molecule.",
"Cyan fluorescent proteins (CFPs) are variant GFPs that contains the mutation Y66W with respect to Aequorea victoria GFP.",
"Yellow fluorescent proteins (YFPs) are variant GFPs that contain aromatic residues at position 203.Blue fluorescent proteins (BFPs) are variant GFPs that contain a Y66H mutation.",
"A group of GFPs which lack the near-UV excitation peak, but retain the wild-type GFP emission peak, have Ser65 substitutions.",
"Other variants of native GFPs with useful fluorescent properties are known in the art, or can be readily prepared by random or directed mutagenesis of a native GFP.",
"Exemplary pairs of donor and acceptor fluorescent domains include BFP-GFP and CFP-YFP.",
"In another embodiment, the donor and/or acceptor fluorescent domain is a “DsRed,” such as a native DsRed from a Discosoma species, an ortholog of DsRed from another genus, or a variant of a native DsRed with optimized properties (e.g.",
"a K83M variant or DsRed2 (available from Clontech)).",
"As used herein, the term “DsRed variant” is intended to refer to polypeptides with at least about 70%, more preferably at least 75% identity, including at least 80%, 90%, 95% or greater identity to a native DsRed, such as a Discosoma DsRed.",
"Other variants of native DsReds with useful fluorescent properties are known in the art, or can be readily prepared by random or directed mutagenesis of a native DsRed (see, for example, Fradkov et al., FEBS Lett.",
"479:127-130 (2000)).",
"Other exemplary pairs of donor and acceptor fluorescent domains, respectively, include GFF-dsRED2 and YFP-dsRED2.Included within the term “donor fluorescent domain” is a bioluminescent domain, such as luciferase from Renilla, related species, and variants thereof.",
"Renilla luciferase emits blue light in the presence of an appropriate substrate, such as coelenterazin, which can be transferred to an appropriate fluorescent acceptor domain, such as a GFP, in a process called Bioluminescence Resonance Energy Transfer, or BRET.",
"BRET is described, for example, in Angers et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 97:3684-3689 (2000); Xu et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 96:151-156 (1999); and components are commercially available from BioSignal Packard (Montreal, Canada).",
"Those skilled in the art can readily apply the compositions and methods described herein with respect to FRET, to compositions and methods involving BRET.",
"In constructs in which the donor fluorescent domain and the acceptor fluorescent domain are present on the same polypeptide, the fluorescent domains can optionally be separated by a flexible “linker sequence.” An appropriate linker sequence allows the donor and acceptor fluorescent domain to be functionally coupled when the single MMID (FIG.",
"1A), or pair of MMIDs (FIG.",
"1B), are associated with a membrane molecule, such that FRET is high, and functionally uncoupled when the MMIDs are not associated with the membrane molecule, such that FRET is low (FIGS.",
"1D and E).",
"In order to optimize the FRET effect, the average distance between the donor and acceptor fluorescent domains should become less than about 10 nm when the MMID is associated with the membrane molecule (e.g.",
"from 1 nm to 10 nm).",
"The linker moiety preferably is between about 1 and 50 amino acid residues in length, preferably between about 2 and 30 amino acid residues.",
"A preferred linker moiety contains, or consists of, the sequence Gly-Gly, Ser-Gly or Gly-Ser.",
"Linker moieties and their applications are well known in the art and described, for example, in U.S. Pat.",
"Nos.",
"5,998,204 and 5,981,200, and Newton et al., Biochemistry 35:545-553 (1996).",
"The invention provides isolated nucleic acid molecules, which alone or in combination as components of a kit encode membrane molecule indicator polypeptides, including each of the exemplary indicators shown schematically in FIGS.",
"1, 9 and 10 and described above.",
"As used herein, the term “nucleic acid molecule” refers to a polynucleotide comprised of either DNA or RNA; which can be single- or double-stranded; which can optionally contain one or more non-natural nucleotides, such as nucleotides having modifications to the base, the sugar, or the phosphate portion; and which can optionally contain one or more non-natural linkages, such as phosphothioate linkages.",
"As used herein, the term “kit” refers to two or more component nucleic acid molecules packaged or sold for use together.",
"The kit components will be contained either in a single container or separate containers.",
"The kit can further optionally contain written instructions for use of the components in the methods of the invention, and/or buffers and components suitable for such methods.",
"The invention nucleic acid molecules are preferably operatively linked to a promoter of gene expression.",
"As used herein, the term “operatively linked” is intended to mean that the nucleic acid molecule is positioned with respect to either the endogenous promoter, or a heterologous promoter, in such a manner that the promoter will direct the transcription of RNA using the nucleic acid molecule as a template.",
"Methods for operatively linking a nucleic acid to a heterologous promoter are well known in the art and include, for example, cloning the nucleic acid into a vector containing the desired promoter, or appending the promoter to a nucleic acid sequence using PCR.",
"A nucleic acid molecule operatively linked to a promoter of RNA transcription can be used to express membrane molecule indicator transcripts and polypeptides in a desired host cell or in vitro transcription or transcription-translation system.",
"The choice of promoter to operatively link to an invention nucleic acid molecule will depend on the intended application, and can be determined by those skilled in the art.",
"For example, if the encoded polypeptide may be detrimental to a particular host cell, it may be desirable to link the invention nucleic acid molecule to a regulated promoter, such that gene expression can be turned on or off.",
"Alternatively, it may be preferred to have expression driven by either a weak or strong constitutive promoter.",
"Exemplary promoters suitable for mammalian cell systems include, for example, the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, and the Moloney murine leukemia virus (MMLV) promoter.",
"Promoters suitable in yeast include, for example, ADH promoter (S. cerevisiae) and the inducible Nmt promoter (S. pombe).",
"It will be appreciated that a nucleic acid molecule encoding a polypeptide containing a MMID and a donor fluorescent domain, and a nucleic acid molecule encoding a polypeptide containing a MMID and an acceptor fluorescent domain (e.g.",
"FIGS.",
"1B and 1D) can optionally be present on the same vector or under the control of the same promoter.",
"Such constructs are advantageous, for example, in simplifying introducing the nucleic acid molecules into a cell and in ensuring 1:1 stoichiometry of the donor and acceptor in the pair.",
"Alternatively, the nucleotide sequences encoding the two polypeptides can be present on separate vectors or under the control of different promoters.",
"The invention further provides a vector containing an isolated nucleic acid molecule encoding a membrane molecule indicator polypeptide.",
"Exemplary vectors include vectors derived from a virus, such as a bacteriophage, a baculovirus or a retrovirus, and vectors derived from bacteria or a combination of bacterial sequences and sequences from other organisms, such as a cosmid or a plasmid.",
"The vectors of the invention will generally contain elements such as an origin of replication compatible with the intended host cells; one or more selectable markers compatible with the intended host cells; and one or more multiple cloning sites.",
"The choice of particular elements to include in a vector will depend on factors such as the intended host cells; the insert size; whether regulated expression of the inserted sequence is desired; the desired copy number of the vector; the desired selection system, and the like.",
"The factors involved in ensuring compatibility between a host cell and a vector for different applications are well known in the art.",
"For recombinant expression of the encoded polypeptide, the isolated nucleic acid molecules will generally be operatively linked to a promoter of gene expression, as described above, which may be present in the vector or in the inserted nucleic acid molecule.",
"Also provided are cells containing membrane molecule indicators, including each of the exemplary indicators shown schematically in FIGS.",
"1, 9 and 10 and described above, and cells containing nucleic acid molecules encoding such indicators.",
"The cells of the invention can advantageously express the encoded polypeptide(s) and thus be used in screens for agonists, antagonists and inverse agonists of signaling pathways indicated by properties of the membrane molecule; to functionally clone modulatory components of the signal transduction pathway in which the membrane molecule is involved; and to determine or confirm the function of potential modulatory components of the signal transduction pathway in which the membrane molecule is involved.",
"Such applications are described further below.",
"The isolated nucleic acid molecule(s) will generally be contained within an expression vector, but optionally can be expressible DNA or RNA not contained within a vector.",
"The isolated nucleic acid molecule(s) can be maintained episomally, or incorporated into the host cell genome.",
"The cells of the invention can be prepared by introducing the nucleic acid molecules of the invention by any suitable means, including, for example, transfection, transduction, electroporation and microinjection, as well as by transgenic technology.",
"The cells of the invention can be prepared from any organism, including, for example, bacteria (e.g.",
"E. coli), insects (e.g.",
"Drosophila), yeast (e.g.",
"S. cerevisiae, S. pombe, or Pichia pastoris), nematodes (e.g.",
"C. elegans), amphibians (e.g.",
"Xenopus embryos and oocytes) and mammals (e.g.",
"human, rodent or primate primary cells and established cell lines, such as COS, CHO, 3T3, N1E-115, HEK, etc., representing either a normal or diseased state of the mammal).",
"The cells of the invention can further recombinantly express, either stably or transiently, a known or candidate modulator of the membrane molecule, such as a known or candidate agonist, antagonist or reverse agonist peptide; a known or candidate receptor; or a known or candidate effector molecule.",
"As used herein, the term “recombinant expression,” with respect to expression of a signaling polypeptide, refers to transient or stable expression of a polypeptide from a recombinant nucleic acid molecule.",
"Recombinant expression is advantageous in providing a higher level of expression of the polypeptide than is found endogenously, and also allows expression in cells or systems in which the polypeptide is not normally found.",
"The term “recombinant nucleic acid molecule” is intended to refer to a nucleic acid molecule that has been constructed, at least in part, by molecular biological methods, such as PCR, restriction digestion or ligation.",
"A recombinant nucleic acid expression construct generally will contain a constitutive or inducible promoter of RNA transcription appropriate for the host cell or transcription-translation system, operatively linked to a nucleotide sequence that encodes the polypeptide of interest.",
"The expression construct can be DNA or RNA, and optionally can be contained in a vector, such as a plasmid or viral vector.",
"The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques well known in the art and described, for example, in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (most recent Supplement).",
"The nucleotide sequences of various receptors and effectors, and methods of recombinantly expressing the encoded polypeptides in a variety of cell types, are well known in the art.",
"The invention further provides membrane molecule indicator polypeptides.",
"The invention polypeptides include polypeptides recombinantly expressed by the invention nucleic acid molecules, as well as constructs produced by chemically coupling some or all of the component domains, which themselves can be recombinantly produced.",
"The expressed polypeptides can optionally be isolated from a transcription-translation system or cell, by biochemical and immunological purification methods known in the art.",
"To facilitate isolation, the invention polypeptides can optionally be fused to a tag sequence, such as an epitope tag, a GST polypeptide or a 6×His fusion.",
"The invention polypeptides can optionally be introduced into a whole cell, such as by recombinant expression or microinjection, and the cells used in the methods described herein.",
"The invention polypeptides can alternatively be introduced into a lipid bilayer, such as a cellular membrane extract, or an artificial lipid bilayer (e.g.",
"a liposome vesicle).",
"Methods of preparing lipid bilayers containing desired amounts and types of molecules, including the membrane molecules and MMID polypeptides described herein, are known in the art.",
"The invention also provides a method of determining a property of a membrane molecule.",
"The method is practiced by (a) providing a cell or lipid bilayer comprising a membrane molecule indicator; and (b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain, wherein FRET between the donor domain and the acceptor domain is indicative of a property of the membrane molecule.",
"Also provided is a method of identifying a compound that modulates a property of a membrane molecule.",
"The method is practiced by (a) contacting a cell or lipid bilayer comprising a membrane molecule indicator with one or more test compounds, wherein the cell or bilayer further comprises said membrane molecule; and (b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain following contacting, wherein increased or decreased FRET following contacting indicates that the test compound is a compound that modulates a property of the membrane molecule.",
"The lipid bilayer useful in such methods can be a whole cell, which naturally or recombinantly expresses the membrane molecule, or a cellular extract containing the plasma membrane or vesicular membranes.",
"Alternatively, the lipid bilayer can be a lipid vesicle, which can include either natural or synthetic lipids or both, into which a membrane molecule of interest is incorporated.",
"Lipid vesicles are advantageous in that the abundance of the membrane molecule (and, optionally, of other signaling molecules of interest) can be controlled.",
"The methods of the invention are useful in the practice of essentially any application for which a readout of signal transduction mediated through membrane molecules is useful.",
"Such applications are well known in the art.",
"Exemplary applications include 1) identifying test compounds that act as agonists, antagonists, inverse agonists or natural ligands of receptors (described further below); 2) expression cloning of peptide agonists, antagonists and inverse agonists of receptors; 3) expression cloning of novel modulators that affect the abundance, localization, conformation or post-translational modification state of the membrane molecule of interest (e.g.",
"enzymes, enzyme inhibitors, transcriptional regulators, and the like), which themselves can be used as therapeutic drug targets; 4) determining the function of variants of known or predicted modulators of membrane molecules (e.g.",
"determining the effect of SNPs, disease-associated mutations and engineered variations in receptors, effectors and the like); 5) establishing dose-response curves of modulators of membrane molecules (e.g.",
"for predicting effective dose of a therapeutic); and 6) determining alterations in membrane molecules and modulators that reflect disease state, which can be applied to the development of diagnostic methods.",
"Methods of using the compositions and methods described herein for such applications, and other applications relating to signal transduction, will be readily apparent to the skilled person.",
"As an example, the methods of the invention can be used to identify test compounds that are agonists, antagonists, inverse agonists or natural ligands of receptors, including G-protein coupled receptors (described further below), tyrosine kinase receptors (e.g.",
"PDGF, IGF, FGF and EGF receptors and the like) and integrins.",
"In the methods of the invention, the basal level of FRET can be determined in an unstimulated lipid bilayer.",
"The lipid bilayers can then be contacted with a test compound, and FRET compared with an unstimulated bilayer.",
"FRET is advantageous over fluorescent visualization methods in that both increases and decreases, relative to the basal level, can be readily determined.",
"Increased or decreased FRET relative to the basal level is a reflection of the activity of the test compound as an agonist, antagonist (or inverse agonist) of the signaling pathway linked to the membrane molecule.",
"As used herein, the term “agonist” refers to a molecule that selectively activates or increases signal transduction.",
"An agonist can act by any mechanism, such as by binding a receptor at the normal ligand binding site, thereby mimicking the natural ligand and promoting receptor signaling.",
"An agonist can also act, for example, by potentiating the binding ability of the natural ligand, or by favorably altering the conformation of the receptor.",
"The compositions and methods of the invention can advantageously be used to identify agonists that acts through any agonistic mechanism.",
"As used herein, the term “antagonist” refers to a compound that selectively inhibits or decreases signal transduction.",
"An important subset of antagonist compounds that can advantageously be identified by the methods described herein, are referred to as “inverse agonists.” Inverse agonists are antagonists that selectively inhibit or decrease signal transduction below basal levels.",
"An antagonist can act by any antagonistic mechanism, such as by binding to a ligand or receptor, thereby inhibiting their interaction.",
"An antagonist can also act by modifying or altering the native conformation of a receptor.",
"The methods of the invention can advantageously be used to identify an antagonist that acts through any antagonistic mechanism.",
"For therapeutic applications, an agonist preferably has an EC50, and an antagonist or inverse agonist preferably has an IC50, of less than about 10−7 M, such as less than 10−8 M, and more preferably less than 10−9 M. However, depending on the stability, selectivity and toxicity of the compound, an agonist with a higher EC50, or an antagonist with a higher IC50, can also be useful therapeutically.",
"EC50 and IC50 of such compounds can be established by dose-response curves using the methods described herein.",
"As used herein, the term “test compound” refers to any molecule that potentially acts as an agonist, antagonist, inverse agonist or natural ligand of a signaling pathway reported by the membrane molecule indicator compositions and methods of the invention.",
"A test compound can be a naturally occurring macromolecule, such as a polypeptide, nucleic acid, carbohydrate, lipid, or any combination thereof.",
"A test compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic molecule prepared by combinatorial chemistry methods.",
"Methods for preparing large libraries of compounds, including simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Pat.",
"No.",
"5,264,563; Francis et al., Curr.",
"Opin.",
"Chem.",
"Biol.",
"2:422-428 (1998); Tietze et al., Curr.",
"Biol., 2:363-371 (1998); Sofia, Mol.",
"Divers.",
"3:75-94 (1998); Eichler et al., Med.",
"Res.",
"Rev.",
"15:481-496 (1995); and the like.",
"Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources.",
"The number of different test compounds to assay in the methods of the invention will depend on the application of the method.",
"For example, one or a small number of test compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several predicted ligands, agonists or antagonists.",
"However, it is generally understood that the larger the number of test compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay.",
"Additionally, large numbers of compounds can be processed in high-throughput automated screening assays.",
"Therefore, “one or more test compounds” can be, for example, 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds, such as greater than about 103, 105 or 107 different compounds.",
"A lipid bilayer can be contacted with a test compound by any mode, such as by extracellular administration, by intracellular uptake of the compound, or by recombinant expression of the compound (in methods involving an intact cell).",
"The contacting with the test compound can optionally take place in the presence of a known agonist or antagonist of the signaling pathway of interest, and the effect of the compound on agonist or antagonist-mediated signaling can be assessed.",
"In cases in which the test compound is a peptide, the peptide can preferentially be targeted to the membrane location of the membrane molecule of interest, such as the extracellular or intracellular face of the plasma membrane, Golgi, ER or the like, using known methods.",
"For example, test compounds that are peptides can be expressed on the extracellular membrane of a cell or the invention, or on a second cell, by phage display methods known in the art.",
"Alternatively, test compounds that are peptides can be secreted from a cell of the invention, or from a second cell, by expressing the peptide with a secretory signal.",
"As an example, the methods of the invention can be used to screen for G-protein coupled receptor (GPCR) agonists, antagonists and inverse agonists, as well as to identify the natural ligands of orphan GPCRs.",
"GPCRs are seven-transmembrane-domain polypeptides that transduce G-protein coupled signals in response to ligands.",
"The natural agonists of different GPCRs range from peptide and non-peptide neurotransmitters, hormones and growth factors, to lipids, nucleoside-sugars, amino acids, light and odorants.",
"GPCRs are involved in a variety of critical biological functions, including cell proliferation, differentiation and apoptosis.",
"GPCRs have proven to be important targets of pharmaceuticals that affect a variety of diseases, including neurological and psychiatric disorders, vascular diseases, endocrinological disorders, and cancer.",
"It is estimated that over 50% of current drugs are targeted towards GPCRs, and represent about a quarter of the 100 top-selling drugs worldwide.",
"The natural ligands of different GPCRs include peptides, biogenic amines, glycoproteins, nucleotides, ions, lipids, amino acids, light and odorants.",
"Structurally, GPCRs can be divided into three major subfamilies, each of which currently includes orphan receptors as well as receptors whose ligands are characterized (reviewed in Gether, Endocrine Reviews 21:90-113 (2000)).",
"A database containing links to the nucleotide and amino acid sequences of numerous mammalian GPCRs, including orphan GPCRs, is available at http://www.darmstadt.gmd.de/˜gpcrdb/.",
"As used herein, the term “G-protein” refers to a class of heterotrimeric GTP binding proteins, with subunits designated Gα, Gβ and Gγ, that couple to seven-transmembrane cell surface receptors to couple extracellular stimuli to intracellular messenger molecules.",
"G-proteins are distinguished by their Gα subunits.",
"The more than 20 different Gα subunits, encoded by 17 different genes, can be grouped into four major families: Gαs, Gαi, Gαq, and Gα12.Signaling through GPCRs that couple to Gαq-containing G proteins activates PLC enzymes to hydrolyze PIP2 in the plasma membrane to DAG and IP3.The specificity of Gα subunits for GPCRs is determined by the C-terminal five amino acids of the Gα.",
"Thus, a variety of signal transduction pathways can be assayed to determine signaling through a GPCR, by co-expressing a chimeric Gα containing the five C-terminal residues of a Gα known or predicted to couple to the receptor of interest (such as Gαi, Gαs or the promiscuous Gα16), with the remainder of the protein corresponding to a Gα coupled to the GPCR that signals through a membrane molecule of interest (see Conklin et al., Nature 363:274-276 (1993), and Komatsuzaki et al., FEBS Letters 406:165-170 (1995)).",
"For example, in instances in which the membrane molecule indicator polypeptides are designed to indicate abundance of PIP2, cells (or other lipid bilayers) can contain a GPCR of interest, and optionally a Gαq or Gα16 (or chimeric or variant Gα which functions as a Gαq).",
"The basal level of FRET between acceptor and donor fluorescent domains linked to a MMID (or two MMIDS) that associate with PIP2 can be determined.",
"In response to agonist-induced signal transduction through the GPCR, PIP2 is hydrolyzed and FRET is decreased, as exemplified in the cells described in the Example, below.",
"Likewise, antagonistic or inverse agonistic effects can be determined by an increase in agonist-induced, or basal, levels of FRET.",
"In the cells described in the Example, below, in which the MMID associates with PIP2 in the plasma membrane, FRET is high in unstimulated cells.",
"In the presence of a test compound that activates PLC (e.g.",
"bradykinin), FRET is significantly lower than in unstimulated cells, as PIP2 in the membrane is hydrolyzed, and the donor and acceptor fluorescent domains are no longer in close proximity.",
"Thus, the compositions and methods described in the Example, below, can be used to identify and compare test compounds that stimulate the activation of PLC, that decrease the basal level of PLC activation, or that antagonize agonist-induced PLC activation.",
"PIP2 hydrolysis leads to the production of the second messengers DAG and IP3.IP3 mediates the release of Ca2+ from intracellular stores.",
"Ca2+ release has been used as a signaling assay in variety of research, diagnostic and screening applications.",
"The methods described herein, which detect PIP2 hydrolysis, can be used in most applications in which determination of Ca2+ release has proven useful.",
"As disclosed herein, determining PIP2 hydrolysis has certain advantages over determining Ca2+ release, in that PIP2 hydrolysis is more proximal to receptor activation, and is thus less dependent on intermediate signaling steps that may introduce variability.",
"As shown herein, signals that yield similar Ca2+ responses have different PLC activation kinetics, suggesting that PIP2 hydrolysis follows receptor activation more faithfully than Ca2+ responses.",
"Optionally, PIP2 hydrolysis, as determined by the FRET methods described herein, and Ca2+ release, as determined using Ca2+ indicator dyes known in the art, can both be assayed.",
"Because Ca2+ release is downstream of PIP2 hydrolysis, Ca2+ release can be assayed simultaneously with FRET to confirm that the observed FRET reflects PIP2 hydrolysis.",
"Methods of determining and quantitating FRET at the single cell level, or in cell populations, are well known in the art or can be determined by the skilled person.",
"For example, FRET can be measured using dual emission fluorescence microscopy, as described in the Example, below.",
"Alternatively, FRET can be measured using fluorescent microscopy imaging methodology, which allows for simultaneous recordings from multiple cells.",
"As a further example, FRET can be determined with fluorescent lifetime.",
"Briefly, upon excitation with an ultrashort pulse of light (e.g.",
"about 0.01 ns), fluorophores have a characteristic decay in emission that is single exponential, and may last 0.1-10 ns, dependent on the fluorophore and conditions.",
"It has been shown that the presence of a FRET acceptor dramatically shortens the decay time of the donor, which can be detected either using direct monitoring of the decay time (time domain monitoring), or using sine-modulated light, in the frequency domain (see, for example, Verveer et al., Biophys.",
"J., 78:2127-37 (2000)).",
"For high-throughput screening applications, FRET can be measured using fluorescence activated cell sorting (FACS), such as with a HeCd laser or frequency-double diode laser.",
"FACS is advantageous in permitting the analysis of around 50,000 cells per second, which is orders of magnitude faster than visual detection methods.",
"FACS also allows the isolation of cells for further growth, manipulation and identification of nucleic acid molecules encoding compounds that modulate association between membrane molecules and the MMIDs of the invention compositions.",
"Therefore, the compositions and methods of the invention are amenable to high-throughput screening for potential therapeutics.",
"The following examples are intended to illustrate but not limit the present invention.",
"EXAMPLE I Preparation and Use of Membrane Molecule Indicator Compositions This example shows the preparation of two pairs of nucleic acid molecules of the invention.",
"In the first pair, the first nucleic acid molecule encodes a polypeptide containing a membrane molecule indicator domain (PH domain) and a donor fluorescent domain (CFP), and the second nucleic acid molecule encodes a polypeptide containing a membrane molecule indicator domain (PH domain) and an acceptor fluorescent domain (YFP).",
"In the second pair, the first nucleic acid molecule encodes a polypeptide containing a membrane molecule indicator domain (PH domain) and a donor fluorescent domain (CFP), and the second nucleic acid molecule encodes a polypeptide containing a membrane anchoring domain (CaaX) and an acceptor fluorescent domain (YFP).",
"This example also shows the use of the pairs of nucleic acid molecules to determine the abundance of a membrane molecule (PIP2), by determining FRET between the donor and acceptor fluorescent domains.",
"High FRET results from high PIP2 abundance at the plasma membrane, which indicates the resting state of the cell; decreased FRET results from PIP2 hydrolysis, which indicates signaling through a G-protein coupled receptor linked to PLC activation.",
"Experimental Procedures Materials 1-oleoyl LPA, histamine, bradykinin (BK), phenyl-arsine oxide and quercetin were from Sigma Chemical Co. (St. Louis, Mo.",
"); neurokinin A, caged IP3 (cat.",
"#407135) and ionomycin were from Calbiochem-Novabiochem Corp. (La Jolla, Calif.); Myo-[3H]inositol (60 Ci/mmol) was from Amersham-Pharmacia Biotech.",
"Fura-red (K salt) was from Molecular Probes Inc. (Eugene, Oreg.).",
"All other chemicals were of analytical grade.",
"Constructs The pleckstrin homology-domain of human phospholipase C δ1 was obtained from the Superhiro-PLCδ1 PH construct (AA 1-174, obtained from T. Meyer) and cloned into the eukaryotic expression vector pECFP-C1 (Clontech, Calif.).",
"Two primers (PLCδPH1; 5′ CCTGC GGCCG CGGTA CCGAT ATCAG ATGTT GAGCT CCTTC AC 3′ (SEQ ID NO:3) and PLCδPH2; 5′ CCGAA TTCCC GGGTC TCAGC CATGG ACTCG GGCCG GGACT TC 3′ (SEQ ID NO:4)) were designed to generate the PH-domain in frame behind the CFP followed by a stop codon.",
"The PCR-product was cloned into the pECFP plasmid with the restriction sites EcoRI and EcoRV on EcoRI and SmaI, leading to pECFP-PH.",
"YFP was obtained from yellow Cameleon 2.0 (obtained from A. Miyawaki and R. Tsien) and subcloned into cloning vector PGEM3z (Promega), via SacI and EcoRI, and subsequently into pcDNA3 (Invitrogen) via BamHI and EcoRI.",
"PCR on YFP-pcDNA-3 with primers T7 (Promega) and GFP3; 5′ GGCTG AGACC CGGGA ATTCG GCTTG TACAG CTCGT CCATG 3′ (SEQ ID NO:5) was done to remove the stop codon.",
"The PH domain PCR-product, taken between primers PLCδPH1 and PLCδPH2, was cloned in frame behind YFP with EcoRI and NotI, leading to pcDNA3YFPPH.",
"To obtain pcDNA3eGFPPH, YFP was swapped with EGFP, using primers T7 and GFP3 on pcDNA3eGFP and restriction enzymes BamHI and EcoRI.",
"For YFP-CAAX and GFP-CAAX, the membrane localization sequence of K-Ras (KMSKD GKKKK KKSKT KCVIM; SEQ ID NO:6) was obtained by PCR from Bp180-CAAX (GenBank accession number M54968 and M38506), using primers CAAX3 5′CCGAA TTCCC GGGTC AAGAT GAGCA AAGAT GGTAA AAAG 3′ (SEQ ID NO:7), containing an EcoRI site, and CAAX2; 5′ CCTGC GGCCG CGGTA CCGAG ATCTT TACAT AATTA CACAC TT 3′ (SEQ ID NO:8), that contained a NotI-site behind the stop codon.",
"The final constructs were made by exchanging the PH domain from YFP-PH and GFP-PH for the CAAX domain using EcoRI and NotI.",
"All clones were verified by sequence analysis.",
"YFP-CAAX contained a point mutation (V instead of G in the CAAX domain), but this did not influence the membrane localization.",
"Constitutively active mutants of Gαq and Gα12 subunits in pcDNA3 vectors were obtained from Dr. O. Kranenburg (Kranenburg et al., Mol.",
"Biol.",
"Cell 10:1851-1857 (1999)).",
"Cell Culture and Transfections N1E-115 neuroblastoma cells were seeded in 6-well plates at about 25,000 cells per well on 25 mm glass coverslips, and cultured in 3 ml Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FCS and antibiotics.",
"Unless otherwise indicated, constructs were transfected for 6-12 hours using calcium phosphate precipitate, at 0.8 μg DNA/well.",
"Following transfection, cells were incubated in serum-free DMEM for 12-48 hours.",
"For fluorescence detections, coverslips with cells were transferred to a culture chamber and mounted on an inverted microscope.",
"All experiments were performed in bicarbonate-buffered saline (containing, in mM, 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, with 10 mM HEPES added), pH 7.2, kept under 5% CO2, at 37° C. Inositol Phosphate Determinations Preparation, culture and labeling of bovine adrenal glomerulosa cells have been described in Balla et al., J. Biol.",
"Chem.",
"269:16101-16107 (1994).",
"Cells labeled with myo-[3H]inositol for 24-48 hrs were stimulated by angiotensin II (30 nM) for the indicated times in a medium containing either Sr2+ or Ca2+.",
"Reactions were terminated with perchloric acid and inositol phosphates were separated by HPLC essentially as described in Balla et al., supra (1994).",
"Confocal Microscopy and Image Analysis For confocal imaging, a Leica DM-IRBE inverted microscope fitted with TCS-SP scanhead was used.",
"Excitation of EGFP was with the 488 nm Argon ion laserline, and emission was collected at 500-565 nm.",
"For translocation studies, a series of confocal images were taken at 2-10 second intervals and stored on disk.",
"Determination of the ratio of membrane to cytosolic fluorescence by directly assigning regions of interest (ROIs) for membrane and cytosol was hampered by the shape changes of cells during experiments.",
"Using Qwin software (Leica) this ratio was therefore calculated by post-acquisition automated ROI assignment and analysis.",
"In brief, a binary mask of the transfected cell was lined out using a thresholding step on a smoothed image.",
"From this mask, the area corresponding to the membrane was eroded by a selectable amount to delineate the membrane.",
"Further erosion was then applied to reliably separate membrane from cytosol area, and the remaining area was taken to represent cytosol.",
"This mask was updated for each image in a series, and translocation was expressed as ratio of the fluorescence values for membrane and cytosol area, to correct for bleaching.",
"This approach corrects fully for cell movements and shape changes, and was able to reliably detect very minor translocations (using e.g.",
"diluted agonists).",
"Fluorescence Determinations For FRET experiments, cells were transferred to an inverted Zeiss Axiovert 135 microscope equipped with dry Achroplan 63× (NA 0.75) objective.",
"Excitation of CFP was at 425±5 nm, and emission was collected with a 460 nm dichroic mirror.",
"Emission of CFP and YFP was split using an additional 505 dichroic and filtered with 475DF30 and 540DF40 bandpass filters, respectively.",
"Detection was with PTI model 612 analog photomultipliers, and for data acquisition, the FELIX software (PTI Inc.) was used.",
"FRET was expressed as the ratio of CFP to YFP signals, the value of which was set as 1.0 at the onset of the experiment.",
"Changes are expressed as percent deviation from this initial value of 1.0.For detection of intracellular Ca2+, Yellow Cameleon 2.1 was used at the same wavelengths (Miyawaki et al., Nature 388: 882-887 (1997)).",
"For sustained stimulation, agonists and inhibitors were added to the medium from concentrated stocks.",
"Stimulation with short pulses of NKA was performed by placing a glass micropipette (tip diamenter about 2 μm) at about 25 μm from the cell using an Eppendorf microinjection system and applying pulses of pressure for 10 seconds.",
"It was verified using Lucifer Yellow in the pipette that following termination of the pressure pulse the concentration at the cell rapidly dropped towards zero.",
"Loading and Flash Photolysis of Caged IP3 Before electroporation, adherent cells grown on coverslips were washed twice in intracellular buffer (containing in mM: 70 KCl, 70 Kglutamate, 2 MgCl2, 0 CaCl2, 5 phosphate buffer, pH 7.1) and then 70 μl of this intracellular buffer was added to the cells with 20 μM of Fura-red tetrapotassium salt and either 1, 10 or 100 μM of caged IP3.Electroporation was achieved by a series of 15 high-frequency square wave pulses, (1-second spaced, amplitude 150V, frequency 80 kHz, lasting 0.5 ms each) using 2 platina electrodes of 8×3 mm with 2.5 mm spacing.",
"The efficiency of this method was assessed by control permeabilizations that were performed on the stage of a confocal microscope.",
"This protocol caused complete permeabilization (based on equilibration of intracellular calcein concentrations with the extracellular buffer) of the cells in the area between the electrodes.",
"For photorelease of caged IP3, a single cell was illuminated with a short pulse of UV light (340-410 nm) from a 100 W HBO lamp using a shutter.",
"The shutter open time was adjusted to give full release of caged IP3, that is no response being observed with a subsequent illumination.",
"For partial photolysis, the flash intensity was adjusted by using neutral density filters placed in the illumination pathway.",
"Quantitation of Expression Levels For quantitation of expression levels of CFP-PH and YFP-PH, cellular fluorescence was compared to the fluorescence of a solution of known concentration of purified, bacterially expressed CFP-PH or YFP-PH, following the method of Miyawaki et al., “Calcium signaling: a practical approach,” Oxford University Press (in press).",
"In short, CFP-PH and YFP-PH were expressed as GST-fusion proteins, and purified on glutathione sepharose beads.",
"Protein concentration was measured by the BCA* Protein Assay (Pierce, Ill., USA).",
"The solution (4.8 μM) was then introduced in a linear wedge-shaped chamber (0-170 μM thickness) that was placed on the microscope (using NA 0.7 objective), and the position of the chamber was adjusted to give a fluorescence readout that matched that of a single, CFP or YFP expressing cell.",
"The estimate of the fluorescent protein concentration in the cell was obtained by comparing the local thickness of the wedge to that of an average cell (17 μM).",
"Relative amounts of CFP-PH and YFP-PH expression in cells were always determined under conditions of full cytosolic localization of the constructs.",
"At the onset of each experiment, photomultiplier gains (high voltage) were adjusted to give a standard 6V output for the resting cell.",
"Noting that over an extended range of light input, every 2-fold change in intensity corresponds to a 35V change in cathode voltage, cell intensities were measured.",
"By comparing these to the values obtained with the GFPwedge, estimates of expression levels were obtained.",
"Fluorescence Recovery After Photobleaching (FRAP) For FRAP experiments, cells were imaged using a Leica TCS-SP confocal microscope equipped with 63× (NA 1.3) oil immersion objective.",
"The beam from an external ArKr laser (25 mW) was coupled into the backfocal plane of the objective via the epifluorescence excitation port, using a 30/70 beamsplitter, thus allowing simultaneous imaging and spot bleaching.",
"Spots of about 1.3 μm (full width half maximum) were bleached (>95%) in the basal membrane using a single 30 ms pulse from the ArKr laser during data collection in linescan mode at 1000, 500 or 125 Hz.",
"Data were corrected for slight (<7%) background bleaching and fitted with single exponents using Clampfit software (Axon Instruments, CA).",
"Results Fluorescence Resonance Energy Transfer Between Plasma Membrane-Localized PLCδ1PH-CFP and PLCδ1PH-YFP PH-CFP and PH-YFP chimera were transiently transfected into N1E-115 mouse neuroblastoma cells at a 1:1 molar ratio.",
"After 1-2 days, cells were transferred to an inverted epifluorescence microscope and assayed for FRET by simultaneously monitoring the emission of CFP (475±15 nm) and of YFP (530±20 nm), while exciting CFP at 425±5 nm.",
"In resting cells, PH-CFP and PH-YFP reside at the plasma membrane bound to PI(4,5)P2, and the two fluoriphores remain within resonance distance.",
"Upon activation of PLC by the addition of bradykinin (BK), PI(4,5)P2 is rapidly hydrolyzed and consequently PH domains can no longer bind to the plasma membrane.",
"Depending on cell type (surface to volume ratio), it is estimated that the distance(s) between fluoriphores increase about 200-1000 fold), and therefore FRET does not occur (FIG.",
"2).",
"As a result, the donor (CFP) emission intensity increases, while the acceptor (YFP) emission decreases.",
"By taking the ratio of CFP to YFP emission, the FRET signal becomes essentially independent on excitation intensity fluctuations and photobleaching.",
"The kinetics of BK-induced PLC activation in N1E-115 cells as detected by FRET is characterized by a rapid onset, with translocation peaking at 20-30 s after addition of the agonist.",
"The decaying phase is somewhat slower, usually returning to baseline within 1 to 4 minutes.",
"This time course is very similar to that deduced from confocal detection of PLCδ1PH-GFP translocation recorded under identical conditions (FIG.",
"2C).",
"In this latter case, the data were extracted from a time series using post-acquisition automated image analysis (see Experimental Procedures).",
"Similar translocation responses can be obtained by FRET in other cell types, including A431 epidermoid carcinoma cells, HEK293 embryonal kidney cells, and COS monkey kidney cells stimulated with a variety of ligands to Gq-coupled receptors.",
"The above described kinetics with a fast and rather complete translocation induced by BK, suggest that PIP2 depletion after stimulation is quite extensive.",
"While most reports of agonist-induced PIP2 hydrolysis, as detected biochemically from [3H]-inositol-labeled cells, show slower and less pronounced decreases in phosphoinositide levels, considerable agonist- and cell type-dependent variations exist, e.g.",
"(Tilly et al., Biochem.",
"J.",
"252:857-863(1988); van der Bend et al., Biochem.",
"J 285 (Pt I):235-240 (1992); Zhang et al., Mol.",
"Pharmacol.",
"50:864-869 (1996)).",
"Where early time points were also studied, rapid decreases in PIP2 levels have been detected (Wijelath et al., Biochem.",
"Biophys.",
"Res.",
"Commun.",
"152:392-397 (1988); Divecha et al., EMBO J.",
"10:3207-3214 (1991); Stephens et al., Biochem.",
"J.",
"296 (Pt 2):481-488 (1993)).",
"For example, significant bradykinin-induced PIP2 decreases were reported to occur within 10 seconds in bovine aortic endothelial cells (Myers et al., Cell Signal.",
"1:335-343 (1989)), and at 1 minute in bombesin-stimulated 3T3 cells (Divecha et al., supra (1991)).",
"Rapid recovery towards basal levels has also been found.",
"Wijelath et al.",
"reported as much as 85% hydrolysis of PIP2 at 5 seconds after stimulation of macrophages with interleukin, while PIP2 levels had recovered to 50% at 60 seconds (Wijelath et al., supra (1988)).",
"Similar fast recovery was also seen in other cell types (Divecha et al., supra (1991); Stephens et al., supra (1993)).",
"Since biochemical analyses have to rely upon measurements on cell populations, where not all cells give synchronized and identical responses (and many cells may not respond at all), it is not surprising to find differences between the results of measurements with these two alternative approaches.",
"Characterization of Fluorescence Signals During agonist-induced translocation, several factors may affect the fluorescent properties of these PH domain chimeras as well as the transfer of fluorescent energy between them (Tsien, Annu.",
"Rev.",
"Biochem.",
"61:509-544 (1998)).",
"For example, the move away from a compartment adjacent to the lipophilic membrane could alter fluorescent characteristics, and is also likely to alter FRET by increasing the degree of rotational freedom.",
"While the relative influence of increased rotational freedom on the translocation-induced decrease in FRET is difficult to assess in this model sytem, fluorescence changes were analyzed in some further detail.",
"Cells were transfected with only one of the PLCδ1PH-CFP or PLCδ1PH-YFP constructs.",
"After stimulation, a small but consistent transient fluorescence decrease was observed with either the CFP or the YFP-tagged PH domains (FIG.",
"3).",
"The original green construct (PLCδ1PH-GFP) displayed similar behavior (not shown).",
"This transient decrease is likely caused by fluoriphore displacement from the membrane, since it is not observed in cells that express a more stably membrane-anchored GFP-CAAX, nor is it seen in cells that express a mutated PLCδ1PH-GFP (R40L) (Varnai et al., J.",
"Cell Biol.",
"143:501-510 (1998)) that can not bind PI(4,5)P2 and, therefore, is cytosolic throughout the experiment.",
"The precise mechanism that causes this decrease of emission upon cytosolic translocation is unknown; however, influence of the local microenvironment (e.g.",
"hydrophobicity, charged groups, changing ion concentrations etc.)",
"on the spectral properties of GFP seems likely (Tsien, supra (1998)).",
"The “displacement” effect may explain why the translocation-induced decrease in YFP signal usually is somewhat larger than the increase in CFP fluorescence.",
"However, expressing FRET as an emission ratio largely eliminates this effect.",
"FRET could also be measured in cells that coexpress PLCδ1PH-CFP with YFP-CAAX (not shown); however, using this pair, ratioing did not cancel the above mentioned displacement effect.",
"To assess the effects of construct concentrations on FRET, cells expressing various levels of the chimeric proteins were compared.",
"Intracellular fluorescent protein concentrations were estimated by comparing the emission intensities of individual cells to those of a solution of bacterially expressed, purified protein of known concentration (Miyawaki et al., supra (in press); see Experimental Procedures).",
"Based on these estimates, resonance could be observed in cells with expression levels between about 2-200 μM, over a 100-fold concentration range.",
"However, FRET was not observed in cells expressing less than about 1 μM of each of the constructs.",
"Very high expression levels, on the other hand, appeared to be detrimental to the cells (as judged from the appearance of membrane blebs and detachment of cells 2-3 days after transfection).",
"Such cells were excluded from analysis.",
"These data also revealed that PLCδ1PH-CFP expression levels (detected in fully translocated cells) did not differ more than about 2-fold from those of PLCδ1PH-YFP in most cells.",
"It was of interest to determine whether estimates of CFP and YFP concentrations can be used to calculate lipid concentrations and molecular proximity in the cells studied.",
"Assuming a typical attached N1E-115 cell to be a pyramid having a 20×20 μm base and 10 μm height (having 1.3 pl volume and 1100 μm2 surface), and assuming that (I) the concentration of both chimera is 20 μM; (II) 50% of fluoriphores are located at the membrane (complete translocation roughly doubles the fluorescence in the cytosol); (III) the distribution of fluoriphores is homogenous along the membrane; and (IV) fluoriphores are insensitive to the local environment, then the calculated mean distance between fluoriphores is 7-8 nm, which is close to the reported Forster radius (50 Angstrom) for FRET between this pair of fluoriphores (Tsien, supra (1998)).",
"However, it should be emphasized that these assumptions are valid only as first approximations.",
"For example, we and others (Tall et al., Curr.",
"Biol.",
"10:743-746 (2000)) noted that GFP-PH is not homogeneously localized along the plasma membrane.",
"Also, as discussed above, the spectral properties of the fluorescent proteins are sensitive to the microenvironment.",
"Nevertheless, these data set a lower limit for the density of PIP2 molecules available for PH binding at the inner surface of the plasma membrane.",
"GFP-PH Rapidly Shuttles Between Membrane and Cytosol Another important characteristic to address was membrane association and dissociation rates of the PH chimera.",
"These rates directly influence reliability of FRET in reporting rapid changes in PLC activity, and are also relevant to the ability of PLC to hydrolyze PI(4,5)P2 in cells that express high levels of the PLCδ1PH-GFP protein.",
"Accordingly, fluorescence recovery after photobleaching (FRAP) experiments were performed to estimate the binding and dissociation kinetics of PLCSIPH-GFP in the membrane.",
"FIG.",
"4 shows representative results from such FRAP experiments in N1E-115 cells.",
"In panels A and B, the recovery rates are depicted for GFP-CAAX and PLCδ1PH(R40L)-GFP, constructs that are delimited to the plasma membrane and the cytosol, respectively.",
"The former presents the extreme of slow, purely membrane-delimited diffusion (2.81±0.31 s, n=15), and the latter of fast cytosolic diffusion (0.201±0.022 s, n=15).",
"Since FRAP of membrane-localized PLCδ1PH-GFP is significantly faster than that of the membrane-delimited GFP-CAAX (1.22±0.23 s, n=40; p<0.0005; compare panel A and C), its recovery has to be partially through the cytoplasm.",
"Thus, PI(4,5)P2-PH binding is a dynamic process, with on-off rates in the order of seconds.",
"In support of this notion, FRAP times further decreased during agonist-induced partial translocation, when association rates are increased due to the raised cytosolic GFP-PH levels (panel D).",
"The rapid shuttling between membrane and cytosol of individual PLCδ1PH-GFP molecules could explain why PI(4,5)P2 is still available for PLC-mediated hydrolysis or for binding of other proteins in cells expressing these chimeras.",
"Widefield FRET Detection Allows Prolonged Monitoring Independent of Cell Shape Changes.",
"Rapid confocal scanning of cells transfected with PLCδ1PH-GFP leads to considerable photobleaching (within 100 frames) and often causes severe phototoxic damage, manifested as membrane blebbing and loss of membrane integrity within minutes.",
"Using wide-field optical detection and integrating emission from an entire cell (or even clusters of cells) allowed excitation intensity to be dimmed by as much as 100 to >1000 fold, while still retaining acceptable signal-to-noise ratio.",
"Thus, FRET can be followed in single cells for extended periods of time without detectable cell damage.",
"This permits recording of complex stimulation protocols, as shown in FIG.",
"5A.",
"As shown therein, a single N1E-115 cell that is repeatedly stimulated with short pulses of neurokinin A (NKA) from a puffer pipette showed repeated PLC activation.",
"The response to NKA displays incremental partial homologous desensitization of PLC activation, while the response to subsequently added BK is unaltered.",
"Optimizing for low excitation intensity, recordings of several hours can be obtained with sub-second resolution.",
"In N1E-115 and other cells, addition of certain agonists causes rapid and significant shape changes.",
"For instance, LPA causes neurites to retract and the cell soma to round up within 60 seconds (Jalink et al., Cell Growth Differ.",
"4:247-255 (1993)).",
"In contrast, addition of BK has opposite effects, promoting a differentiated phenotype (van Leeuwen et al., Nat.",
"Cell Biol.",
"1:242-248 (1999)).",
"During confocal imaging, such shape changes (as well as the slight drift in focal plane that inevitably occurs over prolonged times) seriously complicate the quantification of GFP-PH translocation.",
"Since FRET analysis uses the total integrated emission from a cell, shape changes and focal drift do not present problems.",
"In very flat and small cell structures such as neurites and lamellipodia (below approximately 2 μm in thickness), confocal imaging cannot detect translocation due to its inherent limit in z-axis resolution.",
"However, in such cases changes in FRET can still be reliably detected as shown by the agonist-induced PLC activation recorded over a single neurite (FIG.",
"5B).",
"FRET can also be recorded from cell populations (FIG.",
"5C) providing with an average response that would need analysis of hundreds of single cell recordings.",
"Thus, detecting resonance between fluorescent protein-labeled PH domains overcomes a number of the limitations that are associated with confocal detection.",
"Determination of Whether FRET Reports Changes in Membrane PI(4,5)P2 or Increases in Cytosolic IP3 While PLCδ1PH-GFP has been introduced as an indicator of membrane PI(4,5)P2 (Stauffer et al., Curr.",
"Biol.",
"8:343-346 (1998); Varnai et al., supra (1998)), it also displays high affinity to IP3 (Hirose et al., Science 284:1527-1530 (1999)) which may exceed its affinity to PI(4,5)P2, although it is difficult to accurately measure the latter as it is displayed in vivo.",
"Based on such relative affinity estimates, Hirose and coworkers recently suggested that PLCδ1PH-GFP actually monitors IP3 increases rather than the changes in lipid levels in MDCK cells (Hirose et al., supra (1999)).",
"They reported that microinjection of IP3 in MDCK cells was sufficient to cause displacement of PLCδ1PH-GFP from the membrane to the cytosol through competition for binding of the fluorescent construct to membrane PI(4,5)P2.They also showed that expression of an IP3-5-phosphatase completely blocked the agonist-induced translocation of the fluorescent protein, and concluded that PI(4,5)P2 changes do not make a significant contribution to the translocation response during stimulation.",
"While FRET analysis effectively monitors the result of PLC activation regardless of whether it is the lipid decrease or the IP3 increase that is more important for the translocation response, this question deserved a more detailed analysis.",
"First it was determined whether intracellular applications of IP3 that generate a Ca2+ signal comparable to that evoked by an agonist would cause translocation of the PLCδ1PH that is similar to what is caused by agonist stimulation.",
"N1E-115 cells were loaded with 20 pM of the calcium indicator Fura red and 100 pM caged IP3 by in situ high frequency electroporation.",
"Unlike microinjection, this technique allows setting of the final concentration of caged IP3 in the cytosol with high precision (see Experimental Procedures), as confirmed by the observation that upon electroporation, intracellular and extracellular fluorescence levels were equal.",
"As shown in FIG.",
"6A, UV flash photolysis of 1 PM of caged IP3 rapidly mobilized Ca2+ from internal stores, with no visible translocation of PLCδ1PH-GFP to the cytosol.",
"Subsequent release of 10 μM of caged IP3 caused a higher Ca2+ response and a small translocation.",
"Only high IP3 concentrations that evoked a large and prolonged Ca2+ increase were able to displace PLCδ1PH-GFP from the plasma membrane.",
"In contrast, BK stimulation caused a larger translocation response than the highest amounts of IP3 with a Ca2+ signal that was comparable to that induced by the smallest amount of IP3 (FIG.",
"6A).",
"In cells electroporated with no caged IP3 in the electroporation buffer, intense UV flashes did not influence intracellular Ca2+ levels, membrane localization of the chimera, or any of the BK-induced changes herein (not shown).",
"Next, the effects of interfering with PI(4,5)P2 resynthesis on the kinetics of translocation in N1E-115 cells was studied.",
"PI(4,5)P2 resynthesis was inhibited by low concentrations (5 μM) of phenyl arsine oxide (PAO) (FIG.",
"6B) or quercetin (Wiedemann et al., EMBO J.",
"15:2094-2101 (1996)), or by depletion of free inositol using prolonged incubation in inositol-free medium (not shown).",
"In PAO-treated cells, BK induced a sustained translocation of PLCδ1PH-GFP to the cytosol, while IP3 increases in such cells are only transient (Hunyady et al., J. Biol.",
"Chem.",
"266:2783-2788 (1991)).",
"In control experiments, these pretreatments did not influence signaling events such as BK-induced Ca2+ signaling (peak Ca2+ values of 870±130 nM in control cells, and 845±114 nM, in PAO pretreated cells, n=6, mean±SEM) or the thrombin- and lysophosphatidate-induced actinomyosin contraction (Jalink et al., supra (1993); Jalink et al., J.",
"Cell Biol.",
"118:411-419 (1992)).",
"Similar observations were made in HEK293 cells (not shown), suggesting that the 15 translocation of PH domains under these conditions reports the depleted PI(4,5)P2 pool rather than the transient IP3 increase.",
"Moreover, when adrenal glomerulosa cells were stimulated with angiotensin II in the presence of Sr2+, a condition under which IP3 metabolism via Ins(1,3,4,5)P4 is greatly reduced (Balla et al., supra (1994)), hence yielding significantly higher Ins(1,4,5)P3- and diminished Ins(1,3,4)P3 increases (FIG.",
"6 C,D), the translocation of PLCδ1PH-GFP was not significantly different (FIG.",
"6 E) from that observed in the presence of Ca2+.",
"Translocation responses of N1E-115 cells in response to BK were also similar in the presence of Ca2+ or Sr2+ (FIG.",
"6 F).",
"Taken together, these results indicate that, at least for the cells and agonists described above, PLCδ1PH-GFP translocation primarily reports changes in membrane PI(4,5)P2 content and not IP3 increases.",
"The reason for the apparently stronger binding of PLCδ1PH to membranes observed in live cells compared to the reported low in vitro affinity (Hirose et al., supra (1999)) to PIP2 containing lipid vesicles or BiaCore surface (Lemmon et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"U.S.A 92:10472-10476 (1995)) is unclear at present, but may indicate a more complex interaction of the PLCδ1PH domain with the native membranes that is not mimicked by the in vitro experiments.",
"However, the finding reported in Hirose et al., supra (1999) that high IP3 levels can make significant contributions to the translocation response was confirmed.",
"Whether such high levels or IP3 occur under the experimental conditions used with intact cells remains to be elucidated.",
"Nevertheless, possible interference from large IP3 increases should be kept in mind during interpretations of the results of such translocation experiments.",
"FRET Reveals Response Heterogeneity to Different GPCR Agonists that is not Reflected in Ca2+ Mobilization.",
"Having characterized the use of FRET between CFP and YFP-tagged PH domains of PLCδ1 to record PLC activation, the kinetics of responses to a set of calcium-mobilizing GPCR agonists were compared.",
"Included in this panel were the peptide agonists BK and NKA, as well as the bioactive lipid lysophosphatidate (LPA), the protease thrombin, and the bioactive amine, histamine.",
"Thrombin and LPA, in addition to inducing Ca2+ mobilizations from internal stores, are also strong inducers of Rho-dependent remodeling of the actin cytoskeleton in these cells (Jalink et al., supra (1993); Jalink et al., supra (1992)).",
"Histamine, on the other hand, does not induce Rho-dependent actin remodeling, but is known to induce Ca2+ oscillations in several cell types (e.g.",
"Paltauf-Doburzynska et al., J.Physiol.",
"(Lond) 524 Pt.3:701-713 (2000); Zhu et al., J. Biol.",
"Chem.",
"275:6063-6066 (2000)) These agonists evoke very similar Ca2+ mobilizations in N1E-115 cells, characterized by a fast onset and rapid termination well within 2 minutes (FIG.",
"7).",
"Estimated peak Ca2+ levels ranged from 0.6-2 μM, and, again, showed no consistent differences between agonists.",
"When PLC activation patterns were recorded by FRET analysis, using the same agonists under identical conditions, several distinct profiles of PLC activation kinetics were obtained (FIG.",
"7).",
"First, both NKA and BK caused fast and near-complete translocation of the probe.",
"This response was transient, returning to baseline within 2-5 minutes.",
"Stimulation with thrombin or LPA evoked a different type of response: these translocations had slower onset and smaller amplitude, averaging 25% of BK response control values (n=22).",
"They also returned to baseline at a slower rate.",
"The response to histamine was much slower and of small amplitude (40% of BK-induced peak values, n=15), but it was long-lasting (at least for 15 minutes, but often much longer).",
"Differences in degree of PIP2 hydrolysis induced by activation of different Gq-coupled receptors have also been reported (van der Bend et al., supra (1992), Tilly et al., Biochem.",
"J.",
"266:235-243 (1990)) in biochemical studies.",
"However, so far only cytosolic Ca2+ responses could be used to analyze receptor activation patterns at the single cell level.",
"On the other hand, the shape of the Ca2+ response is determined by several other factors: it can be triggered at relatively low levels of IP3 and its shape is also determined by the Ca2+-induced Ca2+ release and inactivation properties of the IP3-receptor-channels, as well as by the activities of the various Ca2+ sequestration mechanisms.",
"The present approach provides an opportunity to study a more upstream receptor-mediated event, namely PLC activation, and its regulation in detail at the single cell level.",
"PH Domain Translocation Kinetics Mirror Receptor Activation.",
"These results thus suggest that PLC activation as assessed by FRET is a more faithful index of receptor activity than the more distal Ca2+ transients.",
"However, inactivation could occur at various steps in the signal cascade, including at the levels of receptor, G protein and PLC and, conceivably, also by modulation (upregulation) of PI(4,5)P2 resynthesis.",
"To test whether there is desensitization at the level of PLC, G proteins were directly activated using A1F4− (FIG.",
"8A).",
"While onset of A1F4− induced PLC activation was slow, no desensitization was observed in any of these experiments.",
"Similarly, cells expressing a constitutively active Gαq mutant showed mostly cytosolic localization of PLCδ1 PH-GFP domains for at least 2 days (FIG.",
"8B).",
"Control transfection with activated Gα12 had no effect.",
"At lower expression levels, the activating mutant Gαq induced sustained partial translocation that also persisted for several days.",
"These experiments suggested that no significant desensitization occurs downstream of Gq and PLC.",
"In line with this notion, significant heterologous desensitization between sequentially added agonists was not observed (compare e.g.",
"FIGS.",
"5 and 7, last panel), whereas prolonged exposure of cells to each individual agonist induced complete (homologous) desensitization.",
"To further determine whether such monitoring of PLC activity truly follows receptor activity (in other words coupling and uncoupling between receptors and G proteins), the FRET responses of N1E-115 cells expressing either the wild-type NK2 receptors or a C-terminally truncated form, which is greatly impaired in its ability to desensitize (Alblas et al., J. Biol.",
"Chem.",
"270: 8944-8951 (1995)), were compared.",
"After stimulation of the wild-type human NK2 receptors the translocation response decays towards baseline within minutes (average 50%) recovery time 83±38 s, n=25; compare FIGS.",
"7 and 8C).",
"Application of short pulses of agonist using a puffer pipette resulted in incomplete desensitization, and decayed significantly faster (45±7 s, n=60, FIG.",
"8C) between applications of stimuli due to the rapid dissociation of the ligand from the receptor (Vollmer et al., J. Biol.",
"Chem.",
"274:37915-37922 (1999)).",
"Conversely, stimulation of a C-terminally truncated mutant human NK2 receptor, that was reported to be transforming in Rat-1 fibroblasts, and which has been found to display prolonged coupling to PLC (Alblas et al., supra (1995); Alblas et al., EMBO J.",
"15:3351-3360 (1996); Alblas et al., J. Biol.",
"Chem.",
"268:22235-22238 (1993)) induced a much prolonged cytosolic translocation as assessed in FRET analysis (FIG.",
"8C).",
"However, in the majority of cells, the FRET signal eventually slowly returned to baseline (FIG.",
"8D; note the different time scale), with an average 50% recovery time of 1365±599 s (n=19) in the truncated receptor.",
"This result indicates the existence of an alternative and much slower desensitization mechanism that functions even in NK2 receptors lacking the C-terminus.",
"The kinetics of this slow desensitization closely paralleled those of receptor internalization (not shown), suggesting that one of the main determinants for termination of NKA-induced PLC signaling could be receptor internalization.",
"Analysis of receptor activity by monitoring PLC activity by FRET will greatly aid further studies addressing these questions in more detail.",
"In summary, described above is a fluorescence resonance-based detection scheme of membrane localization of tagged PLCδ1 PH domains for analysis of activation-inactivation kinetics of PLC in single cells with high temporal resolution.",
"This method has a number of significant advantages over confocal detection of membrane localization, including: (i) a significant decrease in excitation intensity allowing prolonged experiments or very fast sampling with little photobleaching and phototoxicity; (ii), suitability for very flat cells such as fibroblasts and motile cells; (iii), extendibility to record from cell populations as well as from small subregions such as neurites; and (iv) a simpler detection hardware.",
"FRET detection of PLC activation is a fairly robust response that can be routinely obtained in a variety of cell types.",
"Analysis of the translocation responses suggests that localization of PLCδ1PH-GFP largely reports PI(4,5)P2 dynamics, although at high concentrations IP3 can also contribute to translocation of the PH domains to the cytosol.",
"Comparison of the Ca2+ and FRET-recorded responses of several agonists of GPCRs suggest that PLC activation detected by FRET is a more faithful reflection of receptor activity than the Ca2+ signal and that little if any “desensitization” or “uncoupling” occurs beyond the levels of G proteins.",
"All journal article, reference and patent citations provided above, in parentheses or otherwise, whether previously stated or not, are incorporated herein by reference in their entirety.",
"Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention."
]
] | Patent_10433245 |
[
[
"Growth of human dendritic cells for cancer immunotherapy in closed system using microcarrier beads",
"A method and apparatus for reproducibly generating dendritic cells are provided.",
"Blood mononuclear cells are loaded into a cell culture container containing microcarrier beads therein.",
"Tissue culture comprising the cells loaded in the container is incubated for a predetermined period.",
"Nonadherent cells and cells adhered to the beads are separated.",
"Dendritic cell culture medium may be prepared and transferred to the container after the cells which adhere to the beads are separated from the nonadherent cells.",
"The tissue culture incubated for the predetermined time period may be washed to remove nonadherent cells.",
"The beads may be allowed to settle and supernatant is expressed off.",
"The container may comprise a gas permeable cell culture bag."
],
[
"1.A method of reproducibly generating dendritic cells, comprising the steps of: (a) loading blood mononuclear cells into a cell culture container containing microcarrier beads therein; (b) incubating for a predetermined time period tissue culture comprising the cells loaded in the container in step (a); and (c) separating nonadherent cells and cells adhered to the beads.",
"2.A method of reproducibly generating dendritic cells, comprising the steps of: (a) loading microcarrier beads into a cell culture container; (b) loading blood mononuclear cells into the container; (c) incubating for a predetermined time period tissue culture comprising the mononuclear cells loaded in the container in step (b); and (d) separating nonadherent cells and cells adhered to the beads.",
"3.The method of claim 1, wherein the container comprises a gas permeable cell culture bag.",
"4.The method of claim 1, wherein the container is a closed vessel.",
"5.The method of claim 1, wherein the tissue culture incubated for the predetermined time period in step (b) is washed to remove nonadherent cells.",
"6.The method of claim 1, wherein after step (b) the beads are allowed to settle and supernatant is expressed off.",
"7.The method of claim 1 further comprising: (d) preparing dendritic cell culture medium; and (e) transferring the dendritic cell culture medium prepared in step (d) to the container after step (c).",
"8.The method of claim 7 further comprising: (f) incubating the container for a second predetermined time period after step (e); (g) agitating contents of the container incubated in step (f); and (h) harvesting cell culture suspension by expression into transfer bags using a sterile connecting device after the beads agitated in step (g) are allowed to settle.",
"9.The method of claim 1, wherein after step (c) samples are removed from the container for quality control.",
"10.The method of claim 9, wherein the quality control includes at least one of viability staining, microbial analysis, cell enumeration, microscopic examination of dendritic cell morphology, and immunophenotyping to determine a purity of the dendritic cell preparation.",
"11.The method of claim 1, wherein the blood mononuclear cells are obtained by apheresis.",
"12.The method of claim 1, wherein a ratio of a combined surface area of the microcarrier beads and the container to a volume of the container volume is a value that allows the container to hold enough media for the predetermined time period of incubation in step (b).",
"13.The method of claim 1, wherein the microcarrier beads comprise styrene copolymer beads.",
"14.The method of claim 1, wherein the microcarrier beads comprise polystyrene copolymer beads.",
"15.An apparatus for reproducibly generating dendritic cells, comprising: a cell culture container; and a plurality of microcarrier beads contained within the cell culture container.",
"16.The apparatus of claim 15, wherein the container comprises a gas permeable cell culture bag.",
"17.The apparatus of claim 15, wherein the container is a closed vessel.",
"18.The apparatus of claim 15, wherein the microcarrier beads comprise styrene copolymer beads.",
"19.The apparatus of claim 15, wherein the microcarrier beads comprise polystyrene copolymer beads.",
"20.The apparatus of claim 15, wherein a ratio of a combined surface area of the microcarrier beads and the container to a volume of the container volume is a value that allows the container to hold enough media for a predetermined time period of incubation."
],
[
"<SOH> BACKGROUND <EOH>The present application relates to a method of growing adherence-dependent hematopoietic cells.",
"In particular, dendritic cells are grown in a closed system using microcarrier beads.",
"Throughout this application, various publications are referenced by author and date.",
"Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims.",
"The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.",
"Dendritic cells (DCs) constitute potent antigen-presenting cells.",
"They may be derived from bone marrow progenitor cells and circulate in small numbers in the peripheral blood.",
"As antigen-presenting cells, DCs are able to induce activation of T-cells with a high degree of efficiency.",
"They are highly specialized and optimally equipped for their task, since dendritic cells express molecules which are required for presenting antigen in large quantity.",
"Important adhesion molecules, which guarantee intimate contact with the target cell, are present on the surface of the dendritic cells.",
"Due to low frequency of DC in peripheral blood, ex vivo expansion and maturation of DC precursors are required for their clinical application (Bartholeyns et al., 1998).",
"There is a need to refine DC culture methods for clinical use in immunotherapy for cancer patients.",
"Most DC culture systems are initiated from the adherent fraction of peripheral blood mononuclear cells, selected using open polystyrene flasks, followed by washing and then culture in serum-free medium containing GM-CSF and IL-4 or IL-7 (as well as other maturational cytokines) {Schuler et al., 1997; Di Nicola et al., 1998}.",
"The open system is labor intensive and poses an increased risk of microbial contamination to the expanded product, the patient and the technician.",
"An alternative to the open flask is a closed system for culturing populations of monocyte enriched peripheral blood mononuclear cells using flexible gas permeable cell culture bags and sterile connecting devices (Glaser et al., 1999).",
"Growing human DC in plastic bags, even under clinical grade and using good manufacturing practices, have poor yields because the surface of the bags is suboptimal."
],
[
"<SOH> SUMMARY <EOH>The application provides a method of reproducibly generating dendritic cells, comprising the steps of: (a) loading blood mononuclear cells into a cell culture container containing microcarrier beads therein; (b) incubating for a predetermined time period tissue culture comprising the cells loaded in the container in step (a); and (c) separating nonadherent cells and cells adhered to the beads.",
"The application also provides a method of reproducibly generating dendritic cells, comprising the steps of: (a) loading microcarrier beads into a cell culture container; (b) loading blood mononuclear cells into the container; (c) incubating for a predetermined time period tissue culture comprising the mononuclear cells loaded in the container in step (b); and (d) separating nonadherent cells and cells adhered to the beads.",
"The application also provides an apparatus for reproducibly generating dendritic cells, comprising: a cell culture container; and a plurality of microcarrier beads contained within the cell culture container.",
"The container may comprise a gas permeable cell culture bag.",
"The container is a closed vessel.",
"The microcarrier beads may comprise styrene copolymer beads.",
"The microcarrier beads may comprise polystyrene copolymer beads.",
"A ratio of a combined surface area of the microcarrier beads and the container to a volume of the container volume preferably is a value that allows the container to hold enough media for a predetermined time period of incubation."
],
[
"This application is a continuation-in-part and claims priority of U.S. Ser.",
"No.",
"09/726,883, filed Nov. 30, 2000, the contents of which are hereby incorporated by reference.",
"BACKGROUND The present application relates to a method of growing adherence-dependent hematopoietic cells.",
"In particular, dendritic cells are grown in a closed system using microcarrier beads.",
"Throughout this application, various publications are referenced by author and date.",
"Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims.",
"The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.",
"Dendritic cells (DCs) constitute potent antigen-presenting cells.",
"They may be derived from bone marrow progenitor cells and circulate in small numbers in the peripheral blood.",
"As antigen-presenting cells, DCs are able to induce activation of T-cells with a high degree of efficiency.",
"They are highly specialized and optimally equipped for their task, since dendritic cells express molecules which are required for presenting antigen in large quantity.",
"Important adhesion molecules, which guarantee intimate contact with the target cell, are present on the surface of the dendritic cells.",
"Due to low frequency of DC in peripheral blood, ex vivo expansion and maturation of DC precursors are required for their clinical application (Bartholeyns et al., 1998).",
"There is a need to refine DC culture methods for clinical use in immunotherapy for cancer patients.",
"Most DC culture systems are initiated from the adherent fraction of peripheral blood mononuclear cells, selected using open polystyrene flasks, followed by washing and then culture in serum-free medium containing GM-CSF and IL-4 or IL-7 (as well as other maturational cytokines) {Schuler et al., 1997; Di Nicola et al., 1998}.",
"The open system is labor intensive and poses an increased risk of microbial contamination to the expanded product, the patient and the technician.",
"An alternative to the open flask is a closed system for culturing populations of monocyte enriched peripheral blood mononuclear cells using flexible gas permeable cell culture bags and sterile connecting devices (Glaser et al., 1999).",
"Growing human DC in plastic bags, even under clinical grade and using good manufacturing practices, have poor yields because the surface of the bags is suboptimal.",
"SUMMARY The application provides a method of reproducibly generating dendritic cells, comprising the steps of: (a) loading blood mononuclear cells into a cell culture container containing microcarrier beads therein; (b) incubating for a predetermined time period tissue culture comprising the cells loaded in the container in step (a); and (c) separating nonadherent cells and cells adhered to the beads.",
"The application also provides a method of reproducibly generating dendritic cells, comprising the steps of: (a) loading microcarrier beads into a cell culture container; (b) loading blood mononuclear cells into the container; (c) incubating for a predetermined time period tissue culture comprising the mononuclear cells loaded in the container in step (b); and (d) separating nonadherent cells and cells adhered to the beads.",
"The application also provides an apparatus for reproducibly generating dendritic cells, comprising: a cell culture container; and a plurality of microcarrier beads contained within the cell culture container.",
"The container may comprise a gas permeable cell culture bag.",
"The container is a closed vessel.",
"The microcarrier beads may comprise styrene copolymer beads.",
"The microcarrier beads may comprise polystyrene copolymer beads.",
"A ratio of a combined surface area of the microcarrier beads and the container to a volume of the container volume preferably is a value that allows the container to hold enough media for a predetermined time period of incubation.",
"BRIEF DESCRIPTION OF THE DRAWINGS The present application would be more readily understood from the following detailed description by referring to the accompanying drawings wherein: FIG.",
"1 shows a block diagram of an apparatus for reproducibly generating dendritic cells, in accordance with an embodiment of the present application; FIG.",
"2A shows a flow chart of a method of reproducibly generating dendritic cells, in accordance with an embodiment of the present application; FIG.",
"2B shows a flow chart of a method of reproducibly generating dendritic cells, in accordance with another embodiment of the present application; FIG.",
"3 shows a flow chart of a method, in accordance with another embodiment, for reproducibly generating dendritic cells; FIG.",
"4 shows a table comparing dendritic cell culture methods; FIG.",
"5 shows a table showing typical immunophenotype of cultured dendritic cells; FIGS.",
"6A through 6D show a manufacturing flow sheet of a method, in accordance with another embodiment of the present application, for reproducibly generating dendritic cells; FIGS.",
"7A-7F show histograms corresponding to experimental data obtained through immunofluorescent flow cytometry, depicting the number of cells exhibiting various fluorescence intensities; FIG.",
"8 shows a XY-scatterplot analysis of log base 2-transformed expression data; FIG.",
"9-13 show dendrograms corresponding to data measured by using a cDNA array with elements representing a plurality of distinct human genes; FIGS.",
"14A and 14B show patterns of expression using semi-quantitative reverse transcription polymerase chain reaction of four genes (CD37, CD81, CD53 and BCL-6); FIG.",
"15 shows a table corresponding to gene expression changes in cultured adherent cells treated with GM-CSF and IL-4; FIG.",
"16 shows a table corresponding to gene expression changes in cultured adherent cells treated with GM-CSF and IL-7; FIG.",
"17 shows a table corresponding to gene expression changes in immature DCs treated with IFN γ; FIG.",
"18 shows a table corresponding to gene expression changes in immature DCs treated with TNF α; and FIG.",
"19 shows a table corresponding to gene expression changes in immature DCs treated with s CD40 L trimer.",
"DETAILED DESCRIPTION The present disclosure provides a novel and unobvious tool for reproducible generation of dendritic cells.",
"Addition of selected, sterile plastic microcarrier beads enhances production of human dendritic cells (DC) in gas permeable cell culture bags.",
"The method also may be adapted for growth of other adherence-dependent hematopoietic cells.",
"The present application, in accordance with an embodiment, provides an apparatus for reproducibly generating dendritic cells comprising a cell culture container and a plurality of microcarrier beads contained within the cell culture container.",
"The present application, in accordance with an embodiment, provides a method of reproducibly generating dendritic cells, comprising the steps of (a) loading blood mononuclear cells into a cell culture container containing microcarrier beads therein, (b) incubating for a predetermined time period tissue culture comprising the cells loaded in the container in step (a), and (c) separating nonadherent cells and cells adhered to the beads.",
"The present application, in accordance with another embodiment, provides a method of reproducibly generating dendritic cells, comprising the steps of (a) loading microcarrier beads into a cell culture container, (b) loading blood mononuclear cells into the container, (c) incubating for a predetermined time period tissue culture comprising the mononuclear cells loaded in the container in step (b), and (d) separating nonadherent cells and cells adhered to the beads.",
"The container may comprise a gas permeable cell culture bag.",
"The container is a closed vessel.",
"The microcarrier beads may comprise styrene copolymer beads and/or polystyrene copolymer beads.",
"The tissue culture incubated for the predetermined time period may be washed to remove nonadherent cells.",
"After the tissue culture is incubated for a predetermined time period, the beads may be allowed to settle and supernatant expressed off.",
"The method further may comprise (d) preparing dendritic cell culture medium, and (e) transferring the dendritic cell culture medium to the container after the nonadherent cells and the cells adhered to the beads are separated.",
"The method further also may comprise (f) incubating the container for a second predetermined time period after step (e), (g) agitating contents of the container incubated in step (f), and (h) harvesting cell culture suspension by expression into transfer bags using a sterile connecting device after the beads agitated in step (g) are allowed to settle.",
"Samples may be removed from the container for quality control after the nonadherent cells and the cells adhered to the beads are separated.",
"The quality control may include at least one of viability staining, microbial analysis, cell enumeration, microscopic examination of dendritic cell morphology, and immunophenotyping to determine a purity of the dendritic cell preparation.",
"The blood mononuclear cells may be obtained by apheresis.",
"An apparatus for reproducibly generating dendritic cells, in accordance with an embodiment, will be described with reference to FIG.",
"1.Apparatus 1 includes a cell culture container 3 and a plurality of microcarrier beads 5.The container 3 may comprise a gas permeable cell culture bag.",
"The container 3 is a closed vessel.",
"The microcarrier beads 5 may comprise styrene copolymer beads and/or polystyrene copolymer beads.",
"The apparatus 1 also may be provided with a tubing harness including connectors 7a and 7b coupled to respective ports in the container 3 which facilitate the loading of cells into the container via a transfer process which is preferably substantially sterile, while maintaining the close environment provided by the container.",
"Loading of the container may be manual or via a transfer pump.",
"To optimize the sterility of the apparatus when loading is not being performed, a cap may be provided.",
"A method of reproducibly generating dendritic cells, in accordance with one embodiment of the present application, will be described with reference to FIGS.",
"1 and 2A.",
"Blood mononuclear cells are loaded into the cell culture container 3 containing the microcarrier beads 5 (step 11).",
"Tissue culture comprising the cells loaded in the container 3 are incubated for a predetermined period (step 12).",
"Nonadherent cells and cells adhered to the beads 5 are separated (step 13).",
"A method of reproducibly generating dendritic cells, in accordance with another embodiment of the present application (FIG.",
"2B), includes first loading the microcarrier beads 5 into the cell culture container 3 (step 21), for example through a valve (7a or 7b) provided in the cell culture container.",
"Blood mononuclear cells then are loaded into the container 3 containing the microcarrier beads 5 (step 22).",
"Tissue culture comprising the cells loaded in the container 3 are incubated for a predetermined period (step 23).",
"Nonadherent cells and cells adhered to the beads 5 are separated (step 24).",
"The subject matter of the present application is illustrated in the Experimental Details section which follows with reference to FIGS.",
"3 through 6D.",
"These sections are set forth to aid in an understanding of the application but are not intended to, and should not be construed to, limit in any way the application as set forth in the claims which follow thereafter.",
"EXPERIMENTAL DETAILS Example 1 One desirable property of blood mononuclear cell (MNC) products suitable for DC culture is collection of a maximum number of monocytes and monocyte precursors with a minimum number of red blood cells, lymphocytes and platelets.",
"This may be accomplished by pheresing donors on an apheresis system (e.g., Spectra, COBE BCT, Lakewood, Colo.) using a mononuclear cell program.",
"Thus, the MNC for the DC culture may be obtained (step 401) by apheresis, under informed consent, from G-CSF mobilized donors, in accordance with one embodiment.",
"Donors may undergo, for example, a 10-liter apheresis.",
"The collection schema may utilize a separation fraction of 250, equivalent to a velocity of 635 rpm at an inlet flow of 50 ml per min.",
"Materials and reagents used for the apheresis and DC culture preferably are sterile and/or endotoxin free and FDA approved for human use.",
"Without use of microcarrier beads, the yield of DCs per unit of culture surface area in closed gas permeable cell culture bags is less than the yield in open flask systems.",
"To improve the yields of DCs in a closed system, styrene copolymer beads (e.g., 90-500 micron diameter, density≧1.04 g/cm3, SoloHill Engineering, Inc., Ann Arbor, Mich.) are introduced into the bags, in accordance with one embodiment of the present application, to increase the available surface area (e.g., by 380 cm2) and supply a surface area similar to that found in the flasks {see, e.g., M. Kiremitci et al., Cell adhesion to the surfaces of polymeric beads, 18 Biomater.",
"Artif.",
"Cells Artif.",
"Organs 599-603 (1990)}.",
"Also, the beads have a density that allows them to sink/settle in due course.",
"In accordance with one (FIGS.",
"3 through 5) of many possible embodiments, 1 gram of gamma radiation sterilized beads and 10×10*8 total cells/bag of MNC product are diluted in 100 mls AIM-V (e.g., from GIBCO, Grand Island, N.Y.) and loaded into gas permeable cell culture bags (e.g., Lifecell X-fold Cell Culture Containers PL2417, 180 cm2, Nexell Therapeutics, Irvine, Calif.), under a biological safety cabinet (step 402).",
"The tissue culture bags then are incubated (step 403), for example, in a humidified 37° C., 5% CO2 atmosphere for approximately four hours.",
"After four hours the contents of the bag are gently resuspended (step 404), the beads are allowed to settle for 5 minutes at 1×g (step 405), and the bag is clamped 1 cm above the settled beads (step 406).",
"The supernatant then is expressed off (step 407) using a transfer bag and a sterile connecting device (e.g, from Terumo Corp., Phoenix, Ariz.).",
"This procedure (steps 402-407) may be repeated three times with 50 mls AIM-V media.",
"As control for adherence, a sample of the expressed cells may be immunophenotyped for monocyte markers (e.g., CD14 and CD11c).",
"Adherence of MNC to the bag and bead surface may be inferred by a decrease in the percent of CD14 and CD11c positive cells in the expressed fraction relative to the apheresis product.",
"After removal of nonadherent cells, 100 ml of AIM-V media containing rh-GM-CSF (e.g., 25 ng/ml, Sargramostim, Immunex, Seattle, Wash.) and rh IL-4 (e.g., 1000 U/ml, Sigma, St. Louis, Mo.)",
"is introduced into the tissue culture bags (step 408).",
"The bags may be placed into a dedicated, Hepa-filtered, humidified 37° C. 5% CO2 incubator (step 409) for 7 days.",
"At day 3 or 4, the bags are visually inspected to check for media color change or bacterial/fungal contamination (step 410).",
"On day 7 (although the culture period may be as little as four days), the tissue culture bags may be removed from the incubator and samples removed therefrom for quality control, e.g., viability staining, microbial analyses, cell enumeration using a hematology analyzer (e.g., from Beckman-Coulter, Hialeah, Fla.), microscopic examination of dendritic cell morphology, and immunophenotyping to determine the purity of the dendritic cell preparation (step 411).",
"Immunophenotyping may be performed using a flow cytometer (e.g., FACSCalibur, Becton Dickinson, San Jose, Calif.) and corresponding software (e.g., CellQuest, Becton Dickinson, San Jose, Calif.).",
"The monoclonal antibody panel may include antibodies to CD45/CD14, CD3/CD19, CD1a, CD11c, HLA-DR, CD83, CD86 and CD123.In experiments using the method described above, plastic beads were not visible in the supernatant on microscopic examination.",
"The yields of DC are improved when compared to the other systems studied (see FIG.",
"4).",
"The immunophenotype of the recovered cells (see FIG.",
"5) meets established DC phenotypes effective in adjuvant vaccine therapy.",
"Culture supernatants are routinely negative for microbial contamination.",
"The quantities of the cells produced are acceptable for adaptive transfer strategies.",
"Current tumor antigen vaccine protocols typically use approximately 107 to 108 total DCs.",
"Using this closed system of culture, a sufficient number of DCs can be harvested for a complete course of therapy using a single 10-liter MNC apheresis and an average of five culture bags.",
"Another embodiment will be described with reference to FIGS.",
"6A through 6D.",
"A dendritic cell culture medium is prepared (step 701) by combining AIM V media (e.g., BB-MF 2557, Life Technologies, Grand Island, N.Y.), rh-IL-4 (e.g., 1000 U/ml, GLP grade, Sigma Aldrich, St. Louis, Mo.)",
"and rh-GM-CSF (e.g., 25 ng/ml, Therapeutic grade, Immunex, Seattle, Wash.).",
"Polystyrene copolymer beads (e.g., 250 micron diameter, density=1.07 g/cm3, BB-MF 3094, Solohill Engineering, Inc., Ann Arbor, Mich.) are obtained and prepared (step 702) for use.",
"For example, the beads may be suspended in a phosphate buffered saline (e.g., EDR9865, therapeutic grade, Nexell, Calif.) [100 gms beads/200 mls saline], placed in an autoclavable glass bottle and capped, and sterilized in an autoclave (e.g., 20 lbs/sq.in.",
"at 121° C. for 1 hour with slow exhaust).",
"The container is sealed and then transferred to a biological safety cabinet.",
"A 1 ml aliquot is removed, placed in the transport tube, and tested for sterility (e.g., Bioscreen Testing Services, Inc., Torrance, Calif.).",
"The polystyrene beads are used if no bacterial growth is detected.",
"Also, a sterile peptide (e.g., HER-2, or another peptide antigen specific to another target tumor) solution is prepared (step 703), using for example synthetic peptide (e.g., GLP grade, American Peptide Company) and phosphate buffered saline.",
"For example, HER-2 synthetic peptide powder is dissolved in saline at a concentration of 200 μg/ml (20×) and sterile filtered through 0.2 micron nylon membrane.",
"The solution is aliquoted in sterile 10 ml vials and stored.",
"A cryoprotectant agent (e.g., DMSO, USP grade, Gaylord Chemical Corporation, Slidell, La.)",
"is obtained and tested for sterility (step 704).",
"Apheresis products are transferred to a transfer bag (step 705).",
"Samples of the apheresis products are run through quality control (e.g., hematology analyzer, Trypan blue viability, CD45/14 immunophenotype)[step 706].",
"If quality control is passed, apheresis products (e.g., 10×108 mononuclear cells/bag×four to five bags) are transferred (step 707) from the transfer bag using a sterile connecting device (e.g., Lifecell transfer set, Nexell Therapeutics, Irvine, Calif.) to gas permeable tissue culture bags (e.g., therapeutic grade, Lifecell X-fold Cell Culture Containers PL2417, 180 cm2, Nexell).",
"The beads that pass quality control (step 702) also are inserted in the bags.",
"The tissue culture bags then are incubated (step 708), for example, in a humidified 37° C., 5% CO2 atmosphere for approximately four hours.",
"At the midpoint, the bag is flipped from one side to the other.",
"After four hours, the tissue culture is washed three times with AIM-V to remove nonadherent lymphocytes, platelets, grans, RBC, etc.",
"(step 709).",
"The wash includes transfer of the AIM-V media and expressing off the supernatant while leaving the beads in the bag.",
"Next, the dendritic cell culture medium (prepared in step 701) is transferred via a sterile process to the tissue culture bags (step 710).",
"The bags are incubated again, for example, in a humidified dedicated 37° C. 5% CO2 incubator (step 711) for 5 to 7 days.",
"At day 4, samples of cell suspension are removed for quality control (step 712).",
"On day 7, the tissue culture bags are moved from the incubator to a biological safety cabinet (step 713).",
"The bags are cooled to room temperature, and the contents are gently agitated for five minutes (step 714).",
"The bags are suspended in an upright position to allow the beads to settle for 5 minutes at 1×g (step 715), and the bag is clamped above the settled beads (step 716).",
"The cell culture suspension is harvested (step 717) by expression into transfer bags (e.g., Stericell bags, Nexell Therapeutics) using a sterile connecting device (e.g, from Terumo Corp., Phoenix, Ariz.).",
"Samples are removed from the transfer bags and run through quality control (step 718).",
"For example, if no beads are present and viability is greater than 95%, then the samples are passed to immunophenotype by flow cytometry.",
"If quality control is passed, cells (e.g., approximately 50×106 cells or any range, such as all of the cells) can be transferred (step 719) to a second bag for cryopreservation and immunological function controls (e.g., seven day proliferation assay using harvested cells as stimulators for lymphocytes from three different individuals).",
"HER-2 peptide solution is added to the transfer bag (step 720) for peptide loading onto the HLA Class I of the DC (final concentration is 10 ug/ml).",
"The transfer bag is incubated (step 721) overnight (e.g., 4 to 12 hours at 37° C., 5% CO2 in a humidified atmosphere of a dedicated incubator).",
"Samples of the peptide loaded DC are tested for mycoplasma (step 722).",
"If the test results are negative for mycoplasma, the peptide loaded DCs are washed three times with therapeutic grade phosphate buffer saline (step 723).",
"In a preclinical phase, samples of the suspension may be removed for quality control analysis, such as for endotoxin (e.g., USP LAL), fluoride (ion specific electrode) and residual organic solvent (GC-MS).",
"Injection formulation is prepared by resuspending the washed DCs (step 724) at a concentration less than 107 cells/ml (e.g., 3, 6, 9 or 12×106 cells/ml) in saline supplemented with 5% autologous serum obtained the same day.",
"Samples are removed for quality control (step 725), such as Gram stain.",
"If quality control is passed, the injection formulation is cleared for administration and injected within four hours of preparation (step 726).",
"The remaining cells are cryopreserved.",
"First, the peptide loaded dendritic cells (also DC without peptide) are suspended (step 731) in a solution of therapeutic grade saline supplemented with 5% autologous serum (5×106/ml).",
"Cryoprotectant agent is added to a final concentration of 10% and placed in sterile NUNC vials (5 ml) [step 732].",
"The cells are placed in a methanol bath at −70° C. overnight (step 733), then placed in vapor phase liquid N2 storage until use (step 734).",
"After two to three days of storage, a vial of peptide loaded DC is retrieved (step 735) from the liquid N2 storage for quality control testing.",
"Vials are thawed at 37° C. in a biological safety cabinet (step 736).",
"The cells are washed with AIM-V to remove cryoprotectant (step 737).",
"Aliquots are removed for the following assays: Viability (>70%) Sterility USP (No growth) Mycoplasma by PCR (negative) MLC test (7 day) (Stimulate proliferative response greater that 3 × BACKGROUND at responder to stimulator ratio of 10 to 1) Endotoxin (USP LAL) <0.06 EU/ml If the quality control test (step 738) is passed, the cryopreserved DC are released for thawing.",
"Vials of cryopreserved peptide loaded DC are thawed (step 739) at 37° C. in the biological safety cabinet.",
"Cells are washed with therapeutic grade saline three times to remove cryoprotectant agent (step 740).",
"Washed DC are resuspended (step 741) in saline supplemented with 5% autologous serum obtained the same day.",
"Samples are removed for quality control testing (step 742), e.g., viability staining and Gram stain.",
"If viability is greater than 70%, then the cells are passed to adjust cell concentration to less than 107 viable cells/ml (e.g., 6, 9 or 12×106 viable cells/ml)×1 ml.",
"Next, a Gram stain is applied.",
"If the Gram stain is passed, the injection formulation is cleared for administration and injected within four hours of preparation (step 743).",
"Example 2 Transcript Profiling of Human Dendritic Cells Maturation-Induced Under Defined Culture Conditions: Comparison of the Effects of Tumor Necrosis Factor Alpha, Soluble CD40 Ligand Trimer and Interferon Gamma Using cDNA arrays, patterns of gene expression were characterized in populations of human dendritic cells (DCs) produced for clinical use.",
"Culture and maturation induction of myeloid adherent cells under serum-free conditions yielded DCs with phenotypes similar to those described in serum-based systems.",
"Analysis of gene expression in DCs treated with tumor necrosis factor alpha, soluble CD40L trimer or interferon gamma, however, showed specific patterns for each factor examined.",
"Expression of several transcripts in DCs and/or differentially regulated according to the differentiation state of the DCs were documented, and suggest important functional differences among the DC populations examined.",
"In addition, DC maturation directs changes in the levels of mRNA specific for transcriptional regulators that effect the production of cytokines (e.g., BCL-6, c-rel).",
"Other changes observed, including alteration in the gene expression profile of adhesion molecules and chemokine receptors such as CD44H, CD 49B, Rants R, CXCR5 and CDS 7, suggest differences in trafficking potential between the populations studied.",
"This broad-based description of DC populations, produced under serum-free conditions, provide better basis for definition of intermediate stages of DC maturation as well as the differentiation-inducing effects of cytokines on these cells.",
"Dendritic cells (DCs) are the most effective antigen-presenting cells (APCs) of the immune system characterized to date.",
"These cells, following an encounter with an antigen, can stimulate both naive and memory T-cell responses (Banchereau et al., 2000).",
"The current understanding of DC biology suggests, however, that the differentiation state of the dendritic cell qualitatively affects their interaction with T lymphocytes (Kalinski et al., 1999; Lanzavecchia, 1999).",
"Depending on the level of maturation, DCs typically elaborate different profiles of chemokines and cytokines [e.g., interleukin 12 (IL-12)], show different antigen-processing abilities and have altered expression of adhesion receptors and co-stimulatory molecules (Thomas & Lipsky, 1994).",
"The use of dendritic cells as adjuvants in cancer immunotherapy is supported by studies showing that injection of tumor antigen-loaded dendritic cells can induce tumor-specific cytotoxic T lymphocyte (CTL) responses and, in some cases, regression of metastases.",
"Clinical trials using dendritic cells as vaccination adjuvants have progressed to phase D efficacy studies (Dhodapkar et al., 1999, 2000; Brinckerhoff et al., 2000; Larsson et al., 2000; Rieser et al., 2000; Tjoa & Murphy, 2000).",
"The populations of DCs used in these trials, however, have not yet been fully characterized.",
"Populations of human DCs can be produced for clinical use by culturing precursor cells in the presence of cytokines, notably granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 (Bernhard et al., 1995; Zhou & Tedder, 1996; Thurner et al., 1999).",
"Populations of DCs produced with GM-CSF and IL-4 are phenotypically immature and characterized by active macropinocytosis, endocytosis (Sallusto et al., 1995) and submaximal expression of MHC gene family products and co-stimulatory molecules (Bender et al., 1996).",
"Immature DCs can be driven to a mature phenotype following exposure to a variety of agents including tumor necrosis factor alpha (TNF α) (Siena et al., 1995; Romani et al., 1996), CD40L (Caux et al., 1994; Cella et al., 1996), monocyte-conditioned medium (Bender et al., 1996), heparan sulphate (Kodaira et al., 2000), bacterial and fungal polysaccharides, nucleic acids (Sparwasser et al., 1998; Granucci et al., 1999; Hartmann et al., 1999; Rescigno et al., 1999) and prostaglandin E (PGE) (Kalinski et al., 1997a).",
"This disclosure describes good manufacturing procedure (GMP)-compatible culture methods for the production of dendritic cells to be used in cancer immunotherapy (see also Maffei et al., 2000).",
"The immunophenotype of DCs obtained by this method suggests that these DCs share characteristics with immature DCs previously described (Ye et al., 1996; Zhou & Tedder, 1996; Morse et al., 1997).",
"The maturation state of DCs may affect how they interact with T cells (Kalinski et al., 1999; Tanaka et al., 2000; Vieira et al., 2000).",
"It was sought to better characterize the DCs produced in this system and examine the phenotypes of DCs that were maturation-induced by recombinant TNF α, soluble CD40 ligand trimer (sCD40 LT), interferon gamma (IFN γ) and IL-7 (Takahashi et al., 1997; Li et al., 2000).",
"Analysis of a broad array of transcripts from each of the DC populations studied revealed both similarities and differences in the patterns of gene expression.",
"The data show that both immature and mature human DCs of a mixed DC1-DC2 phenotype can be produced under well-defined culture conditions.",
"This study also suggests that, dependent on the culture conditions used, DC polarization can be partially selected, resulting in different patterns of DC chemokine, lymphokine and cell surface molecule transcript expression.",
"These differences may be exploitable for improved vaccination strategies in DC-based cancer immunotherapy.",
"Materials and Methods A closed system for culturing populations of monocyte-enriched peripheral blood mono-nuclear cells (MNCs), using is flexible gas-permeable cell culture bags and sterile connecting devices, was used (Maffei et al., 2000).",
"Apheresis MNC products were obtained with informed consent from healthy volunteers.",
"All materials and reagents used for the apheresis and DC culture were sterile and/or endotoxin free (<0.5 Limulus amebocyte lysate U/ml) and approved by the Federal Drug Administration (FDA) for human use with the exception of the plastic beads, H-4 and Nycoprep media.",
"The MNC products were harvested on a Spectra apheresis system (Spectra, COBE BCT, Lakewood, Colo., USA) using a cell collection program (Glaser et al., 1999).",
"These products were additionally purified by buoyant density centrifugation over Nycoprep 1068 media (Boyum, 1983).",
"The MNCs (10×8 total cells) were then diluted in 100 ml of AIM-V media (therapeutic grade, Gibco Life Technologies, Grand Island, N.Y., USA) and loaded into gas-permeable cell culture bags (Lifecell X-fold cell culture containers PL2417, 180 cm2, Nexell Therapeutics, Irvine, Calif., USA) containing styrene co-polymer beads (1 g, 90-125 μm diameter, density=1·05 g/cm3, SoloHill Engineering, Ann Arbor, Mich., USA).",
"The tissue culture bags were then incubated in a humidified 37° C., 5% CO2 atmosphere.",
"After 4 hours, the contents of the bag were gently resuspended, the beads allowed to settle and the supernatant was then removed.",
"This procedure was repeated three times with 50 ml of AIM-V media (Gibco).",
"After removal of non-adherent cells, 100 ml of AIM-V media containing recombinant human GM-CSF (rh-GM-CSF; 50 ng/ml, Sargramostim, Immunex, Seattle, Wash., USA) and rh IL-4 (1000 U/ml, Sigma, St. Louis, Mo., USA) or rh IL-7 (32 U/ml, Sigma) was introduced into the tissue culture bags (Romani et al., 1996).",
"Neither fetal calf serum nor autologous human serum was added to the culture medium.",
"The bags were placed into a dedicated, HEPA-filtered, humidified 37° C., 5% CO2 incubator.",
"On d 3 of culture, 50 ml of fresh AIM-V media with GM-CSF and IL-4 (or GM-CSF and IL-7 or GM-CSF alone) was added to each bag (cultures designated G4, G7 or G respectively).",
"On d 6 of culture, fresh medium with cytokines was again added.",
"In addition, the following maturation factors were added to DCs cultured in GM-CSF and IL-4: 25 ng/ml rh TNF α (R & D Systems, Minneapolis, Minn., USA) (cultures designated G4T), 5 μg/ml recombinant soluble CD40L trimeric fusion protein (a kind gift of Immunex Corporation) (cultures designated G4CD40L) or 1000 U/ml IFN γ-1b (Actimmune, Intermune Pharmaceuticals, Palo Alto, Calif., USA) (cultures designated G4IFN).",
"On d 8, the cell suspensions were harvested, washed and pelleted.",
"Aliquots of the cells were removed for phenotyping using immunofluorescent flow cytometry and the remaining cells were used for RNA isolation.",
"Approximately 10 ml of cell culture was retained in the bags and additional fresh medium containing only GM-CSF (25 ng/ml) was added.",
"A repeat immunophenotyping was performed on d 10 of culture.",
"Cell pellets were stored at −80° C. until use.",
"Total RNA was prepared from cell cultures initiated with apheresis products from three different individuals using the TREZOL Reagent (GibcoBRL) according to the manufacturers recommendations.",
"To completely remove RNases, the RNA samples were phenol-chloroform extracted twice.",
"Samples were then precipitated and resuspended in RNase-free water.",
"To eliminate potential genomic DNA contamination, an aliquot of 50 μg of RNA for each sample was incubated with 5 units of RQ1 RNase-free DNase (Promega, Madison, Wis., USA) for 30 min at 37° C., using the buffer recommended by the manufacturers.",
"Finally, the RNA samples were again phenol-chloroform extracted, then precipitated and resuspended in RNase-free water at a concentration of 1 μg/μl.",
"The quality of the RNA prepared was confirmed by analyzing the samples by electrophoresis on a 1·6% agarose gel in Tris-Acetate-EDTA buffer.",
"RNA samples were stored at −80° C. until further use.",
"Large-scale expression probing of immature and maturation-induced human dendritic cells was performed using the Atlas Human Hematology/Immunology cDNA Expression Arrays (Clontech Laboratories, Palo Alto, Calif., USA).",
"Each nylon membrane array was spotted in duplicate with cDNA fragments representing 408 known genes and several housekeeping genes or control sequences.",
"Each cDNA fragment was 200-600 bp long and was amplified from a region of the transcript that lacked the poly A tail, repetitive elements or other highly homologous sequences, to minimize cross-hybridization and the non-specific bindings of the cDNA probe.",
"A list of these genes, including array coordinates and GenBank accession numbers, is available (Clontech Laboratories, 2001).",
"For side-by-side array hybridizations, 5 μg of total RNA from each cell population was reverse-transcribed in the presence of 5 μl of [γ-32P]-dATP (111 TBq/mmol; 370 kBq/ml) (Amersham Pharmacia Biotech; Arlington Heights, Ill., USA) using the reagents and the protocol provided in the Atlas cDNA Expression Array kit to synthesize [32P]-radio-labeled cDNA probes.",
"Radiolabeled probes were denatured under basic conditions, neutralized in the presence of 5 μg of Cot-1 DNA (GibcoBRL) and then added to separate 5 ml aliquots of ExpressHyb hybridization solution (Clontech) containing 100 ng/ml of heat-denatured sheared salmon testes DNA (Sigma), to reach a final probe concentration of approximately 2·5×105 cpm/ml.",
"Hybridization/cDNA probe solutions were applied to prehybridized Atlas Array membranes (1 hour in ExpressHyb with 100 ng/ml of heat-denatured sheared salmon testes added at 68° C. in the absence of a labeled probe) and hybridized overnight at 68° C. After hybridization, membranes were washed twice with 200 ml of 2× saline sodium citrate (SSC), 1% sodium dodecyl sulphate (SDS) solution at 68 T for 30 min.",
"followed by two 30-min washes in 200 ml of 0·1× SSC, 0·5% SDS, at 68° C. Finally, the membranes were rinsed in 2×SSC and exposed overnight to phosphor screens.",
"Hybridized Atlas Arrays were visualized and quantified using a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif., USA) at a pixel resolution size of 80 μm.",
"A grid matrix was generated and applied to the phosphoimage of each Atlas Array, which identified the duplicated target location for each of the 406 known genes as well as the nine genes defined as housekeeping genes and the 12 negative control sequences.",
"The intensity of hybridization signal for each gene sequence was the average of the values determined for both spots in the target location and corrected for background using the intensity values of pixels surrounding the spot areas.",
"Calculated intensities correlated linearly with the amount of message in the total RNA sample, as the target cDNA fixed to the membrane was in excess and the backgrounds were sufficiently low.",
"For assessing differences in gene expression between arrays, the intensity values of each known gene were normalized to the intensity of designated housekeeping genes [i.e.",
"glyceraldehyde phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase (HPRT)].",
"These two genes were selected for normalization over other so-called housekeeping genes (e.g., HLA-C and cytoplasmic β actin), which had been demonstrated to change expression levels during differentiation of DCs in preliminary experiments.",
"Following this normalization, the sums of the intensity values of all the genes on a given array were within one standard deviation of the mean summed intensity for all arrays studied.",
"Comparison between mRNA populations of various DC populations was performed on Atlas arrays of the same batch.",
"Genes that showed an average increase or reduction of greater or equal to fourfold were tabulated.",
"As a measure of consistency in gene expression analysis a scatter plot analysis, in which each point represents a particular gene and its coordinates, as determined from its normalized expression value, was performed (FIG.",
"8).",
"In each scatter plot, points that lie close to the main diagonal represent genes that are expressed at similar levels in the various cell populations studied.",
"For genes that lie away from the diagonal, the perpendicular distance from the diagonal represents the degree of differential expression between the two populations studied.",
"The data were analyzed and displayed (Eisen et al., 1998).",
"Briefly, the hierarchical clustering methodology produces a table of results wherein the elements of the array representing specific genes are grouped based on similarities in their patterns of gene expression (FIGS.",
"9-13).",
"The same methodology was then applied to cluster the data from each population of dendritic cells according to the similarities in their overall patterns of gene expression.",
"The data tables are presented graphically as black and white images.",
"Along the vertical axis, the genes analyzed are arranged as ordered by the clustering methodology, so that the genes with the most similar patterns of expression are placed adjacent to each other.",
"Along the horizontal axis, experimental samples are similarly arranged such that those with the most similar patterns of expression across all genes are placed adjacent to each other.",
"The grey scale value of each square in the table image represents the measured expression of each gene.",
"Where grey scales values are presented, white represents a high level of expression relative to lower levels (indicated in dark purple).",
"After DNase I treatment, 1 μg of total RNA from each sample was used as template for the reverse transcription reaction.",
"cDNA was synthesized using Oligo(dT)15 primer and AMV reverse transcriptase (Reverse Transcription System from Promega, Madison Wis., USA).",
"All oligonucleotides primers for the semi-quantitative polymerase chain reaction (PCR) were synthesized by Life Technologies, Rockville, Md., USA.",
"The following primers were used: (a) GAPDH, AACGGATTTGGTCGTATTGGGC (G3-F) and TCGCTCCTGGAAGATGGTGATC (G3-R); (b) BCL-6, CCTTAATCGTCTCCGGAGTCG (BCL6-F) and CCATCTGCAGGTACATAGCCGT (BCL6-R); (c) CD37, TTTGTGGGCTTGGCCTTCGTGC (CD37-F) and TAGGATTGTGGAGTCGTTGGTCGCC (CD37-R); (d) CD81, GCGCCCAACACCTTCTATGTAGGC (CD81-F) and AGCACCATGCTCAGGATCATCTCG (CD81-R); and (e) CD53, GCTGGGCAATGTGTTTGTCATCG (CD53-F) and CAATCTGGCAGTTCAGGGTCAGTGC (CDS3 F).",
"The PCR reaction was performed in 30 μl with 20 mmol/l Tris-HCl (pH 8·4), 50 mmol/l KCl, 1·5 mmol/l MgCl2, 200 μmol/l of each dNTP, 20 units of recombinant Taq DNA polymerase/ml (PCR SuperMix from Life Technologies), in the presence of 100 pmol of each of the two appropriate primers.",
"The same conditions were used for GAPDH, CD37, CD53 and CD81: the reaction mix was denaturated for 5 min at 94° C., followed by a program consisting of three steps, 40 s at 94° C., 40 s at 55° C. and 90 s at 72° C. These conditions were used for either 20 or 30 amplification cycles (Ferrer et al., 1998).",
"The PCR with the primers for BCL-6 was performed under similar conditions, substituting an annealing temperature of 64° C. rather than 55° C. To ensure the correct conditions for the semi-quantitative PCR, it was necessary to determine the optimum amount of cDNA and number of cycles for linear amplification.",
"To this end, reverse transcription polymerase chain reaction (RT-PCR) was performed with the GAPDH primers on all samples, testing three different amounts of starting total RNA (1, 2·5 and 5 μg) and analyzing, for each amount, the PCR products obtained with three different number of cycles (10, 20 and 30) (data not shown).",
"Linearity was preserved when 1 μg of total RNA was used as starting material for cDNA synthesis and 1/20th of the reaction product was used for PCR.",
"Electrophoresis of the PCR product was performed on a 2% agarose gel containing 1 μg/ml of ethidium bromide.",
"Images from the ethidium bromide-stained gel were captured with a Kodak DC120 Zoom digital camera and light intensity of the bands was quantified using Kodak Digital Science ID image analysis software (Eastman Kodak, Rochester, N.Y., USA).",
"Immunophenotyping of the cultured cells was performed using a FACSCalibur flow cytometer and CellQuest software.",
"The monoclonal antibody (mAb) panel used included fluorochrome-conjugated antibodies to CD45/CD14, CD3/CD19, CD1a, CD11c, HLA-DR, CD83, CD86 and CD123.Staining, washing and analysis was performed as per manufacturers recommendations (Becton Dickinson).",
"Results The dendritic cell populations obtained from the culture bags were immunophenotyped using a series of fluoro-chrome-conjugated mAbs.",
"The data are shown as histograms in FIGS.",
"7A-7F depicting the number of cells exhibiting various fluorescence intensities.",
"The dotted lines represent isotype-matched negative control antibody.",
"The solid lines represent staining with specific antibodies.",
"Results are representative of two independent experiments.",
"Flow cytometric measurements showed nearly unimodal distributions of the cell surface markers studied.",
"Monocytes cultured in GM-CSF were CD 14 high, HLA class II low, CD 80 negative, CD 86 low, CD 83 negative and CD 123 positive (Row G).",
"The same cell populations cultured for 8 d with GM-CSF and IL-4 (Row G4) or GM-CSF and IL-4 plus IFN γ (Row G4IFN) lost expression of CD 14 and CD 123, but displayed enhanced expression of HLA class II, CD 86 and CD 83 (Rows G4 and G4IFN).",
"This is consistent with the immunophenotype of immature dendritic cells previously described (Romani et al., 1996).",
"Cells cultured with GM-CSF and IL-7 (Row G7) displayed an immature immunophenotype with intermediate expression of CD 14, CD 83, CD 123, but a high level of MHC class II, and a low level of CD 80 or CD 86.Immature dendritic cells, following culture in either TNF α (Row G4T) or s CD 40L trimer (Row G4CD40L), showed further enhanced expression of HLA class II, CD 80, CD 86 and CD 83.The cell populations studied contained no CD 3-, CD 19-, CD 20- or CD 56-positive cells (data not shown).",
"The immunophenotype of IFN γ, s CD 40L trimer and TNF α-treated cells was repeated on d 10.On d 10 these mature DCs maintained a similar pattern of expression of the cell surface markers studied (data not shown).",
"The arrays used in these experiments displayed 406 genes, of which 40% were expressed in the dendritic cell populations studied.",
"In a scatter plot analysis in which different mRNA preparations from different dendritic cell populations were compared, the profiles and levels of the expressed genes represented in each population were similar (FIG.",
"8).",
"Many of the specific transcripts measured in DCs were distributed along the diagonal line of ‘identity.’ This indicates that cell culture, RNA isolation, reverse transcription for probe preparations and hybridization conditions were reproducible.",
"To identify genes that were differentially expressed in the DC populations studied, hybridizations of cDNA probes synthesized from RNA isolated from all populations of maturation-induced DCs and immature DCs were compared side-by-side.",
"A scatter plot comparison of the gene expression data from monocytes cultured in GM-CSF (G) and immature DCs obtained from cultures containing GM-CSF and IL-7.",
"(G7) is shown in FIG.",
"8.A pair-wise comparison of gene expression was performed by XY-scatterplot analysis of log base 2-transformed expression data.",
"Expression profiles were obtained from monocytes cultured in GM-CSF alone and monocytes cultured with GM-CSF and IL-7.Each point represents the normalized expression of an individual gene within both mRNA populations.",
"The thick line represents a predicted line of identity.",
"The thin lines indicate thresholds of greater than twofold or less than one-half expression ratios.",
"Although many of the expressed genes lie relatively close to the diagonal line of ‘identity’, other genes exhibited a greater than twofold change in expression levels (FIGS.",
"15-19).",
"The marked differences in gene expression profiles between immature and mature dendritic cells were corroborated by the pattern of expression of many genes whose regulation in DCs and monocytes have been previously characterized (Hashimoto et al., 1999, 2000).",
"For the analysis performed in this study, these genes can be considered as control ‘sentinel genes’ (e.g., TARC, MDC, SMMHC, etc.).",
"In addition, no T- or B-cell lineage-specific transcripts were detected in these arrays (e.g., CD 3, CD 152, CD 7, CD 19, CD 20).",
"The data presented in FIGS.",
"15-19, summarizing the most differentially expressed genes, are a subset of the larger data set that includes genes that showed smaller but significant changes in specific mRNA levels.",
"To better understand the relationships between the different growth conditions (i.e.",
"GM-CSF cultured monocytes, GM-CSF and IL-4, GM-CSF and IL-7, GM-CSF and IL-4 plus IFN γ, GM-CSF and IL-4 plus TNF α GM-CSF and IL-4 plus s CD40 LT), gene expression and phenotype of the dendritic cells produced, the cDNA hybridization data were analyzed using hierarchical cluster analysis.",
"Nineteen genes known to act as soluble immune mediators were selected from the larger group of expressed genes and analyzed by clustering (FIG.",
"9).",
"Data were measured by using a cDNA array with elements representing approximately 400 distinct human genes.",
"Genes were selected for this analysis if their expression level deviated from background by at least threefold in one or more of the different conditions studied.",
"The dendrograms and tables were calculated as described in the text: the grey scale ranges from dark purple (lowest levels of expression) to white (highest levels of expression) with grey values indicating intermediate levels of expression.",
"Each gene is represented by a single row of boxes a single column represents each culture condition.",
"Clustering (and correlation statistics) was performed on groups of genes with known functions of chemokines and cytokine (r=0.95).",
"Examination of the dendrogram (x axis in FIG.",
"9) obtained by cluster analysis shows that the cell populations studied could be categorized into two main families.",
"The first family, GM-CSF cultured monocytes (G) and immature DCs (G4 or G7) were characterized by their increased expression of IL-12 beta, CX3C chemokine, IL-6, IL-3 and IL-1 beta.",
"Cluster analysis shows a significant increases in expression of MDC, TARC, Rants, IL-1 RA and IL-10 genes in G4 DCs relative to the GM-CSF cultured monocytes (FIGS.",
"9 and 15).",
"Similar to the intermediate phenotype revealed by FACs analysis, the pattern of gene expression in DCs grown in GM-CSF and IL-7 occupied an intermediate position in the dendrogram.",
"These cells showed increased expression of MIF, IL-8, NAP-2 and GCP 2 relative to DCs obtained from all other culture conditions.",
"A second cluster observed in mature DCs (treated with either TNF α or s CD40 LT), showed increased expression of IL-14, MIPS beta, MIG, TPO, MDC and TARC.",
"The expression of these genes were further increased beyond the levels seen in immature DCs and, relative to the other populations studied, reached the highest levels (designated in white in FIG.",
"9).",
"Increased quantities of transcripts for TARC, Rants, IL-1 receptor antagonist, IL-10 and MIF were detected in DCs grown in GM-CSF, IL-4 and induced with IFN γ. Twenty genes with known functions in cell-to-cell contact and/or that are involved in APC-effector cell communication were selected from the larger group of expressed genes and analyzed by clustering (FIG.",
"10).",
"The gene expression patterns in the various cell populations studied could be categorized into three families.",
"The first family, GM-CSF cultured monocytes (G) and immature DC (G4) were characterized by their increased expression of CD30L, CD 5 and CD 49B.",
"Cluster analysis did not show a significant elevation of CD 83 and CD 86 gene expression in immature DCs (G4) relative to the cultured monocytes (G) (FIG.",
"15).",
"A second family, formed by G4IFN and G7 DCs, were characterized by increased expression of tsa-1/sca-1, CD 53, CD 11a, CD11c, CD 44H and CD 147.DCs with the highest levels of MHC class II protein expression (FIG.",
"7) (G4T and G4CD40L) were clustered together on the basis of their increased expression of CD 11a, CD 86, CD 83 and CD54/ICAM1 (FIG.",
"10, r=0.91).",
"The expression of 19 molecules representing various receptors for cytokines, chemokines and lymphokines were also examined (FIG.",
"11).",
"GM-CSF cultured monocytes and immature DCs (G4) could be distinguished on the basis of their increased expression of GPR5, CXCR5, MIP1α receptor, CD25 and IL-5 R. GM-CSF cultured monocytes and those cultured with additional IL-7 were grouped on the basis of increased expression of IL-2 R gamma subunits, CD 14 and CD 55/DAF, CD 21 and loss of BLR1/CXCR5 expression (FIGS.",
"11 and 16, r=0.92).",
"The expression of 19 molecules representing various kinases and G proteins (FIG.",
"12, r=0.97) and 39 transcription factors (FIG.",
"13) were analyzed using the above methods.",
"While the relationship between receptor signaling and post-translational modifications of various kinases is well known, the relationship between receptor signaling and the expression of many signaling intermediates remains unexplored in DCs.",
"GM-CSF cultured monocytes and immature dendritic cells (G and G4) clustered together by virtue of their common increased expression of STAM, jnk 2, STAT 5a+STAT 5b, BTK and SLP-76.Mature dendritic cells (G4T, G4IFN and G4CD40L) and those cultured in IL-7 were grouped together.",
"DCs induced with INF γ or TNF α showed increased expression of MEK3, 14-3-3 tau, vav 2, ctk, DAPK1, tec, p21-rac2.An additional cluster formed by DCs treated with s CD40LT and G7 showed increased expression of CAML, lyn, JAK3, raf and RGS1.Although the gene expression patterns of the transcription factors were complex, correlation among gene expression and DCs maturation could be discerned (FIG.",
"13, r=0.99).",
"Monocytes cultured in GM-CSF and immature DCs (G7) formed one cluster, displaying increased expression of erg B, aml-1 and LM02.In cultured monocytes (G) and G4 DCs, tan-1, BMI-1, BCL-6, EWS, homeobox protein prl and numatrin showed higher expression.",
"The pattern of transcription factor expression in maturation-induced DCs (G4T, G4CD40L and G4IFN) appeared to be more specific for the type of DC, although the hierarchical cluster analysis did group DCs treated with sCD40L T and TNF α together.",
"In this grouping, these mature DCs showed enhanced expression of c-rel, homeobox protein HOX-A5, helix loop helix protein, IRF-4 and spi-1/pu-1.Notably, this group also showed decreased expression of transcriptional regulators such as BCL6, AML-1 and CREB.",
"The pattern of down-regulated BCL6 expression in human DC maturation-induced with s CD40 LT was confirmed in parallel experiments using the cDNA array methods previously described (Eisen et al., 1998), in which the patterns of gene expression in G4 DCs and DCs maturation induced with s CD 40 LT were compared (data not shown).",
"DCs, maturation-induced by INF γ, showed a unique cluster of increased gene expression formed by homeobox pbx3, IRF2, host cell factor cl, spi-1/pu-1, dead box protein 6, Ikaros and IRF 5, and C/EBP gamma.",
"To confirm the results on the identification of differentially expressed genes obtained by cDNA array hybridization, the level of expression was determined using semi-quantitative RT-PCR of four genes (CD37, CD81, CD53 and BCL-6) whose expression in mature and immature DCs had not been previously characterized (FIGS.",
"14A and 14B).",
"All band intensities were normalized to the expression of GAPDH, the same internal control used for the normalization in the array experiments.",
"When the cDNAs from immature and maturation-induced DCs were tested with the CD37 primers, an abundant accumulation of specific transcripts in cultured monocytes (G) and immature G7 DCs (G7), as well as a net decrease in the amount of this mRNA in the mature DCs treated with CD40L (G4CD40L) and TNF a (G4T) was detected.",
"These results parallel the cDNA array hybridization findings.",
"The PCR amplification with the CD 81 and CD 53 primers also confirmed the results obtained with the cDNA hybridization on the Atlas arrays.",
"Semi-quantitative PCR analysis of BCL-6 expression in the populations of DCs studied here revealed the following pattern: GM-CSF cultured monocytes, immature DCs (G4) and IFN γ-treated DCs maintained a high level of BCL-6-specific mRNA.",
"Dendritic cells maturation-induced with IL-7, TNF α or s CD40 LT, expressed significantly lower amounts of BCL-6 transcripts, similar to findings in the cDNA hybridization experiments.",
"In separate experiments, using mature and immature DCs from other normal donors, the patterns of specific mRNA accumulation of BCL-6, CDS7, CD81 and CD53 were maintained (data not shown).",
"Discussion As revealed by cluster analysis, the patterns of gene expression ‘in the populations of DCs, maturation-induced with either TNF α or s CD40 LT, were closely related.",
"This finding is consistent with the structural similarity between these members of the TNF α gene super family, the sharing of many intermediates in their signaling pathways and regulation of transcription (Gruss, 1996).",
"The pattern of cytokine gene expression in these mature DCs is also consistent with previous reports on the effects of TNF α and CD40L on immature DCs (Sallusto et al., 1999a).",
"Dendritic cells cultured with s CD40 LT showed increased expression of chemokine genes active on memory and Th 2-type T cells (MDC, TARC, rants), as well as cytokines active on naive T cells (MIP3-β/ELC and IL-8) and Th 1-type T cells (MIG).",
"In this same population of DCs, down-regulated expression of the genes for H-12 was found, and a series of pro-inflammatory cytokines such as MIF, NAP 2, IL-1 beta, IL-6 was also observed.",
"The expression of the anti-inflammatory cytokine genes IL-10 and IL-1 RA was also reduced.",
"A similar pattern of cytokine gene expression was observed in DCs maturation-induced with TNF α. BCL6 is a transcriptional repressor of chemokine gene expression in murine macrophages (Toney et al., 2000).",
"In BCL-6−/−knockout mice macrophages show increased expression of several Th2 type cytokines such as MCP-1 and MRP-1.Also demonstrated by the authors was the presence of BCL6 binding sites in the 5′ untranslated regions of the IL-8 and CD 23 genes.",
"Both IL-8 (LYNAP) and CD 23 showed reciprocal expression with BCL6 in the maturation-induced DCs studied in the experiments (FIGS.",
"9, 11 and 13).",
"Cluster analysis further showed that maturation-induced DCs (G4T and G4CD40L) had increased expression of helix-loop-helix id2, c-rel, IRF-4, HOX-5 and BTG-1 transcription factors.",
"An observation of increased c-rel expression following maturation induction confirms a previous study of human dendritic cells (Neumann et al., 2000).",
"As expected, DCs treated with s CD40 LT showed increased cell surface expression of MHC class II, CD 80, CDS 6 and CD 83 proteins.",
"Expression analysis showed increased levels of transcripts for CD 54 (ICAM-1), CD58 (LFA3), CD 86, CD 83, CD1 1a, CD 23.The level of mRNA specific for several molecules involved in cell-to-cell contact was reduced in DCs following CD 40 ligation.",
"These molecules included CD 44H, CD 49B, CD 43, CD lie, CD18 and several transcripts encoding proteins with accessory functions, such as tsa-1/sca-1 (Saitoh et al., 1995), tetraspanins CD 9, 37, 53 and 81, and the inhibitory co-stimulatory molecule CD 153(CD30L) (Gattei et al., 1999), CXC3 fractalkine (Papadopoulos et al., 1999).",
"The differential expression of CD 37 and other tetraspanin molecules in immature and mature DCs is another novel finding.",
"In the studies, abundant accumulation of mRNA for CD37 was detected in cultured monocytes and immature G7 DCs.",
"Following maturation induction, however, the message for CD37 and four other tetraspanins is significantly reduced.",
"CD 37 has been previously detected on mature B cells (Schwartz-Albiez et al., 1988) and has a putative role in T-cell-B-cell interactions (Knobeloch et al., 2000).",
"CD37 is closely associated with MHC class II molecules and is selectively enriched (along with tetraspanins CD53 and CD81) in exosomes (Escola et al., 1998).",
"Exosomes are formed when specialized endocytic vesicles containing processed antigen and MHC class II molecules (MIICs) fuse with the plasma membrane and are released in the extracellular space.",
"Exosomes are able to prime T lymphocyte-dependent anti-tumor responses in vivo (Zitvogel et al., 1998).",
"Following maturation, DCs are less able to present intact antigen to T cells (Sallusto & Lanzavecchia, 1994; Koch et al., 1995; Mellman et al.",
"1998).",
"Down-regulation of genes coding for constituents of exosomes, such as CD 37, CD S3 and 81, may in part explain these observations.",
"Based on the expression profiles for several cytokines, lymphokines, transcription factors and cell surface molecules, the populations of mature DCs (induced with either s CD40L or TNF α) characterized in these studies share many characteristics with DCs polarized towards the DC2 type (Ria et al., 1998; Kapsenberg & Kalinski, 1999; Hashimoto et al., 2000).",
"However, these populations also retained some characteristics of type 1 DCs.",
"This conclusion is suggested by the detection in these DCs of transcripts specific for MlG, a chemokine active on Th 0- and Th 1-type T cells (Sallusto et al., 1998), MIP 3 β, a chemokine targeted to memory Th 1 (Randolph et al., 1999; Sallusto et al., 1999b), and IL-8 whose CXCR1 receptors are preferentially expressed on Th 1 cells (Bonecchi et al., 1998).",
"Whether the mixed DC1-DC2 phenotype was as a result of heterogeneity in the DC populations or, alternatively, caused by incomplete maturation (Langenkamp et al., 2000) or polarization, could not be answered by the methods used.",
"If the mixed DC1-DC2-type phenotype is common to other DC populations that have been used for adoptive transfer in humans (Dhodapkar et al., 1999), the successful induction of Th 1 and cytotoxic T-cell responses may have been driven by immature DC1-type subpopulation (Koch et al., 1995).",
"Comparison of expression data from G and G4 DCs with maturation-induced DCs (G4T and G4CD40L) showed reciprocal expression of many of the genes studied.",
"Immature DCs showed increased expression of a cluster of genes formed by IL-12, IL-10, CX3C, IL-6 and IL-1 beta.",
"In this cluster, maturation-induced DCs showed down-regulated expression of this same set of genes.",
"Conversely, a second cluster of low expression, formed by IL-14, MIP-3 beta, MIG and TPO, was found in immature DCs.",
"Following maturation, this group of genes was up-regulated.",
"Similar to previous reports of gene expression in dendritic cells grown from CD14+-adherent monocytes (Hashimoto et al., 2000; Ishii et al., 2000), the studies showed that G4 DCs increased expression of the DC2-type chemokine genes (i.e.",
"MDC, TARC and rants) relative to the monocyte population.",
"If however, IL-12 production by DCs is the dominant force driving Th 1-type T-cell development (Kalinski et al., 1997b; Vieira et al., 2000), then the immature G4 DCs that still maintained detectable levels of IL-12 mRNA may be better able to initiate Th 1-type T-cell responses than the mature DCs populations that were characterized.",
"Lastly, relative to the monocyte population (G), the expression of transcripts for the pro-survival Bcl-2 homologue Al (Lin et al., 1996) were decreased following culture with IL-4 or maturation induction.",
"This observation is in accord with findings demonstrating that DCs maturation was accompanied by increased apoptotic susceptibility to HLA-DR-mediated apoptosis (Bertho et al., 2000).",
"Two populations of DCs, those cultured with IL-7 or induced by IFN γ, shared gene expression patterns with both mature and immature DCs examined in this study.",
"Overall, the gene expression pattern of GM-CSF cultured monocytes and immature DCs cultured in IL-7 or IL-4 were closely related.",
"Both IL-4 and IL-7 receptors share the common receptor subunit responsible for mediating differentiation action of their ligands (He et al., 1995).",
"By cluster analysis, immature DCs treated with IFN γ appeared to have a distinct pattern of gene expression relative to the other mature DCs studied.",
"Again, this observation is consistent with the current understanding of the distinct signaling pathways used, by cells responding to INF γ.",
"In the context of DC-based cancer immunotherapy, the immature dendritic cells grown in IL-7 or induced with IFN γ may represent populations that are more polarized towards the DC1 phenotype.",
"For example, DCs grown in IL-7, despite expressing less MHC class II and co-stimulatory molecules, relative to mature DCs, are effective stimulators of cytotoxic T-cell and mixed-lymphocyte responses (Takahashi et al., 1997: Li et al., 2000).",
"In addition, DCs grown in IL-7 show lower levels of transcription of DC2-type chemokines including MDC, TARC and the chemokine receptor CXCR5 (Legler et al., 1998; Ansel et al., 2000), while maintaining detectable IL-12 mRNA levels.",
"Similarly, DCs induced with IFN γ have high cell surface levels of MHC class II molecules, CD 83 and CD 86.Relative to the other maturation-induced cells.",
"DCs maturation-induced with IFN γ retain IL-12 mRNA expression.",
"Dendritic cells, exposed to the Th 1-type lymphokine IFN γ, may themselves be more effective in priming Th 1 responses (Macatonia et al., 1995: Vieira et al., 2000) because they have not undergone the final maturational steps that lead to IL-12 transcript down-regulation (Ebner et al., 2001).",
"The culture conditions used in these studies provide the basis for studying DCs in an immature or intermediate stage of development.",
"In the context of DC-based immunotherapy, anti-tumor DC vaccines based on a single HLA class I (e.g., HLA-A2)-restricted peptide epitope appear promising but have serious shortcomings, such as providing a selective pressure for the escape of tumors that lose HLA-2 expression and are not applicable to HLA-A2-negative patients.",
"One solution might be to return to the use of multiple epitope constructs or full-length antigenic proteins.",
"Under this latter scenario, it is desirable that the DC population to maintain their antigen-processing functions.",
"In this case it may be more effective to load antigen on DCs grown in IL-7 or maturation-induced with IFN γ for adoptive transfer.",
"DC populations are being tested functionally to determine whether the hypotheses generated from these gene expression studies are valid.",
"The use of polystyrene beads in closed containers has a number of advantages over the use of open flasks for reproducibly generating dendritic cells, including sterility, risks of exposure for workers, higher yield, etc.",
"Because of these factors, a closed system, e.g., flexible gas permeable plastic tissue culture bags, is preferred over the open flasks.",
"The bags alone, however, do not provide an ideal surface for the attachment of DC precursor cells (monocytes).",
"The introduction of selected polystyrene beads into the bags provides a surface that the monocytes easily adhere to.",
"Once the monocytes have matured into DC, their adherence to the polystyrene surface, provided by the beads, is significantly reduced.",
"At the end of the culture period, DCs no longer adhere to the beads and are harvested in the supernatant.",
"The beads are selected based, in part, on their size.",
"Since more surface area is desirable, smaller beads in a larger quantity is preferred to larger beads in a smaller quantity.",
"Also, the specific gravity of the beads which allows them to settle after a period also contributes to their utility in the above-described methods.",
"Since the monocytes adhered to the beads settle with the beads and thereby separate from the undesired cells (e.g., lymphocytes, platelets, etc.)",
"which are removed by expressing off the supernatant.",
"Quality control is applied in the methods described above to comply with good manufacturing practices criteria.",
"While the application has been described with reference to specific embodiments, it should be understood that the description is not meant to be construed in a limiting sense, and the application is not limited to the precise embodiments described herein.",
"For example, while the closed system in the embodiments described above use a cell culture bag, other cell culture vessels may be used for the closed container.",
"The container need not have any particular shape.",
"It is preferable, but not essential, to have more than one port on the container to facilitate the transfer of materials in and out of the container.",
"It is important, however, that the container is gas permeable to, for example, O2 and CO2.Also, it is important to maintain a ratio of (beads and container) surface area to container volume that allows the container to hold enough media to support the culture period, so that culture only needs to be fed once, rather than repeatedly.",
"Improvements and modifications which become apparent to persons of ordinary skill in the art after reading this disclosure, the drawings and the appended claims are deemed within the spirit and scope of the present application.",
"It is therefore contemplated that the appended claims would cover any such modifications or improvements.",
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]
] | Patent_10433387 |
[
[
"Spinal intervertebral implant adjustable in situ",
"The invention concerns a spinal intervertebral implant (100) comprising at least a first element (101′) having a first end (112′), and a second element (101) having a second end (112), each end having successive ramps (108, 116), the ramps of the two ends being adapted to co-operate mutually to vary one dimension of the implant depending on the relative position of the elements.",
"The invention is characterised in that the ramps of each end are arranged along a circle."
],
[
"1.A spinal intervertebral implant (100; 200; 300) comprising at least a first element (101′; 201; 301) having a first end (112; 214; 314), and a second element (101; 203; 307) having a second end (112; 230; 370), each end having successive ramps (108, 116; 308, 316), the ramps of the two ends being able to cooperate mutually in order to vary one dimension of the implant depending on the relative position of the elements, characterized in that the ramps of each end are arranged along a circle.",
"2.The implant as claimed in claim 1, characterized in that some of the successive ramps are offset with respect to one another in the same sense in the direction of the dimension which is able to be varied.",
"3.The implant as claimed in claim 1 or 2, characterized in that the successive ramps of one end form groups (110, 118; 310, 318) of adjacent ramps comprising an identical number of ramps.",
"4.The implant as claimed in claim 3, characterized in that the groups are identical to one another.",
"5.The implant as claimed in claim 3 or 4, characterized in that the groups are uniformly distributed along the circle.",
"6.The implant as claimed in one of claims 3 through 5, characterized in that the circle comprises at least two groups of ramps.",
"7.The implant as claimed in one of the preceding claims, characterized in that the ends complement one another.",
"8.The implant as claimed in one of the preceding claims, characterized in that each element comprises lateral orifices (106; 206).",
"9.The implant as claimed in one of the preceding claims, characterized in that it comprises a central orifice extending along the dimension which is able to be varied.",
"10.The implant as claimed in one of the preceding claims, characterized in that it comprises stabilizing means (204) which are able to hold the elements relative to one another with respect to a direction of relative movement.",
"11.The implant as claimed in claim 10, characterized in that the stabilizing means comprise a member (204) which can be received in the central orifice.",
"12.The implant as claimed in claim 10, characterized in that the stabilizing means comprise at least one supporting element integral with at least one of the ends.",
"13.The implant as claimed in one of the preceding claims, characterized in that, with one of the two elements (101′; 201; 301), preferably the first one; having a third end (114; 212; 312) with ramps (116; 308), the implant comprises at least a third element (101″; 202; 306) having a fourth end (114; 220; 360) with ramps able to cooperate with the ramps of the third end in order to vary the dimension of the implant depending on the relative position of the first and third elements.",
"14.The implant as claimed in claim 13, characterized in that the orientation of the ramps of the first end is mirror-symmetrical to that of the ramps of the third end, in a plane perpendicular to the direction of the dimension which is able to be varied.",
"15.The implant as claimed in one of the preceding claims, characterized in that it comprises terminal ends (224, 234) having teeth (222, 232) profiled and parallel to one another.",
"16.The implant as claimed in one of claims 1 through 14, characterized in that it comprises terminal ends (322, 324) having a face (322, 324) and points (320) protruding from the face."
],
[
"The invention concerns implants of the intervertebral cage type, or of the type for replacement of vertebral bodies, intended for the spinal column.",
"The document U.S. Pat.",
"No.",
"5,865,848 discloses an intervertebral cage intended to replace a damaged intervertebral disk and comprising two parts which complement one another.",
"These two complementary parts are able to move relative to one another in the direction of their greatest length.",
"Height adjustment is made possible by the presence of contact surfaces, between the two parts of the cage, comprising ramps toothed in the direction of movement.",
"The disadvantage of such a configuration is that it has a limit stop upon adjustment, necessitating a reverse movement in the event of an error.",
"This reverse movement is awkward and difficult during the surgical intervention and risks causing instability between the vertebrae which are being operated on, said instability being prejudicial to achieving fusion between these operated vertebrae.",
"It is an object of the invention to permit height adjustment without reverse movement being necessary.",
"For this purpose, the invention provides a spinal intervertebral implant comprising at least a first element having a first end, and a second element having a second end, each end having successive ramps, the ramps of the two ends being able to cooperate mutually in order to vary one dimension of the implant depending on the relative position of the elements, and the ramps of each end being arranged along a circle.",
"Thus, the height of the implant is adjusted by rotating one of the elements on an axis passing through the center of the circle of ramps.",
"The adjustment is continuous because it has no limit stop: it is possible to return to the initial position in the same sense of rotation, even in the event of an error.",
"Advantageously, some of the successive ramps are offset with respect to one another in the same sense in the direction of the dimension which is able to be varied.",
"Advantageously, the successive ramps of one end form groups of adjacent ramps comprising an identical number of ramps.",
"Thus, upon height adjustment, a constant minimum number of supports is ensured.",
"Advantageously, the groups are identical to one another.",
"Advantageously, the groups are uniformly distributed along the circle.",
"Advantageously, the circle comprises at least two groups of ramps.",
"Advantageously, the ends complement one another.",
"Advantageously, the implant comprises lateral orifices.",
"Advantageously, the implant comprises a central orifice extending along the dimension which is able to be varied.",
"Advantageously, the central orifice is able to receive substance promoting bone growth.",
"Advantageously, the implant comprises stabilizing means which are able to hold the elements relative to one another with respect to a direction of relative movement.",
"Thus, the stability of the implant is optimal.",
"Advantageously, the stabilizing means comprise a member which can be received in the central orifice.",
"Advantageously, the stabilizing means comprise at least one supporting element integral with at least one of the ends.",
"Advantageously, with one of the two elements, preferably the first one, having a third end with ramps, the implant comprises at least a third element having a fourth end with ramps able to cooperate with the ramps of the third end in order to vary the dimension of the implant depending on the relative position of the first and third elements.",
"Advantageously, the orientation of the ramps of the first end is mirror-symmetrical to that of the ramps of the third end, in a plane perpendicular to the direction of the dimension which is able to be varied.",
"Thus, height adjustment requires only the movement of the intermediate element situated between the two others which thus remain immobile relative to the vertebrae operated on.",
"This ensures better anchoring of the implant in the vertebrae.",
"Advantageously, the implant comprises terminal ends having teeth which are profiled and parallel to one another.",
"Advantageously, the implant comprises terminal ends having a face and points protruding from the face.",
"Provision is also made, according to the invention, for a surgical method which comprises the steps of fitting the implant at the implantation site and adjusting a dimension of the implant in situ by modifying a relative position of at least one of the elements of the implant.",
"Advantageously, the surgical method additionally comprises a step of filling the implant with a substance promoting bone growth.",
"Other characteristics and advantages of the invention will become evident from the following description of three preferred embodiments of the invention which are given as nonlimiting examples.",
"In the attached drawings: FIG.",
"1 is a view, in three dimensions, of a basic element in a first embodiment of the invention; FIG.",
"2 is a view, in three dimensions, of the stacking of a certain number of basic elements from FIG.",
"1 in order to form an implant according to the first embodiment of the invention; FIG.",
"3 is a view, in three dimensions, of a second embodiment of the invention in the position of minimum height; FIG.",
"4 is a view, in three dimensions, of the second embodiment in the position of maximum height; FIG.",
"5 is a view, in three dimensions, of a third embodiment of the invention; and FIG.",
"6 is a view, in three dimensions, of the intermediate member of the third embodiment from FIG.",
"5.The first embodiment of the invention will be described with reference to FIGS.",
"1 and 2.This first embodiment comprises a basic element 101 which, when stacked together with other identical basic elements 101, will constitute an implant 100 of the intervertebral cage type which is adjustable in situ.",
"The basic element 101 is in the form of a ring with an outer face 102 and an inner face 104, both of them cylindrical.",
"The inner face 104 delimits a central orifice.",
"The basic element 101 additionally comprises a first contact surface or end 112 and a second contact surface or end 114, the two surfaces 112 and 114 being mirror-symmetrical in a median transverse plane perpendicular to an axis 1 of revolution of the ring forming the basic element 101.Each of the contact surfaces 112 and 114 comprises a plurality of ramps 108 and 116, respectively, identical to each other.",
"The ramps 108 are organized in groups 110 of adjacent ramps, here numbering four per group 110.All the groups 110 are identical to one another and are uniformly distributed along the upper surface 112.The ramps have a top point 119 and a bottom point 120, these two points forming the ends of each of the ramps 108.The ramps 108 within a group 110 are arranged in such a way that the top point 119 of one ramp is situated above the top point of the preceding ramp, but below that of the following ramp, the succession of ramps within a group 110 being in the opposite direction to the hands of a clock.",
"The top point 119 of one ramp is connected to the bottom point 120 of the following ramp by a vertical wall 121.The bottom point 120 of the first ramp of the group 110 forms the bottom point 122 of the group 110, while the top point 120 of the last ramp of the group 110 forms the top point 124 of the group 110.The top point 124 of one group 110 is connected to the bottom point 122 of the following group by a vertical wall 123.Similarly, but in a manner which is mirror-symmetrical in relation to a transverse plane perpendicular to the axis 1 of the ring 1, the ramps 116 are organized in groups 118 of adjacent ramps, these groups being uniformly distributed along the lower surface 114.The ramps 116 have a top point 132 and a bottom point 130 and are connected to one another by a vertical wall 131 within a group.",
"Moreover, the bottom point of one group 118 is connected to the top point 126 of the following group by a vertical wall 133.In an optimal manner, each group 118 on the lower face is positioned substantially in line with a group 110 on the upper face: thus, the top point 124 is vertically in line with the bottom point 128, thereby defining the maximum height of the basic element 101; likewise, the bottom point 122 is situated vertically in line with the top point 126, thereby defining the minimum height of the basic element 101.The arrangement of the various groups 110 along the surface 112 and that of the various groups 118 along the surface 114 are such that the ramps 108 and 116 respectively describe a circular trajectory whose axis coincides with that 1 of the ring forming the basic element 101.Moreover, the outer face 102 comprises a plurality of through-orifices 106: these orifices open out, on the inner face 104, into the central orifice delimited by this face 104.Having described the basic element 101, we will now discuss its use.",
"Once the surgeon has accessed the implantation site and then prepared said implantation site, he forms an implant 100 by stacking together a plurality of basic elements 101.Here, by way of illustration, there are five basic elements 101, 101′, 101″, 101′″, 101″″.",
"Two elements are respectively stacked on one another by turning the second element around relative to the first element in order to bring the contact surface 112, 114 of the first basic element into contact with its counterpart 112, 114, respectively, of the second basic element.",
"Thus, the basic element 101′ is turned round so that its upper surface 112 comes into contact with the upper surface 112 of the basic element 101.In this way, the two surfaces complement one another.",
"Then, the basic element 101″ is stacked on the basic element 101′ in such a way that the lower surface 114 of the basic element 101″ comes into contact with the lower surface 114 of the basic element 101′.",
"This procedure is continued to stack the subsequent basic elements.",
"Once the implant 100 has been formed, the surgeon fills the central orifice delimited by the inner face 104 of each of the basic elements 101 with an osteoinductive or osteoconductive substance such as a bone graft (allograft or autograft), hydroxyapatite, or tricalcium phosphate (TCP), etc.",
"The surgeon then places the implant 100 in the implantation site.",
"He impacts the ramps of the lower face of the basic element 101 into the upper plateau of the lower vertebra of the site.",
"Likewise, he impacts the ramps of the upper face of the basic element 101″″ into the lower plateau of the upper vertebra of the site.",
"The ramps thus serve in both cases as anchoring means for the implant 100.The surgeon is then able to adjust the height of the implant 100 in situ by rotating one or more of the various intermediate basic elements 101′, 101″, 101′″.",
"He thus obtains the desired height between the two vertebrae, i.e.",
"the upper vertebra and lower vertebra, which delimit the implantation site.",
"The final step involves closing the access route.",
"A second embodiment of the invention will now be described with reference to FIGS.",
"3 and 4.The implant 200 of the intervertebral cage type according to this second embodiment comprises an intermediate element 201, an upper plate 202, a lower plate 203, and stabilizing means 204.The intermediate element 201 is in the form of a ring and is quite similar to the basic element 101 in the previous embodiment.",
"It comprises an upper surface 212 with uniformly distributed groups of ramps, describing a circular trajectory and similar to the groups 110 in the previous embodiment.",
"Likewise, it comprises a lower surface 214 with uniformly distributed groups of ramps, describing a circular trajectory and similar to the groups 118 in the previous embodiment.",
"Like the basic element 101, the intermediate element 201 comprises lateral orifices 206 passing through the thickness of the ring which forms said intermediate element and opening into the central orifice.",
"The upper plate 202 is in the form of a ring having the same internal and external diameters as those of the intermediate element 201.It comprises a lower surface 220 which is able to come into contact with the upper surface 212 of the intermediate element 201.This surface 220 is complementary to the surface 212.The plate 202 additionally comprises an upper face 224 with anchoring means 222 for anchoring to bone material such as vertebral plateaus.",
"These anchoring means 222 are in this case teeth which are of triangular profile with slopes at 45° with respect to the horizontal and perpendicular to one another.",
"The teeth are, furthermore, parallel to one another.",
"The lower plate 203 is the symmetrical counterpart of the upper plate 202, these being mirror-symmetrical in a transverse plane perpendicular to the axis 1 of the ring.",
"It thus comprises an upper surface 230 which is able to come into contact with the lower surface 214 of the intermediate element 201, with which surface it is complementary.",
"The plate 203 additionally comprises a lower face 234 having means 232 for anchoring to bone material which are identical to the anchoring means 222 of the upper plate 202.The stabilizing means 204 comprise a component of revolution whose external diameter is substantially equal to the internal diameter of the ring forming the plates 202, 203 and the intermediate element 201.Thus, the stabilizing means 204 are able to be received with sliding along the axis of revolution in the central orifice of the implant 200.The component of revolution is in this case a tube comprising two end faces 240, namely upper face and lower face, and an inner face 242 delimiting a through-hole 246.The wall of the tube is openworked with openings 244.Thus, the lateral orifices 206 can still communicate with the inside of the implant 200 as represented by the hole 246.The stabilizing means 204 have the role of preventing any sliding, in a substantially radial direction, of one of the plates 202, 203 relative to the intermediate element 201.The positioning of such an implant 200, in a surgical intervention, is similar to that described for the previous embodiment.",
"After accessing and preparing the implantation site, the surgeon forms an implant 200 which he positions at its minimum height as illustrated in FIG.",
"3: the surface 220 is totally in contact with the surface 212, and the surface 230 is totally in contact with the surface 214.The end faces 240 are therefore flush with the summits of the profiled teeth 222 and 234, respectively.",
"The implant is said to be in its lowered configuration.",
"The hole 246 is then filled with an osteoinductive or osteoconductive substance.",
"The implant is then placed in the implantation site.",
"The surgeon impacts the toothed faces of the plates 202, 203 into the vertebral plateaus delimiting the implantation site from above and below.",
"then he sets the desired height by maneuvering the intermediate element 201 in rotation.",
"It is of course possible to obtain greater heights by stacking several intermediate elements 201 between the two plates 202 and 203.This stacking is done identically to that of the basic elements 101 in the previous embodiment.",
"Referring to FIGS.",
"5 and 6, a third embodiment of the invention will now be discussed.",
"the implant 300 of the intervertebral cage type in this third embodiment is almost identical to the previous embodiment in that it has an intermediate element 301 and two plates, namely an upper plate 306 and a lower plate 307.The intermediate element 301 is in the form of a ring and has a lower surface 314 and an upper surface 312.The upper surface 312 comprises a plurality of groups 310 of ramps 308 uniformly distributed on the surface 312 and identical to one another and arranged in the same way as the groups 110 and 210 in the previous embodiments.",
"Likewise; the lower surface 314 comprises a plurality of groups 318 of ramps 316 uniformly distributed on the whole surface 314 and identical to one another and arranged in the same way as the groups 118 and 218 in the previous embodiments.",
"The intermediate element 301 additionally comprises an outer face 302 and an inner face 304 delimiting a continuous central orifice.",
"The width of the ring, that is to say the distance between the inner face 304 and outer face 302 forming the intermediate element, is greater than that of the rings forming the basic element 101 and intermediate element 201.Thus, the lower and upper surfaces are larger and make it possible to obtain a much more stable support, as will be seen below.",
"The upper plate 306 is in the form of a ring with internal and external diameters almost identical to those of the ring forming the intermediate element 301.The plate 306 has a lower surface 360 able to come into contact with the upper surface 312 of the intermediate element 301.The lower surface 360 of the plate 306 is complementary to the upper surface 312 of the intermediate element 301.Moreover, the upper plate 306 has an upper face 322 which is plane and perpendicular to the axis 1 of the ring forming the plate.",
"However, in an alternative embodiment, this face 322 can be inclined relative to a plane perpendicular to the axis of the ring.",
"The plate 306 additionally comprises anchoring means 320 which in this case are points protruding from the face 322 and of circular cross section.",
"The points 320 protrude perpendicularly to the face 322.The lower plate 307 is the mirror-symmetrical counterpart of the upper plate 306 with respect to a transverse plane perpendicular to the axis 1 of the ring.",
"It thus has an upper surface 370 symmetrical to the surface 360 and able to come into contact with the lower surface 314 of the intermediate element 301 in a complementary fashion.",
"The lower plate 307 has a lower face 324 symmetrical to the face 322.The use and positioning of an implant 300, during a surgical intervention, is similar to the use and positioning of the implant 200 in the previous embodiment.",
"It should be noted that, upon impaction into the upper and lower vertebral plateaus delimiting the implantation site, it is the points 320 which are driven into the bone of said vertebral plateaus.",
"It is of course possible for numerous modifications to be made to the present invention without departing from the scope of the latter.",
"The faces comprising the anchoring means can be inclined with respect to a plane perpendicular to the main axis of the implant.",
"The stabilizing means can comprise a supporting element formed integrally on at least one of the contact surfaces of each pair and protruding in the direction of bearing on one of the faces, inner or outer, of the elements constituting the implant.",
"An implant can be provided which comprises three elements, i.e.",
"an intermediate element whose ends are able to come into contact with one of the ends of each of the other two elements.",
"It is possible to conceive of any cam system with cam follower other than those described above, without departing from the present invention."
]
] | Patent_10433531 |
[
[
"Seal for a loading dock bumper",
"At a loading dock for a truck, a compressible bumper seal extends across the front face of a dock bumper to help seal an air gap that may otherwise exist between the bumper face and the rear of the truck.",
"Without the seal, the gap may be created by the truck stopping just short of reaching the bumper or by the truck “bouncing off” the bumper before stopping.",
"If left unsealed, such a gap can create a draft into a building that has the loading dock."
],
[
"1.A bumper assembly for use at a loading dock for receiving an impact from a vehicle moving in a rearward direction, comprising: a bumper installed at the loading dock and including a front face that faces away from the rearward direction, wherein the bumper is compressible to at least partially absorb the impact; and a bumper seal extending across the front face of the bumper and being adapted to be engaged by the vehicle, wherein the bumper seal is more compressible than the bumper.",
"2.The bumper assembly of claim 1, wherein the bumper seal includes a foam core.",
"3.The bumper assembly of claim 2, further comprising a weather resistant cover overlaying the foam core.",
"4.The bumper assembly of claim 3, further comprising a slipcover overlaying the weather resistant cover.",
"5.The bumper assembly of claim 4, further comprising a protective insert disposed between the slipcover and the foam core.",
"6.The bumper assembly of claim 5, wherein the protective insert is sandwiched between the slipcover and the weather resistant cover.",
"7.The bumper assembly of claim 5, wherein the protective insert is a sheet of material that less pliable than the weather resistant cover.",
"8.The bumper assembly of claim 4, wherein the weather resistant cover is more pliable than the slipcover.",
"9.The bumper assembly of claim 1, wherein the bumper seal comprises a collapsible core that includes a flexible sheet of material.",
"10.The bumper assembly of claim 9, wherein the flexible sheet of material is in the shape of a tube.",
"11.The bumper assembly of claim 9, further comprising a slipcover overlaying the collapsible core.",
"12.The bumper assembly of claim 1, wherein the bumper seal includes point of attachment for attaching the bumper seal at the loading dock, wherein the point of attachment is at a position that is further rearward than the front face of the bumper.",
"13.The bumper assembly of claim 1, wherein the bumper seal is attached to the bumper.",
"14.A bumper seal for sealing a gap between a front face of a bumper and a rear surface of a vehicle, wherein the bumper is installed at a loading dock and the front face is adapted to receive an impact created by the vehicle, the bumper seal comprising: a rear section extending across the front face of the bumper and being adapted to engage the front face of the bumper; a front section connected to the rear section and adapted to be engaged by the rear surface of the vehicle; and a central section interposed between the front section and the rear section, wherein the central section is more compressible than the bumper.",
"15.The bumper seal of claim 14, wherein the central section includes a foam core.",
"16.The bumper seal of claim 14, further comprising a weather resistant cover overlaying at least one of the rear section, the front section, and the central section.",
"17.The bumper seal of claim 16, further comprising a slipcover overlaying the weather resistant cover.",
"18.The bumper assembly of claim 17, further comprising a protective insert disposed between the slipcover and the foam core.",
"19.The bumper assembly of claim 18, wherein the protective insert is sandwiched between the slipcover and the weather resistant cover.",
"20.The bumper assembly of claim 18, wherein the protective insert is a sheet of material that less pliable than the weather resistant cover.",
"21.The bumper seal of claim 17, wherein the weather resistant cover is more pliable than the slipcover.",
"22.The bumper seal of claim 14, wherein the bumper seal comprises a collapsible core that includes a flexible sheet of material.",
"23.The bumper seal of claim 22, wherein the flexible sheet of material is in the shape of a tube.",
"24.The bumper seal of claim 22, further comprising a slipcover overlaying the collapsible core.",
"25.The bumper seal of claim 14, wherein the bumper seal includes point of attachment for attaching the bumper seal at the loading dock, wherein the loading dock has a dock face that is closer to the point of attachment than the front face of the bumper.",
"26.A method of at least partially sealing a gap between a front face of a compressible bumper and a rear surface of a vehicle, comprising: installing a bumper seal so that the bumper seal overlays the front face of the compressible bumper; and compressing the bumper seal between the front face of the compressible bumper and the rear surface of the vehicle.",
"27.The method of claim 26, compressing the bumper, but to a lesser extent than that which the bumper seal is compressed.",
"28.The method of claim 26, further comprising attaching the bumper seal to the compressible bumper.",
"29.A sealing assembly for use at a doorway of a loading dock for a vehicle, the sealing assembly comprising: a side seal disposed along a lateral edge of the doorway; a bumper having a front face for receiving an impact of the vehicle, wherein the bumper is spaced apart from the side seal to define a gap therebetween; and a side-sealing member bridging the gap by engaging the bumper and the side seal.",
"30.The sealing assembly of claim 29, wherein the gap is a vertical gap.",
"31.The sealing assembly of claim 29, wherein the gap is a horizontal gap.",
"32.The sealing assembly of claim 29, further comprising a face-sealing member extending from the side-sealing member and overlaying the front face of the bumper, wherein the face-sealing member is more compressible than the bumper.",
"33.The sealing assembly of claim 29, wherein the side seal includes a draft pad near the bottom of the side seal."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The subject invention generally pertains to loading docks, and more specifically to a device that helps seal an air gap that may exist between a rear surface of a vehicle and the front face of a bumper that is attached to the dock.",
"2.Description of Related Art A typical loading dock of a building includes an exterior doorway with an elevated platform for loading and unloading vehicles, such as trucks and trailers.",
"To compensate for height differences between the loading dock platform and an adjacent bed of a truck or trailer, many loading docks have a dock leveler.",
"A typical dock leveler includes a deck, also known as a ramp or dockboard, which is pivotally hinged along its back edge to vary the height of its front edge.",
"An extension plate, or lip, extends outward from the deck's front edge to span the gap between the rear of the truck bed and the front edge of the deck.",
"The lip is usually moveable between a stored position to an extended, operative position.",
"In the extended, operative position, the lip extends from the deck's front edge and rests upon the truck bed to form a bridge between the two.",
"This allows personnel and material handling equipment to readily move on and off the vehicle during loading and unloading operations.",
"To protect the building and the dock leveler from direct vehicle impact, loading docks often include bumpers.",
"Bumpers also help establish a predetermined minimum distance between the rear of the vehicle and the dock leveler, so the dock leveler can first raise and then lower its lip upon the rear of the vehicle.",
"Bumpers are usually installed near the bottom of the doorway, adjacent either side of the dock leveler lip and protrude a few inches out from the face of the dock, where they can be abutted by the rear of the vehicle.",
"To help block out rain, snow and outside air from entering the building through air gaps between the back of the vehicle and the exterior face of the building, usually either a dock seal or a dock shelter is installed around the perimeter of the doorway.",
"Dock shelters often have projecting members that protrude outwardly from the face of the building and run generally along the top and lateral sides of the doorway.",
"From a protruding edge of the projecting members, a top extending member and two laterally extending members may extend inward and generally parallel to the building face to help seal against the truck's top and sides, respectively.",
"The laterally extending members are often made of a fabric or flexible foam.",
"Two inner bottom corners of the dock shelter at the lower back sides of the truck are often partially sealed by draft pads attached to the lower ends of the projecting members.",
"The dock leveler lip resting upon the rear of the vehicle is often relied upon to seal most of the doorway's lower edge.",
"Typical dock seals comprise a resiliently compressible foam core protected by a fabric outer cover.",
"They are usually mounted to the face of a building, along the top and both sides of the doorway.",
"With dock seals, sealing is provided by backing the truck directly up against the seal.",
"The seal then compressively conforms to the shape of the truck's rear edges.",
"The foam core provides the necessary compliance and resilience to repeatedly conform to the shape of various trucks, while the outer cover protects the foam core from dirt, water and abrasion.",
"As with dock shelters, dock seals also rely on the dock leveler lip to seal most of the doorway's lower edge.",
"With conventional dock seals and shelters, an air gap may still exist between the rear of the vehicle and the front face of the bumpers.",
"This can occur when a vehicle backing into the dock “bounces off” the bumpers or simply stops just short of reaching the bumpers.",
"Such an air gap can be a few inches across and can allow a significant air draft into the building.",
"A gap can also exist between a bumper and the underside of a dock shelter or the underside of a dock seal.",
"For dock seals, such a gap may be due horizontal or vertical displacement between the bumper and the portion of the dock seal that runs along the vertical edge of the doorway.",
"For dock shelters, the gap can be due to horizontal or vertical displacement between the bumper and dock shelter's draft pad."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In some embodiments, a relatively soft bumper seal overlays the front face of loading dock bumper.",
"In some embodiments, the bumper seal includes a foam core that is softer or more compressible than the bumper.",
"In some embodiments, the foam core is at least partially covered by a pliable, weather resistant cover.",
"In some embodiments, the weather resistant cover is protected by a tough slipcover that can be replaced when necessary.",
"In some embodiments, the slipcover is stiffer than the weather resistant cover.",
"In some embodiments, a structure of flexible sheets provides a collapsible bumper seal that overlays the front face of a loading dock bumper.",
"In some embodiments, the flexible sheets comprise a plurality of tubes.",
"In some embodiments, the bumper seal is mounted adjacent to the bumper.",
"In some embodiments, the bumper seal is attached to a vertical face of a loading dock.",
"In some embodiments, the bumper seal is attached directly to the bumper.",
"In some embodiments, the bumper seal comprises a face-sealing member and a side-sealing member, wherein the face-sealing member seals a gap between the bumper and the rear of a vehicle, and the side-sealing member seals a gap between the bumper and a lower portion of a dock seal or a dock shelter's draft pad.",
"In some embodiments of bumper seal that include a compressible or collapsible core with a weather resistant cover and a semi-rigid slipcover over that, a protective semi-rigid sheet of material is inserted between the slipcover and the core to help evenly distribute vehicle-imparted forces across the core."
],
[
"BACKGROUND OF THE INVENTION 1.Field of the Invention The subject invention generally pertains to loading docks, and more specifically to a device that helps seal an air gap that may exist between a rear surface of a vehicle and the front face of a bumper that is attached to the dock.",
"2.Description of Related Art A typical loading dock of a building includes an exterior doorway with an elevated platform for loading and unloading vehicles, such as trucks and trailers.",
"To compensate for height differences between the loading dock platform and an adjacent bed of a truck or trailer, many loading docks have a dock leveler.",
"A typical dock leveler includes a deck, also known as a ramp or dockboard, which is pivotally hinged along its back edge to vary the height of its front edge.",
"An extension plate, or lip, extends outward from the deck's front edge to span the gap between the rear of the truck bed and the front edge of the deck.",
"The lip is usually moveable between a stored position to an extended, operative position.",
"In the extended, operative position, the lip extends from the deck's front edge and rests upon the truck bed to form a bridge between the two.",
"This allows personnel and material handling equipment to readily move on and off the vehicle during loading and unloading operations.",
"To protect the building and the dock leveler from direct vehicle impact, loading docks often include bumpers.",
"Bumpers also help establish a predetermined minimum distance between the rear of the vehicle and the dock leveler, so the dock leveler can first raise and then lower its lip upon the rear of the vehicle.",
"Bumpers are usually installed near the bottom of the doorway, adjacent either side of the dock leveler lip and protrude a few inches out from the face of the dock, where they can be abutted by the rear of the vehicle.",
"To help block out rain, snow and outside air from entering the building through air gaps between the back of the vehicle and the exterior face of the building, usually either a dock seal or a dock shelter is installed around the perimeter of the doorway.",
"Dock shelters often have projecting members that protrude outwardly from the face of the building and run generally along the top and lateral sides of the doorway.",
"From a protruding edge of the projecting members, a top extending member and two laterally extending members may extend inward and generally parallel to the building face to help seal against the truck's top and sides, respectively.",
"The laterally extending members are often made of a fabric or flexible foam.",
"Two inner bottom corners of the dock shelter at the lower back sides of the truck are often partially sealed by draft pads attached to the lower ends of the projecting members.",
"The dock leveler lip resting upon the rear of the vehicle is often relied upon to seal most of the doorway's lower edge.",
"Typical dock seals comprise a resiliently compressible foam core protected by a fabric outer cover.",
"They are usually mounted to the face of a building, along the top and both sides of the doorway.",
"With dock seals, sealing is provided by backing the truck directly up against the seal.",
"The seal then compressively conforms to the shape of the truck's rear edges.",
"The foam core provides the necessary compliance and resilience to repeatedly conform to the shape of various trucks, while the outer cover protects the foam core from dirt, water and abrasion.",
"As with dock shelters, dock seals also rely on the dock leveler lip to seal most of the doorway's lower edge.",
"With conventional dock seals and shelters, an air gap may still exist between the rear of the vehicle and the front face of the bumpers.",
"This can occur when a vehicle backing into the dock “bounces off” the bumpers or simply stops just short of reaching the bumpers.",
"Such an air gap can be a few inches across and can allow a significant air draft into the building.",
"A gap can also exist between a bumper and the underside of a dock shelter or the underside of a dock seal.",
"For dock seals, such a gap may be due horizontal or vertical displacement between the bumper and the portion of the dock seal that runs along the vertical edge of the doorway.",
"For dock shelters, the gap can be due to horizontal or vertical displacement between the bumper and dock shelter's draft pad.",
"SUMMARY OF THE INVENTION In some embodiments, a relatively soft bumper seal overlays the front face of loading dock bumper.",
"In some embodiments, the bumper seal includes a foam core that is softer or more compressible than the bumper.",
"In some embodiments, the foam core is at least partially covered by a pliable, weather resistant cover.",
"In some embodiments, the weather resistant cover is protected by a tough slipcover that can be replaced when necessary.",
"In some embodiments, the slipcover is stiffer than the weather resistant cover.",
"In some embodiments, a structure of flexible sheets provides a collapsible bumper seal that overlays the front face of a loading dock bumper.",
"In some embodiments, the flexible sheets comprise a plurality of tubes.",
"In some embodiments, the bumper seal is mounted adjacent to the bumper.",
"In some embodiments, the bumper seal is attached to a vertical face of a loading dock.",
"In some embodiments, the bumper seal is attached directly to the bumper.",
"In some embodiments, the bumper seal comprises a face-sealing member and a side-sealing member, wherein the face-sealing member seals a gap between the bumper and the rear of a vehicle, and the side-sealing member seals a gap between the bumper and a lower portion of a dock seal or a dock shelter's draft pad.",
"In some embodiments of bumper seal that include a compressible or collapsible core with a weather resistant cover and a semi-rigid slipcover over that, a protective semi-rigid sheet of material is inserted between the slipcover and the core to help evenly distribute vehicle-imparted forces across the core.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a perspective view of a loading dock that includes a bumper seal.",
"FIG.",
"2 is similar to FIG.",
"1, but showing a truck having backed into the dock.",
"FIG.",
"3 is similar to FIG.",
"1, but showing a bumper seal being installed over the face of a bumper, wherein the seal's slipcover is partially pealed back to show a mounting bracket.",
"FIG.",
"4 is a cross-sectional view taken along line 4-4 of FIG.",
"1.FIG.",
"5 is similar to FIG.",
"4, but with the vehicle pressing hard against the bumper.",
"FIG.",
"6 is similar to FIG.",
"5, but with the vehicle having moved slightly away from the bumper.",
"FIG.",
"7 is a perspective view of a bumper seal's core.",
"FIG.",
"8 is a perspective view of the core of FIG.",
"7 covered by a weather resistant cover.",
"FIG.",
"9 is a perspective view of the core and cover of FIG.",
"8 with the covered core being further covered by a tough slipcover.",
"FIG.",
"10 is a perspective view of an uncovered bumper seal whose core is collapsible.",
"FIG.",
"11 is similar to FIG.",
"10, but with the bumper seal protected by a slipcover.",
"FIG.",
"12 is a perspective view of another bumper seal with a collapsible core.",
"FIG.",
"13 is a schematic top view of one embodiment of a bumper seal that includes a face-sealing member and a side-sealing member.",
"FIG.",
"14 is similar to FIG.",
"8, but of another embodiment.",
"FIG.",
"15 is similar to FIG.",
"9, but of another embodiment.",
"FIGS.",
"16, 17 and 18 correspond respectively to FIGS.",
"4, 5 and 6 and illustrate another embodiment where a bumper seal attaches directly to a bumper.",
"FIG.",
"19 is a front view of a bumper seal that includes a side-sealing member for sealing a vertical gap.",
"FIG.",
"20 is a front view of the bumper seal of FIG.",
"19, but showing the side-sealing member sealing a horizontal gap.",
"FIG.",
"21 is a similar to FIG.",
"19, but showing the bumper seal sealing a vertical gap between a bumper and a dock shelter.",
"FIG.",
"22 is similar to FIG.",
"20, but showing the bumper seal sealing a horizontal gap between a bumper and a dock shelter.",
"DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to FIGS.",
"1 and 2, a loading dock 10 comprises an elevated doorway 12 of a building 14.A vehicle 16, such a truck or trailer, can move in a rearward direction 18 into dock 10 to load or unload cargo through doorway 12.In this example, a conventional dock leveler 20 with an extendible lip 22 is used to bridge the gap between the lower edge of doorway 12 and a lower rear edge of vehicle 16.To protect building 14 and dock leveler 20 from direct vehicle impact, loading dock 10 is provided with one or more shock-absorbing bumpers 24 that protrude about four to six inches out from a face 26 of the dock.",
"Bumpers 24 also help establish a predetermined minimum distance between the vehicle's back edge 28 and the front of dock leveler 20, so dock leveler 20 can first lift and then lower lip 22 upon the bed of vehicle 16.Bumpers 24 can be installed near the bottom of doorway 12, adjacent either side of dock leveler lip 22, where the bumpers can be abutted by the rear of vehicle 16.Alternatively, bumpers 24 can be attached or coupled to dock leveler 20 and in some cases may even be movable between retracted and operative positions.",
"To help block out rain, snow and outside air from entering building 14 through air gaps between the exterior face of building 14 and the upper and two side edges along the rear of vehicle 16, a dock seal 30 may be installed along the perimeter of doorway 12.For example, dock seal 30 includes two vertical side pads 32 and a head pad 34, which compressively conform to the shape of a vehicle's rear edges as the vehicle backs up against pads 32 and 34.It should be appreciated, however, that the use of a dock shelter instead of a dock seal is also well within the scope of the invention.",
"Regardless of whether a dock shelter or dock seal is used, lip 22 extending out over the bed of truck 16 can be used to help cover the gap adjacent the rear lower edge of vehicle 16.In cases where a loading dock does not have a dock leveler, a lower pad may be installed along the lower edge of the doorway to help seal gaps in that area.",
"The subject invention does not have to be used in combination with a dock leveler.",
"Sometimes, vehicle 16 may stop without its rear edge 28 tightly up against a front face 34 of the bumpers.",
"For instance, vehicle 16 may “bounce off” the bumpers before actually stopping.",
"This can create an air gap between the vehicle's rear edge 28 and the front face of the bumpers.",
"To help seal such an air gap, a bumper seal 36 having a compressible or collapsible face-sealing member 38 can be installed in front of the bumpers, as shown in FIGS.",
"3 and 13.The actual method of mounting can vary, as face-sealing member 38 can be mounted directly to the bumper, or face-sealing member 38 may be associated with additional mounting structure.",
"For example, face-sealing member 38 itself can comprise an entire bumper seal as depicted in FIGS.",
"16-18, or face sealing member 38 may be part of the bumper seal 36 that also includes a side-sealing member 51 (see FIG.",
"13), wherein side-sealing member 51 helps in the mounting of face member 38, as depicted in FIGS.",
"7-9.A rear section 35 of seal 36 engages front face 34 of bumper 24, a front section 37 is adapted to be engaged by vehicle 16, and a central section 39 (which is more compressible than bumper 24) is between sections 35 and 37.Then, if vehicle 16 backs into dock 10 and bounces off bumpers 24, bumper seal 36 can respond in the sequence shown in FIGS.",
"4, 5 and 6.In FIG.",
"4, the face-sealing member 38 of bumper seal 36 is in a normally expanded state as vehicle 16 backs toward bumper 24.In FIG.",
"5, vehicle 16 tightly compresses face-sealing member 38 of seal 36 between the vehicle's rear edge 28 and the bumper's front face 34.The impact of vehicle 16 is transmitted to bumper 24, which may compress bumper 24 a certain amount as the bumper absorbs at least some of the impact.",
"However, face-sealing member 38 is softer or more compressible than bumper 24, so seal 36 is compressed to a much greater extent as vehicle 16 tightly compresses seal 36 between the vehicle's rear edge 28 and the bumper's front face 34.If vehicle 16 bounces away from bumper 24, as shown in FIG.",
"6, seal 36 may expand to fill the gap between bumper 24 and vehicle 16.To mount face-sealing member 38, seal 36 can be constructed as shown in FIGS.",
"7, 8 and 9 to include a side-sealing member 51 that also mounts the face-sealing member adjacent bumper 24.FIG.",
"7 shows bumper seal 36 having a compressible core 40 adjacent a mounting plate, such as a wood backer 43.Backer 43 provides structure that enables a bracket 42 (FIG.",
"3) to attach seal 36 to the face of building 14 and to thus properly locate face-sealing member 38 over the front face of bumper 24.In some embodiments, core 40 comprises one or more compressible foam pads, such as pads 44, 46 and 48.Pad 44 helps seal between backer 43 and a bumper mounting bracket 50 (which connects bumper 24 to building 14, as shown in FIG.",
"4), pad 48 helps seal between the bumper's front face 34 and the vehicle's rear edge 28, and pad 46 helps ensure that the vehicle's impact is transmitted more to bumper 24 than to backer 43.To help protect core 40 from dirt and weather, or to help hold various parts of core 40 together, core 40 can be at least partially covered by a weather resistant cover 52, as shown in FIG.",
"8.The term, “weather resistant” refers to any material that provides at least some shield against wind or rain.",
"Cover 52 may consist of a pliable vinyl or some other type of fabric that is sufficiently pliable to allow core 40 to be compressed by vehicle 16.Cover 52 preferably includes some venting feature (e.g., a round hole at the bottom of bumper seal 36) that allows air to enter and escape from within cover 52 as core 40 is compressed or decompressed.",
"To help protect cover 52 from being cut, excessively worn or otherwise damaged by the impact of vehicle 16, a tough slipcover 54 extends over areas of cover 52 that are most susceptible to damage.",
"Slipcover 54 can be made of PVC or a similar material that is tougher and less pliable than cover 52.In some embodiments, slipcover 54 is made of a polyethylene, cross-linked, closed-cell foam that is formed, compressed and heated to provide a desired density and rigidity.",
"In some cases, the rigidity of slipcover 52 may vary, with certain areas of slipcover 52 being more rigid than others.",
"For example, front section 37 may be more rigid than other areas to help evenly distribute the compressive forces that vehicle 16 may exert against core 40.Preferably, slipcover 54 is readily replaceable by wrapping the slipcover around core 40 and cover 52, as shown in FIG.",
"9.A looped end 56 of slipcover 54 can be sewn, bonded, welded or otherwise connected to itself at a seam 58, and another end 60 of the slipcover can be stapled, screwed, hooked or otherwise attached to backer 43.End 60 can be clamped between bracket 42 and backer 43, or a part 62 of end 60 can overlay bracket 42 as shown in FIG.",
"4.Slipcover 54 can be replaced (or switched from a righthand bumper seal to a lefthand bumper seal) by detaching end 60 from backer 43 and slipping looped end 56 off core 40.Alternate, but similar, embodiments of a bumper seal are shown in FIGS.",
"10, 11 and 12.In FIGS.",
"10 and 11, a bumper seal 64 includes a collapsible core 66 having a collapsible structure made of a flexible sheet of material, such as PVC.",
"The actual structure can be of various shapes and configurations.",
"For example, the flexible sheets of material can be in the form of tubes 68 that resiliently flatten out upon being crushed between bumper 24 and the vehicle's rear edge 28.Accordingly, the volume of core 66 that extends inward (to the left in the sense of FIG.",
"10) from pads 44 and 46 forms a face-sealing member 38′ according to this embodiment.",
"In some cases, backer 43 and pads 44 and 46 can be protected by a cover 68 similar to cover 52, and slipcover 54 can help protect core 66.FIG.",
"12 shows a bumper seal 70 that includes a collapsible core 72 made of flexible sheets of material that form a series of foldable boxes.",
"The boxes resiliently fold into a flattened shape upon vehicle 16 backing up against bumper 24.Seal 70 may be provided with cover 68 and/or slipcover 54.Referring to FIGS.",
"14 and 15, to help evenly distribute the compressive forces that vehicle 16 may exert against core 40 (FIG.",
"7), a protective insert 80 can be installed in front of a bumper seal's core.",
"FIG.",
"14 is the same as FIG.",
"15, but with slipcover 54 removed.",
"In this example, a bumper seal 36′ includes insert 80 installed between slipcover 54 and core 40 (e.g., between slipcover 54 and cover 52).",
"Insert 80 can be made of any semi-rigid sheet of material, such as HDPE (high density polyethylene).",
"Insert 80 can be held in place by any suitable manner, such as by sewing, glueing, etc.",
"Just as FIGS.",
"4, 5 and 6 illustrate in sequence the operation of bumper seal 36, respective FIGS.",
"16, 17 and 18 illustrate the structure and an operational sequence of a bumper seal 82.Bumper seal 82 comprises a face-sealing member 84 that includes a compressible or collapsible core 98 with a pliable cover 100.Face-sealing member 84 attaches directly to a bumper 86 without the use of side-sealing member 51 as in the previous embodiments.",
"It should be appreciated by those skilled in the art that the actual structure of bumper 86 and bumper seal 82 could vary widely without departing from the basic concept of mounting a face-sealing member directly to a bumper.",
"Nonetheless, in this particular example, bumper 86 comprises a hard rubber compressible core 88 contained within a telescoping metal housing.",
"The housing includes one piece 90 that can move relative to another piece 92, so the two pieces 90 and 92 can compress core 88 under an impact of vehicle 16.Bumper seal 82 can be attached to bumper 86 in any suitable manner to properly position face-sealing member 84 relative to the front face of bumper 86.For example, seal 82 may include straps or material 94 that can attach to bumper 86 in any one of various ways including, but not limited to, hooking onto bumper 86, wrapping around bumper 86, being fastened with screws, adhesive, etc.",
"The various means of attachment are schematically represented by element 96.In another embodiment, shown in FIGS.",
"19 and 20, a bumper seal 102 comprises a side-sealing member 104 that mounts a face-sealing member 105 in proper position relative to bumper 24, and that also helps seal a gap between bumper 24 (shown in phantom behind seal 102) and vertical side pad 32 of dock seal 30, wherein side pad 32 is just one example of a side seal disposed along a lateral edge of a doorway.",
"Side-sealing member 104 is shown sealing a vertical gap 106 in FIG.",
"19 and shown sealing a horizontal gap 108 in FIG.",
"20.To achieve this capability, side-sealing member 104 has a greater vertical extent than face-sealing member 102.For example, one could modify the embodiment of FIG.",
"7 so that pads 44 and 46 were taller than pad 48.Also, pad 48 could have a section adjacent pad 46 of a greater height as well.",
"One would preferably make backer 43 taller as well.",
"This is depicted schematically in FIGS.",
"19 and 21.FIGS.",
"21 and 22 are similar to FIGS.",
"19 and 20 respectively.",
"However, instead of dock seal 30, the embodiment of FIGS.",
"21 and 22 have a dock shelter 30′ that includes a draft pad 110.Draft pad 110 helps seal a lower inner corner of dock shelter 30′.",
"Further details of sample dock shelters and draft pads are disclosed in U.S. Pat.",
"Nos.",
"6,014,844; 4,885,881; and 3,792,559, which are specifically incorporated by reference herein.",
"For dock shelter 30′, bumper seal 102 includes a face-sealing member 105 for sealing the front face of the bumper and a side-sealing member 104 for mounting member 105 and that helps seal a gap between bumper 24 and draft pad 110 of vertical side seal 32′, wherein vertical side seal 32′ is just one example of a side seal disposed along a lateral edge of a doorway.",
"Side-sealing member 104 is shown sealing a vertical gap 106′ in FIG.",
"21 and shown sealing a horizontal gap 108′ in FIG.",
"22.Although the invention is described with respect to a preferred embodiment, modifications thereto will be apparent to those skilled in the art.",
"Therefore, the scope of the invention is to be determined by reference to the claims, which follow."
]
] | Patent_10433636 |
[
[
"Method, computer program product and use of a computer program for stabilizing a multiphase flow",
"The present invention relates to a method for stabilizating multiphase flow through a pipeline, riser or well flow line (8a), characterized by implementation of a dynamic feedback controller (9) by controlling a control valve or choke (2) at the outlet (13) of the pipeline or riser (8a)."
],
[
"1.A method for stabilizing a multiphase flow in a flow line, where instability of the multiphase flow is caused by at least one slug, comprising measuring continuously one pressure variable upstream of the point where the main part of the slug is generated, supplying the pressure variable to a dynamic feedback controller, wherein the pressure variable upstream of the slug is an input to the dynamic feedback controller, calculating continuously an output of the dynamic feedback controller, and controlling a control valve at said flow line by means of the output from the dynamic feedback controller, wherein the multiphase flow is stabilized through out the flow line.",
"2.A method according to claim 1, characterized in that the dynamic feedback controller is a multivariable input, single output controller, acting upon periodic variations in measured variables.",
"3.A method according to claim 2, characterized in that one of the input variables of the dynamic feedback controller is a set-point for desired pressure at the same position in the flow line as the pressure variable.",
"4.A method according to claim 3, characterized in that the pressure variable is positioned at the flow line inlet.",
"5.A method according to claim 4, characterized in that the continuous calculation of output of the dynamic feedback controller is aiming at controlling the control valve in such way that the variation around the set point for desired pressure at the flow line inlet is minimized.",
"6.A method according to claim 5, characterized in that the pressure variable is either measured by a pressure measurement means or calculated from a temperature measurement.",
"7.A method according to claim 6, characterized in that the control valve is positioned at the outlet of the flow line.",
"8.A method according to claim 7 characterized in that said pressure variable is calculated based on a first measurement made at the flow line inlet and a second measurement made upstream or downstream of the control valve.",
"9.A method according to claim 8, characterized in that additional measured variables, used as input variables to the dynamic feedback controller, comprises at least one variable situated at the outlet upstream of the control valve.",
"10.A method according to claim 9, characterized in that the variable situated at the outlet is a pressure variable.",
"11.A method according to claim 10, characterized in that stabilization of the multiphase flow is enhanced by including additional measurements of flow, pressure, and temperature, or any combination thereof.",
"12.A method according to claim 11, characterized in that the dynamic feedback controller comprises a feedback stabilization calculation 14 which is described by an equation as Δu2,k=K(Δef,k+Ts/T1ef,k+T2/TΔΔef,k) where Ts is the sampling time, K1 T1 and T2 are tuning parameters and the operator Δ means Δmk=m−mk−1 and ef,k is a filtered control error.",
"13.A method according to claim 12, characterized in that the dynamic feedback controller has a built-in anti-windup prevention logic.",
"14.A method according to claim 13, characterized in that the dynamic feedback controller comprises a slug choking controller which is described by an equation as K ( s ) = k s ( τ 1 s + 1 ) ( τ 2 s + 1 ) , u 3 ( s ) = K ( s ) e 2 ( s ) where K(s) is the Laplace transform of the slug chocking controller, U3 is the controller output, e2 is the filtered nominal value for the pressure variable upstream the control valve minus the pressure variable upstream the control valve, the frequencies f1=1/τ1; and f2=1/τ2 are tuning parameters, and k is the controller gain.",
"15.A method according to claim 1, characterized in that along with the dynamic feedback controller, a calculation for slug detection is applied.",
"16.A method according to claim 15, characterized in that the slug detection calculation utilizes a pressure measurement downstream of the control valve.",
"17.A computer program product containing software code means loadable into the internal memory of a computer or a process controller in a computerized control system, characterized in that said computer program product has means to make said computer or process controller carry out the steps of a method according to claim 1.18.A computer program product containing software code means loadable into the internal memory of a computer or a process controller in a computerized control system, characterized in that said computer program product has means to make said computer or process controller carry out the steps of a method according to claim 2.19.Use of a computer program product according to claim 17 to stabilize a multiphase flow at a production facility for oil and gas."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Oil that is produced offshore is transported through pipelines as a complex mixture of oil, gas water and sand.",
"One common flow regime is known as slug flow, in which the mixture flows intermittently along the pipelines and comprises a concentrated mass in the form of a liquid plug.",
"Such a concentrated mass in movement is hereafter called a slug.",
"In multiphase pipelines/risers/well flow lines at reduced flow rates and/or changing gas oil ratio, compared to design specifications, instabilities in terms of terrain-induced and riser-induced slug flow often occur.",
"Such a slug flow is a bulk of liquid moving in the pipeline followed by an amount of gas.",
"Terrain and riser-induced slug flow is often referred to as severe slugging.",
"A publication by Yehuda Taitel, “Stability of Severe Slugging”, Int.",
"Journal of Multiphase Flow, Vol.",
"12, No.",
"2, pp.",
"203-217, 1986, describes the phenomena of slugging.",
"Terrain and riser-induced slug flow is induced periodically as liquid in terms of oil and water is accumulated in lower parts of the pipeline/riser, see FIG.",
"11 -IV.",
"At a certain time the liquid will restrict the passage for the gas.",
"In this situation a small amount of gas bubbles through the liquid plug, however the main part of the gas accumulates upstream of the liquid plug which causes the pressure to increase (see FIG.",
"11 -I).",
"In this situation the pressure upstream of the liquid plug is equal to the pressure downstream of the liquid plug plus the hydraulic pressure across the liquid plug (applying a static force balance).",
"But when the pressure increase upstream of the liquid plug becomes larger than the pressure increase downstream the liquid plug, the liquid plug starts moving (see FIG.",
"11 -II), and then forms a slug, which accelerates.",
"It should be noted that this condition might be fulfilled before the front of the liquid plug reaches the downstream maximum point in the pipeline profile.",
"Depending on operating conditions and pipeline profile, the slug may die out or it may be transported to the outlet of the pipeline/riser.",
"In a situation where the tail of the liquid plug enters the vertical parts of the flow line ( FIG.",
"11 -III), a rapid increase in the liquid flow rate occurs due to the unstable situation where the pressure head, due to the liquid column, decreases.",
"A slug is formed and the slug get transported to the outlet of the pipeline, and when the gas behind the slug escapes the pipeline/riser, the remaining liquid in the vertical parts returns to the bottom of the riser or dips in the pipeline profile.",
"Then the whole process is repeated, and the result is an unstable multiphase flow pattern/cycle where the liquid flow rate varies from zero to a significant value, as the slug passes a fixed point in the pipeline, in a short period of time.",
"The flow pattern is characteristic of severe slugging (terrain/riser-induced slugging).",
"For terrain-induced slug flow the corresponding liquid plugs are caused by terrain effects reflected in the pipeline profile (offshore and onshore), whereas riser induced slug flow is caused by the pipeline leaving the seabed on its way to the surface (offshore).",
"For long risers, special dynamic effects might occur due to phase transition from liquid to gas due to a considerable pressure decrease in the riser.",
"Different riser shapes may also affect the dynamics in riser-induced slugging.",
"Unstable flow causes considerable problems for production in the upstream wells and operation of the downstream processing plant: Large disturbances to the separator train, causing: Limiting separation capacity due to the need for larger operating margin to achieve the desired separation.",
"Poor separation (water carry over to export pipeline) due to rapidly varying separator feed rates.",
"Poor separation results in varying quality of water outlet from separators, causing large problems in the downstream water treatment system and possible violation of environmental restrictions.",
"Large and rapidly varying compressor loads, causing: Inefficient compressor operation.",
"Limiting compression capacity due to the need for a larger margin to handle gas holdup behind the liquid.",
"Spurious flaring from limited compression capacity.",
"Limited production from the upstream wells.",
"The pressure variations at the pipeline or riser inlet 1 are also visible in the upstream wells, resulting in limited production from wells suffering from reduced lifting capacity.",
"For gas lifted oil wells a problem referred to as casing heading might occur.",
"Applications of gas lifted oil wells are different than slug flow in pipelines, risers and wells in the following sense: The dynamic interactions in the casing heading are between the casing (conducting the gas to the injection point) and the tubing (flowline).",
"For gas lifted wells the gas injection rate (at some point) can be utilized for control, which gives additional degree of freedom.",
"There are four main categories of principles for avoiding or reducing the effects of slug flow: 1.Design changes 2.Operational changes 3.Procedures 4.Control methods: Feed forward control to separation unit Slug choking Active slug control An example of a typical slug handling technique that involves design changes is a technique that requires installation of slug catchers (onshore).",
"Such design changes also have the disadvantage of that substantial capital investment is needed.",
"Another example of such a technique is to increase the size of the first stage separators to provide buffer capacity.",
"For already existing installations where problems with slug flow are present, and for compact separation units, these design changes have limited effects on flow stability.",
"Still, using this technique, compressors may trip due to large rapid variations in the feed rates to the separators caused by unstable multiphase flow.",
"An example of an operational change is to choke the pipeline to such an extent that the operation point is outside the unstable flow regime.",
"But such an operational change may have the disadvantage of decreasing the output flow to a level substantially lower than the capacity of the pipeline.",
"Severe variations of pressure along different positions of the pipeline may also occur.",
"Procedures are rule-based calculations applied by the operator.",
"These are often used during pipeline, riser or well flow line startup.",
"Such rule-based calculations may some extent decrease the magnitude of slugging and reduce the variations in pressure in the flow-line.",
"But a problem is that the approach with rule-based calculations may only decrease the magnitude of slugging at certain operating conditions.",
"And the operating conditions may vary widely.",
"Prior art control methods include: Feed-forward control to a separation unit process control system.",
"In this approach the slug is coped with inside the separation unit.",
"U.S. Pat.",
"No.",
"5,256,171 shows a method, which utilizes process measurements inside the separation unit.",
"U.S. Pat.",
"No.",
"5,544,672 shows slug choking, which utilizes measurements downstream of the point of slug generation and chokes the pipeline control valve in the presence of a slug.",
"By conventional control methods one usually refers to feed-forward or slug choking.",
"Conventional control methods may reduce the negative effects of slugging and flow variations in a pipeline.",
"A remaining problem by applying conventional control methods is to stabilize the multiphase flow in a complete flow line and not only reducing the effect of slugging at a single point of the flow line, typically the outlet of the flow line.",
"Another problem with conventional control methods is that they have not proven to be efficient enough when it comes to stabilize multiphase flow and particularly not in flow-lines comprising remote well-head platforms and subsea wells.",
"In the light of the problems mentioned above, the inventor has found that there is a need for a more efficient method for stabilizing multiphase flow caused by slugging."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>An object of the invention is to provide a method, which stabilize a multiphase flow, in a flow line, where flow instability is caused by at least one slug.",
"Measurement of pressure or temperature is taken upstream the point where the main part of the slug is generated.",
"The pressure measurement, or an estimated pressure calculated from the temperature measurement, is supplied to a dynamic feedback controller which dynamic feedback controller calculates an output controlling at least one control valve, wherein the multiphase flow is stabilized throughout the complete flow line.",
"By a flow line is meant a line conducting transport of a multiphase mixture of oil, gas and water such as, a pipeline, riser or a well flow line.",
"Another object of the invention is to provide a computer program product, which performs the steps of the above-mentioned method including applying a control program or control law.",
"By a control valve is meant a valve or a choke equipped with an actuator suitable for automatic control, such as an electric motor as actuator with positioning control, a stepping motor or a pneumatic actuator.",
"Another advantage of the present invention is that it improves the stability of operating conditions in production facilities for oil and/or gas.",
"In particular the invention significantly reduce the disturbances in feed to the separation process by avoiding flow variation at the outlet of the multiphase flowline that connect wells and remote installation of the processing unit.",
"Another advantage of the invention, compared with a conventional control method, is that the invention reduces the pressure fluctuations at the flow line inlet.",
"This stabilizes the multiphase flow line, which makes it possible not only to reduce the effects of slugging, as in the case of conventional methods, but also to entirely avoid slugging.",
"An advantage of the invention, compared with a conventional control method, is that it stabilizes the multiphase flow trough out the complete flow line.",
"This also means that by applying the invention the production rate, for instance at a production platform, may be increased.",
"An advantage of the invention is that, compared with conventional methods, it reduces the number of occurrences and formations of slugs in a flow line.",
"In this way the invention enables energy to be used in a more efficient way in the flow line in order to transport a multiphase mixture of oil, gas and water.",
"This in contrast of letting energy out of the system in the form of blowouts as slugs leaves the outlet of flow line, such as the outlet of a riser.",
"Another advantage of the invention, compared with conventional methods using design changes, is that it does not require new process equipment to be installed.",
"Hence, substantial capital investment is avoided.",
"It is however assumed that there is at least one available pressure or temperature measurement along the flow line upstream the point where the main part of the slug is generated.",
"It is also assumed there is at least one control valve in the flow line.",
"It is an aim of the invention to supply a method, which applies a dynamic feedback controller, which is a multivariable input, single output controller, acting upon periodic variations in measured variables.",
"It is a further aim of the invention to stabilize the multiphase flow at any point of the flow line.",
"The above mentioned objects and aims are met by the invention as defined by the accompanying patent claims 1 - 16 .",
"In particular, a method for stabilizing a multiphase flow through a flow line where instability of the multiphase flow is caused by at least one slug is defined by independent claim 1 , and a computer program product is defined by independent claim 17 , and use of a computer program product according to independent claim 19 .",
"These and other advantages with the present invention and aspects of it will become apparent from the detailed description and from the accompanying drawings."
],
[
"FIELD OF THE INVENTION The present invention relates to a method for applying a dynamic feedback controller to stabilize multiphase flow in a flow line, such as a pipeline, riser or well production line, where slugs cause instability of the multiphase flow.",
"The invention also relates to a computer program product, which carries out the steps of the method.",
"BACKGROUND OF THE INVENTION Oil that is produced offshore is transported through pipelines as a complex mixture of oil, gas water and sand.",
"One common flow regime is known as slug flow, in which the mixture flows intermittently along the pipelines and comprises a concentrated mass in the form of a liquid plug.",
"Such a concentrated mass in movement is hereafter called a slug.",
"In multiphase pipelines/risers/well flow lines at reduced flow rates and/or changing gas oil ratio, compared to design specifications, instabilities in terms of terrain-induced and riser-induced slug flow often occur.",
"Such a slug flow is a bulk of liquid moving in the pipeline followed by an amount of gas.",
"Terrain and riser-induced slug flow is often referred to as severe slugging.",
"A publication by Yehuda Taitel, “Stability of Severe Slugging”, Int.",
"Journal of Multiphase Flow, Vol.",
"12, No.",
"2, pp.",
"203-217, 1986, describes the phenomena of slugging.",
"Terrain and riser-induced slug flow is induced periodically as liquid in terms of oil and water is accumulated in lower parts of the pipeline/riser, see FIG.",
"11-IV.",
"At a certain time the liquid will restrict the passage for the gas.",
"In this situation a small amount of gas bubbles through the liquid plug, however the main part of the gas accumulates upstream of the liquid plug which causes the pressure to increase (see FIG.",
"11-I).",
"In this situation the pressure upstream of the liquid plug is equal to the pressure downstream of the liquid plug plus the hydraulic pressure across the liquid plug (applying a static force balance).",
"But when the pressure increase upstream of the liquid plug becomes larger than the pressure increase downstream the liquid plug, the liquid plug starts moving (see FIG.",
"11-II), and then forms a slug, which accelerates.",
"It should be noted that this condition might be fulfilled before the front of the liquid plug reaches the downstream maximum point in the pipeline profile.",
"Depending on operating conditions and pipeline profile, the slug may die out or it may be transported to the outlet of the pipeline/riser.",
"In a situation where the tail of the liquid plug enters the vertical parts of the flow line (FIG.",
"11-III), a rapid increase in the liquid flow rate occurs due to the unstable situation where the pressure head, due to the liquid column, decreases.",
"A slug is formed and the slug get transported to the outlet of the pipeline, and when the gas behind the slug escapes the pipeline/riser, the remaining liquid in the vertical parts returns to the bottom of the riser or dips in the pipeline profile.",
"Then the whole process is repeated, and the result is an unstable multiphase flow pattern/cycle where the liquid flow rate varies from zero to a significant value, as the slug passes a fixed point in the pipeline, in a short period of time.",
"The flow pattern is characteristic of severe slugging (terrain/riser-induced slugging).",
"For terrain-induced slug flow the corresponding liquid plugs are caused by terrain effects reflected in the pipeline profile (offshore and onshore), whereas riser induced slug flow is caused by the pipeline leaving the seabed on its way to the surface (offshore).",
"For long risers, special dynamic effects might occur due to phase transition from liquid to gas due to a considerable pressure decrease in the riser.",
"Different riser shapes may also affect the dynamics in riser-induced slugging.",
"Unstable flow causes considerable problems for production in the upstream wells and operation of the downstream processing plant: Large disturbances to the separator train, causing: Limiting separation capacity due to the need for larger operating margin to achieve the desired separation.",
"Poor separation (water carry over to export pipeline) due to rapidly varying separator feed rates.",
"Poor separation results in varying quality of water outlet from separators, causing large problems in the downstream water treatment system and possible violation of environmental restrictions.",
"Large and rapidly varying compressor loads, causing: Inefficient compressor operation.",
"Limiting compression capacity due to the need for a larger margin to handle gas holdup behind the liquid.",
"Spurious flaring from limited compression capacity.",
"Limited production from the upstream wells.",
"The pressure variations at the pipeline or riser inlet 1 are also visible in the upstream wells, resulting in limited production from wells suffering from reduced lifting capacity.",
"For gas lifted oil wells a problem referred to as casing heading might occur.",
"Applications of gas lifted oil wells are different than slug flow in pipelines, risers and wells in the following sense: The dynamic interactions in the casing heading are between the casing (conducting the gas to the injection point) and the tubing (flowline).",
"For gas lifted wells the gas injection rate (at some point) can be utilized for control, which gives additional degree of freedom.",
"There are four main categories of principles for avoiding or reducing the effects of slug flow: 1.Design changes 2.Operational changes 3.Procedures 4.Control methods: Feed forward control to separation unit Slug choking Active slug control An example of a typical slug handling technique that involves design changes is a technique that requires installation of slug catchers (onshore).",
"Such design changes also have the disadvantage of that substantial capital investment is needed.",
"Another example of such a technique is to increase the size of the first stage separators to provide buffer capacity.",
"For already existing installations where problems with slug flow are present, and for compact separation units, these design changes have limited effects on flow stability.",
"Still, using this technique, compressors may trip due to large rapid variations in the feed rates to the separators caused by unstable multiphase flow.",
"An example of an operational change is to choke the pipeline to such an extent that the operation point is outside the unstable flow regime.",
"But such an operational change may have the disadvantage of decreasing the output flow to a level substantially lower than the capacity of the pipeline.",
"Severe variations of pressure along different positions of the pipeline may also occur.",
"Procedures are rule-based calculations applied by the operator.",
"These are often used during pipeline, riser or well flow line startup.",
"Such rule-based calculations may some extent decrease the magnitude of slugging and reduce the variations in pressure in the flow-line.",
"But a problem is that the approach with rule-based calculations may only decrease the magnitude of slugging at certain operating conditions.",
"And the operating conditions may vary widely.",
"Prior art control methods include: Feed-forward control to a separation unit process control system.",
"In this approach the slug is coped with inside the separation unit.",
"U.S. Pat.",
"No.",
"5,256,171 shows a method, which utilizes process measurements inside the separation unit.",
"U.S. Pat.",
"No.",
"5,544,672 shows slug choking, which utilizes measurements downstream of the point of slug generation and chokes the pipeline control valve in the presence of a slug.",
"By conventional control methods one usually refers to feed-forward or slug choking.",
"Conventional control methods may reduce the negative effects of slugging and flow variations in a pipeline.",
"A remaining problem by applying conventional control methods is to stabilize the multiphase flow in a complete flow line and not only reducing the effect of slugging at a single point of the flow line, typically the outlet of the flow line.",
"Another problem with conventional control methods is that they have not proven to be efficient enough when it comes to stabilize multiphase flow and particularly not in flow-lines comprising remote well-head platforms and subsea wells.",
"In the light of the problems mentioned above, the inventor has found that there is a need for a more efficient method for stabilizing multiphase flow caused by slugging.",
"SUMMARY OF THE INVENTION An object of the invention is to provide a method, which stabilize a multiphase flow, in a flow line, where flow instability is caused by at least one slug.",
"Measurement of pressure or temperature is taken upstream the point where the main part of the slug is generated.",
"The pressure measurement, or an estimated pressure calculated from the temperature measurement, is supplied to a dynamic feedback controller which dynamic feedback controller calculates an output controlling at least one control valve, wherein the multiphase flow is stabilized throughout the complete flow line.",
"By a flow line is meant a line conducting transport of a multiphase mixture of oil, gas and water such as, a pipeline, riser or a well flow line.",
"Another object of the invention is to provide a computer program product, which performs the steps of the above-mentioned method including applying a control program or control law.",
"By a control valve is meant a valve or a choke equipped with an actuator suitable for automatic control, such as an electric motor as actuator with positioning control, a stepping motor or a pneumatic actuator.",
"Another advantage of the present invention is that it improves the stability of operating conditions in production facilities for oil and/or gas.",
"In particular the invention significantly reduce the disturbances in feed to the separation process by avoiding flow variation at the outlet of the multiphase flowline that connect wells and remote installation of the processing unit.",
"Another advantage of the invention, compared with a conventional control method, is that the invention reduces the pressure fluctuations at the flow line inlet.",
"This stabilizes the multiphase flow line, which makes it possible not only to reduce the effects of slugging, as in the case of conventional methods, but also to entirely avoid slugging.",
"An advantage of the invention, compared with a conventional control method, is that it stabilizes the multiphase flow trough out the complete flow line.",
"This also means that by applying the invention the production rate, for instance at a production platform, may be increased.",
"An advantage of the invention is that, compared with conventional methods, it reduces the number of occurrences and formations of slugs in a flow line.",
"In this way the invention enables energy to be used in a more efficient way in the flow line in order to transport a multiphase mixture of oil, gas and water.",
"This in contrast of letting energy out of the system in the form of blowouts as slugs leaves the outlet of flow line, such as the outlet of a riser.",
"Another advantage of the invention, compared with conventional methods using design changes, is that it does not require new process equipment to be installed.",
"Hence, substantial capital investment is avoided.",
"It is however assumed that there is at least one available pressure or temperature measurement along the flow line upstream the point where the main part of the slug is generated.",
"It is also assumed there is at least one control valve in the flow line.",
"It is an aim of the invention to supply a method, which applies a dynamic feedback controller, which is a multivariable input, single output controller, acting upon periodic variations in measured variables.",
"It is a further aim of the invention to stabilize the multiphase flow at any point of the flow line.",
"The above mentioned objects and aims are met by the invention as defined by the accompanying patent claims 1-16.In particular, a method for stabilizing a multiphase flow through a flow line where instability of the multiphase flow is caused by at least one slug is defined by independent claim 1, and a computer program product is defined by independent claim 17, and use of a computer program product according to independent claim 19.These and other advantages with the present invention and aspects of it will become apparent from the detailed description and from the accompanying drawings.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a schematic overview of the topology of a flow line, such as a pipeline, between a wellhead platform and a production platform.",
"FIG.",
"2a shows a schematic overview of an implemented control calculation for active feedback control of slug flow through a flow line.",
"Further the figure shows that the measurement of a pressure variable is positioned upstream the position where the main part of a slug is generated.",
"A method according to the invention use the active feedback controller to close/open a control valve, which control valve is also shown in the figure.",
"FIG.",
"2b shows a schematic overview of an alternative embodiment of an implemented control calculation for active feedback control of slug flow through a flow line.",
"FIG.",
"3 shows the different blocks involved in an implemented control calculation for active feedback control of a flow line which control calculation is used by a method according to the invention.",
"FIG.",
"4 shows the different controller modes and the transitions between them in an implemented control calculation for active feedback control in order to stabilize a multiphase flow in occurrence of slugs through a flow line.",
"FIG.",
"5 shows multiphase flow simulations of terrain induced slug flow.",
"The upper part of the figure shows a flow inlet, such as a pipeline inlet, PT1 and outlet PT2 pressure and the lower part shows flow line outlet flow rates.",
"This figure shows that the multiphase flow is unstable in the case of a slugging and prior art technique is used.",
"FIG.",
"6 shows multiphase flow simulations of terrain induced slug flow.",
"Each line represents the pipeline profile plot of liquid volume fraction.",
"The sampling rate is one minute.",
"This figure shows that the multiphase flow is unstable in the case of a slugging and prior art technique is used.",
"FIG.",
"7 shows how an active feedback controller based on the invention stabilizes a terrain-induced slug.",
"The upper part of the figure shows pipeline inlet PT1 and outlet PT2 pressure, the middle part show pipeline outlet flow rates and the lower part shows the controller output U1 to the control valve 2.FIG.",
"8 shows the effect of on flow stability by applying an active feedback controller according to the invention on the same flow line and same case as in FIG.",
"7.There are 240 plots in the figure, which represents the liquid volume fraction but they appear as one.",
"FIG.",
"8 should be compared with the FIG.",
"6 where no dynamic feedback control is used and slugging occurs.",
"FIG.",
"9 shows a dynamic feedback control startup and operation of a flow line, such as a pipeline.",
"The upper part of the figure shows pipeline inlet PT1 and outlet PT2 pressures, together with 12 h moving average in the same variables.",
"The figure is based on the results and measurements made when applying the invention at an off-shore oil production facility.",
"FIG.",
"10 shows a dynamic feedback control startup and operation of a flow line, such as a pipeline.",
"Upper part shows pipeline inlet gas flow rate.",
"Lower part shows pipeline inlet liquid flow rate.",
"Both 30 minute and 8 hour moving average are plotted.",
"FIG.",
"11 shows the four stages in a severe slug cycle.",
"FIG.",
"12 shows pipeline profile for deep-water riser example.",
"FIG.",
"13 shows a severe riser induced slug flow cycle in a deep-water riser.",
"This figure shows that the multiphase flow is unstable in the case of a slugging and prior art technique is used.",
"FIG.",
"14 shows the effect of choking of severe riser induced slug flow.",
"This figure shows that the multiphase flow is unstable in the case of a slugging and prior art technique is used.",
"FIG.",
"15 shows the effect of choking of severe riser induced slug flow.",
"This figure shows that the multiphase flow is unstable in the case of a slugging and prior art technique is used.",
"Chocking the control valve has a mitigating effect on slugging, but the flow is not stabilized.",
"FIG.",
"16 shows pipeline profile plots of liquid volume fraction through a severe riser induced slug flow cycle.",
"FIG.",
"17 shows the effect of choking of severe riser induced slug flow.",
"FIG.",
"18 (upper part) shows pipeline profile plots of liquid volume fraction through a severe riser induced slug flow cycle.",
"The figure (lower part) also shows the height of the flow line.",
"FIG.",
"19 shows how a multiphase flow during startup, operation and controller stop of a deep-water riser.",
"This as a method based on the invention is applied using a dynamic feedback controller.",
"FIG.",
"20 (upper part) shows pipeline profile plots of liquid volume fraction with an active feedback controller according to the invention in operation.",
"The figure (lower part) also shows the height of the flow line.",
"FIG.",
"21 shows a dynamic feedback controller startup, operation and controller stop of a deep-water riser.",
"FIG.",
"22 shows pipeline profile plots of liquid volume fraction with a dynamic feedback controller according to the invention in operation (upper part of figure).",
"DETAILED DESCRIPTION OF THE INVENTION FIG.",
"1 shows a schematic topologic overview of a flowline control system 3 based on the invention.",
"The figure also shows that as an example the flowline 8a could be a pipeline between a wellhead platform 1 and a production platform 7.Several flow lines 4 may operate in parallel.",
"In the example where the flowline 8a connects to a production platform 7 the platform comprises a compressor 6 and a first 5a and second stage 5b separator.",
"A method according to the invention for stabilizing a multiphase flow in a flow line 8a, where instability of the multiphase flow is caused by at least one slug 8b, or slugging, comprises the following steps: measuring continuously one pressure variable 26 upstream the point where the main part of the slug 8b is generated, supplying at least the pressure variable mentioned above to a dynamic feedback controller, calculating continuously an output 11 of the dynamic feedback controller 9, controlling the opening of a control valve 2 at said flow line 8a, wherein the multiphase flow is stabilized through the flow line 8a.",
"FIG.",
"2a shows an overview of a dynamic feedback controller 9 for stabilization of a multiphase flow through a flowline such as a pipeline and/or a riser 8a.",
"As an example the flowline connects a wellhead platform 1 and a production platform 7.The multiphase flow comprises liquid and gas.",
"Such an embodiment is not limited to pipelines from wellhead platforms to production platforms, but is applicable to any flow line such as from a subsea manifold.",
"FIG.",
"2a also shows a schematic image of generation of a slug 8b.",
"The dynamic feedback controller 9 is implemented in such a way that it stabilizes the flow through the flowline 8a, as indicated in FIG.",
"2a, by use of a control valve 2, which is automatically controlled.",
"A method according to the invention uses a pressure variable PT1 26 upstream of any liquid plug.",
"The variable PT1 is a pressure measurement or as an alternative a temperature measurement can be used from which the pressure is calculated.",
"Yet, another alternative to is to calculate the pressure variable on other measurements.",
"As an example one measurement at the flow line inlet and a measurement upstream or down stream the control valve is used as input to calculate/estimate the pressure variable.",
"Pressure measurements PT1 26 at inlet 12, or at some point upstream of the control valve 2, measuring pressure variations upstream of the liquid plug 8b.",
"FIG.",
"2b shows an overview of an alternative dynamic feedback controller 9 for stabilization of a multiphase flow through a pipeline or a riser 8a between a wellhead platform 1 and a production platform 7.Each of the indicated measured variables improves the performance of an embodiment of the invention: Pressure measurements PT2 27 at the outlet 13, upstream of control valve 2.Pressure difference across the pipeline 8a, or a part of the pipeline, based on the two measurements PT1 26 and PT2 27.Flow measurements FT1 28 at the inlet 12, or at some point upstream of the outlet 13, measuring flow variations upstream of the liquid plug 8b.",
"Flow measurements FT2 29 at the outlet 13, upstream of the control valve 2.Measurements can be made topside as well as subsea.",
"Enhanced control can be achieved by including additional measurements (flow, density and temperature).",
"In case flow measurements are included, estimates of flow conditions in the pipeline/riser/well flow line 8a will be improved.",
"A distinct difference between the dynamic feedback control disclosed by the invention compared with traditional control methods is the use of measurements upstream of the position of the slug formation.",
"Whereas the other methods have a mitigating effect, the dynamic feedback control improves the stability of the pipeline flow and in a much more efficient way reduces the occurrence of slugs.",
"That is achieved at a higher mean flowrate than what is possible with traditional control methods.",
"Further the dynamic feedback controller according to the invention reduces the variation of flow line inlet pressure.",
"In case of any kind of failure, output 11 will go to a pre-defined value.",
"The dynamic feedback controller 9 has proved to stabilize the flow both by simulation and also by field tests.",
"The dynamic feedback controller 9 allows stabilization of pipeline variables (pressure, temperature, holdup, and flow), at any point in the pipeline/riser 8a.",
"This means that the controller 9 is not limited to stabilization only at the outlet 13 of the flow line.",
"In an alternative embodiment of the invention, a calculation for slug detection is used together with dynamic feedback controller.",
"In addition to utilizing the same measurements as the dynamic feedback controller 9, the slug detection calculation also uses the pressure upstream of the control valve as input.",
"The output from the slug detection calculation is typically used for information purposes, notifying personnel about an upcoming slug 20-30 minutes in advance.",
"The information is for instance displayed on computer screens in the control room.",
"In one embodiment of the invention the slug detection calculation is used in order to stabilize the multiphase flow even further.",
"The Dynamic Feedback Controller The dynamic feedback controller 9 is preferably a multivariable input, single output controller.",
"The dynamic feedback controller is preferably described in a state-space form and implemented in a control program for a control means.",
"A control means comprises at least one out of an industrial controller, a programmable logic computer (PLC), an industrial purpose computer, a general-purpose computer or a purpose built device for stabilization of multiphase flow.",
"A particularly useful and efficient implementation is achieved by using structured text according to the IEC 1131 program language standard.",
"This and other ways of implementation a control program, based on a state-space form is well known to a person skilled in the art.",
"FIG.",
"3 shows the dynamic feedback controller in more detail.",
"The controller consists of the following blocks: 14: Feedback stabilization ensures that the pipeline flow is stabilized.",
"15: Slug choking throttles the control valve in the presence of a slug (pressure rise upstream of the pipeline choke 2).",
"16-19: Filters for input signals 20: Manual/auto switch 21-25: Max and min function blocks The input signals to the dynamic feedback controller are: PT1: Pressure 26 at the pipeline inlet, 12.Set point PT1: desired/nominal value (SP PT1) 10 for pressure at pipeline inlet, 12.The operator typically enters the nominal value of the set point PT1.As an alternative a computer program may calculate the set point PT1.PT2: Pipeline pressure 27 upstream of the choke, 13.Set point PT2: nominal value (SP PT2) 28 for pipeline pressure upstream of the choke, 13.Output High Limit (OHL): upper limit 33 for the controller output U1 and U2.Output Low Limit (OLL): lower limit 34 for the controller outputs U1 and U2.Manual Output Value (MOV): output value 35 from the controller when it is in manual mode.",
"Mode: operator defined controller mode 36.The output signals from the dynamic feedback controller shown in FIG.",
"3 include: Filtered PT1, filtered pressure measurement one 37.Filtered PT2, filtered pressure measurement two 38.U1: Controller output 39 to be wired to the flow line, such as a pipeline, control valve (2).",
"U2: Controller output 40 based on measurement PT1 26.U3: Controller output 41 based on measurement PT2 27.Additional output signals from the controller (not shown in FIG.",
"3) include: Filtered set-point for PT1 Filtered nominal value for PT2 Controller state Controller ‘I am alive signal’ Alarm signals from the controller (not shown FIG.",
"3) include: High output limit alarm Low output limit alarm Read fail alarm The block diagram in FIG.",
"3 shows the following interconnections: 1.The feedback U2 40 and U3 41 from the two measurements PT1 and PT2 are calculated independently.",
"2.The output U2 40 from Feedback stabilization 14 is restricted to be within the upper 21 and lower 22 output limits.",
"3.The output U3 41 from the slug choking calculation 15 is restricted to be below zero 23.4.The sum of U2 40 and U3 41 is one of the two inputs to the controller mode switch 20.Depending on the operator-defined controller mode, the output from the switch is either the sum of U2 and U3 or the operator-defined Manual Output Value 35.5.The output U1 from the controller mode switch 20 is then restricted by the maximum and minimum function blocks to be within the upper 33 and lower 34 output limits.",
"The next paragraphs describe the feedback stabilization 14, slug choking 15, filters 16-19 and the combination of the blocks into the complete dynamic feedback controller.",
"The description of each part is on three levels.",
"Level one is an overview where the purpose of the block is clarified.",
"Level two is a comprehensive mathematical description of the block, and level three discloses a detailed description of the implementation.",
"Feedback Stabilization The inventor has found that a calculation of the feedback stabilization 14 is beneficial in order to stabilize terrain and riser induced slug flow.",
"Level One.",
"The purpose of the calculation of the feedback stabilization is to achieve control of the pipeline inlet pressure, which is the main contributor to the driving force of the flow through the pipeline or riser.",
"The feedback stabilization 14 acts upon slow periodic variations in the pressure measurement PT1 by including control action at low frequency, e.g.",
"integral action.",
"The feedback stabilization 14 may have built-in logic to prevent anti-windup.",
"The anti-windup scheme is implemented by preserving the old control output in the state of the controller, and by this only a smaller set (subspace) of these states are limited.",
"Level Two.",
"An example of how to calculate feedback stabilization 14 is described by the following difference equation: Δu2,k=K(Δef,k+Ts/T1ef,k+T2/TΔΔef,k) where Ts is the sampling time, K, T1 and T2 are tuning parameters, the operator Δ means Δmk=mk−mk−1 and ef,k is filtered error (set-point minus measurement).",
"The filter to obtain the filtered control error is a first-order filter of the type described below.",
"The feedback stabilization controller is implemented based on a state-space form and the state in the controller has been given special ordering so that integral windup is easily implemented.",
"Level Three.",
"In an embodiment of the invention the calculation of the feedback stabilization 14, is implemented based on the state-space realization: [ x FS , k + 1 ( 1 ) x FS , k + 1 ( 2 ) x FS , k + 1 ( 3 ) x FS , k + 1 ( 4 ) ] ︸ x FS , k + 1 = [ 1 K p ( 1 + T s / T I + T d / T s ) - K p ( 1 + 2 T d / T s ) K p T d / T s 0 ⅇ ( - T s / T f ) 0 0 0 1 0 0 0 0 1 0 ] ︸ Φ n [ x FS , k ( 1 ) x FS , k ( 2 ) x FS , k ( 3 ) x FS , k ( 4 ) ] ︸ x FS , k + [ 0 0 - ( 1 - ⅇ ( - T s / T f ) ) 1 - ⅇ ( - T s / T f ) 0 0 0 0 ] ︸ Γ FS [ y k ( 1 ) y k ( 3 ) ] u k + 1 ( 2 ) = 1 0 0 0 ︸ C FS [ x FS , k + 1 ( 1 ) x FS , k + 1 ( 2 ) x FS , k + 1 ( 3 ) x FS , k + 1 ( 4 ) ] ︸ x FS , k + 1 The first element in the state vector xFS,k(1) contains the previous feedback stabilization controller output.",
"The states xFS,k(2) to xFS,k(4) contain the current and the two previous filtered control errors.",
"The state of the feedback stabilization xFS is updated when the controller is running into the high and low output limits.",
"That is, the non-linear saturation logic functions 21 and 22 interfere with the state of the feedback stabilization calculation.",
"The same kind of interference also appears when the controller is in the manual and the starting mode.",
"Slug Choking Level One.",
"The purpose of the slug choking calculation 15 is to throttle the control valve 2 of the flowline in the presence of a slug, i.e.",
"when the slug approaches the outlet of the pipeline or riser.",
"The pressure measurement PT2, upstream of the flowline control valve 2, increases rapidly whenever a liquid plug approaches the point of measurement PT1.The slug-choking calculation 15 therefore uses PT2 as input and acts upon pressure increase in this measurement by choking the control valve 2.By choking the controller is able to reduce the velocity of the liquid plug.",
"Experience from simulation and field-tests shows that moderate use of choking in the presence of the slug has a stabilizing effect.",
"Level Two.",
"On continuous form the slug choking feedback controller 15 has the following form: K ( s ) = k s ( τ 1 s + 1 ) ( τ 2 s + 1 ) , u 3 ( s ) = K ( s ) e 2 ( s ) where K(s) is the Laplace transform of the slug chocking controller, u3 is the controller output (the control valve opening) and e2 is the filtered nominal value for PT2 minus PT2.Tuning parameters are the frequencies f1=1/τ1 and f2=1/τ2, and the controller gain k. Level Three.",
"With sampling time Ts and Zero Order Hold (ZOH) a discrete time state-space description of the controller K(s) is: [ x sc , k + 1 ( 1 ) x sc , k + 1 ( 2 ) ] ︸ x sc , k + 1 = [ ⅇ - T s / τ 1 0 0 ⅇ - T s / τ 2 i ] ︸ Φ sc [ x sc , k ( 1 ) x sc , k ( 2 ) ] ︸ x sc , k + [ k p ( 1 - ⅇ T s / τ 1 ) k p ( 1 - ⅇ - T s / τ 2 ) ] ︸ Γ sc ( y k ( 2 ) - y k ( 4 ) e k ( 2 ) ) u k + 1 ( 3 ) = [ 1 τ 2 - τ 1 - 1 τ 2 - τ 1 ] ︸ Csc [ x sc , k + 1 ( 1 ) x sc , k + 1 ( 2 ) ] ︸ x sc , k + 1 Filters Level 1.The purpose of the filters 16-19 and the filter inside the feedback stabilization calculation 14, is to filter the error signal to ensure smooth changes in operation point and filtering of high frequency noise.",
"Level 2.The filters are all first-order low pass filters with unity steady-state gain.",
"The common form of these filters is: F ( s ) = 1 T f s + 1 , y f ( s ) = F ( s ) y ( s ) Level 3.With sampling time Ts and ZOH, a discrete time state-space description of the continuous filter F(s) is: x f , k + 1 = ⅇ - T s / T f ︸ Φ f x f , k + ( 1 - ⅇ - T s / T f ) ︸ Γ f y k , y f , k + 1 = 1 ︸ C f x f , k + 1 The Full State-Space Dynamic Feedback Controller Level 1.The individual dynamic parts of a dynamic feedback controller are described above.",
"These parts are put together into one state-space description of the dynamic feedback controller.",
"Level 3.In an embodiment of the invention a state-space an example of representation of the dynamic feedback controller 9 is: xk+1=Φxk+Γyk, uk+1=Cxk+1+Dyk, where xk is the state-space vector of the controller yk is the input vector to the controller (measurements and set points/nominal values etc.)",
"uk is the output vector from the controller The elements in the input vector yk are: yk(1) pipeline inlet pressure 26, PT1 yk(2) pressure upstream the control valve 2, PT2 yk(3) set-point 10 for pipeline inlet pressure, PT1 yk(4) nominal value for pressure upstream of the control valve 2, PT2 The elements in the state vector xk are: xk(1) previous control value, i.e.",
"uk−1(2) xk(2) filtered control error at time k, i.e.",
"ef, k xk(3) filtered control error at time k−1, i.e.",
"ef, k−1 xk(4) filtered control error at time k−2, i.e.",
"ef, k−2 xk(5) Filtered PT1 xk(6) Filtered PT2 xk(7) Filtered set-point to PT1 xk(8) Filtered nominal value for PT2 xk(9) State one in the slug choking calculation xk(10) State two in the slug choking calculation xk(11) Controller state 0 Manual 1 Starting 2 Auto The elements in the output vector uk are: uk(1) Output 39 from the dynamic feedback controller uk(2) Output 40 from the feedback stabilization 14 uk(3) Output 41 from the slug choking 15 uk(4) Filtered pressure measurement 37 PT1 uk(5) Filtered pressure measurement 38 PT2 uk(6) Filtered set-point 16 for pressure measurement PT1 uk(7) Filtered nominal value 18 for pressure measurement PT2 The matrices φ, Γ,C,D are: Φ = [ Φ FS 0 4 × 4 0 4 × 2 0 4 × 1 0 4 × 4 Φ filter 0 4 × 2 0 4 × 1 0 2 × 4 0 2 × 4 Φ sc 0 2 × 1 0 0 0 1 × 2 1 ] , Γ = [ Γ FS Γ filter Γ sc 0 ] C = [ 0 1 × 4 0 1 × 4 0 1 × 2 0 C FS 0 1 × 4 0 1 × 2 0 0 1 × 4 0 1 × 4 C sc 0 0 4 × 4 I 4 × 4 0 4 × 2 0 4 × 1 ] , D = 0 7 × 4 Φ filter = [ ⅇ - T s / T 1 0 0 0 0 ⅇ - T s / T 2 0 0 0 0 ⅇ - T s / T 3 0 0 0 0 ⅇ - T s / T 4 ] , Γ filter = [ 1 - ⅇ - T s / T 1 0 0 0 0 1 - ⅇ - T s / T 2 0 0 0 0 1 - ⅇ - T s / T 3 0 0 0 0 1 - ⅇ - T s / T 4 ] , where 0m×n is a m×n matrix with zeros, T1 to T4 are filter time constants and Ts is the sampling interval.",
"Fixed Controller Output and State Update Whenever the controller output is fixed, due to 1.Manual mode 2.Startup mode 3.Low or high saturation the controller state is updated so that it goes smoothly out of the fixed output or the saturation.",
"This is achieved by updating the previous controller output in the feedback stabilization calculation.",
"Dynamic feedback controller modes and transitions FIG.",
"4 shows three different modes.",
"The description below regarding controller modes may be of use when implementing the controller.",
"The three modes are: 1.Manual 42: The controller is running, but the output from the controller to the control valve (2) is kept constant.",
"2.Startup 43: The controller is running but does not update the output to the control valve (2).",
"It tests for certain process and controller conditions to appear in order to initiate a transition from starting to auto.",
"3.Auto 44: The controller is running and updates the controller output to the control valve (2).",
"The operator initiates the transition 45 from Manual 42 to Startup 43 mode by setting the controller mode input 36 to Auto.",
"The transition 46 from Startup 43 to Auto 44 depends of the process measurements and the calculated output from the controller.",
"The controller will only go from startup to auto if: 1.The controller input mode 36 is in Auto and 2.process measurement PT1 26 is decreasing and 3.the calculated output U1 39 is decreasing.",
"The Startup mode 43 is included to ensure the best possible condition for flow stabilization.",
"The transitions from Startup 43 to Manual 42 and from Auto 44 to Manual 42 are initiated by changing the mode input 36 from Auto to Manual.",
"One alternative when implementing the controller, compared with the description of transitions between modes as described above, is to implement the controller to go directly from Manual to Auto mode.",
"Controller Startup Depending on the characteristic of an individual flowline the controller startup should be adjusted to handle specifications and limitations of that flowline.",
"Below is one example of a controller startup.",
"According to the above section “Dynamic feedback controller modes and transitions”, as an operator changes the operator-defined mode 36 from Manual (zero) to Auto (one), the controller first goes into a Startup mode 43.In the Startup mode, the controller updates all of its internal states, however the controller output to the control valve 2 remains constant.",
"When certain process and controller conditions are fulfilled the controller starts to update the control valve 2.The reason for the special startup sequence is that it is not possible to stabilize the pipeline flow from an arbitrary pipeline state.",
"As the controller goes from Manual mode to Startup, it initially decreases the opening of the control valve.",
"Changes in Operating Point It is advantageous to use filters for changes of the operating point.",
"An operator request to change the operating point, by a change of the set-point/nominal values of the pressure, is filtered by the two filters 16 and 18.The filters are included to ensure smooth transition from one operating point to another.",
"Controller Operation The controller set-point 10 variation depends on the mean (typically 2 to 8 hours moving average) pipeline input flow measurement FT1 and/or the mean (typically 1 to 4 hours moving average) control valve opening 2.The feedback stabilization calculation 14 adjusts the opening of the control valve 2 to achieve the desired pressure (the set-point 10) at the pipe inlet PT1.The slug choking calculation 15 closes the control valve 2 whenever the pressure measurement PT2 upstream of the control valve 2 increases rapidly.",
"This control action returns to zero over some time (typically a few minutes) whenever PT2 stops increasing or decreasing.",
"The control action in the slug choking 15 and the feedback stabilization 14 act on the control valve at different frequencies by proper adjustment of the tuning factors: Tf in the feedback stabilization calculation.",
"τ1 and τ2 in the slug choking calculation.",
"The feedback stabilization calculation 14 acts upon slow to medium periodic variations (typically in the range of 5 minutes to several hours) in the measurement PT1 26 by including control action of low frequency, e.g.",
"integral action.",
"The feedback stabilization 14 has built-in logic to prevent anti-windup.",
"The anti-windup scheme is implemented by giving the controller states a specific ordering, and by this only a smaller set (subspace) of these states are limited.",
"Pipeline Simulations FIG.",
"11 is included in order to facilitate a better understanding of what a typical slug cycle in a flow line looks like.",
"The details of FIG.",
"11 are described in the section below.",
"Simulations are made by use of the simulation environment OLGA.",
"The models used for simulations are based on fluid mechanics well known to a person skilled in the art.",
"The upper part of FIG.",
"5 shows the result of multiphase flow simulations of terrain-induced slug flow.",
"The position of the control valve 2 is constant, in other words there is no feedback control applied to the simulations.",
"The lower part of FIG.",
"5 shows that the liquid flow rate 52 at the pipeline output is zero for a period of about 2 hours.",
"During this time, the pressure upstream of the liquid plug builds up, FIG.",
"11-I.",
"When the inlet pressure 51 exceeds the hydrostatic pressure of the liquid column in the riser, the liquid accelerates and the outlet pressure 50 rises at the pipe outlet, FIG.",
"11-II and FIG.",
"11-III.",
"The liquid is transported to the outlet of the pipe and the pressure decreases as the gas and liquid escape through the pipeline, FIG.",
"11-IV.",
"The process then repeats itself.",
"The variation in gas flow 53 and liquid flow 52 rate corresponding to the slug flow is shown in the lower part of FIG.",
"5.FIG.",
"6 shows the pipeline profile plot of the liquid volume fraction 54, sampled each minute during one slug cycle.",
"FIG.",
"6 corresponds to one of the slug cycles in FIG.",
"5.It is not what happens at different time points that is important here, but what happens at different positions in the pipeline during the cycle.",
"The position in the pipeline is indicated on the x-axis and the corresponding liquid volume fraction is indicated on the y-axis.",
"Positions where the difference between the maximum and the minimum liquid volume fractions are large have the potential to initiate terrain-induced slug flow.",
"FIG.",
"7 shows the simulated performance of the dynamic feedback controller according to the invention at hand.",
"During the first eight hours the controller is in manual mode and the multiphase flow is unstable as indicated by the characteristic pressure swings in the pipeline inlet pressure 56 and outlet pressure 57.The controller starts at t=28 h and spends the next 5 to 7 hours stabilizing the pipeline.",
"The slugging behavior disappears.",
"From FIG.",
"7 it may be concluded that the controller has stabilized the multiphase flow and holds the valve opening 58 at a constant output value at t=38 h. However, that the valve opening is held at a constant value is not entirely true.",
"As the controller output 58 is magnified, it becomes clear that the controller constantly makes small movements in the output around its mean value.",
"Those movements controlled by the dynamic feedback controller are necessary to keep the multiphase flow stable.",
"Further in FIG.",
"7, the controller is set to manual at t=45 h, with its output equal to the mean value of the control valve opening over the previous 3 hours.",
"It is seen from FIG.",
"7 that with the controller set back to manual the multiphase flow once again becomes unstable.",
"However, the transition into an unstable multiphase flow is slow.",
"Still, FIG.",
"7 shows that with the dynamic feedback controller turned off the slug flow once again builds up.",
"This despite of that the output of the controller, which sets the control valve opening 58, seemed to be at an optimum level.",
"To stabilize the multiphase flow, at the indicated gas and liquid flow rates, the dynamic feedback controller according to the invention is needed.",
"From FIG.",
"7 it seems reasonable to assume that the complete flowline is stabilized as the dynamic feedback controller is started.",
"This since the input pressure 56 and at the output pressure 57 are stabilized.",
"However, due to the length of the pipeline, it could be claimed that internal instability might occur in it.",
"FIG.",
"8 shows the profile plots of the resulting liquid volume fraction for the case same case as in FIG.",
"7.The profile plots of FIG.",
"8 corresponds to the time period from t=41 to 45 h, i.e., which is part of the period in FIG.",
"7 where the multiphase flow is stabilized.",
"The profile plots of the liquid volume fraction are sampled at 60-second intervals.",
"The number of profile plots of the liquid volume fraction are 240, all lying on top of each other.",
"In this context, stabilization of the multiphase flow is defined by the liquid volume fraction at any point of the flow line showing less than 5% variation around its mean value.",
"The mean value is calculated based on measurements taken during a time interval, where a time interval corresponds to the time of at least one complete slug cycle in the flow line without applying control.",
"Indeed, FIG.",
"8 shows that the multiphase flow in the pipeline is stabilized according to this definition, and not only in one single point but also through the complete flow line.",
"FIG.",
"8 should be compared with FIG.",
"6 where no dynamic feedback control is used.",
"In contrast to FIG.",
"8, FIG.",
"6 shows large variations in the liquid volume fraction.",
"When comparing the two figures it is clear that in this case the dynamic feedback controller clearly stabilizes the multiphase flow throughout the flowline.",
"Riser Simulations This section presents results from a deep-water pipeline-riser system, which is simulated by use of the simulation environment OLGA.",
"FIG.",
"12 shows the pipeline geometry.",
"The total length is approximately 6500 m with a 5000 m flow-line on the seabed and a 1500 m long riser.",
"The inclination from the wellhead to the riser base is 1° downward slope.",
"The sea depth at riser base is 1320 m. A source is located at the inlet of the pipeline.",
"The boundary conditions at the inlet are closed and at the outlet the pressure is set equal to 15 Bar.",
"A control valve is located at the top of the riser.",
"This control valve will be used to control the multiphase flow in the riser.",
"Two different inlet condition cases are simulated.",
"The first case is at the start of the production profile.",
"The input flow rate is in case one set to 6000 Sm3/d and the gas fraction is read from the PVT table with the pressure and temperature in the inflow section (the first section in the pipeline) as input to the table.",
"This means that the gas fraction and thereby also the Gas Oil Ratio GOR may vary to some degree.",
"The GOR varies around 125 Sm3/Sm3.In the second case the GOR is set equal to 250 Sm3/Sm3 and the flow rate is set equal to 2000 Sm3/d.",
"In both case one and case two the water cut is zero, hence two-phase simulations are considered.",
"Case One—Using a Traditional Control Method FIG.",
"13 to FIG.",
"16 show simulations of riser-induced slug flow (severe slugging) with an input flow rate of 6000 Sm3/d with GOR around 125 Sm3/Sm3.The simulation is applied on the flowline with the profile according to FIG.",
"12.FIG.",
"13 shows a severe riser induced slug cycle.",
"It is worth to note the following: 1.The substantial variations in the pipeline inlet pressure 75 and outlet pressure 76.2.The outlet oil flow rate 77 is nonzero for a large portion of the time as opposed to terrain-induced slug flow.",
"The reason is that the liquid plug extends far into the nearly horizontal pipeline before the riser and it takes some time to produce the liquid in the pipeline, cf.",
"FIG.",
"11-II.",
"3.The simulations show a considerable amount of flashing, which gives rise to a gas-lift effect.",
"The gas-lift effect gives a rapid increase in the outlet oil flow rate 77 observed as the first peak in the slug flow cycle.",
"The latter peak in the outlet oil flow rate 77 is related to the blow-out of the riser, i.e., emptying the riser, cf.",
"FIG.",
"11-III.",
"FIGS.",
"14 and 15 show the effect of a stepwise closing of the control valve from 100% to 20%.",
"It should be noted that: 1.In order to get out of the region with unstable riser induced slug flow and to some level stabilize the outlet flow rate 82 and the outlet flow rate 87, it is seen that the control valve 2, according to the plotted control valve position 83 and 88, needs to be closed more than 20%.",
"2.In order to reduce the peak in the outlet oil flow rate significantly by constant choking, the valve needs to be closed more than 40%.",
"FIG.",
"15 shows that the control valve needs to be closed as much as 14% to achieve stable out let flow conditions by choking a pipeline inlet pressure of approximately 135 bar.",
"FIG.",
"16 shows profile plots of liquid volume fraction through one slug flow cycle.",
"The shaded area corresponds to 900 lines laid on top of each other.",
"There are 10 seconds between each line.",
"The profile plots illustrate the span-in the amount of liquid in different parts of the pipeline.",
"The following conclusions can be made: 1.The liquid plug covers a distance of 1300 m upstream of the riser base.",
"2.The liquid volume fraction in the local maximum point in the S-shaped riser is never larger than 50%.",
"Case One—Active Dynamic Feedback Control FIGS.",
"19 and 20 show active dynamic feedback control according to the invention, which is applied to remove the occurrence of slugging in the multiphase flow in the pipeline.",
"Initially, the controller is in manual mode with a control valve opening of 70%.",
"Then, at time t=5h 100a, the dynamic feedback controller is activated.",
"The controller waits for the best startup condition to occur.",
"This condition occurs at approximately time t=6 h and at this point the controller starts updating the output (control valve).",
"During dynamic feedback control based on the invention the gas flow 99 and oil flow 98 is stabilized, and from FIG.",
"19 the controller eventually seems to reach a constant output 100b of about 43%.",
"However, with a higher resolution of the plotted trend, it becomes clear that the controller constantly makes small movements (varying in the range of 43.1-43.2%) around its mean value.",
"The small movements in the control valve position are necessary to keep the flow stable.",
"Further FIG.",
"19 shows that at time t=16 hours the controller input mode is set back to manual mode with its previous output (43%) as the manual output value.",
"With the controller in manual mode and with the control valve in what seems to be the most optimum position the riser induced slug flow again builds up.",
"No other changes are made.",
"Slug flow with approximately the same control valve opening (40%) is also predicted from the simulations without control.",
"FIG.",
"19 shows that the invention results in a far better stabilization of multiphase flow compared with a prior art technique.",
"FIG.",
"19 also shows that the inlet pressure with the controller in operation is lower (103 Bar), than the case without control, but with maximum opening of the choke and at the same time stable flow (136 Bar).",
"This is an observation made also in other simulated cases.",
"FIG.",
"19 further shows the following important points: Less pressure drop over pipeline with active feedback control than the mean pressure drop with traditional control (this also applies to terrain induced slug flow).",
"Increased pressure upstream of the control valve with active feedback control results in larger pressure drop over the control valve.",
"This is necessary to have an effect of the movements in the control valve.",
"With active feedback control, liquid plugs do not occur, only minor movements in the profile plot of liquid volume fraction can be observed during control.",
"FIG.",
"20 shows a profile plot of liquid volume fractions during slug control.",
"The plot shows the corresponding plot area of 360 profile plots, 10 seconds apart.",
"They all lie on top of each other, implying that the pipeline indeed is stable.",
"It is important to note that field tests where the innovation is applied show the same advantages and similar effects compared with traditional control methods as in a simulated environment.",
"Case Two—Using a Traditional Control Method The deep-water riser in case two has an input flow rate of 2000 Sm3/d and a GOR of 250 Sm3/Sm3.The higher GOR compared to case one means that this case related to a lighter fluid with more gas and less oil.",
"FIG.",
"17 shows the effect of stepwise closing of the pipeline control valve from 50% to 20%.",
"Further FIG.",
"17 shows that: 1.The characteristics of the riser induced slug cycles are different from case one.",
"From FIG.",
"17, it is seen that the flashing blowout is small, almost missing in this case.",
"The mass transportation period with constant outlet flow rate is also missing.",
"However, still large oscillations in the inlet and outlet pressure appear, which is similar to case one using a traditional control method.",
"2.The liquid flow rate is zero for a larger portion of the time.",
"The slug cycles in this case compared to case one are much more similar to terrain induced slug flow.",
"FIG.",
"17 shows that the control valve needs to be closed as much as 10% to achieve stable flow conditions by choking with a pipeline inlet pressure of approximately 65 bar.",
"The reason for the lower pipeline inlet pressure in this case is the larger GOR.",
"FIG.",
"18 shows profile plots (900 lines) of liquid volume fraction through one slug flow cycle.",
"The profile plots are 10 seconds apart.",
"The profile plots illustrate the span in the amount of liquid in different parts of the pipeline.",
"FIG.",
"18 indicates that: 1) The liquid plug covers a distance of 7000 meter upstream the riser base.",
"2) The liquid volume fraction in local maximum point in the S-shaped riser does not exceed 70%.",
"By comparing FIG.",
"18 of case two with FIG.",
"16 of case one, it is found that the slug does not extend to the same extent into the horizontal part of the pipeline for case two as it did for case one.",
"Case Two—Using Active Feedback Control FIGS.",
"21 and 22 show active feedback control of case two, with an input flow rate of 2000 Sm3/d and GOR 250 Sm3/Sm3.FIG.",
"21 shows that initially the controller is in manual mode with a control valve opening of 100%.",
"Then, at time t=8 h the active feedback controller is activated.",
"The controller waits for the best startup condition to occur.",
"This condition occurs at approximately time t=8.5 h, at which point the controller starts updating the output (control valve).",
"During slug control the flow is stabilized, and again, from FIG.",
"21 the controller seems to reach a constant output of about 34.5%.",
"However, if the controller output is magnified, it becomes clear that the controller constantly makes small movements (varying in the range from 34 to 35%) around its mean value.",
"Also in this case the small movements in the control valve position are necessary to keep the flow stable.",
"This is proved by, at time t=24 hours, setting the controller input mode at manual with its previous output (32.5%) as the manual output value.",
"With the controller in manual and with the control valve in this position the riser induced slug flow again builds up.",
"Again, no other changes are made.",
"Slug flow with approximately the same control valve opening (30%) is also predicted from the simulations without control, FIG.",
"17.It should again be noted that the inlet pressure with the controller in operation is lower (41 Bar), than the case without control, but with maximum opening of the control valve (10%) and at the same time stable flow (65 Bar).",
"Further FIG.",
"21 shows the following effects and advantages by applying active feedback control according to the invention: 1.There is less pressure drop over the pipeline with control compared with the mean pressure drop using a traditional control method.",
"2.The pressure increases upstream of the choke with control, which results in larger pressure drop over the control valve.",
"This is necessary to have an effect of the movements in the control valve.",
"3.When applying the invention on the flow line, no liquid plugs occur.",
"Only minor movements in the profile plot of liquid volume fraction can be observed during control.",
"FIG.",
"22 shows a profile plot of liquid volume fractions during slug control.",
"The plot includes 360 profile lines, again 10 seconds apart.",
"They all lie on top of each other, implying that the pipeline is stable.",
"By comparing FIG.",
"22 with FIG.",
"20, it is found that there is much less liquid preserved in the riser for case two than for case one.",
"This is also the reason for the lower inlet pressures.",
"If the peaks of flow rate in FIG.",
"19 are compared with the peaks of flow rate in FIG.",
"21, it is clear that the peaks are larger for case two than for case one.",
"We noted in the sections presenting the simulations without control that the peaks in the flow rates are much larger for case two than for Case one.",
"In addition, the fraction of time in which the output flow rate is zero is greater for Case two.",
"However, which of these two that represents the largest and most troublesome problem for the downstream production plant is not clear and depends on the plant configuration.",
"It should be clear though that there are several benefits with applying the invention in both cases.",
"In the latter case it is interesting to observe how much the pipeline inlet pressure is reduced compared to pressure variations in slug flow.",
"This pressure reduction represents increased production rate from the wells due to a lower backpressure.",
"EXAMPLE OF OPERATION IN THE FIELD, OFF-SHORE PIPELINE FIG.",
"9 shows a dynamic feedback controller during startup and operation.",
"The upper part of the figure shows pipeline inlet PT1 and outlet PT2 pressure.",
"The lower part shows controller output U1.The controller is initially in manual mode.",
"After eight hours, the manual input is changed from 20% to 25%, and the mode is changed to auto.",
"The controller then goes into startup mode.",
"Just before time 12 hours the controller goes from startup to auto.",
"The controller starts to close the control valve but then opens the control valve to lower the pipeline inlet pressure.",
"The controller stabilizes the multiphase flow in the pipeline.",
"FIG.",
"9 shows that as the dynamic feedback controller is active there is a significant decrease of the swings in pressure.",
"Due to slugging in the wells connected to the platforms the controller uses the control valve to keep the pipeline pressure PT1 and PT2 within bounds.",
"The pipeline inlet flow is shown in FIG.",
"10.",
"(Remark: the missing flow measurements at the beginning of the plotted trend are due to the fact that the wells were produced bypassed the test-separator at the platform.)",
"The mean inlet gas flow 70 is shown in the upper part of FIG.",
"10.The mean inlet liquid flow is shown in the lower part of FIG.",
"10.As shown in FIG.",
"10, the input flow rates vary.",
"These variations represent quite large disturbances, to the dynamic feedback controller.",
"Even though there are large disturbances the dynamic feedback controller significantly reduces the slugging.",
"It is to be noted that the invention described above and shown in the drawings is to be regarded as a non-limiting example of the invention and that the scope of protection is defined by the patent claims."
]
] | Patent_10433653 |
[
[
"System and method for the management of genomic data",
"A system and method is disclosed for managing users' genomic data, including providing and offering access to genomic-based services, routing genomic data to providers of genomic-based services, brokering financial transactions related to the management of genomic data, securing for users best prices for genomic-based services, allowing users to earn money for the use of their genomic and other data, and using genomic data for marketing and developing products in particular geographic regions or for particular populations."
],
[
"1.A method for recruiting a new user for a genome management service, comprising: obtaining a cell sample from a person; waiting a period of time; after the period of time has elapsed, seeking from the person final permission to have his or her genomic data managed; analyzing at least a portion of the person's genome; and storing the resultant genomic data electronically.",
"2.The method of claim 1 wherein said cell sample is obtained via a cheek swab.",
"3.The method of claim 1 wherein said sample is obtained in a mobile unit.",
"4.The method of claim 1 wherein said sample is obtained in a kiosk.",
"5.The method of claim 1 wherein said sample is obtained in a physician's office.",
"6.The method of claim 1 wherein said period of time is one week.",
"7.The method of claim 1 wherein the resultant genomic data is stored on a data card.",
"8.The method of claim 7 wherein said data card is kept by the user.",
"9.The method of claim 7 wherein said data card is the only location where said genomic data is stored.",
"10.The method of claim 1 wherein the genomic data is stored on a secure server.",
"11.A device for maintaining an individual's genomic data, comprising: a data storage unit in which the individual's genomic data is stored; and a self destruct unit, which deletes said data on said device when a trigger event occurs.",
"12.The device of claim 11, wherein said trigger event is an attempt to copy the data stored on said device.",
"13.The device of claim 11, wherein said trigger event is an unauthorized attempt to read data from the device.",
"14.The device of claim 11, wherein said data is stored in an encrypted format.",
"15.The device of claim 11, wherein said data storage device is kept by the individual.",
"16.A data card for maintaining an individual's genomic data, comprising a data storage unit in which the individual's genomic data is stored.",
"17.The data card of claim 16, further comprising a self-destruct unit, which deletes said data on said device when a trigger event occurs.",
"18.The data card of claim 17, wherein said trigger event is an attempt to copy the data stored on said device.",
"19.The data card of claim 17, wherein said trigger event is an unauthorized attempt to read data from the card.",
"20.The data card of claim 16, wherein said data is stored in an encrypted format.",
"21.The data card of claim 16, wherein said data card is kept by the individual.",
"22.A method for providing product usage advice for an individual, comprising: receiving the individual's genomic data; using said genomic data to consult a database or table, the database or table correlating genomic data with responses to products; and creating a report containing product usage advice for one or more products.",
"23.The method of claim 22, wherein said correlations are obtained by using a computer program.",
"24.The method of claim 22, wherein said receiving further includes receiving any necessary additional information.",
"25.The method of claim 22, wherein said using step further includes consulting the database or table using additional information.",
"26.The method of claim 22, wherein said database or table further correlates additional information with responses to products.",
"27.The method of claim 22, further including the step of updating said report when said product is purchased.",
"28.The method of claim 22, wherein said receiving is performed in conjunction with a point of sale operation.",
"29.The method of claim 28, wherein said point of sale operation is a transaction using a data card.",
"30.The method of claim 28, wherein said point of sale operation is a transaction using a cash register.",
"31.The method of claim 28, wherein said point of sale operation is an online purchase.",
"32.The method of claim 22, wherein said product usage advice is a prediction of the individual's response to one or more products.",
"33.The method of claim 22, wherein said product usage advice is a dosage recommendation for one or more products.",
"34.The method of claim 22, wherein said product usage advice is a prediction of side effects for one or more products.",
"35.The method of claim 24, wherein said additional information includes a proposed usage suggestion for one or more products.",
"36.The method of claim 35, wherein said product usage advice is a prediction of the individual's response to the proposed product or products.",
"37.The method of claim 35, wherein product usage advice is a recommendation of one or more alternative products.",
"38.The method of claim 22 wherein said report is provided to the individual.",
"39.The method of claim 22 wherein said report is provided to a healthcare provided authorized by the individual.",
"40.The method of claim 22 wherein said report is provided to an expert assisting the individual, but the individual's genomic data is not.",
"41.The method of claim 22 wherein a fee is charged for each report created.",
"42.The method of claim 22, wherein said database or table correlates the individual's genomic data with a response to certain drugs.",
"43.The method of claim 22, additionally including the steps of: receiving feedback concerning said individual's actual response to one or more of said products; and updating said database or table based on said feedback.",
"44.A method for producing marketing data, comprising: receiving from a group of individuals their genomic data; receiving from said group of individuals data concerning their purchasing or consumption habits; determining correlations between said genomic data and said purchasing or consumption habits; and making a prediction concerning an individual's purchasing or consumption habits based on that individual's genomic data.",
"45.The method of claim 44, wherein said correlations are stored in a database or table.",
"46.The method of claim 44, wherein said correlations are obtained by using a computer program.",
"47.The method of claim 44, wherein said correlations are statistical.",
"48.The method of claim 44, wherein the said correlations contain no personally-identifying data related to said individuals.",
"49.The method of claim 44, with the additional step of selling said correlations to interested parties.",
"50.The method of claim 44, wherein members of said group are paid for their participation.",
"51.The method of claim 44, wherein said data concerning purchasing habits is received when a data card is used to make a purchase.",
"52.The method of claim 44, additionally including the steps of: receiving feedback relating to the accuracy of said prediction and/or correlations; and updating said prediction and/or correlations based on said feedback.",
"53.A method for marketing products to individuals based on their genomic data, comprising: receiving from a group of individuals their genomic data; receiving from said group of individuals data concerning their purchasing or consumption habits; determining correlations between said genomic data and said purchasing or consumption habits; making a prediction concerning an individual's purchasing or consumption habits based on that individual's genomic data; and making a product suggestion.",
"54.The method of claim 53, wherein said correlations are stored in a database or table.",
"55.The method of claim 53, wherein said correlations are obtained by using a computer program.",
"56.The method of claim 53, wherein said correlations are statistical.",
"57.The method of claim 53, wherein the said correlations contain no personally-identifying data related to said individuals.",
"58.The method of claim 53, with the additional step of selling said correlations to interested parties.",
"59.The method of claim 53, with the additional step of selling said product suggestions to interested parties.",
"60.The method of claim 53, with the additional step of offering said product suggestions to said individuals.",
"61.The method of claim 53, wherein members of said group are paid for their participation.",
"62.The method of claim 53, wherein said data concerning purchasing habits is received when a data card is used to make a purchase.",
"63.The method of claim 53, additionally including the steps of: receiving feedback relating to the appeal of the suggested product; and updating said suggestion and/or correlations based on said feedback.",
"64.A method of providing a gaming experience to an individual based on his or her genomic data, comprising: receiving the genomic data of said individual; and affecting gameplay using said genomic data; whereby the individual's gaming experience is due at least in part to his or her genomic data.",
"65.The method of claim 64, wherein said affecting involves assigning the individual to a team.",
"66.The method of claim 64, wherein said affecting involves the manipulation of visual gameplay aspects.",
"67.The method of claim 64, wherein said affecting involves the manipulation of aural gameplay aspects.",
"68.The method of claim 64, wherein said affecting involves giving game characters strengths or weaknesses.",
"69.The method of claim 64, additionally including the steps of: receiving feedback relating to said gaming experience; and revising said affecting based on said feedback.",
"70.A method of providing an individual with lifestyle advice related to his or her genomic data, comprising: using an individual's genomic data to consult a database or table which correlates genomic data with lifestyle advice; and receiving, as a result of said consultation, said lifestyle advice.",
"71.The method of claim 70 wherein said correlations are obtained using a computer program.",
"72.The method of claim 70 wherein said genomic data is haplotypes or haplotype pairs.",
"73.The method of claim 70 wherein said lifestyle advice comprises recommendations on taking preventative steps against the onset of an illness.",
"74.The method of claim 70 wherein said lifestyle advice comprises diet recommendations.",
"75.The method of claim 70 wherein said lifestyle advice comprises exercise, recommendations.",
"76.The method of claim 70 wherein said lifestyle advice comprises information about unique genotypical aspects of the individual.",
"77.The method of claim 70, additionally including the step of providing the services of a genetic counselor to explain said lifestyle information.",
"78.The method of claim 70, additionally including the steps of: receiving feedback relating to the accuracy of said lifestyle advice; and updating said database or table based on said feedback.",
"79.A method of providing an individual with lifestyle advice related to his or her genomic data, comprising: using an individual's genomic data to consult a database which correlates genomic data with information related to that genomic data; receiving, as a result of said consultation, information related to said genomic data; and providing lifestyle advice related to said information.",
"80.The method of claim 79 wherein said correlations are obtained using a computer program.",
"81.The method of claim 79 wherein said genomic data is haplotypes or haplotype pairs.",
"82.The method of claim 79 wherein said lifestyle advice comprises recommendations on taking preventative steps against the onset of an illness.",
"83.The method of claim 79 wherein said lifestyle advice comprises diet recommendations.",
"84.The method of claim 79 wherein said lifestyle advice comprises exercise recommendations.",
"85.The method of claim 79 wherein said lifestyle advice comprises information about unique genotypical aspects of the individual.",
"86.The method of claim 79, additionally including the step of providing the services of a genetic counselor to explain said lifestyle information.",
"87.The method of claim 79, additionally including the steps of: receiving feedback relating to the accuracy of said lifestyle advice; and updating said advice and/or said database or table based on said feedback.",
"88.A method of designing products based on an individual's genomic data, comprising: obtaining the individual's genomic data; and creating a design for said product based on said genomic data.",
"89.The method of claim 88 wherein said creating involves consulting a database or table which correlates certain genomic data with certain designs.",
"90.The method of claim 89, additionally including the steps of: receiving feedback relating to the appeal of said design; and updating said database or table based on said feedback.",
"91.The method of claim 88 wherein said creating involves using a computer program which correlates certain genomic data with certain designs.",
"92.The method of claim 91, additionally including the steps of: receiving feedback relating to the appeal of said design; and updating said computer program based on said feedback.",
"93.The method of claim 88 wherein said creating involves executing a design algorithm which takes said genomic data as an input.",
"94.The method of claim 93, additionally including the steps of: receiving feedback relating to the appeal of said design; and updating said algorithm based on said feedback.",
"95.The method of claim 88 wherein said product is a food.",
"96.The method of claim 88 wherein said product is artwork.",
"97.The method of claim 88 wherein said product is wearing apparel.",
"98.The method of claim 88 wherein said product is a perfume.",
"99.The method of claim 88 wherein said product is jewelry.",
"100.The method of claim 88 wherein said product is music.",
"101.A method for marketing an individual's genomic data, comprising: contacting a party interested in using an individual's genomic data; negotiating with the party to determine the terms of use for said data; seeking the individual's consent to allow said party to use said data under the determined terms of use; and if consent is received, providing, under the determined terms of use, said genomic data to said party.",
"102.The method of claim 101 wherein said providing is performed in such a manner that the party is not allowed to permanently keep said genomic data.",
"103.The method of claim 101 wherein said negotiations involves determining a price that said party will pay said individual for use of said genomic data.",
"104.The method of claim 101 wherein said negotiations involves determining the portions of the individual's genomic data that will be used by the party.",
"105.A method for providing an individual with low price genomic-based services, comprising: receiving from the individual a request for a genomic-based service; negotiating with a plurality of parties capable of providing said service in order to determine which party of said parties is willing to offer said service at a lower price than the remainder of said parties; and upon receiving the individual's consent, allowing said party which offered said lower price to perform said service.",
"106.The method of claim 105 wherein said service is performing a medical test based on said individual's genomic data.",
"107.The method of claim 105 wherein said service is providing information based on said individual's genomic data.",
"108.The method of claim 105 wherein said service is providing artwork whose design is based on said individual's genomic data.",
"109.The method of claim 105, including the additional step of charging the individual a fee.",
"110.The method of claim 105, including the additional step of charging said service provider a fee.",
"111.The method of claim 105, wherein said negotiating step includes considering the quality of the providers.",
"112.The method of claim 111, wherein said receiving step further includes receiving from the individual quality requirements.",
"113.The method of claim 111, wherein the management company sets quality requirements.",
"114.A billing method for a genomic data managing service, comprising: charging a management fee; and charging a fee for each access of said data.",
"115.The method of claim 114, wherein said management fee is a periodic fee for maintaining said data.",
"116.The method of claim 115 wherein said periodic fee is a fee charged each time a predetermined interval elapses.",
"117.The method of claim 114, wherein said management fee is a fee for setting up a new account.",
"118.The method of claim 114, wherein said management fee is a fee for adding or deleting genomic data.",
"119.The method of claim 114, wherein said management fee is a fee for adding or deleting non-genomic data.",
"120.A method for providing an individual's genomic data to a party, comprising: receiving from a party a request for an individual's genomic data; negotiating with the party to determine the terms of use for said data; seeking the individual's consent to allow said party to use said data under the determined terms of use; and if consent is received, providing, under the determined terms of use, said genomic data to said party.",
"121.The method of claim 120 wherein said providing is performed in such a manner that the party is not allowed to hold or posses said genomic data.",
"122.The method of claim 120 wherein said negotiating involves determining a price that said party will pay said individual for use of said genomic data.",
"123.The method of claim 120 wherein said negotiating involves determining which portions of the individual's genomic data will be used by the party.",
"124.A method for securely transmitting an individual's genomic data to a party, comprising: storing an individual's genomic data on a data card; and physically transporting said data card to said party.",
"125.The method of claim 124, wherein said data card deletes the data it carries when a trigger event occurs.",
"126.The method of claim 125 wherein said trigger event is an attempt to read the data on the card using an incorrect decryption key.",
"127.The method of claim 125 wherein said trigger event is an attempt to use the data for a purpose other than the one agreed upon.",
"128.The method of claim 125 wherein said trigger event is the expiration of a count-down timer.",
"129.The method of claim 125 wherein said trigger event is said party failing to acknowledge receipt of said data.",
"130.The method of claim 124 wherein said data is stored in an encrypted manner.",
"131.A method for securely transmitting an individual's genomic data to a party, comprising: creating a data package, said data package containing the individual's genomic data; and allowing said party to download said package over a network.",
"132.The method of claim 131 wherein said package deletes the data it carries when a trigger event occurs.",
"133.The method of claim 132 wherein said trigger event is an attempt to read the data in the package using an incorrect decryption key.",
"134.The method of claim 132 wherein said trigger event is an attempt to use the data for a purpose other than the one agreed upon.",
"135.The method of claim 132 wherein said trigger event is the expiration of a count-down timer.",
"136.The method of claim 132 wherein said trigger event is said party failing to acknowledge receipt of said data.",
"137.The method of claim 131 wherein said package contains said data in an encrypted format.",
"138.The method of claim 131, wherein the party is selected from the group consisting of the individual, the individual's physician, the individual's genetic counselor, the individual's hospital, the individual's physician's office, the individual's pharmacy and the individual's pharmacist.",
"139.A system for providing product usage advice for an individual, comprising: a memory having program code stored therein; a database or table correlating genomic data with responses to products; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: receiving the individual's genomic data; using said genomic data to consult the database or table; and creating a report containing product usage advice for one or more products.",
"140.The system of claim 139 wherein said correlations are obtained by using a computer program.",
"141.The system of claim 139, wherein said receiving further includes receiving any necessary additional information.",
"142.The system of claim 139, wherein said using step further includes consulting the database or table using additional information.",
"143.The system of claim 139, wherein said database or table further correlates additional information with responses to products.",
"144.The system of claim 139, further including the step of updating said report when said product is purchased.",
"145.The system of claim 139, wherein said receiving is performed in conjunction with a point of sale operation.",
"146.The system of claim 145, wherein said point of sale operation is a transaction using a data card.",
"147.The system of claim 145, wherein said point of sale operation is a transaction using a cash register.",
"148.The system of claim 145, wherein said point of sale operation is an online purchase.",
"149.The system of claim 139, wherein said product usage advice is a prediction of the individual's response to one or more products.",
"150.The system of claim 139, wherein said product usage advice is a dosage recommendation for one or more products.",
"151.The system of claim 139, wherein said product usage advice is a prediction of side effects for one or more products.",
"152.The system of claim 141, wherein said additional information includes a proposed usage suggestion for one or more products.",
"153.The system of claim 152, wherein said product usage advice is a prediction of the individual's response to the proposed product or products.",
"154.The system of claim 152, wherein product usage advice is a recommendation of one or more alternative products.",
"155.The system of claim 139 wherein said report is provided to the individual.",
"156.The system of claim 139 wherein said report is provided to an expert assisting the individual, but the individual's genomic data is not.",
"157.The system of claim 139 wherein a fee is charged for each report created.",
"158.The system of claim 139, wherein said database or table correlates haplotypes or haplotype pairs with a response to certain drugs.",
"159.The system of claim 139, additionally including the steps of: receiving feedback concerning said individual's actual response to one or more of said products; and updating said database or table based on said feedback.",
"160.A system for producing marketing data, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: receiving from a group of individuals their genomic data; receiving from said group of individuals data concerning their purchasing habits; determining correlations between said genomic data and said purchasing habits; and making a prediction concerning an individual's purchasing habits based on that individual's genomic data.",
"161.The system of claim 160 wherein said correlations are stored in a database or table.",
"162.The system of claim 160 wherein said correlations are obtained by using a computer program.",
"163.The system of claim 160, wherein said correlations are statistical.",
"164.The system of claim 160, wherein the said correlations contain no personally-identifying data related to said individuals.",
"165.The system of claim 160, with the additional step of selling said correlations to interested parties.",
"166.The system of claim 160, wherein members of said group are paid for their participation.",
"167.The system of claim 160, wherein said data concerning purchasing habits is received when a data card is used to make a purchase.",
"168.The system of claim 160, additionally including the steps of: receiving feedback relating to the accuracy of said prediction and/or correlations; and updating said prediction and/or correlations based on said feedback.",
"169.A system for marketing products to individuals based on their genomic data, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: receiving from a group of individuals their genomic data; receiving from said group of individuals data concerning their purchasing habits; determining correlations between said genomic data and said purchasing habits; making a prediction concerning an individual's purchasing habits based on that individual's genomic data; and making a product suggestion.",
"170.The system of claim 169 wherein said correlations are stored in a database or table.",
"171.The system of claim 169 wherein said correlations are obtained by using a computer program.",
"172.The system of claim 169, wherein said correlations are statistical.",
"173.The system of claim 169, wherein the said correlations contain no personally-identifying data related to said individuals.",
"174.The system of claim 169, with the additional step of selling said correlations to interested parties.",
"175.The system of claim 169, with the additional step of selling said product suggestions to interested parties.",
"176.The system of claim 169, with the additional step of offering said product suggestions to said individuals.",
"177.The system of claim 169, wherein members of said group are paid for their participation.",
"178.The system of claim 169, wherein said data concerning purchasing habits is received when a data card is used to make a purchase.",
"179.The system of claim 169, additionally including the steps of: receiving feedback relating to the appeal of the suggested product; and updating said suggestion and/or correlations based on said feedback.",
"180.A system for providing a gaming experience to an individual based on his or her genomic data, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: receiving the genomic data of said individual; and affecting gameplay using said genomic data; whereby the individual's gaming experience is due at least in part to his or her genomic data.",
"181.The system of claim 180, wherein said affecting involves assigning the individual to a team.",
"182.The system of claim 180, wherein said affecting involves the manipulation of visual gameplay aspects.",
"183.The system of claim 180, wherein said affecting involves the manipulation of aural gameplay aspects.",
"184.The system of claim 180, wherein said affecting involves giving game characters strengths or weaknesses.",
"185.The system of claim 180, additionally including the steps of: receiving feedback relating to said gaming experience; and revising said affecting based on said feedback.",
"186.A system for providing an individual with lifestyle advice related to his or her genomic data, comprising: a memory having program code stored therein; a database or table correlating genomic data with lifestyle advice; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: using the individual's genomic data to consult the database or table; and receiving, as a result of said consultation, said lifestyle advice.",
"187.The system of claim 186, wherein said correlations are obtained using a computer program.",
"188.The system of claim 186, wherein said genomic data is haplotypes or haplotype pairs.",
"189.The system of claim 186, wherein said lifestyle advice comprises recommendations on taking preventative steps against the onset of an illness.",
"190.The system of claim 186, wherein said lifestyle advice comprises diet recommendations.",
"191.The system of claim 186, wherein said lifestyle advice comprises exercise recommendations.",
"192.The system of claim 186, wherein said lifestyle advice comprises information about unique genotypical aspects of the individual.",
"193.The system of claim 186, additionally including the step of providing the services of a genetic counselor to explain said lifestyle information.",
"194.The system of claim 186, additionally including the steps of: receiving feedback relating to the accuracy of said lifestyle advice; and updating said database or table based on said feedback.",
"195.A system for providing an individual with lifestyle advice related to his or her genomic data, comprising: a memory having program code stored therein; a database or table which correlates genomic data with information related to that genomic data; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: using the individual's genomic data to consult the database or table; receiving, as a result of said consultation, information related to said genomic data; and providing lifestyle advice related to said information.",
"196.The system of claim 195, wherein said correlations are obtained using a computer program 197.The system of claim 195, wherein said genomic data is haplotypes or haplotype pairs.",
"198.The system of claim 195, wherein said lifestyle advice comprises recommendations on taking preventative steps against the onset of an illness.",
"199.The system of claim 195, wherein said lifestyle advice comprises diet recommendations.",
"200.The system of claim 195, wherein said lifestyle advice comprises exercise recommendations.",
"201.The system of claim 195, wherein said lifestyle advice comprises information about unique genotypical aspects of the individual.",
"202.The system of claim 195, additionally including the step of providing the services of a genetic counselor to explain said lifestyle information.",
"203.The system of claim 195, additionally including the steps of: receiving feedback relating to the accuracy of said lifestyle advice; and updating said advice and/or said database or table based on said feedback.",
"204.A system for designing products based on an individual's genomic data, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: obtaining the individual's genomic data; and creating a design for said product based on said genomic data.",
"205.The system of claim 204, wherein said creating involves consulting a database or table which correlates certain genomic data with certain designs.",
"206.The system of claim 205, additionally including the steps of: receiving feedback relating to the appeal of said design; and updating said database or table based on said feedback.",
"207.The system of claim 204, wherein said creating involves using a computer program which correlates certain genomic data with certain designs.",
"208.The system of claim 207, additionally including the steps of: receiving feedback relating to the appeal of said design; and updating said computer program based on said feedback.",
"209.The system of claim 204, wherein said creating involves executing a design algorithm which takes said genomic data as an input.",
"210.The system of claim 209, additionally including the steps of: receiving feedback relating to the appeal of said design; and updating said algorithm based on said feedback.",
"211.The system of claim 204, wherein said product is a food.",
"212.The system of claim 204, wherein said product is artwork.",
"213.The system of claim 204, wherein said product is wearing apparel.",
"214.The system of claim 204, wherein said product is a perfume.",
"215.The system of claim 204, wherein said product is jewelry.",
"216.The system of claim 204, wherein said product is music.",
"217.A system for marketing an individual's genomic data, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: contacting a party interested in using an individual's genomic data; negotiating with the party to determine the terms of use for said data; seeking the individual's consent to allow said party to use said data under the determined terms of use; and if consent is received, providing, under the determined terms of use, said genomic data to said party.",
"218.The system of claim 217 wherein said providing is performed in such a manner that the party is not allowed to permanently keep said genomic data.",
"219.The system of claim 217 wherein said negotiations involves determining a price that said party will pay said individual for use of said genomic data.",
"220.The system of claim 217 wherein said negotiations involves determining the portions of the individual's genomic data that will be used by the party.",
"221.A system for providing an individual with low price genomic-based services, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: receiving from the individual a request for a genomic-based service; negotiating with a plurality of parties capable of providing said service in order to determine which party of said parties is willing to offer said service at a lower price than the remainder of said parties; and upon receiving the individual's consent, allowing said party which offered said lower price to perform said service.",
"222.The system of claim 221 wherein said service is performing a medical test based on said individual's genomic data.",
"223.The system of claim 221 wherein said service is providing information based on said individual's genomic data.",
"224.The system of claim 221 wherein said service is providing artwork whose design is based on said individual's genomic data.",
"225.The system of claim 221, including the additional step of charging the individual a fee.",
"226.The system of claim 221, including the additional step of charging said service provider a fee.",
"227.The system of claim 221, wherein said negotiating step includes considering the quality of the providers.",
"228.The system of claim 227, wherein said receiving step further includes receiving from the individual quality requirements.",
"229.The system of claim 227, wherein the management company sets quality requirements.",
"230.A billing system for a genomic data managing service, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: charging a management fee; and charging a fee for each access of said data.",
"231.The system of claim 230, wherein said management fee is a periodic fee for maintaining said data.",
"232.The system of claim 231, wherein said periodic fee is a fee charged each time a predetermined interval elapses.",
"233.The system of claim 230, wherein said management fee is a fee for setting up a new account.",
"234.The system of claim 230, wherein said management fee is a fee for adding or deleting genomic data.",
"235.The system of claim 230, wherein said management fee is a fee for adding or deleting non-genomic data.",
"236.A system for providing an individual's genomic data to a party, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: receiving from a party a request for an individual's genomic data; negotiating with the party to determine the terms of use for said data; seeking the individual's consent to allow said party to use said data under the determined terms of use; and if consent is received, providing, under the determined terms of use, said genomic data to said party.",
"237.The system of claim 236, wherein said providing is performed in such a manner that the party is not allowed to hold or posses said genomic data.",
"238.The system of claim 236, wherein said negotiating involves determining a price that said party will pay said individual for 108 of said genomic data.",
"239.The system of claim 236, wherein said negotiating involves determining which portions of the individual's genomic data will be used by the party.",
"240.A system for securely transmitting an individual's genomic data to a party, comprising: a memory having program code stored therein; a data card interface; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: storing an individual's genomic data on a data card; and arranging for the physical transport of said data card to said party.",
"241.The system of claim 240, wherein said data card deletes the data it carries when a trigger event occurs.",
"242.The system of claim 241, wherein said trigger event is an attempt to read the data on the card using an incorrect decryption key.",
"243.The system of claim 241, wherein said trigger event is an attempt to use the data for a purpose other than the one agreed upon.",
"244.The system of claim 241, wherein said trigger event is the expiration of a count-down timer.",
"245.The system of claim 241, wherein said trigger event is said party failing to acknowledge receipt of said data.",
"246.The system of claim 241, wherein said data is stored in an encrypted manner.",
"247.A system for securely transmitting an individual's genomic data to a party, comprising: a memory having program code stored therein; and a processor connected to said memory for carrying out instructions in accordance with said stored program code; wherein said program code, when executed by said processor, causes said processor to perform the steps of: creating a data package, said data package containing the individual's genomic data; and allowing said party to download said package over a network.",
"248.The system of claim 247, wherein said package deletes the data it carries when a trigger event occurs.",
"249.The system of claim 248, wherein said trigger event is an attempt to read the data in the package using an incorrect decryption key.",
"250.The system of claim 248, wherein said trigger event is an attempt to use the data for a purpose other than the one agreed upon.",
"251.The system of claim 248, wherein said trigger event is the expiration of a count-down timer.",
"252.The system of claim 248, wherein said trigger event is said party failing to acknowledge receipt of said data.",
"253.The system of claim 247, wherein said package contains said data in an encrypted format.",
"254.A method for reimbursing a physician for the care of a patient comprising the steps of: determining whether the physician prescribed a drug that the management company recommended for the patient based on the patient's therapeutic needs and the patient's genomic data; and reimbursing the physician if the physician prescribed a recommended drug to the patient.",
"255.A method of marketing a product in a geographic region of interest, comprising: obtaining information relating to correlations between users' response to the product and a haplotype profile; determining the frequency of the haplotype profile in the population living in the geographic region; and making a marketing decision for the geographic region based on the determined frequency of the haplotype profile.",
"256.The method of claim 255, wherein the product is a drug or biologic.",
"257.The method of claim 256, wherein the marketing decision is to proceed with marketing the product if the determined frequency of the haplotype profile is at least 25%.",
"258.The method of claim 256, wherein the marketing decision is to proceed with marketing the product if the determined frequency of the haplotype profile is at least 50%.",
"259.The method of claim 256, wherein the geographic region is a state or territory of the United States of America.",
"260.The method of claim 256, wherein the geographic region is a country.",
"261.A method for developing a new product to satisfy a particular unmet demand or need of a population, comprising: identifying a haplotype profile that is correlated with the unmet demand or need in the population; determining a functional cause for the correlation between the haplotype profile and the unmet need or demand; and developing a new product designed to avoid the functional cause.",
"262.The method of claim 261, wherein the unmet demand or need is weight management.",
"263.The method of claim 261, wherein the unmet demand or need is addiction to smoking.",
"264.The method of claim 261, wherein the unmet demand or need is addiction to alcohol.",
"265.The method of claim 261, wherein the unmet demand or need is a treatment for schizophrenia.",
"266.The method of claim 261, wherein the unmet demand or need is a treatment for dyslipidemia.",
"267.The method of claim 261, wherein the unmet demand or need is a treatment for diabetes.",
"268.A method for marketing a drug for inclusion in a formulary, comprising: identifying a haplotype profile that is correlated with a good therapeutic profile for the drug; determining the frequency of the haplotype profile in the population served by the formulary; and making a marketing decision based on the determined frequency of the haplotype profile.",
"269.The method of claim 168, wherein the marketing decision is to pursue inclusion in the formulary if the determined frequency of the haplotype profile is at least 25%.",
"270.A method for choosing a drug for inclusion in a formulary, comprising: identifying a group of drugs that are prescribed to treat or alleviate the same medical condition, symptoms or disease; obtaining for each drug a haplotype profile that is correlated with an acceptable therapeutic response profile for that drug; and determining in the population served by the formulary the frequency of each obtained haplotype profile; and chosing a drug for the formulary based on the determined haplotype profile frequencies.",
"271.The method of claim 271, wherein the chosing step comprises selecting the drug whose correlated haplotype profile has the highest frequency.",
"272.The method of claim 271, wherein the chosing step comprises selecting each drug whose correlated haplotype profile has a frequency greater than 25%."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Recent advances in the understanding of the human genome portend great potential benefit to the population at large.",
"It is known, for example, that there are genetic markers that indicate susceptibility to certain diseases.",
"If an individual learns of such a susceptibility through genetic testing, she may be able to alter her lifestyle to prevent or delay the disease's onset, or to ameliorate its effects.",
"Genomic analysis can also be used to allow a couple to make an informed reproductive decision, by determining the likelihood of children of that couple inheriting a genetic disease.",
"Genetic variation among individuals has also been found to be relevant to their responses to pharmaceuticals.",
"Correlations have been found between certain genetic markers, such as haplotypes, and responses to drugs.",
"If such correlations were used to produce genetic-based prescribing information, then prescriptions could be written with an individual's genetic makeup in mind.",
"This could improve individuals' lifestyles by lessening side-effects and increasing efficacy.",
"Non-medical uses of genomic data have also been found.",
"For example, certain manufacturers of candy and cosmetics have become interested in how genetic diversity accounts for people's varying perceptions of taste and smell.",
"This has the potential of allowing a person to purchase candy that is particularly appealing to her genetically-determined sense of taste.",
"However, despite the great potential benefits of doing so, few individuals have taken advantage of genomic-based services.",
"One reason for this is the public's concern for the security and privacy of its genomic data.",
"People fear, for example, that they could be denied employment, denied insurance, and otherwise discriminated against if the details of their genomic makeup became public.",
"Another reason is convenience.",
"With emerging Internet and communications technologies, people are used to being able to get information quickly and with little inconvenience.",
"However, genomic-based services such as genetic testing have been heretofore inconvenient to use.",
"For example, an individual may have to travel to a distant location for a test.",
"If several tests performed at different locations were required, an individual would likely have to give a genetic sample to each location.",
"Similarly, genomic-based prescription information is not available to patients and medical professionals in such a way that it can be unobtrusively incorporated into the average medical office or pharmacy.",
"Further, genomic-based services, such as tests for disease susceptibility, can be expensive.",
"Individuals are used to enjoying low prices for products and services due to competition and the assistance of Internet services.",
"However, such price-lowering has not yet come to genomic-based services.",
"Thus, for at least these reasons, advances in genomic knowledge have fallen short of realizing their potential benefits to the population."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the invention features a system and method for managing users' genomic data.",
"Therefore, one object of the invention is to provide a system and method for providing and offering access to genomic-based services.",
"Another object of the invention is to provide a system and method for routing genomic data to providers of genomic-based services.",
"Still another object of the invention is to provide a system and method for brokering financial transactions related to the management of genomic data.",
"A further object of the invention is to provide a system and method for securing a user a best price for a genomic-based service.",
"Still another object of the invention is to provide a system and method for allowing users to earn money for the use of their genomic and other data.",
"Still further objects of the invention are to provide a system and method for using genomic data to market a product in a geographic region of interest, and a system and method of using genomic data in developing new products to satisfy unmet demands or needs of a population.",
"Accordingly, in one embodiment the invention concerns a method for recruiting a new user for a genome management service, comprising obtaining a cell sample from a person, waiting a period of time, after the period of time has elapsed seeking from the person final permission to have his or her genomic data managed, analyzing at least a portion of the person's genome, and storing the resultant genomic data electronically.",
"In a second embodiment the invention concerns a method for maintaining an individual's genomic data, comprising a data storage unit in which the individual's genomic data is stored and a self-destruct unit, which deletes the data on the device when a trigger event occurs.",
"In a third embodiment the invention concerns a data card for maintaining an individual's genomic data, comprising a data storage unit in which the individual's genomic data is stored.",
"In a fourth embodiment the invention concerns a method for providing product usage advice for an individual, comprising receiving the individual's genomic data, using the genomic data to consult a database or table which correlates genomic data with responses to products, and creating a report containing product usage advice for one or more products.",
"In a fifth embodiment the invention concerns a method for producing marketing data, comprising receiving from a group of individuals their genomic data, receiving from the group of individuals data concerning their purchasing habits, determining correlations between the genomic data and the purchasing habits, and making a prediction concerning an individual's purchasing habits based on that individual's genomic data.",
"In another embodiment the invention concerns a method for marketing products to individuals based on their genomic data, comprising receiving from a group of individuals their genomic data, receiving from the group of individuals data concerning their purchasing habits, determining correlations between the genomic data and the purchasing habits, making a prediction concerning an individual's purchasing habits based on that individual's genomic data, and making a product suggestion.",
"In an additional embodiment the invention concerns a method of providing an individual with lifestyle advice related to his or her genomic data, comprising using an individual's genomic data to consult a database or table which correlates genomic data with information related to the genomic data, receiving, as a result of the consultation, information related to the genomic data, and providing lifestyle advice related to the information.",
"In yet another embodiment, the invention concerns a method of marketing a product in a geographic region of interest, comprising obtaining information relating to correlations between users' response to the product and a haplotype profile, determining the frequency of the haplotype profile in the population living in the geographic region, and making a marketing decision for the geographic region based on the determined frequency of the haplotype profile.",
"In yet another embodiment, the invention provides a method for developing a new product to satisfy a particular unmet demand or need of a population, comprising identifying a haplotype profile that is correlated with the unmet demand or need in the population, determining a functional cause for the correlation between the haplotype profile and the unmet need or demand, and developing a new product designed to avoid the functional cause.",
"In still another embodiment the invention concerns a method of providing a gaming experience to an individual based on his or her genomic data, comprising receiving the genomic data of the individual and affecting gameplay using the genomic data, whereby the individual's gaming experience is due at least in part to his or her genomic data.",
"In a further embodiment the invention concerns a method of designing products based on an individual's genomic data, comprising obtaining the individual's genomic data and creating a design for the product based on the genomic data.",
"In another embodiment the invention concerns a method for marketing an individual's genomic data, comprising contacting a party interested in using an individual's genomic data, negotiating with the party to determine the terms of use for the data, seeking the individual's consent to allow the party to use the data under the determined terms of use, and if consent is received, providing, under the determined terms of use, the genomic data to the party.",
"In still another embodiment the invention concerns a method for providing an individual with low price genomic-based services, comprising receiving from the individual or his or her representative a request for a genomic-based service, negotiating with a plurality of parties capable of providing the service in order to determine which party of the parties is willing to offer the service at a lower price than the remainder of the parties, and upon receiving the individual's or representative's consent, allowing the party which offered the lower price to perform the service.",
"In a further embodiment the invention concerns a billing method for a genomic data managing service, comprising charging a management fee and charging a fee for each access or update of the data.",
"In an additional embodiment the invention concerns a method for providing an individual's genomic data, comprising receiving from a party a request for an individual's genomic data, negotiating with the party to determine the terms of use for the data, seeking the individual's consent to allow the party to use the data under the determined terms of use, and, if consent is received, providing, under the determined terms of use, the genomic data to the party.",
"Another embodiment of the invention concerns a method for securely transmitting an individual's genomic data to a party, comprising storing an individual's genomic data on a data card and physically transporting the data card to the party.",
"Still another embodiment of the invention concerns a method for securely transmitting an individual's genomic data to a party, comprising creating one or more data packages containing the individual's genomic data and allowing the party to download the package over a network.",
"A further embodiment of the invention concerns a method for allowing a user to make use of his or her genomic data, comprising receiving from the user a request for an operation he or she wishes to be performed making use of his or her genomic data and performing the operation.",
"The scope of the invention should not be considered as being limited by these objects and embodiments.",
"Additional aspects, objects, and embodiments will become clear upon a reading of the disclosure and the claims that follow it."
],
[
"FIELD OF THE INVENTION This invention relates to systems and methods for the management of genomic data and to the use of genomic data in developing and marketing products and services to consumers and the healthcare industry.",
"BACKGROUND OF THE INVENTION Recent advances in the understanding of the human genome portend great potential benefit to the population at large.",
"It is known, for example, that there are genetic markers that indicate susceptibility to certain diseases.",
"If an individual learns of such a susceptibility through genetic testing, she may be able to alter her lifestyle to prevent or delay the disease's onset, or to ameliorate its effects.",
"Genomic analysis can also be used to allow a couple to make an informed reproductive decision, by determining the likelihood of children of that couple inheriting a genetic disease.",
"Genetic variation among individuals has also been found to be relevant to their responses to pharmaceuticals.",
"Correlations have been found between certain genetic markers, such as haplotypes, and responses to drugs.",
"If such correlations were used to produce genetic-based prescribing information, then prescriptions could be written with an individual's genetic makeup in mind.",
"This could improve individuals' lifestyles by lessening side-effects and increasing efficacy.",
"Non-medical uses of genomic data have also been found.",
"For example, certain manufacturers of candy and cosmetics have become interested in how genetic diversity accounts for people's varying perceptions of taste and smell.",
"This has the potential of allowing a person to purchase candy that is particularly appealing to her genetically-determined sense of taste.",
"However, despite the great potential benefits of doing so, few individuals have taken advantage of genomic-based services.",
"One reason for this is the public's concern for the security and privacy of its genomic data.",
"People fear, for example, that they could be denied employment, denied insurance, and otherwise discriminated against if the details of their genomic makeup became public.",
"Another reason is convenience.",
"With emerging Internet and communications technologies, people are used to being able to get information quickly and with little inconvenience.",
"However, genomic-based services such as genetic testing have been heretofore inconvenient to use.",
"For example, an individual may have to travel to a distant location for a test.",
"If several tests performed at different locations were required, an individual would likely have to give a genetic sample to each location.",
"Similarly, genomic-based prescription information is not available to patients and medical professionals in such a way that it can be unobtrusively incorporated into the average medical office or pharmacy.",
"Further, genomic-based services, such as tests for disease susceptibility, can be expensive.",
"Individuals are used to enjoying low prices for products and services due to competition and the assistance of Internet services.",
"However, such price-lowering has not yet come to genomic-based services.",
"Thus, for at least these reasons, advances in genomic knowledge have fallen short of realizing their potential benefits to the population.",
"SUMMARY OF THE INVENTION In one aspect, the invention features a system and method for managing users' genomic data.",
"Therefore, one object of the invention is to provide a system and method for providing and offering access to genomic-based services.",
"Another object of the invention is to provide a system and method for routing genomic data to providers of genomic-based services.",
"Still another object of the invention is to provide a system and method for brokering financial transactions related to the management of genomic data.",
"A further object of the invention is to provide a system and method for securing a user a best price for a genomic-based service.",
"Still another object of the invention is to provide a system and method for allowing users to earn money for the use of their genomic and other data.",
"Still further objects of the invention are to provide a system and method for using genomic data to market a product in a geographic region of interest, and a system and method of using genomic data in developing new products to satisfy unmet demands or needs of a population.",
"Accordingly, in one embodiment the invention concerns a method for recruiting a new user for a genome management service, comprising obtaining a cell sample from a person, waiting a period of time, after the period of time has elapsed seeking from the person final permission to have his or her genomic data managed, analyzing at least a portion of the person's genome, and storing the resultant genomic data electronically.",
"In a second embodiment the invention concerns a method for maintaining an individual's genomic data, comprising a data storage unit in which the individual's genomic data is stored and a self-destruct unit, which deletes the data on the device when a trigger event occurs.",
"In a third embodiment the invention concerns a data card for maintaining an individual's genomic data, comprising a data storage unit in which the individual's genomic data is stored.",
"In a fourth embodiment the invention concerns a method for providing product usage advice for an individual, comprising receiving the individual's genomic data, using the genomic data to consult a database or table which correlates genomic data with responses to products, and creating a report containing product usage advice for one or more products.",
"In a fifth embodiment the invention concerns a method for producing marketing data, comprising receiving from a group of individuals their genomic data, receiving from the group of individuals data concerning their purchasing habits, determining correlations between the genomic data and the purchasing habits, and making a prediction concerning an individual's purchasing habits based on that individual's genomic data.",
"In another embodiment the invention concerns a method for marketing products to individuals based on their genomic data, comprising receiving from a group of individuals their genomic data, receiving from the group of individuals data concerning their purchasing habits, determining correlations between the genomic data and the purchasing habits, making a prediction concerning an individual's purchasing habits based on that individual's genomic data, and making a product suggestion.",
"In an additional embodiment the invention concerns a method of providing an individual with lifestyle advice related to his or her genomic data, comprising using an individual's genomic data to consult a database or table which correlates genomic data with information related to the genomic data, receiving, as a result of the consultation, information related to the genomic data, and providing lifestyle advice related to the information.",
"In yet another embodiment, the invention concerns a method of marketing a product in a geographic region of interest, comprising obtaining information relating to correlations between users' response to the product and a haplotype profile, determining the frequency of the haplotype profile in the population living in the geographic region, and making a marketing decision for the geographic region based on the determined frequency of the haplotype profile.",
"In yet another embodiment, the invention provides a method for developing a new product to satisfy a particular unmet demand or need of a population, comprising identifying a haplotype profile that is correlated with the unmet demand or need in the population, determining a functional cause for the correlation between the haplotype profile and the unmet need or demand, and developing a new product designed to avoid the functional cause.",
"In still another embodiment the invention concerns a method of providing a gaming experience to an individual based on his or her genomic data, comprising receiving the genomic data of the individual and affecting gameplay using the genomic data, whereby the individual's gaming experience is due at least in part to his or her genomic data.",
"In a further embodiment the invention concerns a method of designing products based on an individual's genomic data, comprising obtaining the individual's genomic data and creating a design for the product based on the genomic data.",
"In another embodiment the invention concerns a method for marketing an individual's genomic data, comprising contacting a party interested in using an individual's genomic data, negotiating with the party to determine the terms of use for the data, seeking the individual's consent to allow the party to use the data under the determined terms of use, and if consent is received, providing, under the determined terms of use, the genomic data to the party.",
"In still another embodiment the invention concerns a method for providing an individual with low price genomic-based services, comprising receiving from the individual or his or her representative a request for a genomic-based service, negotiating with a plurality of parties capable of providing the service in order to determine which party of the parties is willing to offer the service at a lower price than the remainder of the parties, and upon receiving the individual's or representative's consent, allowing the party which offered the lower price to perform the service.",
"In a further embodiment the invention concerns a billing method for a genomic data managing service, comprising charging a management fee and charging a fee for each access or update of the data.",
"In an additional embodiment the invention concerns a method for providing an individual's genomic data, comprising receiving from a party a request for an individual's genomic data, negotiating with the party to determine the terms of use for the data, seeking the individual's consent to allow the party to use the data under the determined terms of use, and, if consent is received, providing, under the determined terms of use, the genomic data to the party.",
"Another embodiment of the invention concerns a method for securely transmitting an individual's genomic data to a party, comprising storing an individual's genomic data on a data card and physically transporting the data card to the party.",
"Still another embodiment of the invention concerns a method for securely transmitting an individual's genomic data to a party, comprising creating one or more data packages containing the individual's genomic data and allowing the party to download the package over a network.",
"A further embodiment of the invention concerns a method for allowing a user to make use of his or her genomic data, comprising receiving from the user a request for an operation he or she wishes to be performed making use of his or her genomic data and performing the operation.",
"The scope of the invention should not be considered as being limited by these objects and embodiments.",
"Additional aspects, objects, and embodiments will become clear upon a reading of the disclosure and the claims that follow it.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a schematic diagram of a system according to one embodiment of the invention.",
"FIG.",
"2 illustrates one embodiment of the process of adding a new user's genomic data to the system.",
"FIG.",
"3 illustrates one embodiment of the process of fulfilling a user's request for a service.",
"FIG.",
"4 illustrates one embodiment of the process of providing food, drug and nutritional supplement guidance.",
"FIG.",
"5 illustrates one embodiment of the process of finding a best price compensation for use of a user's genomic or other data.",
"FIG.",
"6 illustrates one embodiment of the process of securing for a user a best price for a particular service.",
"FIG.",
"7 illustrates one embodiment of the process of allowing a service provider access to a user's genomic or other data.",
"DETAILED DESCRIPTION OF THE INVENTION A. Definitions The following definitions are used herein: Candidate Gene—A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.",
"Genetic marker—A variation from a reference genomic or mitochondrial DNA sequence that occurs in at least one individual in a population.",
"As used herein genetic markers include polymorphisms, haplotypes, haplotype pairs, DNA methylation patterns, and other types of markers that are presently known or subsequently discovered.",
"Genotype—An unphased 5′ to 3′ sequence of nucleotide pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual.",
"Haplotype—A sequence of nucleotides found at one or more of the polymorphic sites in a locus in a single chromosome of an individual.",
"Haplotype pair—The two haplotypes found for a locus in a single individual.",
"Haplotype profile—A combination of one or more haplotypes (or haplotype pairs) that are correlated with a particular phenotype, including consumer purchasing habits, disease susceptibility, drug therapeutic profiles, patient compliance with prescribed or recommended dosing regimens.",
"Locus—A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.",
"Nucleotide pair—The nucleotides found at a polymorphic site on the two copies of a chromosome from an individual.",
"Polymorphic site—A nucleotide position within a locus at which the nucleotide sequence varies from a reference sequence in at least one individual in a population.",
"Sequence variations can be substitutions, insertions or deletions of one or more bases.",
"Polymorphism—The sequence variation observed in an individual at a polymorphic site.",
"Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.",
"Polymorphism data—Information concerning one or more of the following for a specific gene: location of polymorphic sites; sequence variation at those sites; frequency of polymorphisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.",
"Polymorphism Database—A collection of polymorphism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.",
"Reference Population—A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population living in a defined geographic region.",
"In preferred embodiments, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.",
"Single Nucleotide Polymorphism (SNP)—A polymorphism in which a single nucleotide observed in a reference individual is replaced by a different single nucleotide in another individual.",
"Therapeutic Profile—A plot of the response (e.g., level of efficacy and or number of adverse events) exhibited by a group of individuals to a particular drug or therapy.",
"Unphased—As applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, unphased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus (i.e., located on a single DNA strand) is not known.",
"B.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS A system in accordance with an exemplary embodiment of the invention is shown in FIG.",
"1.A “management company”, which manages people's genomic data and/or offers them genomic-based services, could operate a management device 100.Management device 100, as shown, consists of interconnected main components storage device 102 and routing/intelligence device 101.In some embodiments, storage device 102 may be implemented using one or more secure servers or general purpose computers, while routing/intelligence device 101 may be implemented using one or more general purpose computers.",
"In other embodiments, the functions of these two components may be combined into one component.",
"For example, management device 100 may be implemented as one or more general purpose computers, with each computer providing the functionality of storage device 102 and/or routing/intelligence device 101.Alternately, the functionality of these two components may be spread among two or more components.",
"The phrases “general purpose computer” and “computer,” as used herein, include, but are not limited to, an engineering workstation or PC.",
"“General purpose computer” and “computer” also include, but are not limited to, one or more processors operatively connected to one or more memory units, wherein the memory may contain data, algorithms, and/or program code, and the processor or processors may execute the program code and/or manipulate the program code, data, and/or algorithms.",
"Management device 100 is connected to one or more I/O workstations 104 without data card interfaces, one or more I/O workstations with data card interfaces 105 (e.g., I/O workstations operatively connected to data card interfaces), one or more “home” genome-based services 103 provided by the management company, and optionally one or more genome-based services 106 provided by third parties.",
"The I/O workstations equipped with card readers are capable of reading from, and in some embodiments writing to, data cards 107.These connections may be made in a variety of ways well-known in the art such as using the Internet, private lines such as leased T1 lines, or a local or areawide wireless network.",
"In the case where private lines are used, the management device may be additionally connected to the Internet as shown in connection 108.The I/O workstations may take several forms depending on the specific tasks they will be used for.",
"For example, an I/O workstation used by a member to access home and third party services may be a web browsing device located in that person's home, such as a personal computer connected by the Internet.",
"As a second example, an I/O workstation for entering a new sequence might be a browser-equipped engineering workstation connected to the Internet, further interfaced with a smart card reader/writer and laboratory equipment.",
"As a third example, an I/O workstation in a physician's office, pharmacy, health food store, supermarket, restaurant, cyber cafe or the like might be a browser-equipped personal computer or computerized cash register connected to the Internet, further interfaced with a smart card reader.",
"Other embodiments of I/O workstations will be obvious to those skilled in the art in light of the remainder of this disclosure and its appended claims.",
"The genetic material of an individual who desires or is in need of having her genomic data stored, managed or analyzed for correlations with phenotype would first need to be submitted to the management company.",
"Genetic material may be submitted by the individual who desires genomic services, or may be submitted by a skilled intermediary, such as a physician or physician's assistant.",
"In a preferred embodiment, this genetic material would be submitted in the form of cells obtained during a cheek swab.",
"Although other cell and tissue samples, such as fibroblasts or blood, would provide the needed genetic material, the cheek swab has the benefit of being painless and noninvasive.",
"In certain embodiments, specific cell types will be obtained.",
"For example, in some embodiments it might be desired to obtain B or T cells.",
"There are several conditions under which the cheek cells could be obtained.",
"In one embodiment, the patient could perform the procedure in her own home.",
"A kit could be provided which would include instructions and the materials needed for the obtaining and shipping the sample.",
"In a preferred embodiment, the individual comes to a collection center where the sample will be collected by trained personnel.",
"Such a scheme has several benefits, among which are not only ensuring a properly harvested sample, but also the trained personnel being able to provide information and emotional support to the individual.",
"This support is important because much of the general population has many questions concerning genetic information, as well as fear concerning the privacy of this data.",
"In order to make it easy for individuals to submit their samples, sample collection centers could be set up in a number of locations.",
"In one embodiment, collection kiosks could be set up in public areas like malls and airports.",
"In another embodiment, a mobile collection van could travel to certain areas where people congregate.",
"For example, the van might park outside a busy office building at lunch time.",
"In a third embodiment, collection could be performed at the office of a healthcare professional such as a physician.",
"These collection centers could be “branded.” In other words, the collection centers could have distinct colors, designs, interior layouts, exterior shapes, and the like.",
"Such branding has several benefits, which are well known to those versed in the art.",
"Among these are advertising, close binding of the company and its service, and establishing a corporate image.",
"For example, designs could be chosen which make users feel that the company is “professional,” courteous, and concerned about genetic privacy.",
"When these designs are also distinctive, customers would be able to easily tell the company apart from its competitors.",
"Such designs would also likely stick in people's minds, hence acting as a sort of advertising.",
"In some embodiments, the sample would be processed as soon as possible after collection.",
"Such an embodiment could be employed, for example, when there exists an immediate medical need to make use of an individual's genomic data.",
"In other embodiments, a period of time would elapse between collection of the sample and its processing.",
"For example, this period of time could be one week.",
"The customer, or her authorized agent such as her physician, could contact the service during this period of time to have the sample destroyed or to delay further processing.",
"If the customer did not contact the service during this period of time, the company could contact the user at the end of the period seeking permission to have the genetic sample processed.",
"The user could choose to grant permission, deny permission, or to delay processing.",
"If the user denied permission, or no response was received from the customer, the sample could be destroyed.",
"Preferably, a user who chose to delay processing could choose to do so either indefinitely or until a certain date.",
"If the user chose to delay until a certain date, the management company would contact the user on that date seeking permission to process the sample.",
"At this time the user would again have the choice of granting permission, denying permission, or further delaying processing.",
"If the user choose to delay indefinitely, the management company would store the sample until it received explicit instructions to process or destroy the sample.",
"In some embodiments, the management company would contact the user periodically, asking permission to proceed.",
"If the user did not answer, the sample would continue to be stored but would not be processed.",
"In other embodiments, the company would not contact the user, but instead would wait to be contacted by the user.",
"In certain embodiments, the user would be charged a fee for the storage service.",
"For example, the user may be charged a monthly fee if the sample is stored without being processed for more than six months.",
"Further, the management company may impose an upper limit on how long it would be willing to store the sample without processing it.",
"For example, the management company might set as a policy that all samples which sit unprocessed for three years are destroyed.",
"Many customers are reluctant to make big decisions, and might be hesitant to join the service for fear they would regret the decision later.",
"The “waiting period” would give customers opportunity to initially submit a sample without fear, knowing that they had a period of time in which to change their mind without any consequence, with the possible exception of being charged a small processing fee.",
"Once the “waiting period” elapses, and final consent is given, the genetic sample provided is processed so as to yield the individual's genomic data.",
"Such genomic data includes, but is not limited to, data relating to the individual's genes, genotype, genomic sequence or a portion thereof, haplotypes or haplotype pairs, the data describing one or more of the individual's polymorphisms, such as SNPs and RFLPs, data describing B or T cell DNA rearrangements, and data describing DNA modifications, such as methylation.",
"The term “genomic data,” as used herein, also includes data from non-genomic DNA, such as data describing mitochondrial DNA.",
"The processing may be done using conventional techniques well-known in the art.",
"In some embodiments, individuals can either give final consent or have their consent presumed if they have not asked for the sample to be destroyed within a certain period of time.",
"This processing may be performed by the management company or a third party under contract with the management company.",
"The resultant genomic data can easily be stored on digital media.",
"For example, the sequence of base pairs that makes up an individual's genomic sequence is effectively a string of characters.",
"The system will write to a storage location such resultant genomic data corresponding to a new user.",
"In some embodiments, the user or her authorized agent can opt to have this data deleted at a later time.",
"FIG.",
"2 is a flow chart showing one exemplary embodiment of the above-described procedure.",
"In step 201, the sample is collected.",
"In step 202, it is determined if the customer has requested to further delay processing.",
"If the customer has requested to further delay processing, flow proceeds to step 210 where the new expiration date is received from the user, and the period is set to end on this date.",
"If the customer has not requested to further delay processing, flow proceeds to 203 where it is determined if the customer has requested to destroy the sample.",
"If it is determined that the customer has requested to destroy the sample, flow proceeds to step 209 where the sample is destroyed.",
"If the customer has not requested to destroy the sample, flow proceeds to step 204 where it is determined if the period has expired.",
"If the answer is “no,” flow proceeds back to step 202.If the answer is “yes,” flow proceeds to step 205 where a response is sought from the user as to whether he or she wants to further delay processing, to process the sample, or to destroy the sample.",
"The response is received in step 206 and depending on the response, flow proceeds to step 210 (if the customer chooses to further delay processing), 207 (if the customer chooses to process the sample), or 209 (if the customer chooses to have the sample destroyed).",
"In step 207 the sample is processed, after which the resultant data is written to a storage location (step 208).",
"In a preferred embodiment the party performing the processing of the sample, whether it be a third party or employees of the management company, would not know the identity of the individual who submitted the sample.",
"One method of achieving this is for the management company to correlate a temporary identification number with the individual.",
"Such a temporary identification number might include numerals, letters, or other characters.",
"This correlation may be done by generating a semi-random temporary identification number and associating this number in a lookup table with the individual's identity.",
"“Semi-random” refers to the fact that the randomly-generated identification number might have to meet certain requirements in order to be acceptable, and if not found to be acceptable would be regenerated.",
"For example, the system might require that the generated number not be one which is currently in use.",
"In other embodiments, no lookup table would be used, and the correlation would be done using a cryptographic algorithm which would translate between actual identities and temporary identification numbers.",
"Methods for formulating such algorithms, generating such semi-random numbers, and building such lookup tables are well known to those versed in the art.",
"This temporary identification number, but no personally identifying information, would be included with the genetic sample for submission to the party performing the processing.",
"This party would return the resultant genomic data, along with the corresponding temporary identification number, to the management company.",
"In some embodiments, the party may do this by entering the information into an I/O workstation 104.The management company, upon receipt of the information, would ascertain from the returned temporary identification number which individual's genomic data had been received.",
"This ascertaining step might be done using a lookup table.",
"In another example, this ascertaining step might be done using a cryptographic algorithm to decode the identification number into an actual identity.",
"Next, the genomic data would be stored and the temporary identification number would be de-correlated from the individual so that the number could be reused.",
"Alternately, the sample may be delivered to the party performing the processing of the sample, and the resulting genomic data may be received therefrom, in a manner according to pending application Ser.",
"No.",
"09/611,654 “Methods and Apparatus for Ensuring The Privacy and Security of Personal Medical Information” (filed Jul.",
"7th, 2000), incorporated herein by reference.",
"This application discloses a method of ensuring the security of data from a medical test.",
"The method includes providing the patient with a medical data card issued by a secure information provider, and having a unique patient identification number (PID), a public key encryption private key (Key 1), and a public key encryption public key (Key 2).",
"The medical data card is used to generate a first test request card that accompanies the test specimen taken from the patient to the secure information provider.",
"The first test request card includes an encrypted identification of the patient and the test, a code identifying the health care provider, the patient identification number, public encryption public key (Key 2), and an identification of the test type.",
"The secure information provider uses the first test request card to generate a second test request card to forward the patient's specimen to a testing laboratory.",
"The second test request card and the specimen are forwarded to the laboratory.",
"The second test request card bears an encryption of the patient's unique identification number, but does not otherwise bear any indicia that would identify the patient.",
"The laboratory performs the prescribed test and generates a first test results card.",
"The results, together with the patient's unique identification number, are provided to the secure information provider that issued the medical data card.",
"The secure information provider provides the encrypted test results onto a second test results card, and forwards the card to the health care provider.",
"The test results on the second test results card are decrypted using the patient's medical data card.",
"The methods described in this application could be used for the non-medical uses described herein as well.",
"In another embodiment of the invention, the genomic services requested of the management company relate to providing therapeutic guidance to an individual, or preferably her healthcare professional (e.g., physician, pharmacist, etc), in connection with the treatment of the individual for a particular disease or condition.",
"In this case, the individual or her healthcare professional may submit a sample for processing as described above, or alternatively, the individual or her healthcare profession may already be in possession of the genomic data that is relevant to the advice being sought and such data is submitted to the management company who would perform, or have a third party perform, the requested genomic services.",
"The delivery of the individual's genomic data to the management company may be done by any methods for securely transmitting data that are disclosed herein, as well as by other methods known in the art.",
"In one embodiment, all or part of the genomic data is stored on a secure server, such as storage 102, preferably in an encrypted manner.",
"In such a case, the genomic data that resulted from processing would be entered into or transferred to an I/O terminal 104.The data would then be routed to secure storage 102 by routing/intelligence module 101.Such routing could be achieved using signals.",
"Secure storage 102 is managed by routing/intelligence 101 so as to carefully restrict who has access to an individual's genomic data, the guiding principle being that no one would have access to an individual's data without that individual's explicit permission.",
"Optionally, information connecting an individual's identity to her genomic data may be separately stored, secured, and managed.",
"In one embodiment, the genomic data would be stored in a secure database which correlated genomic data with identification numbers rather than with identities.",
"A separate secure database, perhaps located at another location, would correlate the identification numbers with the actual identities.",
"In another embodiment, a cryptographic algorithm would be used to translate between identification numbers and actual identities.",
"In another embodiment, all or part of the genomic data would reside on a data card 107 rather than on storage 102.In such a case, the genomic data that resulted from processing is entered into or transferred to an I/O terminal with data card reader/writer 105, and subsequently written to a data card 107.In certain embodiments, this would be done so that the management company would not view or posses the genomic data.",
"For example, the party processing the sample could directly write the resultant genomic data to the data card 107.The party could than affix the temporary ID number to the outside of the card.",
"The card would then be forwarded to the management company by secure messenger.",
"Upon receipt of the card, the management company could, using the affixed temporary ID number, determine the user whose card has been received and forward the card to that user, perhaps by secure messenger.",
"The management company could do this without accessing the contents of the card.",
"In further embodiments, the party doing the processing could write the data to the card in an encrypted manner, wherein the key to unlock the data would be provided to the user but not the management company.",
"In another embodiment, the management company could write the genomic data to the data card 107 through a secure network connection between the management company and an I/O terminal with data card reader/writer 105 located at the healthcare professional's office.",
"In another embodiment, the data card 107 would include some or all of the individual's medical records, in particular information relating to the requested genomic services, e.g., medical history, diagnosis, clinical or physical measurements, adverse drug responses and the like.",
"The genomic and medical information on the data card could be updated as further information becomes available.",
"Data card 107 would preferably be credit card sized so as to easily fit in an individual's wallet, billfold, purse, or the like.",
"Several types of storage cards of this type exist.",
"Among these are magnetic strip cards, “Smartcards,” flash-memory cards, and the like.",
"Smartcards are available from numerous venders, one such vender being Siemens of Munich, Germany.",
"Preferably, personally identifying information such as the individual's name would neither be stored on the card nor printed on its exterior.",
"As one example of the functionality of data card 107, a patient could go to her physician with a card encoded with her genomic data.",
"The card could be swiped through a reader that taps into a database of drug information maintained by the management company or other service provider.",
"The information could advise the physician which medication and/or which dose of a medication the patient should take—or avoid—for a particular illness, based on the person's genetic makeup.",
"Alternately, the reader could be located at the patient's pharmacy and the pharmacist consults the drug information database in connection with filling a prescription written by the patient's physician.",
"If the pharmacist receives any drug response information from the service provider that is inconsistent with the prescription, the pharmacist could communicate such information to the physician and request a revised prescription.",
"In certain instances, the physician or pharmacist may be aware of a reason why the patient should take or avoid a particular drug.",
"If so, that knowledge could optionally be added to the database.",
"As researchers learn more about who is genetically likely to have a good or bad reaction to approved drugs, the database of drug information could be updated.",
"In another embodiment, the reader can comprise a handheld computer such as a Palm™ Handheld (manufactured by Palm Incorporated) or IPAQ™ Pocket PC (manufactured by Compaq Corporation).",
"The physician may use the handheld device for other purposes, such as patient scheduling, accessing patient's records, taking notes, and the like.",
"The hand-held computer may contain a database of drug information, or may be in communication with such a database operated by the management company or other service provider, either directly via a wireless connection or indirectly via a base unit station located in the physician's office or hospital.",
"The base unit station could be connected to the database at the management company via the Internet or a private network or other type of direct link.",
"At the end of the day, the physician may place the hand-held computer in the base unit station so that it can be recharged as well as upload data to and/or download data from the management company's database.",
"In a preferred embodiment, the storage card would be a smart card or a smartcard like device because of the extra features they provide.",
"For example, smartcards have on-board processing units.",
"Such extra features make it easier to add additional functionality to the data card.",
"One example of such additional functionality would be a self destruct function by which the card would destroy its data under certain conditions.",
"Examples of such conditions include the card being tampered with, an attempt to copy the card, an attempt to perform an unauthorized read of data from the card, and an attempt to perform an unauthorized write to the card.",
"In another example, the processing unit of the card could perform encryption and decryption functions on board, a function of smart cards well-known in the art.",
"In another embodiment, the card could have the additional functionality of being able to operate in the manner of a credit card or bank card such that purchases could be made using the card.",
"In still another example of additional functionality, a patient's medical record could be stored on the card, preferably in an encrypted format, in addition to her genomic data.",
"In an alternative embodiment, portable data storage devices other than cards can be used.",
"For example, a touch-memory device, such as the “i-button” produced by Dallas Semiconductor of Dallas, Tex.",
"could be used.",
"Such touch-memory devices can be easily incorporated into objects such as jewelry.",
"Further, the data storage device may be implemented so that it communicates wirelessly with the routing/intelligence device.",
"Such functionality could be achieved using IEEE 802.11 wireless networking technology, as well as using other wireless communication methods well-known to those versed in the art.",
"Although the data card represents an alternative to storing genomic data on storage device 102, in some embodiments the user could opt to have the information on his data card “backed up” on a storage device such as storage device 102.Preferably, the data would be stored in an encrypted manner.",
"Further, such a backup vault would preferably not be connected to public networks, such as the Internet, so as to decrease the likelihood of data theft, tampering, and manipulation.",
"A function of the system is to provide users easy access to services based on their genomic data.",
"Some of these services may be provided by the management company itself (“home services” 103), others may be provided by third parties identified by or under contract with the management company, while still others could be offered by both.",
"In some embodiments, the user would have the option to download, receive or view her own genomic data.",
"A user might wish to do this, for example, if she wished to do her own research on her genomic data.",
"Further, the user may upload tests, programs, or algorithms which she wants performed or executed on her on data.",
"For example, the user may write or execute a program which searches her genomic data for certain haplotype pairs.",
"In another example, the user may write or execute a program which creates a musical rendition based on her genomic data.",
"In one embodiment, a user accessing the system through I/O workstation 104 for the purpose of using a service would be provided with menu options.",
"This menu may take several forms, a preferred embodiment of which is a web page.",
"An exemplary top-level menu is shown below: Home Functions 1) Health and Life Style Advice 2) Games And Learning 3) Food, Drug and Nutritional Supplement Guidance 4) Genetic Tests 5) Participate in a Test—Medical 6) Participate in a Test—Non-medical 7) Purchase A Product based on your Genomic Data 8) Access Current Medical Information 9) Subscribe to Medical Information Updates Third Party Functions 1) Genetic tests 2) Participate in a Test—Medical 3) Participate in a Test—Non-medical 4) Purchase A Product Based on your Genomic Data 5) Access Current Medical Information 6) Subscribe to Medical Information Updates User Functions 1) View my Genomic Data 2) Receive my Genomic Data on an Encrypted Data Card, delivered by Secure Messenger 3) Download my Genomic Data to the Inserted Data Card or Other Media 4) Route my Genomic Data to a Specified Party In such an embodiment, a user clicking on a menu option would be dropped to a lower level menu.",
"Clicking on an option in the lower level menu might result in a still lower level menu, and so on until a final choice was chosen.",
"For example, a user clicking on “genetic tests” would be given a choice of the available tests.",
"After clicking on the desired test, the user would be given a menu listing providers of the test.",
"In a preferred embodiment, prices would be listed next to each choice.",
"Further, quality ratings may be listed next to each choice, perhaps using a rating system of one to four stars or a numerical ranking system which orders the providers based on quality.",
"These ratings could be based on user feedback, expert evaluation, or the like.",
"In some embodiments, such user feedback and/or expert evaluation could be obtained by having a user and/or expert enter the feedback and/or evaluation into an I/O terminal connected to management device 100 via the Internet.",
"For example, a user and/or expert could provide feedback and/or an evaluation by answering questions on a survey and returning it to the management company.",
"In such embodiments routing/intelligence device 101 could route a survey from the management company to a user and/or expert's I/O terminal and, after the user and/or expert completed the survey using the I/O terminal, route the completed survey to the management company.",
"In alternate embodiments, feedback, evaluations, and/or surveys could be transported between the management company and the expert or user using a courier.",
"The survey would preferably include questions seeking both quantitative and qualitative information.",
"For example, the survey could include questions concerning the accuracy of the service, as well as questions concerning whether the experience with the provider was a satisfactory one.",
"Such a survey could be designed using methods well known in the field of surveys.",
"For example, the survey could include alternate forms of questions which seek to obtain the same, or similar, answers.",
"Inconsistent answers could be rejected and/or tagged for follow-up by the service provider for clarification or validation.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"In some embodiments, a quality seal of approval from a recognized authority which monitors compliance with certain standards could be listed next to providers which had been awarded the approval.",
"In a further preferred embodiment “find the best price” and “find the provider with the highest quality rating” could be options on the menu of providers.",
"Upon the user selecting a provider, or a best price or quality provider being suggested to the user by the system, the system would ask the user to confirm the choice of selected or suggested provider.",
"Preferably, this confirmation would involve the user entering a password or the like.",
"In response to the user's confirmation, the system would route the user's genomic data to the appropriate service provider, be it the management company or a third party provider.",
"Such routing could be achieved using signals.",
"Depending on the service requested, the provider might require or prefer that additional information be provided in addition to the genomic data.",
"For example, a provider who was to perform a genetic test for the user, or provide therapeutic guidance to the user's healthcare professional, might also require family history data, dietary data, medical data, lifestyle data, and the like.",
"In such a case, the system would retrieve the required additional information and route it to the provider.",
"In some embodiments the system would retrieve the additional information by having the user or her authorized healthcare professional enter it on her I/O terminal.",
"In other embodiments, the system would retrieve the data from a secure database on storage unit 102.In cases where additional information is to be sent to another party, the system seeks permission from the user, and does not send the additional information to the party unless permission is granted.",
"In certain embodiments, the additional information could be compartmentalized so that only subsets of data could be retrieved, depending on the instructions of the user.",
"If the chosen service provider is a third party, the routing/intelligence module 101 routes the data to the appropriate third party 106.Alternately, if the chosen service is a “home service” provided by the management company, the data is forwarded to the appropriate home service provider 103.Once the provider completes the service, the routing/intelligence unit makes the resultant data available to the user.",
"In one embodiment, the intelligence unit would retrieve the results from the service provider and temporarily store them on storage device 102 in an encrypted format, and the system would notify the user that the results were available.",
"The user could then download, decrypt, and view the results the next time she logged on to the system.",
"In some embodiments, the user would have the option of transferring the results to her data card or I/O terminal.",
"Once the results were transferred or read, they could be deleted from the storage device 102.In another embodiment, the system would not store the results.",
"Instead it would notify the user that results were ready and available next time the user logged on.",
"When the user logged on and requested the results, the intelligence module would route the results data from the provider directly to the user for reading or transfer to a data card or the like.",
"In a preferred embodiment, the data would be routed in a secure and encrypted format.",
"This embodiment may allay some users' fears of mishandling of their data, because the results would not even temporarily be stored on the system.",
"FIG.",
"3 is a flow chart showing one exemplary embodiment of the above-described procedure.",
"These steps may be performed, for example, by routing/intelligence device 101.In step 301, user confirmation of the chosen or suggested provider is received.",
"Flow then proceeds to step 302 where the appropriate genomic data and additional information is routed (or provided) to the chosen or suggested provider, and then to step 303 where it is determined if the service has been completed.",
"If the answer is “yes,” flow proceeds to step 304 where the resultant data is made available to the user.",
"If the answer is “no,” flow returns to step 303.In some embodiments, a provision may be made to cancel the service with the initially chosen provider and chose a new provider and route data to that new provider, for example, when the first provider is taking too long to complete the service.",
"In such cases, the initially chosen provider's access to the data would preferably be revoked.",
"Notification of available results to the user could take several forms.",
"For example, the system might flash a “results available” message the next time the user logged onto the system with an I/O terminal, or send an e-mail message.",
"In another embodiment, the user could periodically call a telephone number to determine if her test was ready.",
"In some embodiments, the number would connect to a live operator.",
"In other embodiments, the number would connect to an automated voice system.",
"In both cases, the user would preferentially have to enter a password to learn if her results were ready.",
"In some embodiments additional security methods, such as voice print identification, may be used.",
"In certain embodiments, the user could hear her test results via the telephone.",
"Alternately, the system might make a voice telephone call to the user using speech synthesis.",
"In still another embodiment, the system might prompt a live telephone operator to call the individual and inform her that her test was ready.",
"In some embodiments, the user would need to enter a password or to pass voice print identification before learning that a test was ready.",
"In some embodiments, the user could choose to hear her results over the phone.",
"It is conceivable that in certain cases, due to illness or other factors, a user might be unable to retrieve her results and may wish to have a friend, family member, medical professional, or the like do it for her.",
"In other cases, government regulation may require that the results be made available to a medical professional intermediary who is qualified to counsel the user as to the meaning and/or implications of the results.",
"To provide such functionality, in some embodiments the user may specify additional parties who may access test results.",
"A user would be able to designate such parties as being able to access the results of all tests, certain types or classes of tests, or one or more particular test instances.",
"Further, parties may be granted conditional access.",
"For example, a user may choose to grant her brother access to some or all of her test results, but only if she is critically ill or injured.",
"In some embodiments, a user may choose that results could be protected using a fingerprint reader, such that results could not be accessed unless the user's finger, hand, or the like was placed in the reader.",
"Such an embodiment could decrease the likelihood of unauthorized access to the results while, for example, allowing a physician to access results for an unconscious user by placing the user's finger, hand, or the like in the reader.",
"In another embodiment, the user of the system may be a medical professional, such as a physician or pharmacist, who is authorized by the patient to submit her genetic material or genomic data to the management company in connection with requesting genomic services relating to the patient's healthcare.",
"For example, a physician or pharmacist may seek therapeutic guidance from the management company relating to which drug or dosage regimen is likely to be optimal for a particular patient based on that patients' genomic data, and preferably medical data.",
"The physician could then choose to use the therapeutic guidance received from the management company when prescribing a drug or other therapy for the patient and the pharmacist could use such therapeutic guidance in connection with filling a prescription.",
"In another embodiment, the user of the system is the patient's healthcare payer, e.g., insurance company or health maintenance organization (HMO), who is authorized by the patient to access the system to determine if the most cost effective therapy has been prescribed for the patient.",
"In this case, the healthcare payer might not be able to access to the patient's genomic data and would only be able to access information relating to the efficacy and/or safety of different treatment options.",
"It is further conceivable that a parent might want to have tests done for her minor child, infant, fetus, or the like.",
"In certain embodiments, the system may allow the parent to choose which parties can request tests for the child and view the results of those tests.",
"For example, a mother would be able to decide that she and her husband, but no other parties, would have the power to request tests and view test results.",
"As is alluded to by the sample top level menu, many genomic-based services are made available to the user.",
"Each of these services could potentially be offered by a third party, the management company, or both.",
"Many of these services employ databases or tables in which genomic data (including, but not limited to, haplotypes, haplotype pairs, SNPs, or methylation patterns) and/or additional information is correlated, perhaps statistically, with phenomena such as responses to foods or medications or susceptibility to diseases.",
"For example, a database could correlate genomic data and/or additional information with responses to medications so as to produce a therapeutic guidance model, perhaps accessible via the Internet, that could be used by a physician for prescribing purposes.",
"In some embodiments, functionality could be added for updating these databases, for example, by using feedback such as feedback evaluating the quality and/or accuracy of the provided service.",
"For example, a service provider whose database correlated genomic data and responses to food might, after reporting to a user a potential reaction to a food, ask the user what her actual response was.",
"The user's reported response, along with her genomic data and/or additional information, could be used to update the database.",
"In some embodiments, such feedback could be obtained by having a user, expert and/or professional enter the feedback into an I/O terminal connected to management device 100 via the Internet or private network.",
"For example, a user, expert and/or professional could provide feedback by answering questions on a survey and returning it to the management company.",
"In such embodiments routing/intelligence device 101 could route a survey from the management company to a user, expert and/or professional's I/O terminal and, after the user, expert and/or professional completed the survey using the I/O terminal, route the completed survey to the management company.",
"In alternate embodiments, evaluations, and/or surveys could be transported between the management company and the user, expert and/or professional using a courier.",
"The survey would preferably include questions seeking both quantitative and qualitative information.",
"For example, the survey could include questions concerning the accuracy of the service and/or advice given, as well as questions concerning whether the experience with the provider was a satisfactory one.",
"Such a survey could be designed using methods well known in the field of surveys.",
"For example, the survey could include alternate forms of questions which seek to obtain the same, or similar, answers.",
"Inconsistent answers could be rejected and/or tagged for follow-up by the service provider for clarification or validation.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"In further embodiments, functionality could be added for dealing with database “misses.” For example, a user seeking advice from a service provider whose database correlated genomic data and responses to food might, for example, ask about a food which was not processed in the database.",
"This would constitute a database “miss.” As a result, the service provider might process collected genomic data and collected responses to various foods, find correlations between the genomic data and responses to the food which was not in the database, and add data concerning response to the food to the database.",
"In other cases, in response to a database miss an expert may be contacted.",
"For example, if a user's request for disease susceptibility information from a service provider led to a database miss, the service provider might employ the services of a genetic disease expert to answer the user's query and/or update the database.",
"In some embodiments, service providers may employ algorithms which act upon, or take as input, all or part of a user's genomic data.",
"In some embodiments, service providers may use one or more devices to perform the above-described tasks such as employing algorithms, contacting genetic experts, and updating, maintaining, and consulting databases.",
"Such devices may include general purpose computers known in the art, such as PC's and engineering workstations.",
"In some embodiments, these devices would be connected to management device 100, perhaps via the Internet.",
"Genomic and related services described herein that employ databases may make use of the teachings of pending PCT International Application PCT/US00/7540 “Methods for Obtaining and Using Haplotype Data” (filed Jun.",
"26th, 2000; WO 01/01218), incorporated herein by reference.",
"This application discloses methods, computer programs and databases to analyze and make use of gene haplotype information.",
"These include methods, programs, and databases to find and measure the frequency of haplotypes in the general population; methods, program, and database to find correlation's between an individuals' haplotypes or genotypes and a clinical outcome; methods, programs, and databases to predict an individual's haplotypes from the individual's genotype for a gene; and methods, programs, and databases to predict an individual's clinical response to a treatment based on the individual's genotype or haplotype.",
"Similarly, such services may employ the teachings of pending PCT International Application PCT/US01/12831 “Method and System for Determining Haplotypes from a Collection of Polymorphisms” (filed Apr.",
"18, 2001; WO 01/80156), incorporated herein by reference.",
"This application discloses methods, computer programs and databases for identifying the haplotypes that exist in a population and methods, programs and databases for predicting an individual's haplotype for a gene from the individual's genotype for that gene.",
"As was illustrated in the above example, one example of a service is a genetic test which returns a result explaining a susceptibility to a disease.",
"Another such service is a “custom product” service.",
"A custom product service produces products based on one's genome.",
"An example of this would be the production of music, jewelry or clothing whose design is derived from an individual's genomic data.",
"For example, one-of-a-kind tee shirts or quilts could be designed by employing an algorithm that created a unique graphic based on a person's genomic data.",
"Alternately, the shirts or quilts could be designed by accessing a database which correlates graphic designs or design components with genomic features such as haplotypes.",
"A further example of a custom product would be a food that was produced so as to be especially appealing to the purchasing individual's genetically-determined sense of taste, as indicated by her genomic data.",
"To achieve this, the service provider might, for example, maintain a database correlating, perhaps statistically, the presence of certain haplotypes or haplotype pairs in ones genome with liking certain flavors or textures.",
"The service provider, upon receiving a food or meal request, would use the user's genomic data to consult the database in order to create a food or meal that the purchaser was likely to enjoy.",
"A similar example of a custom product would be a food or meal that was produced so as to be particularly appropriate for the purchasing individual's genetically-determined nutritional needs.",
"In this case, the service provider might, for example, maintain a database correlating, perhaps statistically, the presence of certain haplotypes or haplotype pairs in one's genome with certain dietary needs.",
"The service provider, upon receiving a food or meal request, would use the user's genomic data to consult the database in order to create a food or meal that would be a good fit for the purchaser's nutritional needs.",
"In some embodiments, recipes and menus for the food or meal would be provided to the user.",
"Alternately, the food or meal could be prepared for and delivered to the user.",
"Still another example of a custom product would be a musical composition that was designed so as to be especially appealing to the purchasing individual's genetically-determined sense of what is aurally pleasing, as indicated by her genomic data.",
"To achieve this, the service provider might maintain a database correlating, perhaps statistically, the presence of certain haplotypes or haplotype pairs in one's genome with liking certain musical styles or constructions.",
"The service provider, upon receiving a musical composition request, would consult the database in light of the user's genomic data in order to create a musical composition that the purchaser was likely to enjoy.",
"In some embodiments, functionality could be added for updating the database and/or algorithm, for example, by using feedback such as feedback evaluating the appeal of the provided product, using methods such as those described above, including surveys.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"Another service illustrated in the exemplary web page menu is for a user to access current medical information or subscribe to medical information updates.",
"For example, a user may choose to access current or up-to-date medical information such as scientific articles, news articles, lectures, and films about diseases, drugs, nutritional supplements, and the like.",
"Such information could be provided online, by e-mail, by physical delivery or other methods.",
"A user choosing to subscribe to such information might, for example, select to receive each week all scientific articles relating to a specific disease.",
"In some embodiments, users may choose to receive information based on their genomic data and/or additional information.",
"For example, a user might choose to receive each week all scientific articles relating to the genetic diseases for which, according to her genomic data and/or additional information, she is at risk.",
"In such an embodiment, the system could determine which articles would be appropriate for the user by consulting a database in which genomic data and/or additional information was correlated with increased likelihood of certain genetic diseases.",
"In some embodiments, functionality could be added for updating the database, for example, by using feedback such as feedback evaluating the appropriateness of the provided articles, using methods such as those described above, including surveys.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"Still another service illustrated in the exemplary web page menu is for a user to route her genomic data to a Specified Party.",
"For example, a user might use this feature to route her genomic data and/or additional information to a service provider not listed on any of the web page menus.",
"Also as seen in the sample web page menu, another service is for the user to participate in a test.",
"One example of this would be a test run by medical researchers (including, but not limited to, a clinical trial), while another would be one run by market researchers.",
"This is another case where the provider might require that additional information be routed along with the genomic data.",
"For example, for a market researcher the additional information might be the food preference of the user.",
"Such a provider's goal may be to statistically correlate food preference with the presence in the genome of certain markers.",
"On the other hand, for a medical researcher, the additional information might be information relating to childhood illness suffered by the individual, the individual's lifestyle activities, or the individual's dietary habits.",
"As is the case with all of the services, this service could conceivably be provided by the management company, a third party, or both.",
"The system may provide for reimbursement of the user in exchange for the information as well as payment of a processing fee to the management company.",
"Another example of genomic-based services are games in which gameplay is based on and/or affected by a user's genomic makeup.",
"For example, a multi-player game might employ an algorithm by which the player's genomic data would change the gameplay scenario, manipulate on-screen images, produce audible events, give game characters strengths or weaknesses (including illnesses) or otherwise affect the player's capabilities, or determine team assignment.",
"In some embodiments, functionality could be added for updating the algorithm, for example, by using feedback such as feedback evaluating the appeal of the gameplay experience, using methods such as those described above, including surveys.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"Still another example of a genomic-based service is to provide health and lifestyle information and guidance on various topics based, at least in part, on a person's genomic data.",
"For example, a person choosing “health and lifestyle advice” from the main menu might be shown the below sub-menu: 1) What diseases do I have a genetic propensity for, and what preventative steps can I take now?",
"2) What is a recommended diet for me?",
"3) What is a recommended exercise program for me?",
"4) What sports would I be best at?",
"5) What genetic risks would my children face based on my genomic data and/or family history?",
"6) (Two users only) What enetic risks would our children face based on our genomic data and/or family histories?",
"To execute such selections, a user's genomic data, along with any necessary additional information such as the user's weight, age, and family history, could be considered by using a database which correlates genomic data, and in some cases additional information, with lifestyle advice.",
"Such lifestyle advice might include advice on taking preventative steps against the onset of a genetic illness or a condition to which an individual is genetically predisposed, diet recommendations, exercise recommendations, or the like.",
"For example, a consultation of the database may yield the advice that the user should stop smoking because she is prone to arteriosclerosis.",
"In alternate embodiments, the database would correlate genomic elements, and in some cases “additional information,” with information concerning diseases, lifestyle outcomes, and the like instead of advice.",
"In such an embodiment, the system would need to take additional steps to provide advice related to this information.",
"In one embodiment, a second database could be used to yield the actual advice.",
"For example, a consultation or the first database may show that the user is prone to arteriosclerosis.",
"This information may be used to consult a second database, which would yield the advice that the user should stop smoking.",
"For example, a user might ask about what sports she would be best at.",
"A consultation of the database in light of her genomic data might yield the answer that she had a higher than average percentage of white muscle tissue and thus would be better at sports that require sprinting than endurance.",
"The system might also recommend appropriate exercises if she were more interested in improving her performance in other types of sports.",
"Another user, asking “What diseases do I have a genetic propensity for, and what preventative steps can I take now?” might learn that he was particularly susceptible to the effects of cigarette smoking and should quit immediately.",
"In some embodiments, the system could provide estimated risks, which could vary as further information was obtained and integrated into the database.",
"In certain embodiments, two users could jointly ask a question of the system and have their genomic data jointly compared.",
"For example, a couple planning on having children could have their genomic data jointly compared so that they could determine what genetic risks their planned children would face.",
"This service would provide more functionality than simple genomic testing, because results would preferably not just be a simple “yes” or “no” but would include lifestyle advice.",
"In some embodiments, a counselor would be on call (by phone, on-line, etc.)",
"to answer any questions the user had-about the results provided by the database.",
"In preferred embodiments, the results provided by the database would be “dynamic”.",
"This is to say that the results provided would change as more information were obtained from various sources (such as users) and analyzed.",
"A related service provides users with trivial information concerning their genomes.",
"For example, a user who selected from the menu system the question “what's genetically unique about me?” might learn that she lacked a psoas minor, one of the five muscles of the human body which are most frequently absent.",
"In some embodiments, functionality could be added for updating the database, for example, by using feedback received in connection with previously provided genomic services such as feedback evaluating the accuracy and/or utility of the provided health and/or lifestyle information and/or guidance, using methods similar to those described above, including surveys.",
"For example, a survey could include questions concerning the accuracy of health and/or lifestyle information and/or guidance given, as well as questions concerning whether the provided information lead to a perceived lifestyle improvement and answers to these questions could be used to update the database.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"Still another service is a food, drug and nutritional supplement guidance service.",
"In one embodiment, a user, preferably working with her physician, pharmacist, or nutritional supplement expert selects “Food, Drug and Nutritional Supplement Guidance” from the menu system of an I/O terminal.",
"Such an I/O terminal, perhaps connected to management device 100 via the Internet, could be located in a physician's office, health food store, or pharmacy, so that the genetically-guided prescription or usage suggestion of drugs and nutritional supplements could be unobtrusively incorporated into clinical or pharmaceutical practice or store operation.",
"The user's genomic data, along with necessary additional information such as the proposed prescription or usage suggestion and perhaps demographic information such as the user's weight, age, and family history, is then considered in light of the content of a database.",
"The database preferably correlates genomic data (such as haplotypes, haplotype pairs, SNPs, methylation patterns, and the like), and perhaps certain additional information, with responses to specific over-the counter drugs, prescription-only drugs, nutritional supplements, and similar products.",
"In preferred embodiments, the database can be updated as new information is obtained.",
"Using the result of the database consultation, the system creates a report with product and dosage advice, adverse reaction warnings, and the like.",
"In some embodiments, the routing/intelligence unit would, as above, make the report available only to the user.",
"It would be the user's responsibility to pass the report to the physician, pharmacist or nutritional supplement expert.",
"In other embodiments, the user's doctor or pharmacist receives the report, but has no access to any genomic data of the patient.",
"Preferably, the physician or pharmacist would receive the report after accessing the system using the pharmacy or medical office I/O terminal and entering a password.",
"In still other embodiments, the report would be delivered to the physician or pharmacist, but in such a manner that it could not be read by the physician or pharmacist without the presence or consent of the patient.",
"For example, the user might need to enter a password on the medical office I/O terminal in order to allow the physician or pharmacist to view the report.",
"In another embodiment, the user's consent or presence would be determined by taking the user's fingerprint, voiceprint or retinal scan.",
"Alternatively, the patient might choose to entrust the physician or pharmacist with the password.",
"In certain embodiments, there may be different passwords for access to different information or data.",
"For example, the system may create or allow the use of a password which offered a physician access to results of a test that she ordered for a user, but to no other information regarding that user.",
"In another embodiment, the physician or pharmacist would be able to access the report without the user's permission, e.g, if the report was deemed critically necessary for providing appropriate medical treatment to an unconscious or mentally incapacitated user or to a relative of a deceased user.",
"In a preferred embodiment, when it is time for a product refill, the database is re-consulted and a new report is created so that the prescription can be changed to reflect any updated genomic-based prescription advice.",
"In some embodiments, the user, doctor, pharmacist, or other professional could forward the patient's actual drug reaction to the service provider so that the database could be updated to take into account the reported drug reaction when giving future advice.",
"The service provider could use this information to refine statistical correlations of drug responses with genomic data and/or other data.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the patient.",
"In certain embodiments routing/intelligence device 101 could forward the patient's reaction from the professional to the service provider.",
"Such behavior could be achieved, for example by having the professional enter the reaction into an I/O terminal connected to management device 100 via the Internet.",
"In some embodiments, the professional would report the drug reaction by answering questions on a survey and returning it to the service provider.",
"In such embodiments routing/intelligence device 101 could route a survey from the service provider to the professional's I/O terminal and, after the professional completed the survey using the I/O terminal, route the completed survey to the service provider.",
"In alternate embodiments, results and/or surveys could be transported between the service provider and the professional using a courier.",
"The survey would preferably include questions seeking both quantitative and qualitative information.",
"For example, the survey could include questions concerning actual physical test results or responses, as well as questions concerning whether the response was sufficient for the physician to decide to maintain the patient on the drug.",
"Such a survey could be designed using methods well known in the field of surveys.",
"For example, the survey could include alternate forms of questions which seek to obtain the same, or similar, answers.",
"Inconsistent answers could be rejected and/or tagged for follow-up by the service provider for clarification or validation.",
"Further, the report could be updated at the time the product was refilled.",
"This update could be based on additional information which was added to the database since it was last consulted, such as the user's response to the prescribed product and dose, the user's response to other product, as well as information from other users or other sources.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"In additional embodiments, the food, drug and/or nutritional supplement guidance report may not only give information for a drug or nutritional supplement chosen by a physician or pharmacist, but may also suggest that a different product be used.",
"For example, a physician may ask for a food, drug and nutritional supplement guidance report for drug A for a particular patient.",
"The consultation of the database would yield not only potential lack of efficacy or adverse reactions, but also the advice that Drug B would be a better choice based on one or more of the patient's genetic profile, lifestyle information, and other additional information.",
"In further embodiments, a physician, pharmacist or other professional would not have to submit a proposed prescription.",
"Instead, the professional would submit, or request the retrieval from storage of, any required additional information such as diagnostic values, symptoms, and/or a diagnosis of the patient's condition.",
"Based on the genomic data and any additional information, a profile would be created which would suggest one or more drugs, preferably noting for each one a suggested dose and/or efficacy rating.",
"In a preferred embodiment, probable side effects would also be listed.",
"For example, a physician may submit, or request the retrieval from storage of, a patient's blood lipid level, age, gender or other relevant information relating to a patient's cardiovascular disease.",
"The report created based on this information and the patient's genomic data might contain the prediction that the patient had a 90% chance of responding to a first lipid-modulating drug, a 80% chance of responding to a second lipid-modulating drug, and a 20% chance of responding to a third lipid-modulating drug, along with a dosage suggestion for each drug.",
"The physician would then be able to choose which of the three drugs she wished to use for the patient.",
"For example, the physician may choose the second drug if it is in a hospital's formulary, or the patient's HMO formulary, but the first drug is not.",
"In one embodiment, a physician may choose to prescribe to a patient a particular drug or dosage regimen for such drug even though the report predicts that the patient will exhibit an adverse response to that drug or dosage regimen.",
"In this case, the physician would monitor the patient for the adverse response while on the drug, and afterwards, if medically appropriate.",
"Because an adverse response to the drug may affect the patient's compliance with the prescribed dosage regimen, the physician may also choose to more closely monitor how and when the patient is consuming the drug.",
"FIG.",
"4 is a flow chart showing one exemplary embodiment of the operation of the above-described food, drug, and/or nutritional supplement guidance service.",
"These steps may be performed, for example, by a general purpose computer.",
"In step 401 an individual's genomic data, and optionally additional information, is received.",
"In step 402, the database is consulted.",
"Next a report is produced (step 403) and made available to the user and/or a professional assisting the user (step 404).",
"In step 405, it is determined if the user's (and/or other user(s)') response to the product has been received.",
"If the answer is “yes,” flow proceeds to step 406 where the database is updated using the user's (and/or other user(s)') response and then returns to step 402, where the database is consulted and an updated report is produced based on data incorporating the user's (and/or other user(s)') response.",
"If the answer in step 405 is “no,” flow proceeds to step 407 where it is determined if it is time for a product refill.",
"If the answer is “yes,” flow proceeds to step 402.If the answer is “no,” flow proceeds to step 405.In a second embodiment, the food, drug, and nutritional supplement guidance service would operate in a manner similar to the first embodiment, the main difference being that it would provide advice concerning the use of foods instead of drugs and nutritional supplements.",
"In one instance of this embodiment a user, preferably working with a nutritionist or healthcare professional, would submit a suggested food, menu, or diet, along with any necessary additional information, in the manner discussed in the first embodiment.",
"Alternately, this embodiment is compatible with a food market or restaurant environment wherein a clerk, server, or the like would enter a proposed meal or food purchase along with any necessary additional information into an I/O workstation located on the premises.",
"The submitted genomic and additional information would be considered in light of a database which correlated genomic data with dietary restrictions and dietary guidance, and an advisory report would be created.",
"In a third embodiment, the food, drug and nutritional supplement guidance service would operate in a manner similar to the first two embodiments, but the I/O workstation used to enter data would be in the form of a computerized cash register or similar device located in a food market, health food store, pharmacy, restaurant or the like.",
"Alternately, the I/O workstation could be a web browser used to make online purchases.",
"This embodiment could provide genetically-guided point of sale advice concerning the purchase of food, drugs, nutritional supplements and other products.",
"For example, a mother whose child had PKU (pkenylketonuria) might attempt to purchase for her child a soft drink bottle at a store whose cash registers functioned as I/O workstations.",
"The child's genomic data, along with perhaps additional information, would be considered in light of a database that correlated genomic data and perhaps additional information with responses to foods and other products.",
"As a result of the database consultation the system might advise the mother that the purchase was not advised because the soft drink contained phenylalanine, a substance which would be harmful to the child due to her genetic makeup.",
"This point of sale embodiment could also advise users against the purchases of foods they were allergic to or that were problematic for reasons such as inborn errors of metabolism, or advise against the purchase of over-the-counter drugs or nutritional supplements which were not ideally genetically compatible or were genetically problematic.",
"In some embodiments, the system could suggest alternate foods, drugs, or other products that were more genetically compatible or less genetically harmful than the products that the user was attempting to purchase.",
"Still another function of the system is to broker financial transactions related to management of genomic data.",
"In order to implement this feature, the routing/intelligence device 101 could execute one or more of the below-described billing schemes.",
"Billing records could be held on the storage unit, while monetary transfer could be achieved using electronic finds transfer (EFT) and credit card billing techniques well known in the art.",
"In one aspect, individuals storing their genomic data in the system may be charged a fee by the management company for various actions.",
"For example, individuals may be charged a fee for collection and/or processing of their biological sample so as to yield genomic data.",
"Individuals may further be charged a fee for initially establishing a management account and/or a fee for maintaining the account.",
"The latter fee, for example, might take the form of a monthly or annual fee.",
"Individuals may also be charged for further processing of their DNA or initial sample, or for processing of an additional sample, to yield additional genomic data.",
"For example, a fee may be charged to a user who had initially paid to have only five genes or haplotypes recorded as her genomic data, but later chose to have additional genes or haplotypes recorded.",
"Similarly, users may be charged for adding, deleting, or otherwise revising non-genomic data.",
"In embodiments of the invention which use data cards, individuals may be charged an initial issue fee for a data card, and another fee if the card is lost or damaged and needs to be replaced.",
"Fees may be charged for the routing of genomic data and/or other information to service providers, both third party providers and those run by the management company.",
"For example, in some embodiments an individual may be charged a fee when the system routes her genomic data to a service provider.",
"If the service provider is a third party, the third party, instead of the individual, may be charged the fee.",
"In still other embodiments, both the third party provider and the individual are charged.",
"In other embodiments, the management company may pay a fee or credit the account of a user for use of her genomic or other information.",
"In some embodiments, users may opt to pay a one-time fee rather than being charged for various actions and/or for having their genomic data and/or other information routed to service providers.",
"For example, a one-time fee might entitle a user to have her sample collected and processed, her management account established and maintained for life, and her genomic data and/or other information routed to service providers without additional cost.",
"In some embodiments, there may be limitations to what one is entitled to after paying a one-time fee.",
"For example, the payment of the one-time fee might entitle one to a certain number of routings a year with additional routings being available for an extra cost.",
"Further, fees may be charged for the services themselves.",
"For example, for services provided by third parties, the system may act as an intermediary and collect money from the individual on behalf of the third party.",
"In preferred embodiments, the third party would be charged a fee for this billing and collection service.",
"For “home services” provided by the management company, the management company may directly bill the individual.",
"For example, home services which provide lifestyle advice might charge a fee for each piece of advice given.",
"Games may charge a fee per play, or per unit of time the game is played.",
"As a further example, fees may be charged for system functions related to food, drug and nutritional supplement guidance profiles.",
"For example, a fee may be charged for each profile created.",
"This fee may be charged to the individual, the medical or other professional, or both.",
"In some embodiments, drug companies may be charged fees when a drug is prescribed on the advice of a food, drug and nutritional supplement guidance profile.",
"For example, a drug company may be charged a “finder's fee” if a drug is chosen by a doctor on the advice of a food, drug and nutritional supplement guidance profile.",
"For example, a physician may ask for a food, drug and nutritional supplement guidance profile for drug A, produced by company X, for a particular patient, and be advised that Drug B, produced by company Y, would be a better fit based on the patient's genetic profile.",
"If the company X were different from company Y, company Y may be charged a “finder's fee” if there was a business agreement between company Y and the management company.",
"In other embodiments, a healthcare payer (e.g., a health insurance company or HMO) may only reimburse a healthcare provider, or its insured patient, for service fees or drug costs that are incurred in connection with the insured's medical treatment if the healthcare provider prescribes or dispenses a drug or therapy that is predicted to provide the patient with the most medically- and/or cost-effective care based on the patient's genomic data.",
"Thus, for example, if the patient has cardiovascular disease and is in need of a statin and is predicted to have less severe side effects or a greater reduction in LDL-cholesterol to statin A than statin B, an insurance company or HMO would only reimburse the healthcare provider for the patient's care in regard to cardiovascular disease if the healthcare provider prescribes or dispenses statin A to the individual.",
"In another embodiment, the healthcare payer may not require that the healthcare provider prescribe the best drug for the individual (based on the individual's DNA) but one of the better drugs.",
"For example, if the individual is in need of a reduction in cholesterol, and drugs A, B, C, and D are each predicted to cause a 20%, 24%, 32% and 5% decrease, respectively, in cholesterol in the individual, an insurance company or HMO may reimburse the health care provider (or the patient) if the individual is prescribed drug A, B or C, but not D. In some embodiments, the level of medical- and cost-effectiveness of certain therapies that qualifies for reimbursement may be specified in the patient's contract with the healthcare payer.",
"In other embodiments, whether the level of efficacy and/or safety predicted for a proposed therapy qualifies for reimbursement may be determined on a case by case basis by the healthcare payer in consultation with medical experts and/or with the insured's healthcare provider.",
"In another embodiment, the insurance company or HMO may only reimburse the healthcare provider (or the patient) if the patient is prescribed or receives a drug that the management company recommends for the patient based on the patient's genomic data and preferably other patient information relevant to providing therapeutic guidance.",
"The invention contemplates that this recommendation may be transmitted by the management company directly to the healthcare payer, who in some embodiments, may pay the management company a fee for such transmittal.",
"In yet another embodiment, a drug may be indicated for individuals with a certain haplotype profile, and the insurance company would only reimburse the health care provider (or the insured individual) for prescription or purchase of the drug if the insured individual has that haplotype profile.",
"The presence or absence of the haplotype profile in a patient may be ascertained by using any of the genomic services described herein or by the performance of a genetic test that is designed specifically for determining whether a patient belongs to the genetically-defined population that is part of the approved indication for the drug.",
"In all the above embodiments, the healthcare provider may be a physician, group of physician's, hospital, clinic, nurse or physician's assistant.",
"In other embodiments, the healthcare provider may be a pharmacy, pharmacist, or other entity that dispenses medications to individuals.",
"The system also includes methods of facilitating the earning of money by users.",
"In such embodiments, the system may, in response to an individual's request to earn money by participating in a study, seek out or receive requests from one or more third parties interested in using some or all of an individual's genomic data and/or other information for purposes such as research.",
"For example, the intelligence module might maintain a list of pharmaceutical companies, academic institutions, or contract research organizations and periodically e-mail the research directors or other appropriate personnel of these institutions to learn what sort of research participants are currently being sought.",
"Alternately, the management company might advertise itself in medical and scientific journal as a “clearing house” for research participants.",
"In such a case, research groups seeking participants would contact the management company or intelligence module that is requesting participants with certain characteristics.",
"The system bargains with the third parties to decide upon the fee the third party will pay for the use of the genomic data and/or other information of a particular user.",
"This is especially effective in cases where more than one third party is interested in using the data and/or other information, but only one will be awarded use.",
"The brokering might include not only negotiation of price to be paid, but also amount of genomic data and/or other information used.",
"For example, a third party might initially request information about three of an individual's haplotypes, but as result of the negotiation it might be bargained that only two would be used.",
"In some embodiments, once the system had come up with a highest price for the use of an individual's genomic data or other information, the system would inform the individual of how much money she would get, what specific data or other information would be used, and the purpose of the use.",
"The system would seek the user's agreement, and would not execute the transaction with the third party unless agreement was received.",
"In alternate embodiments, the system notifies an individual of a research project seeking participants, informs the individual of the nature of a research project and of the data being sought, and asks the individual if she is interested in having a place in the project negotiated for her, and if so under what conditions.",
"For example, the user might state the condition that she was only interested in participating if she would receive at least $100, or that she was only willing to allow 5 of the 10 requested haplotypes to be used.",
"The system would take such conditions into account during the bargaining process.",
"In some embodiments, users could set default values concerning the conditions under which they would be interested in entering a research project.",
"For example, an individual might set as her default profile that she only wanted to participate in medically-related projects related to a particular disease, such as breast cancer, or projects in which she would receive at least $75.Preferably, as above, once the system had come up with a best-price for the use of an individual's genomic data and/or other information, the system would seek the user's agreement, and would not proceed unless agreement was received.",
"Alternately, however, a user may agree ahead of time to participate in the project so long as her minimum conditions were met.",
"It is understood that, in some cases, higher prices could be paid to the participating users when there is a high level of participation by other users, or approval to use more genomic data or other information that initially estimated.",
"For example, it could be provided that the fee paid to an individual will increase as the amount of data she provided increases or the level of participation by other users increases.",
"In preferred embodiments, the system would charge for its negotiation services.",
"In one embodiment, the system would take a percentage of the money agreed to be paid, while in other embodiments the system would take a flat fee equal to a certain amount of money.",
"Such monies could be collected from the individual, the third party, or both.",
"In some embodiments, the individual or third party might be allowed to choose between the two charging models, preferably before the result of the auction was announced to the user.",
"In some embodiments, the management company might accept access to a provider's services in lieu of money owed to it by that provider.",
"FIG.",
"5 is a flow chart showing one exemplary embodiment of the above-described procedure.",
"These steps may be performed, for example, by routing/intelligence device 101.In step 501, a user's request to participate in a study is received.",
"In step 502, requests are sought out and received for the user's genomic and, optionally, additional information.",
"In step 503, the parties interested in using the user's genomic data and, optionally, additional information are bargained with in order to determine what data will be used and what price will be paid.",
"In step 504, the user is informed of the negotiated price, the data that will be used, and the purpose of the use.",
"In step 505, the user's agreement to the proposed terms is sought.",
"If the user does not agree, flow proceeds to step 507 where the transaction is canceled.",
"If the user does agree, flow proceeds to step 506 where the transaction is executed.",
"As noted above, in some embodiments the system works to find the best price for a particular service when that service is offered by more than one provider.",
"In one embodiment, the intelligence module 101 requests “sealed bids” from each of the providers offering the service.",
"The intelligence module then selects the lowest bid, and forwards this information to the user.",
"Alternately, the system could request that the user name the price she wanted to pay.",
"The system would then forward this information to the providers offering the service, and see if any were willing to provide the service for the noted price.",
"Other auctioning methods of securing a fair market price will be apparent to those skilled in the art.",
"In certain embodiments the system might consider the quality of the providers when finding the best price for a particular service, perhaps by considering quality rankings or seals of approval.",
"In some cases, a user could specify quality requirements when requesting that the system find a best price for a service.",
"For example, a user could specify that she wants the best price among providers with a quality rating of three or more stars.",
"Additionally, the management company could set quality requirements that could be applied to all user requests to find the best price for a service.",
"For example, the management company might decide that the system would, when finding a best price for a customer, only consider providers who had been granted seals of approval.",
"In further embodiments, the management company may charge the user for the service of having been secured a fair market price, charge the provider for the service of having found them a customer, or charge both parties.",
"FIG.",
"6 is a flow chart showing one exemplary embodiment of this procedure.",
"These steps may be performed, for example, by routing/intelligence device 101.In step 601, a request is received to find a best price for a particular service.",
"In step 602, sealed bids are requested from providers offering the service, and in step 603 the bids are received.",
"In step 604, it is determined which provider is offering the lowest bid.",
"As noted above, some embodiments of the data card allow it to be used to make purchases.",
"Such functionality can be easily implemented by the system because it can perform conventional EFT and credit card billing functions in performing the abovementioned financial transactions.",
"In the embodiments of the data card that include financial transaction capability, users might be able to earn money points if they allowed the management company to anonymously record information concerning their purchases.",
"Such information could be recorded, for example, each time a user makes a purchase using her data card.",
"The goal in doing so would be to create databases correlating genomic data such as haplotypes and purchasing or consumption habits.",
"In some embodiments, product and/or purchasing suggestions could be made based on these correlations.",
"These suggestions could be offered to the users.",
"The management company could construct the database in such a way that no personally-identifying information would be included.",
"In this way the user's anonymity would be maintained.",
"In some embodiments, the management company would sell its suggestions and/or statistical correlations, or the use thereof, to third parties.",
"In some embodiments, routing/intelligence device 101 may be configured to perform these functions.",
"In some embodiments, functionality could be added for updating the database, for example, by using feedback such as feedback evaluating the appeal of a suggested product or the accuracy of the provided statistical correlations, using methods described above, including surveys.",
"For example, the survey could include questions concerning the accuracy of the correlations, as well as questions concerning whether the suggested product was appealing.",
"Preferentially, the database would be updated in a manner that ensured the anonymity of the user.",
"Other embodiments might provide this functionality without the use of a data card with financial transaction capability.",
"For example, in some embodiments the purchasing information could be recorded at a cash register and forwarded to the management company physically or electronically.",
"In other embodiments, users may submit their own purchasing information, perhaps by filling out a questionnaire or by submitting copies of store purchase receipts.",
"Such submission could also be done electronically or physically.",
"In other embodiments, the user could give permission for her identity and genomic data relating to her purchasing habits to be disclosed to third parties.",
"As alluded to above, a further function implemented by the system is to route an individual's genomic data, and any appropriate additional information, to a provider of a requested service, such as a third party provider or the management company.",
"As explained previously, a guiding principle behind this function is that an individual's genomic data may only be used by another party with the individual's permission.",
"Accordingly, in preferred embodiments, care is taken that these parties never hold or posses a patient's genomic data, that they are not allowed to download the data, and that they are only allowed to use it for the purposes agreed upon and for the duration of time agreed upon.",
"Further, it is preferred that a party does not know the identity of the individual to whom it is providing a particular service.",
"In certain embodiments, when the management company owns or runs service providers such as for providing the service of correlating an individual's genomic information with drug response, these service providers will face the same restrictions as third party service providers.",
"For example, in preferred embodiments, while certain personnel of the management company will have access to user identities, steps will be taken so the personnel that work in a service provider division of the management company, in other words personnel responsible for performing services, will not know the identities of individuals to whom they provide services.",
"Further, while the management company may store genomic data, it is preferred that service provider divisions of the company will not be allowed to download and keep the data.",
"Thus, in certain embodiments, the genomic data shall be secured by appropriate internal safeguards so that it is only used for authorized uses by appropriate personnel.",
"In order to meet this goal, special techniques are used for the transmission of genomic and/or other data.",
"In a first embodiment, service providers access the genomic data via a transient storage area on storage device 102.In a second embodiment, the service provider downloads a “self-destructing” data package containing the genomic data in an encrypted format.",
"In a third embodiment, the service provider physically receives a “self-destructing” data card containing the genomic data.",
"In a fourth embodiment, the service provider reads the data directly from the data card during the period of time which the card is in the reader.",
"Each of these embodiments will now be discussed in detail.",
"Other embodiments are apparent to those skilled in the art.",
"In an example of the first embodiment, once permission has been secured the genomic data and any additional information is copied to a “transient storage area” to which the service provider is granted access.",
"In a preferred embodiment, a temporary identification number corresponding to the user and a description of the service requested is also copied to the transient storage area.",
"By use of a temporary identification number, the service provider does not know the identity of the individual for whom the service is being performed.",
"In embodiments where the genomic or additional information is stored on a data card, the user places her card in an I/O terminal with card reader 105.The data is copied from the card to the transient storage area.",
"In embodiments where the genomic or additional information is stored on storage device 102, this copying may be actual or virtual.",
"In the actual case, the data is copied to the transient storage area and the service provider is given access to this transient area In the virtual case, the data is not moved from or copied from the original storage area, but instead the service provider is given expiring access to the original storage area.",
"In preferred embodiments, the service provider could only access the data through an active connection, and would be prevented from downloading the data.",
"One method for achieving this would be set restrictions on the file so that it could be read but not copied or written to.",
"Such file restrictions are used in most modern computer operating systems, and thus the methods for implementing them are well known in the art.",
"Further, the data could be encoded in such a way that it would check where it was stored before allowing access to itself.",
"Thus, if it were somehow downloaded to a storage location other than the one it was intended to be stored on, it would not allow itself to be accessed.",
"Techniques such as this are well known in the art, as they are used to ensure, for example, that a program licensed to run on a specific computer only runs on that computer.",
"The temporary identification number functionality may be implemented in a manner similar to the disclosed method for ensuring anonymous processing of the initial genetic sample.",
"Thus the management company may generate a semi-random temporary identification number and associate this number in a lookup table with an individual's identity.",
"Alternately, a cryptographic algorithm may be used.",
"As described above, the service provider would have access to the temporary identification number, the genomic data, any additional information, and description of the requested service.",
"The service provider would return the results of the service, along with the corresponding temporary identification number, to the management company.",
"The management company, upon receipt of the information, would ascertain from the returned temporary identification number which individual's service results had been received.",
"The results would then be made available to the individual, and the temporary identification number would be de-correlated from the individual so that the number could be reused.",
"The phrase “transient storage area” emphasizes the fact the system will delete, or otherwise render unreadable, the data in this storage area under a number of circumstances.",
"For example, the data might be deleted after a certain date has been reached or a certain period of time has elapsed.",
"The data might also be deleted if the service provider attempts to copy it.",
"Further, in cases where the genomic data is stored on a data card, the data might be deleted upon removal of the card from the card reader.",
"The data might also be deleted if the service provider attempts to handle the data in a manner other than the one agreed upon.",
"For example, the data might be deleted if the service provider attempted to perform tests on it other than those agreed upon.",
"In this way, the individual could feel assured that his genomic data would only be used for the purposes she agreed to and the service provider would be prevented from having permanent possession of the user's data.",
"FIG.",
"7 is a flow chart showing one exemplary embodiment of the above-described procedure.",
"These steps may be performed, for example, by routing/intelligence device 101.In step 701 the user's permission is secured.",
"In step 702, the user's genomic data and additional information is copied to the transient storage area.",
"In step 703, the appropriate provider is allowed access to the storage area.",
"In step 704 it is determined if a criterion for deletion has been met.",
"If the answer is “yes,” flow proceeds to step 705 where the data is deleted.",
"If the answer is “no,” flow proceeds to step 704.In the second embodiment, the system would copy the genomic and additional information in the manner disclosed for the first embodiment.",
"However, instead of the data being copied to a transient storage area, a “self-destructing” data package containing the data is created by the system.",
"In preferred embodiments, the identification number and the description of the service requested are also placed in this data package.",
"The system allows the service provider to download this package.",
"The phrase “self-destructing” refers to the fact that the package is capable of deleting itself, or otherwise rendering the data that it carries unreadable.",
"There are a number of circumstances under which the data would self-destruct.",
"These circumstances may include the sample circumstances that led to deletion of data from the transient storage area in the first embodiment.",
"For example, the package might be set with an expiring-self-destruct.",
"In such a case, the package would destroy its data after a certain date had been reached or a certain period of time had elapsed.",
"The data might also be set to self-destruct if the service provider attempted to copy it.",
"Further, in cases where the genomic data is stored on a data card, the data might be set to self-destruct upon removal of the card from the card reader.",
"In these ways, the service provider would be prevented from having permanent possession of the user's data.",
"Further, the package might be set to self-destruct if the service provider attempted to handle the data in a manner other than the one agreed upon.",
"For example, the data might self destruct if the provider attempted to perform tests on it other than those agreed upon.",
"In this way, the individual could feel assured that his genomic data would only be used for the purposes she agreed to.",
"To deal with the eventuality of the package being intercepted in transport, additional self-destruct events could be added.",
"For example, the package could have its data stored in an encrypted format, and be programmed to “self-destruct” if an incorrect key were applied to it.",
"Further, the package could be programmed with a “self-destructing countdown timer”.",
"In such an embodiment, when the card was shipped a self-destruction countdown timer would be set, perhaps for 12 hours.",
"When the service provider claimed receipt of the package, the management company would give the service provider a code with which to “defuse” the self-destruction countdown.",
"Hence, if the package were intercepted in transport, the interceptor would not know the code with which to defuse the countdown, and thus the data on the card would self-destruct after the allotted time elapsed.",
"Such a self-destruction countdown timer should not be confused with the above described expiring-self-destruct feature.",
"An important difference between the two is that the countdown timer can be “defused,” while the expiring-self-destruct cannot.",
"The third embodiment is like the second, but the data package is not made available to the service provider for downloading.",
"Instead, it is placed on a data card that is physically delivered to the service provider.",
"Such a “self-destructing data card” would preferably be a smart card or some other card with on-board computing abilities.",
"The on-board computational device would be programmed with a series of events that should result in “self-destruction” of the data, as well as with a self-destruct routine.",
"These events would include, among others, those described in connection with the above-described self-destructing data package.",
"The computational device would watch for these events, and when one occurred the processor would execute a routine which would delete the card's data contents.",
"In preferred embodiments, the card would also have the features of the above-described permanent data card.",
"For example, the card would preferably have its data stored in an encrypted format, with the card's on-board processor handing the decryption process.",
"Thus, in practice, the genomic data, and any additional information, would be loaded onto the self-destructing data card.",
"The self-destructing data card would be physically delivered, preferably via a secure carrier or messenger, to the service provider.",
"Preferably, the data would be stored in an encrypted format for which the service provider would be given a “key”.",
"In this way if the card were intercepted in transport, or someone took improper possession of the card, the contents of the card would be inaccessible.",
"In the fourth embodiment, the service provider reads the genomic data, and perhaps additional information, directly from a data card.",
"Thus, the provider only has access to this data during the period of time for which the card is in the card reader.",
"Once the card was removed from the reader, access would no longer be possible.",
"In one scenario, the card reader would be located at the third party's physical location, and the individual would go there to insert his card.",
"In another scenario, the individual would insert the card at a reader distant from the third party, such as a reader-equipped I/O terminal 105, and the service provider would read the data from the card via a secure and preferably encrypted network connection.",
"Preferably, the card reader would capture or otherwise lock the card into place in a manner that it could only be removed from the reader by the owner of the card.",
"In this way the individual could go to a card reader location, insert her card, and retrieve it at a later time.",
"In one embodiment, the identification number corresponding to the user and a description of the service requested could be delivered to the provider using one of the first three embodiments, while the genomic data itself would be made available using the method of the fourth embodiment.",
"If the provider required additional information which was not on the data card, the provider might receive this data by having the user enter it on an I/O terminal 105.Alternately, the additional information could be delivered to the provider using one of the first three embodiments.",
"As in the above embodiments, “self-destruct” procedures could be set up to protect the data.",
"For example, the additional information could be sent as a self-destructing data card, and the personal data card would destroy its data if the service provider executed one of the above-described self-destruct events.",
"Alternately, instead of the personal data card destroying its data, it could instead leave its data intact but cut off the provider's access to it.",
"For example, if the third party attempted to access the data more times than was agreed upon, the provider's access to the card would be cut off, but the card would remain undamaged so as to not inconvenience the owner.",
"In other embodiments, the card would shut off not the provider's access to the card, but all read access to the card.",
"In such embodiments the user would have to reactivate the card to make it functional again, perhaps by inserting it into an I/O terminal with card reader and entering a secret reset code.",
"In these embodiments, the data is preferably encrypted such that the service provider needs a key to access the data In some embodiments, each service provider is periodically provided a unique “period key” which will be used to decode all data that the provider is to access during that period.",
"For example, the provider may be given a key each month which is to be used to decode all data during that month.",
"In some embodiments the period key would be transmitted via a secure, encrypted transmission.",
"For example, a security officer at the service provider might have a master key that can be used to decrypt the period keys that are provided.",
"In other embodiments, the period key could be delivered physically by bonded messenger.",
"In some embodiments, the provider may be given a multitude of keys at once.",
"The multitude of keys could be stored in a lookup-table which associates keys with code words.",
"In such an embodiment, the service provider might receive from the management company a daily e-mail or daily physical letter via bonded messenger which contains the code word of the day.",
"For example, the service provider might be informed that “today is an AZSDEFE” day.",
"The service provider would feed “AZSDEFE” into the lookup table in order to receive the day's key.",
"In still other embodiments, the keys would not be sent to the service providers at all.",
"Instead, each provider could be given a algorithm for creating the keys.",
"The management company could use corresponding algorithms to encode data meant to be read by each provider.",
"For further security, the key-generating algorithm could be a “black box” in the eyes of the service providers, which is to say they would not know how the algorithm worked.",
"These methods provide only examples, and other methods known to those versed in the art for encryption and the providing of keys may be used as well.",
"In yet another embodiment, the invention concerns a method of marketing a product in a geographic region of interest, comprising obtaining information relating to at least one correlation between users' positive response to the product and at least one haplotype profile, determining the frequency of the haplotype profile in the population living in the geographic region, and making a marketing decision for the geographic region based on the determined frequency of the haplotype profile.",
"As used herein, the term “marketing” means any activity associated with advertising, offering to sell and selling goods or services.",
"The product may be any good or service for which there is a perceived need or demand in a particular geographic region.",
"Preferably, the good or service is medically related, and more preferably the good or service is a drug or biologic, either of which may be previously approved by an appropriate regulatory agency, may be the subject of a pending application for regulatory approval or may be marketed without regulatory approval.",
"The geographic region of interest may be defined in any one of a number of ways, e.g., by the official or unofficial boundaries of a town, city or section thereof, county, state, multi-state region (e.g., in the United States, the Northeastern, Midwestern, Southern or Western region) country or continent.",
"In one embodiment, the geographic region of interest may be a territory serviced by a healthcare provider such as an individual pharmacy or a pharmaceutical chain.",
"In other embodiments, the geographic region of interest may be a territory serviced by a healthcare payer.",
"The response to the product includes any type of response exhibited by the user or consumer of the product that indicates the product will at least meet the perceived need or demand for such a product in the geographic region, and preferably indicates this need or demand will be met in a way that is superior to the performance of other products marketed in that geographic region for the same need or demand.",
"In preferred embodiments, the product is a drug or biologic and the response to such a product is the therapeutic profile exhibited by the population present in the geographic region of interest or by a suitable reference population therefor.",
"Information relating to the haplotype profile correlation may already exist at the time this marketing method is being performed; such preexisting information might be obtained from a provider of genomic services such as the management company described herein.",
"Alternately, obtaining such correlation information requires performing a new study that is designed to identify any correlations between genetic variation and product response that may exist in the population living in the geographic region.",
"The entity seeking to market the product could conduct this study by itself or could contract the performance of this study with another party, e.g., the management company described herein.",
"The frequency of the correlated haplotype profile(s) in the population in the targeted geographic region may be determined by consulting preexisting genomic data for that population, or a reference population therefor, or by independently testing individuals for the presence or absence of the haplotype profile.",
"The genetic testing may be performed for either most or all individuals of the population of interest to directly determine the frequency of the haplotype profile.",
"Alternately, the haplotype profile frequency in the population of interest may be indirectly determined, or estimated, by the frequency of the haplotype profile that is directly determined for a suitable reference population.",
"As above, the seeking to market the product may determine the frequency of the haplotype profile in the population of interest or may contract with another party, e.g., the management company described herein, to determine this frequency information.",
"The marketing decision is based on the determined frequency of the haplotype profile in the population living in the geographic region of interest and may include a decision to market the product to the geographic region if the frequency of the haplotype profile correlated with a positive response to the product indicates that the product will achieve a sufficient level of market penetration in the targeted geographic region to meet the business goals and/or profit objectives of the entity seeking to market the product in that region.",
"Alternately, if the frequency of the haplotype profile indicates the product will produce low sales in the geographic region of interest, the marketing decision may be to not proceed with marketing the product in that geographic region.",
"In yet another embodiment, the invention provides a method for developing a new product to satisfy a particular unmet demand or need of a population, comprising identifying a haplotype profile that is correlated with the unmet demand or need in the population, determining a functional cause for the correlation between the haplotype profile and the unmet need or demand, and developing a new product designed to avoid the functional cause.",
"Examples of unmet demands or needs to which this invention may be applied include but are not limited to weight management, addictions to harmful substances such as nicotine, alcohol and other drugs, and diseases that are not being adequately treated in the population of interest by existing drugs or therapies.",
"To identify a haplotype profile that is correlated with an unmet demand or need, the frequency of haplotypes for one or more genes in a first population having the unmet need or demand is compared with the frequency of such haplotypes in a second population that either lacks the same need or demand or in which existing products satisfy that need or demand.",
"This frequency information may be obtained using any method known in the art, including those described or incorporated by reference herein, or may be obtained from a genomic data management company such as described herein.",
"The genes evaluated may primarily be those that are candidate genes for the unmet need or demand, or may constitute all genes known to exist in the genome.",
"Any haplotype or combination of haplotypes that is significantly higher in the first population than in the second population is a haplotype profile that may be selected to evaluate in the next step of the method, which is identifying a functional cause for the correlation between that haplotype profile and the unmet need or demand.",
"For example, where the unmet need is a disease that is not adequately treated by existing drugs in the first population, the functional cause may be a haplotype in the haplotype profile that defines an isoform of the target for the existing drugs that has poor binding characteristics for such existing drugs.",
"Or, the functional cause may be a haplotype in the haplotype profile that causes the bearer of that haplotype to be a poor metabolizer of these existing drugs, which may lead to lack of efficacy and/or undesirable side effects.",
"Once the functional cause for the correlation between the haplotype profile and unmet need or demand is determined, a new product may be designed to overcome the functional cause.",
"For example, if the functional cause is poor drug binding characteristics for the isoform of the drug target that is present in the first population, then new candidate compounds for treating the disease may be identified by screening against that isoform of the drug, or alternately, a different target and drug combination with potential efficacy in treating the disease may be sought.",
"Similarly, if the functional cause is the presence of a “poor metabolizer haplotype” in the haplotype profile, a new candidate drug metabolized by another metabolic enzyme or via a different pathway may be evaluated for its ability to overcome this functional cause and thereby meet the unmet need or demand.",
"The invention further provides a method for marketing a drug for inclusion in a formulary controlled by a healthcare provider or healthcare payer.",
"Nonlimiting examples of the healthcare provider and payer are hospitals and HMOs, respectively.",
"In one embodiment, the method comprises identifying a haplotype profile that is correlated with a good therapeutic profile for the drug, determining the frequency of the haplotype profile in the population served by the formulary and making a marketing decision based on the determined frequency of the haplotype profile.",
"In one embodiment, the marketing decision is to pursue inclusion in the formulary if the frequency of the haplotype profile indicates that a significant percentage of the population served by the formulary will exhibit a better response to the new drug than drugs currently in the market, which may include drugs already in the formulary.",
"In another embodiment, the invention provides a method for choosing a drug for inclusion in the formulary.",
"This method comprises identifying a group of drugs that are prescribed to treat or alleviate the same medical condition, symptoms or disease, and determining for each drug a haplotype profile that is correlated with an acceptable therapeutic response profile for that drug.",
"Then, the frequency of each of these haplotype profiles in the population served by the formulary is determined.",
"A drug is chosen for the formulary based on the determined haplotype profile frequencies.",
"For example, the drug whose correlated haplotype profile is the most frequent of the identified haplotype profiles may be the only drug from the group included in the formulary, or alternately, each drug in the group whose haplotype profile is present above a certain percentage in the population served by the formulary may be chosen.",
"It should be noted that many other embodiments are within the spirit of the invention and the preceding is only by way of example, and should not be construed to limit the invention to any of the specific details disclosed above."
]
] | Patent_10433700 |
[
[
"Collapsing chair with solid armrest and tensioned seat",
"A chair (100) with solid armrests (150) and a tensioned seat collapses in a single movement, in which the front legs (110) approximate each other when the front and rear legs (120) pivot towards each other and when the seat and the back pivot towards each other."
],
[
"1.A collapsible chair, comprising: a pair of front legs, a pair of rear legs, and a pair of solid arm rests, wherein at least one of the rear legs is telescoping, and wherein at least one of the front legs is rotatably coupled to at least one of the pair of rear legs; a backrest coupled to a pair of back support rods, and a seat coupled to a pair of seat support rods; and wherein the front legs, the rear legs, the arm rests, the back support rods, and the seat support rods are coupled in a manner such that the chair collapses in a single movement in which the front legs approximate each other when the front legs and the rear legs pivot towards each other and when the seat support rods and the back support rods pivot towards each other.",
"2.The collapsible chair of claim 1 wherein one of the pair of front legs and one of the pair of rear legs are rotatably coupled to each other, and wherein one of the pair of front legs and one of the pair of rear legs are rotatably coupled to one of the pair of arm rests.",
"3.The collapsible chair of claim 1 wherein one of the pair of seat support rods is rotatably coupled to one of the pair of front legs and one of the pair of rear legs.",
"4.The collapsible chair of claim 1 further comprising a first pair of cross braces that couple the pair of seat support rods to the pair of front legs.",
"5.The collapsible chair of claim 4 further comprising a second pair of cross braces that couple the pair of back support rods to the pair of rear legs.",
"6.The collapsible chair of claim 1 wherein at least one of the pair of front legs, the pair of rear legs, the pair of seat support rods, and the pair of back support rods is manufactured from aluminum.",
"7.The collapsible chair of claim 1 wherein the backrest comprises a weather resistant fabric.",
"8.The collapsible chair of claim 1 wherein the backrest comprises Nylon.",
"9.The collapsible chair of claim 1 wherein the backrest is removably attached to the pair of back support rods.",
"10.The collapsible chair of claim 1 wherein the tensioned seat is removably attached to the pair of seat support rods.",
"11.The collapsible chair of claim 1 wherein the seat is coupled to the backrest.",
"12.The collapsible chair of claim 1 wherein the pair of seat support rods pivot upwardly and the pair of rear legs leg pivot towards the pair of front legs, when the chair folds into a closed configuration.",
"13.",
"(canceled) 14.",
"(canceled) 15.",
"(canceled) 16.",
"(canceled) 17.",
"(canceled)"
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Folding chairs are relatively popular, in part because they can be stored at considerably reduced space requirements when compared to non-folding chairs.",
"Exemplary folding chairs with solid arm rests and seat support rods are described in U.S. Pat.",
"No.",
"4,613,185 to Marchesini et al.",
"(Sep. 23, 1986), U.S. Pat.",
"No.",
"5,899,525 to Tseng (May 4, 1999), U.S. Pat.",
"No.",
"5,947,553 to Tseng (Sep. 7, 1999), and U.S. Pat.",
"No.",
"6,062,639 to Hill (May 16, 2000), all of which are incorporated by reference herein.",
"Nevertheless, previously known folding chairs still take up a relatively large space when folded, since the dimension of the folding chair is generally reduced only along one space coordinate (e.g., reduced length).",
"To further reduce the space requirement, collapsible chairs have been developed in which further size reduction is achieved by folding the chair along at least two space coordinates (e.g., length and width).",
"Various collapsing chairs are known in the art.",
"For example, Sparkes describes in U.S. Pat.",
"No.",
"Des 247,618 (Mar.",
"28, 1978) a collapsible stroller with solid armrests and seat support rods that is first folded in a front to back motion and then folded in a side-to-side motion.",
"Although Sparkes' stroller provides substantial space saving over non-collapsible strollers, the collapsing is relatively cumbersome since both halves of the chair have to be rotated relative to each other.",
"Improved collapsibility can be achieved by including a detachable connector element into a structure that couples foldable halves of the stroller as described in U.S. Pat.",
"No.",
"4,317,581 to Kassai (Mar.",
"2, 1982).",
"Uncoupling of the detachable element advantageously simplifies side-to-side folding of the stroller.",
"However, Kassai's stroller nevertheless requires at least two folding operations to collapse the stroller.",
"To avoid at least some of the problems associated with multiple operations of collapsible chairs, Mann describes in U.S. Pat.",
"No.",
"5,058,950 (Oct. 22, 1991) the use of hinges in both armrests and seat support rods.",
"Mann's chair can be collapsed in a single side-to-side and front-to-back motion, however, the hinges in the armrests may be uncomfortable for at least some of the users.",
"Moreover, due to the particular configuration of movable elements in the chair, front and back seat support rods are perpendicular to the legs of a person sitting in the chair, which will likely restrict blood flow in the legs of almost all users over a prolonged period.",
"Although there are various collapsible chairs with solid armrests known in the art, all or almost all of them suffer from one or more disadvantages.",
"Therefore, there is a need to provide improved methods and apparatus for collapsible chairs with solid armrests."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to a collapsible chair that can be collapsed in a single motion, wherein the chair includes a pair of solid armrests and a tensioned seat coupled to a pair of seat support rods.",
"In particular, the collapsible chair has a pair of front legs, a pair of rear legs, and a pair of solid arm rests, wherein at least one of the rear legs is telescoping.",
"A backrest is coupled to a pair of back support rods, and a tensioned seat is coupled to a pair of seat support rods, wherein the front legs, the rear legs, the arm rests, the back support rods, and the seat support rods are coupled in a manner such that the chair collapses in a single movement in which the front legs approximate each other when the front legs and the rear legs pivot towards each other and when the seat support rods and the back support rods pivot towards each other.",
"In one aspect of the inventive subject matter, at least one of the front legs and one of rear legs are rotatably coupled to each other and rotatably coupled to an arm rest.",
"It is further contemplated, that at least one of the seat support rods is rotatably coupled to a front and rear leg.",
"Additional cross braces may couple the seat support rods to the front legs, and further additional cross braces may couple the back support rods to the rear legs.",
"In another aspect of the inventive subject matter, the legs, back support rods and seat support rods are manufactured from a metal, preferably aluminum.",
"It is preferred that the seat and backrest are removably attached to the chair, and are fabricated from a weather resistant material, preferably a synthetic polymer, and more preferably from Nylon.",
"In a further aspect of the inventive subject matter, a method of imparting collapsibility into a chair comprises one step in which a front leg, a telescoping rear leg, a seat support rod, a back support rod, and a solid armrest are provided.",
"In a further step, both the back support rod and the front leg are rotatably coupled with the armrest and the seat support rod to form a quadrilateral.",
"In a still further step, the telescoping rear leg is rotatably coupled to at least one of the armrest and the front leg, and rotatably coupled to at least one of the seat support rod and the back support rod.",
"Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawing, in which like numerals represent like components."
],
[
"FIELD OF THE INVENTION The field of the invention is collapsible furniture.",
"BACKGROUND OF THE INVENTION Folding chairs are relatively popular, in part because they can be stored at considerably reduced space requirements when compared to non-folding chairs.",
"Exemplary folding chairs with solid arm rests and seat support rods are described in U.S. Pat.",
"No.",
"4,613,185 to Marchesini et al.",
"(Sep. 23, 1986), U.S. Pat.",
"No.",
"5,899,525 to Tseng (May 4, 1999), U.S. Pat.",
"No.",
"5,947,553 to Tseng (Sep. 7, 1999), and U.S. Pat.",
"No.",
"6,062,639 to Hill (May 16, 2000), all of which are incorporated by reference herein.",
"Nevertheless, previously known folding chairs still take up a relatively large space when folded, since the dimension of the folding chair is generally reduced only along one space coordinate (e.g., reduced length).",
"To further reduce the space requirement, collapsible chairs have been developed in which further size reduction is achieved by folding the chair along at least two space coordinates (e.g., length and width).",
"Various collapsing chairs are known in the art.",
"For example, Sparkes describes in U.S. Pat.",
"No.",
"Des 247,618 (Mar.",
"28, 1978) a collapsible stroller with solid armrests and seat support rods that is first folded in a front to back motion and then folded in a side-to-side motion.",
"Although Sparkes' stroller provides substantial space saving over non-collapsible strollers, the collapsing is relatively cumbersome since both halves of the chair have to be rotated relative to each other.",
"Improved collapsibility can be achieved by including a detachable connector element into a structure that couples foldable halves of the stroller as described in U.S. Pat.",
"No.",
"4,317,581 to Kassai (Mar.",
"2, 1982).",
"Uncoupling of the detachable element advantageously simplifies side-to-side folding of the stroller.",
"However, Kassai's stroller nevertheless requires at least two folding operations to collapse the stroller.",
"To avoid at least some of the problems associated with multiple operations of collapsible chairs, Mann describes in U.S. Pat.",
"No.",
"5,058,950 (Oct. 22, 1991) the use of hinges in both armrests and seat support rods.",
"Mann's chair can be collapsed in a single side-to-side and front-to-back motion, however, the hinges in the armrests may be uncomfortable for at least some of the users.",
"Moreover, due to the particular configuration of movable elements in the chair, front and back seat support rods are perpendicular to the legs of a person sitting in the chair, which will likely restrict blood flow in the legs of almost all users over a prolonged period.",
"Although there are various collapsible chairs with solid armrests known in the art, all or almost all of them suffer from one or more disadvantages.",
"Therefore, there is a need to provide improved methods and apparatus for collapsible chairs with solid armrests.",
"SUMMARY OF THE INVENTION The present invention is directed to a collapsible chair that can be collapsed in a single motion, wherein the chair includes a pair of solid armrests and a tensioned seat coupled to a pair of seat support rods.",
"In particular, the collapsible chair has a pair of front legs, a pair of rear legs, and a pair of solid arm rests, wherein at least one of the rear legs is telescoping.",
"A backrest is coupled to a pair of back support rods, and a tensioned seat is coupled to a pair of seat support rods, wherein the front legs, the rear legs, the arm rests, the back support rods, and the seat support rods are coupled in a manner such that the chair collapses in a single movement in which the front legs approximate each other when the front legs and the rear legs pivot towards each other and when the seat support rods and the back support rods pivot towards each other.",
"In one aspect of the inventive subject matter, at least one of the front legs and one of rear legs are rotatably coupled to each other and rotatably coupled to an arm rest.",
"It is further contemplated, that at least one of the seat support rods is rotatably coupled to a front and rear leg.",
"Additional cross braces may couple the seat support rods to the front legs, and further additional cross braces may couple the back support rods to the rear legs.",
"In another aspect of the inventive subject matter, the legs, back support rods and seat support rods are manufactured from a metal, preferably aluminum.",
"It is preferred that the seat and backrest are removably attached to the chair, and are fabricated from a weather resistant material, preferably a synthetic polymer, and more preferably from Nylon.",
"In a further aspect of the inventive subject matter, a method of imparting collapsibility into a chair comprises one step in which a front leg, a telescoping rear leg, a seat support rod, a back support rod, and a solid armrest are provided.",
"In a further step, both the back support rod and the front leg are rotatably coupled with the armrest and the seat support rod to form a quadrilateral.",
"In a still further step, the telescoping rear leg is rotatably coupled to at least one of the armrest and the front leg, and rotatably coupled to at least one of the seat support rod and the back support rod.",
"Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawing, in which like numerals represent like components.",
"BRIEF DESCRIPTION OF THE DRAWING FIG.",
"1 is a perspective view of a collapsible chair without seat and backrest.",
"FIG.",
"2 is a perspective detail view of the collapsible chair of FIG.",
"1.FIG.",
"3 is a perspective view of the collapsible chair of FIG.",
"1 with attached seat and backrest.",
"FIG.",
"4 is a perspective view of the collapsible chair of FIG.",
"1 in a collapsed configuration.",
"DETAILED DESCRIPTION It is generally contemplated that a collapsible chair includes a pair of solid armrests, a tensioned seat coupled to a pair of seat support rods, and a backrest coupled to a pair of back support rods.",
"Contemplated chairs further have a pair of front legs, a pair of telescoping rear legs, and can be collapsed in a single motion in which the front legs approximate each other when the front legs and the rear legs pivot towards each other and when the seat support rods and the back support rods pivot towards each other.",
"In a particularly preferred aspect of the inventive subject matter (as depicted in FIG.",
"1), a collapsible chair 100 has a pair of front legs 110, a pair of telescoping rear legs 120, and a pair of seat support rods 130.A backrest (not shown in FIG.",
"1, see FIG.",
"3) is attached to a pair of back support rods 140, which are coupled to the rear legs 120 and the seat support rods 130.A pair of solid armrests 150 is coupled to the front legs 110, telescoping rear legs 120, and the back support rods 140.A first pair of cross braces 160 is rotatably coupled to the front legs and seat support rods, and a second pair of cross braces 170 is rotatably coupled to the rear legs and back support rods.",
"A preferred manner of coupling the armrest, the front leg, the telescoping rear leg, the seat support rod and the back support rod is depicted in FIG.",
"2, which is a detail view of the chair depicted in FIG.",
"1.Here, the front leg 210 is rotatably coupled to both the seat support rod 230 and solid armrest 250, while the back support rod 240 is rotatably coupled to both the armrest 250 and the seat support rod 230.The telescoping rear leg 220 is rotatably coupled to the back support rod 240, the seat support rod 230, the armrest 250, and the front leg 210.The seat support rod 230 is rotatably coupled to the front leg 210 and the telescoping rear leg 220.FIG.",
"3 depicts the collapsible chair of FIG.",
"1 with a seat 380 and a backrest 390, and FIG.",
"4 shows the collapsible chair of FIG.",
"1 in a collapsed configuration.",
"It is contemplated that the seat and the backrest are fabricated from a weather resistant material or fabric, preferably a woven synthetic polymer (e.g., Nylon) and is uniformly colored (e.g., blue).",
"Particularly preferred seats have a width of about 21 inches and an overall length of about 24 inches.",
"However, it should be appreciated that various alternative materials, colors, and sizes are also appropriate.",
"For example, alternative materials may include natural and synthetic fabrics and all reasonable combinations thereof.",
"Contemplated materials may further be woven or non-woven and particularly contemplated materials include polyester, polyvinyl chloride, cotton, hemp, and wool.",
"With respect to the color, it is contemplated that suitable colors need not be restricted to uniform color, but appropriate colors may also include color patterns, prints, no color at all.",
"While it is generally preferred that the chair according to the inventive subject matter is sized and dimensioned to fit an average adult person, it is also contemplated that appropriate chairs may also accommodate a child, a smaller- or larger-than-average adult, or more than a single person.",
"Therefore, alternative chairs may have dimensions that are wider than 21 inches, and suitable widths include 21-24, 24-30, and 30-40 inches, and wider, but also 18-21, 14-18, and 8-14 inches, and narrower.",
"Likewise, the length of appropriate seats may vary between 20-42, 15-10, and 12-15 inches and less, but also between 24-27, 27-30, and more.",
"It should further be appreciated that contemplated seats may also be tapered from the front end to the back end, or vice versa.",
"With respect to the backrest it is contemplated that the backrest is fabricated from the same material as the seat, and that the backrest is removably or permanently coupled to the seat (e.g., sewed, coupled with a zipper, etc.)",
"Thus, it is preferred that the backrest has a width of about 21 inches.",
"A preferred height of the backrest is about 24 inches.",
"With respect to the material and color, it is contemplated that the same considerations as for the seat apply.",
"It is further contemplated that the width and height of suitable backrests may vary, and that width and height will depend among other things on the persons' size and the number of persons to be seated in the chair.",
"Thus, alternative backrests may have a width between 18-12 inches and less, but also between 18-22 and more.",
"Similarly, contemplated backrests may have a height between 12-24 inches and less, but also between 24-30 inches and more.",
"It is generally contemplated that the seat and the backrest may be coupled to the back support rods and seat support rods in numerous ways, including temporary and permanent coupling.",
"Temporary couplings include hook-and-loop type fasteners, snaps, buckles, slidable elements (e.g., a pouch slidably coupled to a post, a ring slidably coupled to a rod, etc.",
"), and threadbly securable elements (e.g., laces threaded through rings).",
"Permanent couplings include sewed or glued elements.",
"For example, the backrest may permanently coupled to the chair via a rivet.",
"On the other hand, the front end of the seat may be temporarily coupled to the front legs via ring-shaped openings slided over the top ends of the front legs.",
"It is further particularly preferred that the seat and the backrest are coupled together.",
"It should further be appreciated that the attachment of the seat and/or the backrest to the chair may be directly or indirectly.",
"As used herein, the term “direct” attachment means that the seat and/or the backrest are in immediate contact with the supporting structure, whereas the term “indirect” means that an additional element connects the seat and/or backrest with the supporting structure.",
"For example, the seat may be directly attached to the seat support rods via a slidable pouch.",
"Alternatively, the seat may be indirectly coupled to the seat support rods via a ring-shaped opening in the seat that slidably engages with the rods.",
"With respect to the legs, back support rods, seat support rods, armrests, and cross braces of contemplated chairs, it should be appreciated that all of these elements may be manufactured from various materials, including metals, metal alloys, natural and synthetic polymers, and any reasonable combination thereof.",
"However, it is preferred that the legs, back support rods, seat support rods, and cross braces are manufactured from black anodized aluminum tubing with a wall strength of about 1/32 inch and an outer diameter of approximately ½ inch.",
"It is also preferred that the solid armrest is fabricated from a recycled molded thermoplastic polymer, and especially preferred alternative materials for the armrest, legs, support rods, and cross braces include stainless steel, fiberglass, and wood.",
"As used herein, the term “solid armrest” means that the armrest is fabricated from a sufficiently stiff material to maintain the shape of the armrest while the chair is being folded or unfolded.",
"For example, a wooden or metal armrest is considered a solid armrest under the scope of this definition, because such armrests are fabricated from a material with sufficient stiffness to maintain the shape of the armrest.",
"In contrast, a woven or textile armrest, is not considered a solid armrest under the scope of this definition because such armrests will deform (i.e.",
"change their shape) while the chair is folded or unfolded.",
"Where one of the armrests, legs, back and seat support rods, and cross braces are rotatably or pivotally coupled to another one of the armrests, legs, back support rods, seat support rods, and cross braces it is generally contemplated, that all known manners of rotatably coupling are suitable for use in conjunction with the teachings presented herein.",
"For example, appropriate manners of rotatably coupling include coupling of two elements via a common axis, coupling via a hinge wherein the hinge may or may not have a slidable connection to another element, coupling via a ball bearing, etc.",
"Similarly, where one of the legs, seat support rods, and cross braces are slidably coupled to another one of the legs, seat support rods, and cross braces, all known slidable couplings are contemplated to be appropriate, and include a sliding sleeve, slide rails, guiding rings, etc.",
"It should further be recognized that contemplated couplings may be realized in various configurations.",
"For example, where the telescoping rear leg and the front leg are coupled to the armrest at substantially the same location, the coupling may involve a single axle, pin, or otherwise rotatable connector.",
"Alternatively, two independent axles, pins, or otherwise rotatable connectors may be employed.",
"Furthermore, rotatable couplings may include rotatable couplings that are also slideable relative to their point of attachment.",
"For example, contemplated alternative rotatable couplings may include a sleeve that may be slideably coupled to e.g., a front leg with an axle which may rotatably couple another element to the coupling.",
"Thus, it should be appreciated that the coupling may be rotatable and slidable.",
"Alternatively, where rotatable and slidable couplings are less desirable, temporary couplings may be employed and suitable temporary couplings include snap connectors, connectors that are secured with a pin or other removable element, etc.",
"It is generally contemplated that various methods and configurations may be employed to impart variability in length to the telescoping rear leg.",
"Thus, a telescoping rear leg need not be limited to a configuration in which a first portion of the leg is at least partially disposed within the lumen of a second portion (see FIG.",
"2).",
"Alternative configurations may include a first portion that is movable relative to a second portion, wherein the first and second portion may or may not directly contact each other.",
"For example, a first portion and a second portion may share a common rail or other sliding mechanism through which the first and second portion are coupled to each other.",
"In another example, the first and second portion may be coupled through a series of hinge members.",
"Alternatively, a first portion and a second portion may be engaged and/or disengaged with each other in a fixed or otherwise predetermined position relative to each other by clamps, bolts, screws, etc.",
"In a still further example, the telescoping rear leg may change its length via compressible, foldable, or otherwise pliable material.",
"While it is generally contemplated that the extent to which the length of the rear leg is variable is not limiting to the inventive subject matter, it is preferred that the rear leg has a minimum length when the chair is in an extended configuration (i.e., unfolded to support a person), and a maximum length when the chair is in a substantially completely collapsed configuration.",
"It should further be appreciated that a collapsible chair may have a self-folding configuration by employing a gas spring as a telescoping element in the rear leg.",
"Thus, it should be recognized that the rear leg may be telescoping in its entirety, or may only include a telescoping portion.",
"In a further aspect of the inventive subject matter, a collapsible chair has two solid armrests, two front legs, two telescoping rear legs, two back support rods, and two seat support rods.",
"A first pair of cross braces couples (a) a first front leg with a first seat support rod on the opposite side of the chair, and (b) the second front leg with the second seat support rod.",
"A second pair of cross braces couples (a) a telescoping rear leg with a first back support rod on the opposite side of the chair, and (b) the second telescoping rear leg with the second back support rod.",
"It is generally contemplated that all of the front legs, rear legs, back and seat support rods are coupled through the cross braces in a manner that allows collapsing the chair in a single movement, i.e., that the front legs approximate each other and pivot towards the rear legs as the arm rests pivot towards the back support rods.",
"As viewed from another perspective, the seat support rods pivot upwardly and the rear legs leg pivot towards the pair of front legs, when the chair folds into a closed configuration.",
"In alternative aspects of the inventive subject matter the number of legs, and/or seat support rods, and/or cross braces may vary considerably.",
"For example, where the chair is sized and dimensioned to accommodate more than one person, three, four, or more legs, and/or seat supports, and/or cross braces may be included.",
"On the other hand, where stability of the seat is particularly desirable, three or more seat support rods may be included in a chair with two front legs and two rear legs.",
"Likewise, the number of cross braces may vary, and while some chairs may have only one pair of cross braces, other chairs may include three, four, or more cross braces.",
"It should be especially appreciated that in contemplated configurations of collapsible chairs, the seat is tensioned when the front legs move apart, and that the seat remains substantially tensioned when the seat supports a person.",
"The term “tensioned seat” means that the seat is substantially level when the chair is in the open configuration, wherein the term “substantially level” means that the vertical distance between any point of the seat and the seat support rod is no more than one 0.75 inch, more preferably no more than 0.5 inch, and most preferably no more than 0.5 inch.",
"The term “open configuration” refers to the configuration of the collapsible chair in which the front legs have a maximum distance from each other when the chair is opened using reasonable force (i.e.",
"without damaging the mechanical structure).",
"The term “remains substantially tensioned” means that the vertical distance between the lowest point of the seat and the seat support rod increases no more than one inch, preferably no more than one inch, more preferably no more than 0.75 inch, and most preferably no more than 0.5 inch.",
"Thus, it should be recognized that the tension of the seat is predominantly determined by the firmness of the material of the seat.",
"While not whishing to be bound by a particular theory, it is contemplated that the tension in the seat remains substantially tensioned due to mechanically coupling an approximating movement of the seat support rods with a simultaneous movement of at least one of the rear legs relative to the front leg and a movement of the front legs relative to each other.",
"View from another perspective, it should be recognized that contemplated modes of coupling the front leg with the rear leg and the seat support rod prevent loss of tension of the seat when a person is supported by the chair.",
"Thus, a method of imparting collapsibility into a chair has one step in which a front leg, a telescoping rear leg, a seat support rod, a back support rod, and a solid armrest are provided.",
"In another step, both the back support rod and the front leg are rotatably coupled with the armrest and the seat support rod to form a quadrilateral.",
"In a further step, the telescoping rear leg is rotatably coupled to at least one of the armrest and the front leg, and rotatably coupled to at least one of the seat support rod and the back support rod.",
"The so formed quadrilateral that is coupled to the telescoping rear leg may then be coupled with a second quadrilateral that is coupled to a second telescoping rear leg via at least one cross brace.",
"The term “quadrilateral” as used herein refers to a configuration of a plurality of elements in which at least four of the plurality of elements are coupled to each other to form a polygon of four sides.",
"For example, a regular or distorted trapezoid made of four elements coupled to each other is considered a quadrilateral under the scope of this definition.",
"Contemplated methods may further include comprising attaching a first cross brace to the seat support rod, comprising attaching a second cross brace to the seat support rod, attaching a backrest to the back support rod, and attaching a seat to the seat support rod.",
"With respect to the front leg, the telescoping rear leg, the seat support rod, the back support rod, the seat, the backrest, and the solid armrest, it should be appreciated that the same considerations as described above apply.",
"Thus, specific embodiments and applications of collapsible chairs have been disclosed.",
"It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein.",
"The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims.",
"Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context.",
"In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced."
]
] | Patent_10433703 |
[
[
"Method for quantifying cytosine methylations in genomic dna that is amplified in a complex manner",
"A method is described for preparing demethylated DNA as reference material for the analysis of cytosine methylations in genomic DNA samples with the use of complex amplification."
],
[
"1.A method for providing demethylated DNA as reference material for the analysis of cytosine methylations in genomic DNA samples with the use of complex amplifications is hereby characterized in that the following method steps are conducted: a) a genomic DNA sample is amplified, wherein either very short or degenerate oligonucleotides or oligonucleotides complementary to adaptors are used each time as primers; in the latter case, the genomic DNA is cleaved with a restriction enzyme prior to the amplification, and adaptors are ligated to the ends of the DNA fragments that are formed; b) the amplificates are chemically treated in such a way that cytosine bases that are unmethylated are converted to uracil, or another base unlike cytosine in hybridization behavior, while the 5-methylcytosine bases remain essentially unchanged; c) the chemically pretreated amplificates are again amplified, whereby either several specifically hybridizing oligonucleotides or oligonucleotides complementary to the adaptors are used each time as primers; in the latter case, the adaptors are also converted according to the rules of step 1b.",
"2.A method for the analysis of cytosine methylations in genomic DNA samples with the use of complex amplifications by means of adaptors, is hereby characterized in that the following method steps are conducted: a) a genomic DNA sample to be investigated is cleaved by means of a restriction enzyme; b) adaptors are ligated to the ends of the DNA fragments and the sample is then divided; the first portion of the sample is amplified by means of oligonucleotides, which are complementary to the adaptors, as primers, whereas the second portion of the sample is not amplified; b) the two portions of the sample are chemically treated separately in such a way that cytosine bases that are unmethylated are converted to uracil, or another base unlike cytosine in hybridization behavior, while the 5-methylcytosine bases remain essentially unchanged; d) the two portions of the sample that are chemically treated are amplified, whereby oligonucleotides complementary to the adaptors after the chemical treatment are used as primers; e) both portions of the sample are analyzed, whereby the first portion of the sample supplies the reference value for a methylation degree of 0%, and the second portion of the sample supplies the measurement value, which essentially corresponds to the degree of methylation in the original genomic DNA sample.",
"3.The method according to claim 1 or 2, further characterized in that a PCR (polymerase chain reaction) is used for the amplification.",
"4.The method according to claim 1 or 2, further characterized in that a heat-stable DNA polymerase is used for the polymerase chain reaction.",
"5.The method according to claim 1 or 2, further characterized in that the amplification of several DNA segments is conducted in one reaction vessel.",
"6.The method according to claim 1 or 2, further characterized in that the chemical treatment is conducted with sodium bisulfite (=hydrogen sulfite, disulfite).",
"7.The method according to claim 1 or 2, further characterized in that after the chemical treatment, the amplificates are separated from reagents and other components of the reaction mixture by binding to a solid phase or to a gel and by washing steps.",
"8.The method according to claim 1 or 2, further characterized in that after the chemical treatment, the reagents and the other components of the reaction mixture are preferably then diluted in such a way that they are no longer troublesome in the subsequent amplification, but the concentration of the treated amplificate is still sufficient for the second amplification.",
"9.The method according to claim 1 or 2, further characterized in that one of the following restriction endonucleases is used: RsaI, DpnI, DpnII, MseI, Sau3AI, AluI, NlaIII, HaeIII, BfaI, Tsp509I, BstUI or MboI.",
"10.The method according to claim 1 or 2, further characterized in that the demethylated reference DNA which is produced is analyzed in the same way as a sample DNA to be investigated and supplies in the analysis the reference value for a methylation degree of 0%.",
"11.The method according to claim 1 or 2, further characterized in that a DNA which is methylated enzymatically and which is treated in the same way as the sample DNA in the following steps, additionally is used as a reference for a methylation degree of 100%."
],
[
"The invention concerns a method for the quantification of cytosine methylations of a genomic DNA sample with unknown methylation status by comparison with a demethylated reference DNA.",
"5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells.",
"For example, it plays a role in the regulation of transcription, genomic imprinting and in tumorigenesis.",
"The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest.",
"5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behavior as cytosine.",
"In addition, in the case of a PCR amplification, the epigenetic information which is borne by the 5-methylcytosines is completely lost.",
"The amplification of DNA by means of PCR is state of the art.",
"Several methods are known which solve these problems.",
"For the most part, a chemical reaction or enzymatic treatment of the genomic DNA is conducted, as a consequence of which cytosine bases can be distinguished from methylcytosine bases.",
"One familiar method is the conversion of genomic DNA with bisulfite, which leads to a conversion of cytosine bases to uracil in two steps after alkaline hydrolysis (Shapiro, R., Cohen, B., and Servis, R. Nature 227, 1047 (1970)).",
"5-Methylcytosine remains unchanged under these conditions.",
"The conversion of C to U leads to a change of the base sequence from which the original 5-methylcytosines can now be determined by sequencing.",
"The modification of the genomic base cytosine to 5-methylcytosine represents up to today the most important and best-investigated epigenetic parameter.",
"Nevertheless, even though there are now methods for determining complete genotypes of cells and individuals, there are still no comparable approaches for also generating and extensively evaluating epigenotypic information.",
"Demethylated DNA is used in the prior art in numerous methods for quantification of DNA methylation.",
"The following are named as representative techniques: “Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension” (Gonzalgo, M. L., Jones P. A. Nucleic Acids Res.",
"25, 2529 (1997)) and “Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA” (Warnecke, P. M., Stirzaker, C., Melki, J. R., Douglas, S. M., Paul, C. L., Clark, S. J.",
"[Journal] 25, 4422 (1997)).",
"This demethylated DNA is obtained, e.g., from cells which are demethylated in the target sequence, or from cells which lack the enzyme, DNA methyltransferase.",
"On the other hand, it is easily possible to produce demethylated DNA fragments by means of PCR, since the methylation information becomes lost during the amplification, i.e., cytosine is always incorporated instead of methylcytosine, if dCTP is utilized as usual in the polymerase reaction.",
"If one would like to investigate cytosine methylation, however, as mentioned above, by means of the bisulfite method, in which all unmethylated cytosine bases are converted to uracil and finally to thymine, then for the quantification, a reference DNA must be produced, which contains thymine, instead of all methylated and unmethylated cytosines.",
"This DNA then serves as reference material for a methylation state of 0%.",
"This DNA can be obtained in the simplest way, for the analysis of individual fragments, by first conducting a PCR of the genomic DNA sample in a first amplification and thus the desired fragment is produced, which then essentially no longer has any methylation.",
"The bisulfite treatment is subsequently conducted and the fragment in question is now amplified for the second time with appropriate, but different primers.",
"This method is not suitable, however, for conducting complex amplifications, which will provide many fragments simultaneously for methylation detection.",
"The problem arises here that it is difficult to ensure that the first amplification also substantially produces the fragments which will be amplified in the second PCR after the bisulfite reaction.",
"This is essential, however, since the sample to be investigated, for whose methylation analysis the reference DNA is produced, is usually amplified exclusively after the bisulfite treatment.",
"Therefore, in a complex PCR reaction, the reference and the sample can no longer be compared, since they potentially contain different fragments.",
"Presentation of the Problem The present invention provides a method for the analysis of cytosine methylations in genomic DNA samples, for the purpose of which, DNA with a methylation degree of 0% is produced as reference material.",
"It is thus possible for the first time to produce a substantially unmethylated reference DNA for complex amplifications.",
"DESCRIPTION The present invention describes a method for providing demethylated DNA as reference material for the analysis of cytosine methylations in genomic DNA samples with the use of complex amplifications.",
"The following method steps are conducted individually for this: a) A genomic DNA sample is amplified with primers, which are either very short or degenerate oligonucleotides or oligonucleotides complementary to adaptors.",
"In the second case, prior to the amplification, the sample is cleaved with a restriction enzyme, and the adaptors, which are understood to be short nucleotide fragments of known sequence, are ligated to the ends of the DNA fragments that are formed.",
"The amplificates are chemically treated in such a way that cytosine bases that are unmethylated at the 5-position are converted to uracil, thymine or another base unlike cytosine in hybridization behavior, while the 5-methylcytosine bases remain essentially unchanged.",
"This is understood in the following as chemical pretreatment.",
"The chemically pretreated amplificates are again amplified.",
"Either several specifically hybridizing oligonucleotides or oligonucleotides complementary to adaptors are used as primers for this purpose.",
"The chemical pretreatment is also conducted for the latter case.",
"b) A genomic DNA sample to be investigated is cleaved by means of a restriction enzyme.",
"Adaptors are ligated to the ends of the DNA fragments and the sample is then divided.",
"The first portion of the sample is amplified with primer oligonucleotides, which are complementary to the adaptors.",
"In contrast, the second portion of the sample is not amplified.",
"The two parts of the sample are chemically pretreated and then amplified separately, whereby primer oligonucleotides, which are complementary to the adaptors, are used.",
"The two portions of the sample are then analyzed.",
"The first portion of the sample thus supplies the reference value for a methylation degree of 0%.",
"In contrast, the second portion of the sample supplies the measurement value that essentially corresponds to the degree of methylation in the original genomic DNA sample.",
"The genomic DNA to be analyzed is obtained preferably from the usual sources for DNA, such as, e.g., cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast, liver, skin or bone marrow, histological slides and all possible combinations thereof.",
"Preferably, the above-described treatment of genomic DNA with bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis, which converts unmethylated cytosine nuleobases to uracil, is used for this purpose.",
"In a particularly preferred variant of the method, the polymerase chain reaction (PCR) is used for the amplification.",
"A heat-stable DNA polymerase is preferably used for the polymerase chain reaction.",
"The amplification of several identical or several different DNA segments is preferably conducted in one reaction vessel.",
"The following are preferably used as restriction endonucleases: RsaI, DpnI, DpnII, MseI, Sau3AI, AluI, NlaIII, HaeIII, BfaI, Tsp509I, BstUI or MboI.",
"After the chemical treatment, the amplificates are separated from the reagents and other components of the reaction mixture by binding to a solid phase or to a gel and subsequent washing steps.",
"The reagents and the other components of the reaction mixture are preferably then diluted in such a way that they are no longer troublesome in the subsequent amplification, but the concentration of the treated amplificate is still sufficient for the second amplification.",
"The demethylated reference DNA which is produced is most preferably analyzed in the same way as a sample DNA to be investigated.",
"This reference DNA supplies in the analysis the reference value for a methylation degree of 0%.",
"Preferably, a DNA which is methylated enzymatically and which is treated in the same way as the sample DNA in the following [steps], additionally serves as a reference for a methylation degree of 100%.",
"The following examples explain the invention: EXAMPLE 1a Preparation of a Demethylated Reference DNA by Means of Multiplex PCR The following example refers to the preparation of a down-methylated DNA sample, which serves as the reference in comparison to an unknown methylated DNA.",
"A genomic DNA sample, which was digested in this case with the restriction enzyme, MssI, is used.",
"Then (1-40 ng) of the cleaved DNA are amplified by a preamplification, by conducting a DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) according to the method of Nelson (V. G. Cheung, S. F. Nelson, PNAS 93, 1476-1479, 1996) with the genomic primer oligonucleotide 5′-CCGACTCGAGNNNNNNATGTG G-3′.",
"The method particularly serves for the purpose of preamplifying very small quantities of genomic DNA, in order to permit a multiple genetic analysis from 2-15 μg (200-1000 bp).",
"All methylcytosines are treated as cytosine bases in the amplification.",
"Reaction batch (50 μl): 1 μl (1-40 ng) DNA 2 μl (2 μM) DOP Primer (5′-CCGACTCGAGNNNNNNATGTG G-3′) 5 μl (200 μM) dNTP's (Fermentas) 5 μl PCR buffer (10×, 15 mM MgCl2) (Qiagen) 0.5 μl (2.5 U) Taq Polymerase (HotstarTaq, Qiagen) 36.5 μl water (for molecular biology, Fluka) The PCR reaction is conducted in the Master Cycler Gradient (Eppendorf, Hamburg) with the following program.",
"The PCR sample is diluted with water (1:10-1:100) and 1 ul of the diluted material is chemically converted with hydrogen sulfite (=bisulfite, disulfite).",
"The DNA is first thermally denatured and then reacted with hydrogen sulfite (=bisulfite, disulfite), a radical trap and a denaturing reagent, and is incubated for a relatively long time at elevated temperature.",
"The bisulfite reaction leads to the conversion of all cytosine bases to uracil.",
"In order to purify the bisulfited DNA, it is bound to a reversed-phase C18 solid phase and is freed of chemicals by washing with a suitable buffer solution.",
"Then the DNA is eluted with a polar solvent, such as, e.g., acetonitrile or water and concentrated to a smaller volume.",
"Alkaline hydrolysis of the fragments treated with hydrogen sulfite (=bisulfit, disulfite) is conducted for 20 min at 96° C. under basic conditions directly prior to the specific amplification.",
"Preferably 1-500 different primer oligonucleotides which do not contain wobble base pairings are utilized in this reaction.",
"In this Example, the specific amplification is conducted with 128 primer oligonucleotides, wherein at least 64 primer oligonucleotides are labeled with Cy5 (Amersham Pharmacia).",
"One primer oligonucleotide of a primer pair is labeled each time.",
"Reaction batch, multiplex PCR (25 μl) 1 μl DNA treated with hydrogen sulfite 2.5 μl PCR buffer (10×, Qiagen) 0.6 μl Primer oligonucleotide mixture (128 primer oligonucleotides, 64 of which are labeled with Cy5, 0.78 pmol/μl of each) 0.8 μl dNTPs (25 mM per dNTP, Gibco-BRL) 3 μl MgCl2 (15 mM) 4.5 μl water (for molecular biology, Fluka) 12.5 μl Tris-HCl (pH 9.5; 100 mM) 0.2 μl Polymerase (1 unit) (HotstarTaq, Qiagen) The PCR reaction is conducted in the Master Cycler Gradient (Eppendorf, Hamburg) with the following program.",
"The PCR amplificates produced were analyzed by agarose gel electrophoresis (1.5% agarose in 0.5×TBE buffer, Sambrook et al.).",
"For this, 4 μl of the PCR batch are subjected to gel electrophoresis.",
"Under the given conditions, 64 genes are successfully amplified simultaneously.",
"EXAMPLE 1B Preparation of an Unknown Methylated DNA Sample by Means of Multiplex PCR The following Example 1b concerns the preparation of an unknown methylated DNA sample, which is compared with the down-methylated reference DNA from Example 1a.",
"A genomic DNA sample is used, which was cleaved in this case with the restriction enzyme, MssI.",
"The sample is then reacted with hydrogen sulfite (=bisulfite, disulfite).",
"Then one can proceed according to 2 different methods.",
"The first method (Olek et al., Nucl.",
"Acids Res.",
"1996, 24, 5064-5066) is a conversion with hydrogen sulfite and a radical trap, wherein the DNA is embedded in agarose.",
"The desulfonation of the DNA is also conducted in agarose.",
"The DNA is used in this case without further purification operations in a preamplification (PEP=primer extension preamplification).",
"Alternatively, the DNA can also be chemically converted without agarose matrix employing hydrogen sulfite (=bisulfite, disulfite) and a radical trap at elevated temperature.",
"An organic reagent, which supports the denaturation, is added, and the batch is incubated at elevated temperature.",
"All cytosine bases are converted to uracil in both methods by the treatment with hydrogen sulfite, whereas methylcytosines remain the same.",
"In order to purify the bisulfited DNA without agarose matrix, it is bound to a reversed-phase C18 solid phase and is freed of chemicals by washing with a suitable buffer solution.",
"Then the DNA is eluted with a polar solvent, such as, e.g., acetonitrile and water and concentrated to a smaller volume.",
"The preamplification of the DNA treated with hydrogen sulfite is conducted with degenerate primer oligonucleotidea (5′-TTATAATGTTTT and 5′-TAATACTAAT).",
"Reaction batch (20 μl): 1 μl bisulphite DNA (0.2-1 ng) 2 μl reaction buffer (10×, Qiagen) 2 μl dNTP's (10 mM per dNTP, Fermentas) 1 μl Primer (TTATAATGTTTT) 25 pmol 1 μl Primer (TAATATACTAAT) 25 pmol 0.2 μl polymerase (1 unit) (HotstarTaq, Qiagen) 12.8 μl water (for molecular biology, Fluka) The following amplification with Cy5-labeled bisulfite-specific primer oligonucleotides is conducted with the 128 primer oligonucleotides described in Example 1a, whereby the same primer oligonucleotide is labeled with Cy5.The amplificates are also subjected to an agarose gel electrophoresis for analysis.",
"EXAMPLE 1c Comparison of the Unknown Methylated DNA Sample with the Down-Methylated Reference DNA The unknown methylated DNA sample is compared with the down-methylated reference DNA, preferably by hybridization on an oligonucleotide array.",
"Fluorescing points are visible corresponding to position on the array.",
"It happens that specific points show a clearly increased or decreased fluorescence relative to other points and to the reference DNA, as long as the amplificates are present in comparable concentration in the individual samples to be investigated.",
"The intensity of the fluorescent dye Cy5 (635 nm) is measured in the individual amplificates.",
"Techniques for the evaluation of fluorescence measurements are known to the person skilled in the art.",
"EXAMPLE 2 Preparation of Demethylated Reference DNA In the first step, a genomic sequence is enzymatically cleaved according to the manufacturer's instructions by addition of a restriction enzyme, here NlaIII (Fermentas), which recognizes the sequence CATG.",
"Thus, fragments of an average 400 bp in size are produced.",
"The cleaved fragments have 3′ overhanging CATG ends and are ligated with the oligomer with the genomic sequence TGTCATCCTGTTGTCATG with the addition of T4-DNA ligase according to standard conditions (Fermentas) at the sequence segments and unligated adaptors are removed according to standard conditions with a purification kit (Qiaquick PCR Purification Kit, Qiagen).",
"Then the single-stranded ends are completed to form the double strand with Klenow enzyme (DNA Polymerase I, Roche Molecular Biochemicals) and dNTP's (FIG.",
"1a).",
"If a reference DNA is to be produced, then the procedure is the following: In the following step, the ligated sequence segments are amplified in a PCR reaction with the addition of primer oligonucleotides with the sequence TGTCATCCTGTTGTCATG and with a heat-stable DNA polymerase.",
"The PCR reaction is conducted in the Master Cycler Gradient (Eppendorf, Hamburg) with the following parameters: Denaturation: 15 minutes (min) at 96° C., [and] the following cycles are repeated 45 times: 60 seconds (sec) at 96° C., 45 sec at 51° C., 60 sec at 72° C. and subsequent incubation for 10 minutes at 72° C. In the next step, the DNA is treated with the use of bisulfite (hydrogen sulfite, disulfite) in such a way that all of the unmethylated cytosines at the 5-position of the base are modified such that a base that is different in its base-pairing behavior is formed, while the cytosines that are methylated in the 5-position remain unchanged.",
"If 1.7 M bisulfite solution is used for the reaction, then an addition occurs at the unmethylated cytosine bases.",
"Also, a denaturing reagent or solvent as well as a radical trap must be present.",
"A subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases to uracil.",
"This converted DNA serves for the detection of methylated cytosines.",
"In the last step of the method, the treated DNA sample is diluted with water or an aqueous solution.",
"A desulfonation of the DNA (20 min, 96° C.) at pH 9 is then preferably conducted.",
"In the last step of the method, the DNA sample is amplified with the primers now complementary to the bisulfite-treated DNA, again in a polymerase chain reaction.",
"The PCR reaction is conducted in the Master Cycler Gradient (Eppendorf, Hamburg) with the following parameters: Denaturation: 15 minutes (min) at 96° C., [and] the following cycles are repeated 45 times: 60 seconds (sec) at 96° C., 45 sec at 42° C., 60 sec at 72° C. and subsequent incubation for 10 minutes at 72° C. (FIG.",
"1b).",
"If a DNA with unknown methylation state is to be investigated (FIG.",
"2), then the cleaved DNA ligated with adaptors (FIG.",
"1a) is to be treated with bisulfite.",
"After the bisulfite treatment, a PCR is conducted, whereby primer oligonucleotides with the sequences TGTTATTTTGTTGTTTAG and TATCATCCTATTATGATA are used.",
"The PCR reaction is conducted in the Master Cycler Gradient (Eppendorf, Hamburg) with the following parameters: Denaturation: 15 minutes (min) at 96° C.; [and] the following cycles are repeated 45 times: 60 seconds (sec) at 96° C., 45 sec at 42° C., 60 sec at 72° C. and subsequent incubation for 10 minutes at 72° C. Both in the case of the down-methylated reference DNA as well as in the case of a DNA with unknown methylation state, the detection of the hybridization product is based on primer oligonucleotides fluorescently labeled with Cy5, which were used for the amplification.",
"A hybridization reaction of the amplified DNA with the oligonucleotide occurs only if a methylated cytosine was present at this site in the bisulfite-treated DNA.",
"Thus the methylation state of the respective cytosine to be investigated decides the hybridization product.",
"Legends to the figures which follow: FIG.",
"1a a) Restriction enzyme, NlaIII b) Adaptor 1 5′-TGTCATCCTGTTGT, ligase e.g.",
"T4-DNA c) Kienow enzyme (DNA Polymerase I), dNTP's, buffer d) Purification (Qiaquick Purification Kit) FIG.",
"1b e) PCR, Primer 5′-TGTCATCCTGTTGTCATG, dNTP's, buffer, TAQ f) Reaction with hydrogen sulfite g) PCR, primer 1 5′-TGTTATTTTGTTGTTATG, primer 2 5′-TATCATCCTATTATCATA FIG.",
"2: a) Reaction with hydrogen sulfite b) PCR, primer 1 5′-TGTTATTTTGTTGTTATG, primer 2 5′-TATCATCCTATTATCATA"
]
] | Patent_10433742 |
[
[
"Spinal intervertebral implant adjustable in situ comprising hard pass point",
"The invention concerns a spinal intervertebral implant (200) comprising at least an upper element (203), a lower element (205), and an intermediate member (201) adapted to co-operate with the upper and lower elements by helical linking means (207, 254) including means (216) forming hard pass points when the helical linkage is carried out."
],
[
"1.A spinal intervertebral implant comprising at least an upper element (203; 403), a lower element (205; 405), and an intermediate member (201; 401) which can cooperate with the upper and lower elements via helical linking means (207, 254; 407, 435, 450), characterized in that the helical linking means include means (216; 408, 416) forming hard pass points when the helical linkage is carried out.",
"2.The implant as claimed in claim 1, characterized in that the helical linking means comprise at least a cam (254; 435, 450) and a cam follower (207; 407) which is able to come into contact with a bearing surface (254; 438, 454) of the cam.",
"3.The implant as claimed in claim 2, characterized in that the bearing surface has zones forming the hard pass points (236, 237; 417, 419).",
"4.The implant as claimed in claim 2 or 3, characterized in that the zones forming the hard pass points comprise pass points (236, 237; 417, 419) protruding from the bearing surface.",
"5.The implant as claimed in claim 4, characterized in that the high pass points are bosses (219; 417, 419).",
"6.The implant as claimed in one of claims 2 through 5, characterized in that the intermediate member comprises the cam.",
"7.The implant as claimed in one of claims 2 through 5, characterized in that the intermediate member comprises the cam follower (207; 407).",
"8.The implant as claimed in one of the preceding claims, characterized in that the intermediate member (401) can be received in one of the upper (403) and lower (405) elements.",
"9.The implant as claimed in claim 8, characterized in that the central element can be received in the other of the upper and lower elements.",
"10.The implant as claimed in one of claims 1 through 7, characterized in that one of the upper (203) and lower (205) elements can be received in the intermediate member (201).",
"11.The implant as claimed in claim 10, characterized in that the other of the upper and lower elements can be received in the intermediate member.",
"12.The implant as claimed in one of the preceding claims, characterized in that the helical linkage between the intermediate member and the upper element has a direction of screwing counter to that of the helical linkage between the central element and the lower element.",
"13.The implant as claimed in one of the preceding claims, characterized in that it additionally comprises means (432) for blocking in position at least one of the upper and lower elements relative to the intermediate member.",
"14.The implant as claimed in claim 13, characterized in that the means for blocking in position comprise a stud (432).",
"15.The implant as claimed in claim 14, characterized in that the stud comprises a part which is eccentric in relation to the main axis of use of the stud.",
"16.The implant as claimed in claim 14, characterized in that the stud comprises a part which can be screwed.",
"17.The implant as claimed in one of the preceding claims, characterized in that it comprises means (222; 422) for anchoring the implant in the vertebral plateaus.",
"18.The implant as claimed in one of the preceding claims, characterized in that the upper (203) and lower (205) elements can fit into one another through being of complementary shape.",
"19.The implant as claimed in claim 18, characterized in that the upper and lower elements fit into one another with sliding.",
"20.The implant as claimed in claim 18 or 19, characterized in that each lower and upper element has a general U-shape, the elements being able to fit into one another with the U-shapes in opposition.",
"21.The implant as claimed in claim 18, 19 or 20 and as claimed in claim 7, characterized in that, with at least one of the upper and lower elements comprising the cam (254), said cam has an opening facing its bearing surface (245)."
],
[
"The invention concerns implants of the intervertebral cage type, or of the type for replacement of vertebral bodies, intended for the spinal column.",
"The document WO 99/56675 discloses an implant intended for the replacement of vertebral bodies and comprising a first, central element with two threads, the pitches of which threads run counter to one another.",
"It also comprises two end elements, that is an upper end element and a lower end element, which can be screwed onto said central element.",
"In this way, the implant can be adjusted in situ by rotating the central element.",
"Thus, the two end elements, that is the upper end element and the lower end element, are moved away from one another or closer to one another depending on the sense of rotation of the central element.",
"However, in the case of this maneuver, it is difficult to know how the adjustment stands.",
"This entails a maneuvering difficulty in surgical operations, the result being a prolongation of the operating time.",
"It is an object of the invention to make available an implant of the intervertebral cage type, or of the type for replacement of vertebral bodies, which it is possible to adjust in situ in a sufficiently quick and precise manner.",
"For this purpose, provision is made, according to the invention, for a spinal intervertebral implant comprising at least an upper element, a lower element, and an intermediate member which can cooperate with the upper and lower elements via helical linking means which include means forming hard pass points when the helical linkage is carried out.",
"Thus, the means forming hard pass points permit indexing of the in situ adjustment in a simple and precise manner for the surgeon.",
"Advantageously, the screwing means comprise at least a helical cam and a cam follower which can come into contact with a bearing surface of the cam.",
"Advantageously, the bearing surface has zones forming the hard pass points.",
"Advantageously, the zones forming the hard pass points comprise pass points protruding from the bearing surface.",
"Advantageously, the upper pass points are bosses.",
"Advantageously, the intermediate member comprises the cam.",
"Advantageously, the intermediate member comprises the cam follower.",
"Advantageously, the intermediate member can be received in one of the upper and lower elements.",
"Advantageously, the intermediate member can be received in the other of the upper and lower elements.",
"Advantageously, one of the upper and lower elements can be received in the intermediate member.",
"Advantageously, the other of the upper and lower elements can be received in the intermediate member.",
"Advantageously, the helical linkage between the intermediate member and the upper element has a direction of screwing counter to that of the helical linkage between the central element and the lower element.",
"Advantageously, the implant additionally comprises means for blocking in position at least one of the upper and lower elements relative to the intermediate member.",
"Advantageously, the means for blocking in position comprise at least one stud.",
"Advantageously, the stud comprises a part which is eccentric in relation to the main axis of use of the stud.",
"Advantageously, the stud comprises a part which can be screwed.",
"Advantageously, the implant comprises means for anchoring the implant in vertebral plateaus.",
"Advantageously, the upper and lower elements can fit into one another through being of complementary shape.",
"Advantageously, the upper and lower elements fit into one another with sliding.",
"Advantageously, each upper and lower element has a general U-shape, the elements being able to fit into one another with the U-shapes in opposition.",
"Advantageously, with at least one of the upper and lower elements comprising the cam, said cam has an opening opposite its bearing surface.",
"Provision is also made, according to the invention, for a surgical method which comprises the steps of positioning the implant at the implantation site and adjusting said implant in situ by using the screwing means between the central element and the end elements, the screwing means having means which form hard pass points.",
"Advantageously, the surgical method additionally comprises a step of filling the implant with a substance that promotes bone growth.",
"Other characteristics and advantages of the invention will become evident from the following description of a preferred embodiment of the invention and of an alternative embodiment given as nonlimiting examples.",
"In the attached drawings: FIG.",
"1 is a view, in three dimensions, of a first embodiment of the invention in the position of minimum height; FIG.",
"2 is a view, in three dimensions, of the first embodiment of the invention in the position of maximum height; FIG.",
"3 is a view, in three dimensions, of one of the bases of the first embodiment from FIG.",
"1; FIG.",
"4 is a view, in three dimensions, of the intermediate element of the first embodiment from FIG.",
"1; FIG.",
"5 is a view, in three dimensions, of a second embodiment of the invention in the position of minimum height; FIG.",
"6 is a view, in three dimensions, of the second embodiment of the invention in the position of maximum height; FIG.",
"7 is a view, in three dimensions, of the intermediate element of the second embodiment from FIG.",
"5; FIG.",
"8 is a view, in three dimensions, of one of the bases of the second embodiment from FIG.",
"5; and FIG.",
"9 is a view, in three dimensions, showing the arrangement of the two base elements of the second embodiment from FIG.",
"5.A first embodiment of the invention will be described with reference to FIGS.",
"1 through 4.The implant 400 of the intervertebral cage type in this present embodiment comprises the following main elements: an upper base 403, a lower base 407, and an intermediate element 401 which can be received with sliding and rotation inside the upper and lower bases.",
"The intermediate element 401 is a tube having an internal diameter and an external diameter.",
"The wall of the tube comprises a plurality of longitudinal openings 410 similar to elongate slots.",
"These openings 410 have their main dimension parallel to a generatix of the tube forming the intermediate element and are parallel to one another.",
"The openings 410 extend radially from the outer face 411 to the inner face 412, these faces delimiting the thickness of the tube forming the intermediate element 401.This intermediate element 401 thus has an internal hollow space which extends from an upper face 413 to a lower face 414 and which is delimited radially by the inner face 412.The internal hollow space can receive any osteoconductive or osteoinductive substance that promotes bone fusion.",
"Moreover, the intermediate element 401 comprises a plurality of studs 407 protruding outward in a radial direction from the outer face 411 of the intermediate element 401.The studs 407 are uniformly distributed about the circumference of the tube in two groups situated, respectively, near the upper end face 413 and lower end face 414.Each group here comprises three studs 407.Each of the studs 407 has what is called a contact surface 474, a plane front face 472 perpendicular to the surface 474, and engagement means 470.The surface 474 is a surface of revolution, here cylindrical, while the face 472 is plane and circular.",
"The height of the studs 407 is such that, once the implant 400 has been mounted, the face 472 is flush with the outer face 473 of the bases 403 and 405, which bases will now be described.",
"Referring to FIG.",
"3, the lower base 405 is, here, a tube having an upper face 427, a lower face 426, an outer face 473, and an inner face 471.The internal diameter of the tube is substantially equivalent to the external diameter of that forming the intermediate element 401.Thus, upon assembly of the implant 400, the face 471 is in contact with the face 411, and the intermediate element 401 is mounted movably with sliding and rotation on the base 405.The upper face 427 is perpendicular to the axis of revolution of the tube forming the base 405.The lower face 426, here substantially parallel to the face 427, comprises a plurality of teeth 422, here profiled with a triangular section and parallel to one another.",
"The teeth 422 in the present case form means of anchoring in the vertebral plateaus with which the face 426 can come into contact.",
"The outer face 473 comprises a plurality of openings 450 of helical form which extend through the thickness of the wall of the tube forming the base 405 and which are uniformly distributed about the circumference of the base 405.Here, they are three in number.",
"Each opening 450 has a smooth face 452 situated toward the plane face 472 of the base 405 and forming a single ramp.",
"The opening 450 additionally has an opposite surface 454, facing and substantially parallel to the face 452.This surface 454, called a contact surface, comprises a group of several ramps 416, here six in number, of concave form, whose radius of curvature is substantially equal to the radius of the surface 474 of the studs 407 with which the ramps 416 can come into contact during use of the implant 400.Each ramp 416 comprises a low end 417 and a high end 419.The arrangement of the ramps 416 within the group is such that a high end of one ramp forms the low end of the following ramp.",
"The ramps 416a and 416b, called end ramps, of the group do not have a low end or high end, respectively, because their surface continues with the same radius of curvature in such a way as to tangentially rejoin the face 452 and thus complete the opening 450.Moreover, the outer face 473 of the base 405 comprises a plurality of orifices 430 of circular cross section which are threaded (the thread is not shown), are situated near the face 427, are uniformly distributed about the circumference of the base 405, and are able to receive a lock 432 which is screwed in.",
"The lock is longer than the thickness of the wall of the base 405 so as to be able to engage in one of the openings 410 of the intermediate element 401.The role of the lock 432 is to secure the assembly of the implant 400, as will be seen later in the description.",
"Similarly, the outer face 473 of the base 405 comprises a plurality of orifices 434 of circular cross section which are smooth, are situated near the face 426 of the base 405, and are uniformly distributed about the circumference of the base 405.These orifices are able to improve the communication between the outside and the inside of the implant 400 when the latter is in the high configuration, as is illustrated in FIG.",
"8.The upper base 403 is, in a transverse plane perpendicular to the axis of revolution of the base, the mirror-symmetrical counterpart of the lower base 405 which has just been described in detail.",
"The base 403 comprises a plurality of openings 435 symmetrical to the openings 450: each opening 435 comprises a smooth face 436 forming a single ramp, a surface 438, called the contact surface, opposite the face 436 and comprising a group of a plurality of ramps 408 similar to the ramps 416.The group comprises ramps 408a and 408b, called end ramps, whose surface continues in the same radius of curvature in order to tangentially rejoin the face 436.The base 403 additionally comprises a face 424 with anchoring means 422, here teeth similar to those present on the face 426 and previously described.",
"At the time of the surgical intervention, the surgeon establishes the access route and then prepares the implantation site.",
"He then assembles the implant 400 by choosing the intermediate element 401 and also a base 403 and a base 405.He joins the bases to the intermediate element.",
"He places the various studs 407 on the element 401 via the helical openings 435 and 450.He fills the inside of an implant 400, thus formed, with an osteoinductive or osteoconductive substance.",
"He then places the implant 400 in the implantation site.",
"He adjusts the height of the implant 400 by turning the intermediate element 401, while the bases 403 and 405 are immobile because of the anchoring means impacted into the vertebral plateaus delimiting the implantation site.",
"Rotation of the implant 401, on account of the helical linkage joining the intermediate element 401 to each base 403, 405, causes the studs 407 to move in their respective openings 435 and 450, their surface 474 passing from one ramp 416, 408 to another, thus permitting adjustment of the height of the implant.",
"Once the desired distraction height has been obtained, the surgeon secures the implant 400 in the distraction position, by using the position-blocking means 432, then closes his access route.",
"By virtue of the discontinuities between the ramps 416, the surgeon knows that each ramp passage corresponds to a fixed increase or decrease in height.",
"This indexing of the height adjustment means that the surgeon knows how the distraction of the implantation site stands.",
"A second embodiment of the invention will be described with reference to FIGS.",
"5 through 9.The implant 200 of the intervertebral cage type in this embodiment comprises three main elements: an upper base 203, a lower base 205, and an intermediate element 201.The upper 203 and lower 205 bases can be received with sliding and rotation inside the intermediate element 201 in order to form a helical linkage as in the aforementioned first embodiment.",
"The intermediate element 201 is a tube having an internal diameter and an external diameter.",
"The wall of the tube comprises a first plurality of openings 210, of helical form, whose helix is screwed in the direction of rotation of the hands of a clock, downward when its axis is vertical.",
"These openings 210, here numbering two, are uniformly distributed about the circumference of the tube and are situated near the lower end face 250.Likewise, the wall of the tube comprises a second series of helical openings 211 whose helix is screwed in the opposite direction to the hands of a clock, downward when its axis is vertical.",
"These openings 211, here also numbering two, are uniformly distributed along the circumference of the tube and are situated near the upper end face 251.The openings 210 and 211 each have a bearing surface 217, 218, respectively.",
"The bearing surfaces can come into contact with position-blocking means (not shown in the figures), as will be seen later in the description.",
"Moreover, near the upper 251 and lower 250 end faces, the intermediate element 201 comprises a plurality of circular orifices 212 uniformly distributed about the circumference of the tube and here numbering two.",
"These orifices 212 extend through from the outer face 204 to the inner face 215.Each of the orifices 212 comprises a threaded part 213 and a countersink 214 whose diameter is greater than that of the threaded part 213.The thread 213 can cooperate with the thread present on a stud 207, which will be described below.",
"Likewise, the countersink 214 can receive the head of the stud 207.Each of the studs 207 comprises a substantially plane and circular face 272 in which engagement means 270 are formed, here in the form of a hexagonal socket.",
"Once in place, the stud 207 is flush with the outer face 204 of the intermediate element 201.Moreover, the length of this stud 207 is much greater than the thickness of the tube forming the intermediate element.",
"This is so that it can come into contact with a part 254 of the upper 203 and lower 205 bases, as will now be described.",
"Referring to FIG.",
"8, only the upper base 203 will be described, because the lower base 205, in a transverse plane perpendicular to the axis of revolution of the base, is the mirror-symmetrical counterpart of the upper base 203 which will now be described in detail.",
"The upper base 203 is, here, a tube having an upper face 224, a lower face 225, an outer face 231, and an inner face 234.The external diameter of the tube is substantially equivalent to the internal diameter of that forming the intermediate element 201.Thus, upon assembly of the implant 200, the face 231 is in contact with the face 215, and the upper base 203 is thus mounted movably with sliding and rotation in the intermediate element 201.The lower face 225 is perpendicular to the axis of revolution of the tube forming the base 203.The upper face 224, here substantially parallel to the face 225, comprises a plurality of teeth 222, here profiled with a triangular cross section having concave sides and uniformly distributed in a circle having, as its center, the axis of revolution of the tube forming the base 203, and having a diameter corresponding to the mean of the internal and external diameters of said tube forming said base 203.The teeth 222 in the present case form means of anchoring in the vertebral plateaus with which the face 224 will come into contact.",
"Moreover, the face 224 has a circular orifice 234 with its center the axis of revolution of the tube forming the base 223.The body of the base comprises two extensions forming branches 273 and 274.These two extensions are symmetrical to one another, on the axis of revolution of the tube forming the base 203.The two extensions 273 and 274 delimit, on either side of the axis, two openings which are each open toward the lower 225 end face of the base 203, said openings comprising a first substantially vertical lateral face 233, an upper face 254, a face 235, and a second substantially vertical lateral face 232 which is substantially parallel to the first lateral face 233.The upper face 254 called the contact surface or bearing surface comprises a group of several ramps 216, here seven in number, of concave shape whose radius of curvature is substantially equal to the radius of that part of the studs 207 with which the face 254 can come into contact upon use of the implant 200.Each ramp 216 comprises a low end 236 and a high end 237.The arrangement of the ramps 216 within the group is such that the high end of one ramp forms the low end of the following ramp; the ramps 216a and 216b, called end ramps, of the group do not have a high end or low end, respectively, because their surface continues with the same radius of curvature so as to tangentially rejoin the face 233 and the face 235, respectively.",
"The high and low ends 237, 236 form a plurality of bosses 219.Moreover, the branch 273, like the branch 274 too, comprises a radially extending through-orifice 230 of circular cross section.",
"This orifice 230 can receive the position-blocking means (not shown in the figures) of the implant 200.Such an arrangement of the branches 273 and 274 of the base 203, and of their counterparts on the base 205 which is mirror-symmetrical to the base 203, as has been indicated above, allows the two bases to be fitted into one another upon assembly and use of the implant, as is illustrated in FIG.",
"9.The two bases 203 and 205 are mounted with sliding relative to one another, the different faces 232 of one of the bases being able to come into contact with the different faces 233 of the other base, respectively.",
"During this fitting together, each extension penetrates into one of the openings.",
"At the time of the surgical intervention, the surgeon, as before, establishes the access route and then prepares the implantation site.",
"He then assembles the implant 200 by choosing an intermediate element 201 and also a base 203 and a base 205.With the different studs 207 already in place on the intermediate element 201, in the orifices 212, he positions the two bases by inserting them inside the intermediate element 201, as is illustrated in FIG.",
"5, the studs 207 being in contact with the ramps 216a of the upper base 203 and of its symmetrical counterpart of the base 205.The implant is now in its lower configuration.",
"He then fits the position-blocking means (not shown) through the slots 211 and 210, respectively, in the orifices 230 of the bases 203 and 205.The position-blocking means can be studs having a thread cooperating with a thread formed in the orifice 230, or studs which can be fitted with rotation in the orifice 230 and whose head received in the slots 210, 211 is eccentric in relation to the axis of the orifice 230.Once the implant 200 has been assembled, the surgeon fills the inside of the implant with an osteoinductive or osteoconductive substance.",
"He then places the implant 200 in the implantation site.",
"Then he adjusts the height of the implant by turning the intermediate element 201 while the bases 203 and 205 are immobile relative to the respective vertebrae, on account of the anchoring means impacted into the vertebral plateaus which delimit the implantation site.",
"Rotating the intermediate element 201 causes the studs 207 to move along the ramps 216 of each of the openings in the bases 203 and 205.The surgeon then uses the position-blocking means, received through the slots 210 and 211, in order to block the implant in position.",
"The surgeon then closes the access route.",
"It is of course possible for numerous modifications to be made to the present invention without departing from the scope of the latter.",
"The faces comprising the anchoring means could be inclined relative to a main perpendicular plane of the implant.",
"It is possible to conceive of any cam system with cam follower other than that described above, without departing from the present invention.",
"It could also be possible to provide an implant having upper and lower elements able to fit into one another through being of complementary form, for example as is illustrated in FIG.",
"9, without having helical linkages between the intermediate member and the upper and lower elements with means forming hard pass points."
]
] | Patent_10433769 |
[
[
"Brush part for electric toothbrush",
"A brush part for an electric toothbrush in which tufts of bristles are arranged in polygons, and in which the tufts of bristles lean, either around the polygons or inwardly and outwardly, so that the tuft alignments converge and preferably cross."
],
[
"1-12.",
"(canceled) 13.A brush part for an electric toothbrush which includes a bristle holder mounted for rotary motion about a rotation axis, a plurality of first tufts projecting from a surface of the bristle holder and having their bases arranged in a polygon around the rotation axis and leaning outwardly away from the rotation axis so that their distal end is further out from the rotation axis than their base, and a plurality of second tufts projecting from a surface of the bristle holder and having their bases arranged in a polygon around the rotation axis, characterized by: the first and second tufts having their bases at different radial distances from the rotation axis, the first and second tufts oriented relative to the rotation axis such that at a point along their base-distal end length the first and second tufts are respectively at radial distances from the rotation axis with a difference in their respective radial distances less than the difference in the respective radial distances of their bases from the rotation axis.",
"14.A brush part according to claim 1 wherein the bases of the second tufts being located at a greater radial distance from the rotation axis than are the bases of the first tufts.",
"15.A brush part according to claim 13 wherein at a point along their base-distal end length, the first and second tufts are respectively at the same radial distances from the rotation axis so that the difference in their respective radial distances is zero.",
"16.A brush part according to claim 15 wherein at a greater distal distance along their base-distal end length the difference in the respective radial distances from the center increases.",
"17.A brush part according to claim 13 wherein by first and second tufts circumferentially adjacent to each other at the point of minimum difference in their respective radial distances, on a circle centered on the rotation axis.",
"18.A brush part according to claim 13 wherein a plurality of second tufts having their bases further radially out from the rotation axis than the bases of the first tufts, and aligned parallel to the rotation axis.",
"19.A brush part according to claim 13 wherein a plurality of second tufts having their bases further radially out from the rotation axis than the bases of the first tufts, and leaning inwardly toward the rotation axis so that their distal ends are at less distance radially from the rotation axis than their base.",
"20.A brush part according to claim 13 wherein a plurality of second tufts having their bases further radially out from the rotation axis than the bases of the first tufts, and leaning outwardly away from the rotation axis so that their distal end is further out from the rotation axis than their base, but at a lesser angle of lean relative to the rotation axis than the first tufts.",
"21.A brush part according to claim 13 wherein the distal ends of the first tufts are further radially out than the distal ends of the first tufts.",
"22.A brush part according to claim 21 wherein the respective lean of the outwardly leaning first tufts and the orientation of the second tufts is such that as viewed along a line tangential to a circle centered on the rotation axis and mid way between the bases of the first and second tufts circumferentially adjacent first and second tufts as seen superimposed are seen to cross each other.",
"23.A brush part according to claim 13 wherein a polygon of bases of the second tufts which lies outside and is concentric with the polygon of bases of the first tufts.",
"24.An electric toothbrush provided with a brush part according to claim 13."
],
[
"This invention relates to bristles for electric toothbrushes, in particular to bristle clusters for electric toothbrushes.",
"Electric toothbrushes generally comprise a handle which contains inter alia a power supply and a drive motor, a neck extending from the handle and containing a transmission means e.g.",
"a drive shaft, and a brush part which includes a bristle holder mounted thereon for rotary motion about a rotation axis, and connected by the transmission means to the motor.",
"A cluster of tufts of bristles is mounted on a surface of the holder, extending generally in a bristle direction, with their end closest to the bristle holder, i.e.",
"their base end, set in the holder and their distal end furthest from the holder.",
"Generally the bristle tufts extend generally parallel to the rotation axis.",
"Generally the handle, neck and bristle holder lie along a longitudinal axis direction, and the rotation axis is at a transverse angle to this axis direction, e.g.",
"generally at right angles.",
"Commonly the bristle holder is replaceable on the handle, often together with the neck.",
"Often the rotary motion is oscillatory, i.e.",
"motion involving reciprocal angular displacement about a mean position, and the term “rotary” herein includes oscillatory rotary motion.",
"Sometimes the rotary motion also involves a reciprocal back and forth movement of the bristle holder along the rotational axis direction.",
"Numerous constructions of electric toothbrush of this type are known, for example the applicant's own Dr BEST “E-FLEX” (™) electric toothbrush launched in 2000 which has a brush part mounted for oscillatory rotary motion.",
"Electric toothbrushes are known from U.S. Pat.",
"No.",
"4,608,968 and DE-G-88 07 968.6 in which the tufts of bristles are arranged leaning outwardly relative to the rotation axis, with progressively less steeply leaning tufts toward the centre of the cluster, and with tufts at the centre of the bristle cluster arranged parallel to the rotation axis.",
"EP-A-0 765 642 discloses electric toothbrush tuft clusters which have their bristle tufts inclined an a non-zero angle to the rotation axis, leaning toward a central longitudinal plane of the toothbrush.",
"WO-A-0041592 discloses an oscillatory rotary electric toothbrush bristle cluster in which the tufts of bristles are inwardly leaning toward the rotation axis in a conical or pyramidal arrangement.",
"For “manual” toothbrushes, i.e.",
"toothbrushes without an electric motor and which operate solely by the motion of the user's hand, bristle configurations are known in which as viewed from certain directions, particularly from the side, the bristle tufts, or their extrapolation, are seen to converge and cross.",
"This is for example disclosed in DE-A-633 556, DE-G-82 02 897.6, FR-A-683 311, FR-A-2,624,360, U.S. Pat.",
"No.",
"2,242,743, U.S. Pat.",
"No.",
"3,085,273, U.S. Pat.",
"No.",
"4,010,509, U.S. Pat.",
"No.",
"4,081,876, U.S. Pat.",
"No.",
"4,776,054, U.S. Pat.",
"No.",
"5,274,873 and WO-A-99/23910.DE-A-44 12 301 and DE-A-35 44 256 disclose electric toothbrushes with bristles which are crossed in an “X” arrangement.",
"These known bristle clusters do not optimise tooth cleaning, particularly the cleaning of the interproximal spaces between the teeth where dirt can collect.",
"It is an object of the present invention to provide an improved bristle cluster for electric toothbrushes of the above-described type.",
"According to a first aspect of this invention a brush part for an electric toothbrush is provided which includes a bristle holder mounted for rotary motion about a rotation axis, a plurality of first tufts projecting from a surface of the bristle holder and having their bases arranged in a polygon around the rotation axis and leaning outwardly away from the rotation axis so that their distal end is further out from the rotation axis than their base, and a plurality of second tufts projecting from a surface of the bristle holder and having their bases arranged in a polygon around the rotation axis, characterised by: the first and second tufts having their bases at different radial distances from the rotation axis, the first and second tufts oriented relative to the rotation axis such that at a point along their base-distal end length the first and second tufts are respectively at radial distances from the rotation axis with a difference in their respective radial distances less than the difference in the respective radial distances of their bases from the rotation axis.",
"According to a second aspect of this invention a brush part for an electric toothbrush is provided which includes a bristle holder mounted for rotary motion about a rotation axis, a plurality of first tufts projecting from a surface of the bristle holder and having their bases arranged in a polygon around the rotation axis, and a plurality of second tufts projecting from a surface of the bristle holder and having their bases arranged in a polygon around the rotation axis, characterised by: the first tufts comprising an inner polygon in which at least one tuft is mounted inclined at an angle such that the distal end of the tuft is further around the polygon than the base end in a first direction, and the second tufts comprising an outer polygon in which at least one tuft is mounted inclined at an angle such that the distal end of the tuft is further around the polygon than the base end, in a second direction opposite to the first direction.",
"In both the first and second aspects of this invention the bristle holder is connectable to a toothbrush handle containing an electric drive motor and capable of being driven in rotary motion by the drive motor when connected thereto about a rotation axis generally transverse to an axis between the brush part and the handle.",
"Such rotary motion may be oscilatory rotary motion.",
"Preferably in the toothbrush head of the first aspect of this invention the bases of the second tufts are located at a greater radial distance from the rotation axis than are the bases of the first tufts.",
"Preferably in this brush part of the first aspect of the invention at a point along their base-distal end length the first and second tufts are respectively at the same radial distances from the rotation axis, i.e.",
"so that the difference in their respective radial distances at this point is zero.",
"Preferably further distally along the base-distal length from this point the difference in their respective radial distances of the first and second tufts increases.",
"Preferably in this brush part of the first aspect of the invention the first and second tufts are circumferentially adjacent to each other at the point of minimum difference, e.g.",
"of zero difference, in their respective radial distances, on a circle centred on the rotation axis.",
"One embodiment of this brush part of the first aspect of the invention comprises a plurality of second tufts having their bases further radially out from the rotation axis than the bases of the first tufts, and aligned parallel to the rotation axis.",
"A second embodiment of this brush part of the first aspect of the invention comprises a plurality of second tufts having their bases further radially out from the rotation axis than the bases of the first tufts, and leaning inwardly toward the rotation axis so that their distal ends are at less distance radially from the rotation axis than their base.",
"A third embodiment of this brush part of the first aspect of the invention comprises a plurality of second tufts having their bases further radially out from the rotation axis than the bases of the first tufts, and leaning outwardly away from the rotation axis so that their distal end is further out from the rotation axis than their base, but at a lesser angle of lean relative to the rotation axis than the first tufts.",
"Preferably in this brush part of the first aspect of the invention the distal ends of the first tufts are further radially out than the distal ends of the first tufts, although the distal ends of the first tufts may be at the same or lesser radial distance than the distal ends of the second tufts from the rotation axis.",
"This allows a preferred arrangement in which the respective lean of the outwardly leaning first tufts and the orientation of the second tufts is such that as viewed along a line tangential to a circle centred on the rotation axis and mid way between the bases of the first and second tufts circumferentially adjacent first and second tufts as seen superimposed are seen to cross each other.",
"In such an arrangement the tufts may cross at the mid point between the base and the distal ends along the tufts, or closer to the base or closer to the distal ends of the tufts.",
"Preferably in this brush part of the first aspect of the invention the second tufts also have their bases arranged in a polygon around the rotation axis in which in such a polygon the bases of the second tufts lie outside and preferably concentric with the polygon of bases of the first tufts.",
"Suitably there may be one or more such polygon of first tufts and/or one or more such polygon of second tufts.",
"For example in this brush part of the first aspect of the invention there may be two or more polygons of first tufts, one within the other preferably concentrically about the rotation axis, the respective first tufts in these polygons leaning outwardly at the same or different angle relative to the rotation axis, and for example having their first tufts arranged on radii from the rotation axis.",
"In such an arrangement there may for example also be two or more polygons of second tufts, one within the other.",
"A preferred arrangement of first and second tufts comprises, proceeding radially outward from the rotation axis: a polygon of bases of first tufts; a polygon of bases of second tufts oriented parallel to the rotation axis; another polygon of bases of first tufts; another polygon of bases of second tufts oriented parallel to the rotation axis.",
"In this brush part of the first aspect of the invention a polygon of second tufts may be the radially outermost tufts of the cluster of bristle tufts.",
"Preferably in this brush part of the first aspect of the invention the angle relative to the rotation axis at which the outwardly leaning first tufts may lean may be between 2.5 to 35°, preferably between 5 to 10°, for example typically around 8-10°.",
"If in this brush part of the first aspect of the invention the second tufts are inwardly leaning as mentioned above, they may be combined in the cluster with first tufts which lean outwardly at between 2.5 to 35°, preferably between 5 to 25°, for example between 10 to 15°, typically around 12°, and the respective first and second tufts may lean respectively outwardly and inwardly at the same or different angles relative to the rotation axis.",
"In the clusters described above in this brush part of the first aspect of the invention where a polygon of bases of outwardly leaning first tufts is within a polygon of bases of second tufts, the tufts may be arranged so that first tufts are radially (relative to the rotation axis) adjacent to a gap between two circumferentially adjacent second tufts.",
"Such an arrangement allows an outwardly leaning first tuft to lean outwardly between flanking pairs of second tufts which are oriented substantially parallel to the rotation axis, and a second tuft oriented substantially parallel to the rotation axis may be between flanking pairs of outwardly leaning first tufts.",
"It alternatively allows an outwardly leaning first tuft to lean outwardly between flanking pairs of inwardly leaning second tufts, and an inwardly leaning second tuft to lean inwardly between flanking pairs of outwardly leaning first tufts.",
"Alternatively it allows an outwardly leaning first tuft to lean outwardly between flanking pairs of second tufts which also lean outwardly, and a second tuft leaning outwardly to be between flanking pairs of outwardly leaning first tufts.",
"With such arrangements of first and second tufts it is appropriate that there are the same numbers of tufts in the respective inner first and outer second polygons.",
"Alternatively in this brush part of the first aspect of the invention the outwardly leaning first tufts may be arranged in groups of two or more circumferentially adjacent first tufts, and/or the second tufts may be arranged in groups of two or more circumferentially adjacent second tufts.",
"In such arrangements, such groups of first tufts may be circumferentially between pairs of second tufts, or vice versa.",
"Outwardly and inwardly leaning tufts in this brush part of the first aspect of the invention may lean inwardly along radii centred at the rotation axis, so that the tufts lean respectively straight away from and toward the rotation axis, i.e.",
"perpendicular to the direction of rotation.",
"In such an arrangement the respective directions of lean in projection converge radially on the axis of rotation.",
"Alternatively the respective directions of lean in projection may be along lines parallel to each other, preferably with a parallel line between the directions of lean passing through the axis of rotation.",
"Turning now to the brush part of the second aspect of the invention, at least one tuft in each of the first and second polygons is inclined around the polygon as described, and preferably all of the tufts in the polygons are inclined.",
"In this brush part of the second aspect of the invention, relative to the axis of rotation the angle at which the at least one, or preferably all the tufts, are inclined such that the end of the tuft which is furthest from the surface of the bristle holder is further around the polygon than the end of the tuft which is mounted in the surface may suitably be in the range 1 to 30°, typically 3 to 15°, especially 7-12°.",
"If the ends of the tuft which are mounted in the bristle holder lie in a plane, e.g.",
"if the surface of the bristle holder is a flat surface defining such a plane, then this angle may be in the range 89 to 60°, typically 87 to 80°, especially 83-78° to this plane.",
"In this brush part of the second aspect of the invention the inclined tuft(s) may be aligned in a direction substantially parallel to an adjacent side of the polygon.",
"Alternatively the tuft(s) may be inclined inwardly or outwardly, relative to the centre of the polygon, for example in a direction in the range 0°-30°, preferably 0°-10°, inwardly or preferably outwardly angled relative to the centre of the polygon as viewed in plan, relative to the direction of an adjacent side of the polygon.",
"The consequence of this latter inclination is that the end of the tuft which is furthest from the surface of the bristle holder is further out or in relative to the centre of the polygon, than is the end of the tuft which is mounted in the surface.",
"In the inner first and outer second polygons in this brush part of the second aspect of the invention, the respective inclined tufts of bristles are inclined in respective opposite directions around the polygon.",
"For example the tuft(s) in the inner first polygon may be inclined such that the distal end of the tuft(s) is further around the polygon than the base end of the tuft in an anti-clockwise direction, whilst the tuft(s) in the outer second polygon may be inclined such that the distal end is further around the polygon than the base end in a clockwise direction, or vice versa.",
"In such first and second polygons of this brush part of the second aspect of the invention, the angles of inclination in the respective directions around the polygon may be the same or different, and/or the tuft(s) of the inner first and outer second polygons may differ in whether they are aligned with an adjacent side of the polygon or whether they are inclined inwardly or outwardly.",
"For example the tuft(s) of the inner polygon may be inclined at a smaller angle relative to the axis of rotation in the direction around the polygon than the outer polygon, For example the tufts of the inner polygon may be inclined parallel to an adjacent side of the polygon, whilst the tufts of the outer polygon are inclined outwardly.",
"As described herein the position of a tuft relative to the axis of rotation refers to the position of the centre of its base or distal end.",
"In the brush part of both the first and second aspects of the invention the above described polygons are preferably regular polygons.",
"In such above described inner and outer polygons of tufts there may be for example be the same or different numbers of first and second tufts in the respective inner and outer polygons.",
"In both the first and second aspects of the invention the distal ends of the first and second tufts of the cluster may all be at the same distance from the surface of the bristle holder, e.g.",
"so that the distal ends lie in a plane perpendicular to the rotation axis.",
"In both the first and second aspects of this invention the first and second tufts may be combined in the cluster with one or more other kinds of tufts, called generally herein “third tufts”.",
"Such one or more third tuft may be provided in the cluster for example to create a suitable density of tufts for effective tooth cleaning.",
"Such one or more third tuft may be located at any position in the cluster.",
"For example the cluster may comprise one or more polygon of tufts which include both one or more first tuft and one or more third tuft, and/or one or more polygon of tufts which include both one or more second tuft and one or more third tuft.",
"Alternatively such third tufts may be provided in one or more third polygon which includes third tufts, and for example may contain only third tufts.",
"Such third polygon may have a common centre with the above-mentioned one or two polygons.",
"For example such third polygon may be situated within and surrounded by, or outside of and surrounding, the above-mentioned first and second polygons.",
"For example such a third polygon may be within and surrounded by the innermost of a polygon of first or second tufts.",
"For example such third polygon may be radially between the inner first and outer second polygon.",
"There may for example be a single third tuft at the centre of the first or second polygon.",
"For example in the brush head of both the first and second aspects of the invention such one or more third tuft may comprise one or more tuft aligned parallel with the rotation axis.",
"For example in the first aspect of the invention such one or more third tuft may comprise one or more tuft leaning in the direction of rotation, e.g.",
"as disclosed in EP-A-0 765 642, or leaning inwardly toward the rotation axis e.g.",
"as disclosed in WO-A-0041592, the contents of which are incorporated by reference.",
"For example in the brush part of the first aspect of this invention, two such above-described respective inner and outer polygons of respectively first and second tufts may comprise the two outermost tufts of the cluster, and there may be such third tufts within the inner polygon, e.g.",
"forming an innermost third polygon.",
"The third tufts may for example extend further from the surface than do the first and second tufts, which all may have their distal ends at the same perpendicular distance from surface.",
"In this arrangement the third tufts form a central peak, and the distal ends of the third tufts may be cut obliquely to their length.",
"For example in the brush part of the second aspect of the invention the cluster of bristles may comprise three polygons of tufts, being a first inner polygon and a second outermost polygon surrounding the first inner polygon, with all of the tufts in these first and second polygons being inclined at an angle such that the distal end of the tuft is further around the polygon than the base end but in respectively opposite directions around the polygon, together with a third innermost polygon of tufts within the first inner polygon, the tufts of this innermost third polygon being parallel to the axis of rotation.",
"These various polygons preferably have a common centre.",
"In this embodiment there may also be a single third tuft at the centre of the polygons being aligned parallel to the axis of rotation.",
"In the brush heads of both aspects of this invention the number of tufts in the various above-mentioned polygons may be determined in practice by the dimensions of the tufts and the bristle holder.",
"Typically bristle tufts as used in electric toothbrushes have a cross sectional dimension of ca.",
"1 mm, and the width of the bristle surface of a rotatable bristle holder of an electric toothbrush is ca.",
"10-12 mm.",
"Typically the outermost polygon of tufts contains 10-20 tufts.",
"Typically a polygon immediately radially inward of such an outermost polygon of tufts 8-12 tufts.",
"The above-mentioned polygons in both aspects of the present invention are preferably regular polygons.",
"The ends of the bristles remote from the surface of the bristle holder may be profiled in various ways.",
"For example as mentioned above in the brush part of the first aspect of this invention, an innermost polygon of third tufts may for example extend further from the surface than do the first and second tufts, which all may have their distal ends at the same perpendicular distance from surface.",
"In this arrangement the third tufts form a central peak, and the distal ends of the third tufts may be cut obliquely to their length.",
"For example in the brush part of the second aspect of this invention the ends of all the tufts of the polygon(s) in which at least one tuft is mounted inclined at an angle such that the distal end of the tuft is further around the polygon than the base end, may be in a plane perpendicular to the axis of rotation.",
"Alternatively for example in the brush part of the second aspect of this invention the ends of the tufts may form a central peak.",
"For example such a cluster may comprise first and second inner and outer polygons of tufts including the inclined tuft(s), with an innermost polygon of third tufts aligned parallel to the rotation axis, with optionally a single central tuft, and the ends of the bristles of the innermost polygon and of the central tuft (if present) may be profiled to form a central peak.",
"The ends of the tufts of the innermost polygon may be cut obliquely to their length so as to incline upwards toward the centre of the polygon.",
"In such a central peak the ends of the tufts may be profiled into a generally conical shape with its apex pointing away from the bristle surface.",
"Alternatively in the brush part of the second aspect of this invention the ends of the tufts may form a concavity about the centre of the cluster, and for example the ends of the tufts may form a central peak about the centre of this concavity so that the height of the polygons dips between the outermost and innermost polygons.",
"The ends of the tufts of this peak may be profiled into a generally domed shape, e.g.",
"a generally hemispherical dome with its radius pointing away from the bristle surface.",
"For example in such an arrangement the tufts of the innermost polygon may be higher that the tufts of the outermost polygon by ca.",
"0.5-1.5 mm, and the tufts of the intermediate polygon may be lower that the tufts of the outermost polygon by ca.",
"0.5-1.5 mm.",
"For example alternatively in another embodiment of the brush part of the second aspect of this invention the ends of all the tufts may be profiled in a concave generally cylindrical surface having the longitudinal axis of the cylinder aligned perpendicular to the axis of rotation.",
"A typical width for the cluster of the brush part is ca.",
"10-12.5 mm measured perpendicular to the bristle direction.",
"With such a width a suitable radius of curvature of the cross section of such a cylindrical surface is ca.",
"8-12 mm, e.g.",
"10-11 mm.",
"The term “conical” as used herein including shapes with other than true circular cross sections across the axis of rotation e.g.",
"pyramid shapes, and with curved sides e.g.",
"ogival shapes.",
"The term “sphere” and derived terms as used herein including shapes with other than true circular cross sections e.g.",
"oblate spheroids and ellipsoidal shapes.",
"The term “cylindrical” as used herein includes true cylinders i.e.",
"a shape having straight longitudinal sides and a circular cross section, with the longitudinal axis of the cylinder passing through the centre of the circular section.",
"The term also includes distorted cylinders, e.g.",
"shapes with convex bulging longitudinal sides e.g.",
"“barrel” shapes, and shapes with concave sides, i.e.",
"wider at the cylinder ends than at a waist partway between the ends, provided however that the radius of curvature of the convex or concave sides is greater than the radius of curvature of the cross section shape.",
"Also the term “cylinder” includes such shapes with oval or oblate circular, or polygonal, including polygonal with rounded corners or sides, cross section.",
"The brush part of this invention appears to be suitable for all electric toothbrushes in which the bristle holder is mounted for and moveable in rotary motion about an axis transverse to, preferably substantially perpendicular to, the longitudinal head—handle axis of the toothbrush, substantially parallel to the bristle direction, and passing through or close to the centre of the bristle pattern.",
"Preferably the rotary motion is oscillatory, i.e.",
"involving rotation through an angle in one rotary direction, alternating with rotation through substantially the same angle in the opposite rotational direction.",
"Preferably this rotary motion is combined with reciprocal motion of the bristles along the bristle direction, i.e.",
"up and down the bristle direction.",
"For example the motion of the bristle holder, combining such oscillatory rotary and reciprocal motion, may be reciprocally helical.",
"Suitable drive mechanisms to drive the bristle holder in this way are known, for example in U.S. Pat.",
"No.",
"5,577,285, WO-A-01/06946 and WO-A-01/06947, the contents of which are included herein by reference.",
"The drive speeds, amplitudes, and oscillation frequencies, e.g.",
"ca.",
"3000-6000 rpm achievable by means of such known drive mechanisms are believed to be suitable for the brush part of both aspects of the present invention.",
"Therefore the invention further provides an electric toothbrush having a brush part as described above.",
"For example the brush part of this invention may be connectable, preferably replaceably connectable, to a toothbrush handle containing an electric drive motor which when so connected can drive the brush part in the above-described rotary motion.",
"For this purpose the brush part suitably comprises part of a toothbrush head which is itself provided with connection means by which the brush part may be connected to the handle and to the motor.",
"The brush part may for example be rotatably mounted on the toothbrush head, for example by known means, such as an axle mounting, many types of which are known.",
"The connection means may comprise a hollow neck part extending longitudinally between the head and the handle and enclosing a drive shaft by which the motor can drive the brush part via suitable transmission means.",
"The neck part may itself be connectable, preferably replaceably, at its end remote from the head, to the handle in a manner which also connects the drive shaft to the motor.",
"Alternatively the head may itself be connectable, preferably replaceably, to the neck part at its end remote from the handle, to the neck part in a manner which also connects the drive shaft to the brush part.",
"Many means by which the motor can drive the shaft, and the shaft can drive the brush part, to achieve such motion are known.",
"Generally an electric toothbrush is an elongate structure which comprises a head (including the brush part) and handle disposed along a head—handle axis being the length of the toothbrush, and for example if the bristle holder performs oscillatory rotary motion the longitudinal axis of the part cylindrical surface may oscillate about a mean alignment perpendicular to the length of the electric toothbrush.",
"The toothbrush bristle holder and the bristles themselves of the brush part of this invention may be made of materials which are conventional in the field of electric toothbrush manufacture, e.g.",
"respectively of plastics materials and bristles of nylon, e.g.",
"Tynex™ material fibres.",
"The plastics material parts of the bristle holder and other plastics material parts of the toothbrush may be made by an injection moulding process, and accordingly the invention further provides a process for making a toothbrush as described herein, comprising injection moulding of plastics material.",
"Further the invention provides an injection mould suitable for use in such a process.",
"The bristle cluster of this invention is believed to provide improved tooth cleaning relative to know bristle clusters.",
"The invention will now be described by way of example only with reference to the following figures which show: FIG.",
"1 shows the overall schematic layout of an electric toothbrush having a brush part as described.",
"FIGS.",
"2 and 3 show a side and a plan view of a tuft cluster of the first aspect of this invention.",
"FIGS.",
"4 and 5 show a side and a plan view of another tuft cluster of the first aspect of this invention.",
"FIGS.",
"6 and 7 show a side and a plan view of another tuft cluster of the first aspect of this invention.",
"FIGS.",
"8 and 9 show a side and a plan view of another tuft cluster of the first aspect of this invention.",
"FIGS.",
"10, 11, 12 and 13 show part views of the clusters of FIGS.",
"2 to 9, and of another cluster of the first aspect of this invention.",
"FIG.",
"14 shows a part view of a cluster of the first aspect of this invention.",
"FIG.",
"15 Shows perspective and plan views of a bristle cluster of the second aspect of this invention.",
"FIG.",
"16 Shows a side, perspective and plan view of another bristle cluster of the second aspect of this invention.",
"FIG.",
"17 Shows a side and perspective view of another bristle cluster of the second aspect of this invention.",
"FIG.",
"18 Shows side and perspective views of another bristle cluster of the second aspect of this invention.",
"FIG.",
"19 Shows a plan view of and a section through another bristle cluster of the second aspect of this invention.",
"FIG.",
"20 Shows a plan at the bristle surface, a plan view and a side view of another bristle cluster of the second aspect of this invention.",
"Referring to FIG.",
"1 an electric toothbrush is shown overall in side view 10.The toothbrush 10 comprises a handle 11 by which it may be held, and which includes a drive motor, batteries, controls etc.",
"(not shown).",
"The handle 11 is replaceably connected at link 12, suitably a bayonet connection, to a replaceable section 13 including a head 14 at the end of the section 13 remote from handle 11 and a neck part 15.The assembly of handle 11, replaceable section 13 and head 14 are disposed along the length direction A—A of the toothbrush 10.In the head 14 is mounted a brush part comprising a bristle holder 16, from which a cluster of bristles 17 extend in a general bristle direction B—B generally perpendicular to length A—A.",
"The holder 16 is driven by the motor (not shown) via drive shaft 18 (shown generally) extending along inside the neck part 15.The bristle holder 16 is mounted on an axle (shown 18 in FIG.",
"3) in head 14 for oscillatory rotation about an axis passing through the centre of the bristle cluster 17 and parallel to the bristle direction B—B and for simultaneous reciprocal movement up and down this axis as shown by the arrows.",
"In use the holder 16 performs simultaneously an oscillatory, i.e.",
"reversing, rotary motion about an axis of rotation parallel to bristle direction B—B and passing through the centre in plan of the cluster of bristles 17, and a reciprocal motion up and down along the bristle direction B—B.",
"The amplitude of the oscillatory rotary motion is ca.",
"30° either side of a mean position, and the amplitude of the reciprocal motion is ca.",
"1.0 mm.",
"Numerous drive mechanisms are known in the art to achieve such motion.",
"Referring to FIG.",
"2 a bristle holder 20 is shown in a side view.",
"FIG.",
"3 shows a plan view looking down onto the surface 21, from which the bristle cluster 17 projects, along the rotation axis 22 about which the holder 20 rotates with oscillating rotation.",
"Mounted on the surface 21 are tufts of bristles 23, 24.These comprise first, outwardly leaning tufts 23 and second tufts 24 which are oriented parallel to the axis 22, i.e.",
"perpendicular to surface 21.The tufts 23 all lean outwardly along a radius from rotation axis 22, making an angle of ca.",
"10° with the orientation of the axis 22 so that their distal ends are radially further from the rotation axis 22 than their bases.",
"Being parallel to the axis 22, the distal ends of the second tufts 24 are at the same radial distance from the axis 22 as their bases.",
"The bases of tufts 23, 24 are arranged in two respective regular polygons each concentric around the rotation axis 22.The bases of all the first tufts 23 are radially inward of the bases of all the second tufts 24 so that the polygon of the bases of the first tufts 23 lies within and concentric with the polygon of bases of the second tufts 24.There are ten tufts 23 and ten tufts 24 in the respective polygons but there may be more or less bases in each polygon.",
"It is also seen that outwardly leaning first tufts 23 are arranged radially inward from gaps between adjacent second tufts 24 so that an outwardly leaning tuft 23 leans outwardly between flanking pairs of second tufts 24, and similarly a second tuft 24 extends parallel to the axis 22 between flanking pairs of outwardly leaning first tufts 23.FIG.",
"10 shows an arrangement similar to FIG.",
"2 and shows a view in the direction of a tangent to a circle radially mid way between the polygons of tufts 23 and 24.It is seen that the distal ends of tufts 24 are slightly further out radially than the distal ends of the tufts 23.Referring to FIG.",
"4 a bristle holder 20 is shown in a side view looking perpendicular to the rotation axis 22.FIG.",
"5 shows a plan view looking down onto the surface 21, from which the bristle cluster 17 projects, along the rotation axis 22.Mounted on the surface 21 are tufts of bristles 23, 24.These comprise first, outwardly leaning tufts 23 and second tufts 24 which are oriented parallel to the axis D—D, i.e.",
"perpendicular to surface 21.The tufts 23 all lean outwardly along a radius from rotation axis 22, making an angle of ca.",
"10° with the orientation of the axis 22 so that their distal ends are radially further from the rotation axis 22 than their bases.",
"Being parallel to the axis 22, the distal ends of the second tufts 24 are at the same radial distance from the axis 22 as their bases.",
"The relative leaning of the tufts 23, 24 is thus similar to FIGS.",
"2 and 3.The cluster shown in FIGS.",
"4 and 5 comprises two polygons of first tufts 231, 232, one 231 within the other 232, concentrically about the rotation axis 22, having their first tufts 231, 232 leaning outwardly at the same angle of ca.",
"8° relative to the rotation axis 22.The first tufts 231, 232 are arranged on radii from the rotation axis 22.In the arrangement shown there are also two polygons of second tufts 241, 242, one 241 within the other 242, the second tufts 241, 242 being oriented parallel to the axis 22.The arrangement therefore comprises, proceeding radially outward from the rotation axis 22: a polygon of bases of first tufts 231; a polygon of bases of second tufts 241 oriented parallel to the rotation axis; another polygon of bases of first tufts 232; another polygon of bases of second tufts 242 oriented parallel to the rotation axis.",
"Each polygon of first tufts 231, 232 contains six tufts so the polygon is a hexagon.",
"The inner polygon of second tufts 241 also contains six tufts.",
"The outer polygon of second tufts 242 contains twelve tufts so is a dodecagon.",
"It is also seen that the outermost second tufts 242 are arranged in groups, i.e.",
"pairs 242A, of circumferentially adjacent second tufts 242, so that first tufts 231 are circumferentially between circumferentially adjacent pairs of these groups of second tufts 242.FIG.",
"11 shows an arrangement similar to FIGS.",
"4 and 5, showing a view in the direction of a tangent to a circle radially mid way between the polygons of tufts 241 and 242.It is seen that the distal ends of the tufts 231 are further out radially than the distal ends of the tufts.",
"241, and that the distal ends of tufts 232 are slightly further out radially than the distal ends of the tufts 242.Referring to FIG.",
"6 a bristle holder 20 is shown in a side view looking perpendicular to the rotation axis 22.FIG.",
"7 shows a plan view looking down onto the surface 21, from which the bristle cluster 17 projects, along the rotation axis 22.Mounted on the surface 21 are tufts of bristles 23, 241, 242.These comprise first, outwardly leaning tufts 23 and second tufts 241,242 which are oriented parallel to the axis D—D, i.e.",
"perpendicular to surface 21.The arrangement is similar to FIGS.",
"2 and 3 above.",
"First tufts 23 are arranged in a regular pentagon, and second tufts 241,242 are arranged in two polygons having respectively their bases further radially out 241, and at the same distance out 242 from the rotation axis than the bases of the first tufts 23, and aligned parallel to the rotation axis.",
"In the outer polygon of second tufts 241 the tufts are arranged in circumferentially adjacent groups, i.e.",
"pairs.",
"Referring to FIG.",
"8 a bristle holder 20 is shown in a side view looking perpendicular to the rotation axis 22.FIG.",
"9 shows a plan view looking down onto the surface 21, from which the bristle cluster 17 projects, along the rotation axis 22.Mounted on the surface 21 are tufts of bristles 23, 24.These comprise first, outwardly leaning tufts 23, which all lean outwardly along a radius from rotation axis 22, making an angle of ca.",
"12° with the orientation of the axis 22 so that their distal ends are radially further from the rotation axis 22 than their bases.",
"The cluster also comprises second inwardly leaning tufts 24, which all lean inwardly along a radius toward rotation axis 22, making an angle of ca.",
"11° with the orientation of the axis 22 so that their distal ends are radially closer to the rotation axis 22 than their bases.",
"The bases of tufts 23, 24 are arranged in two respective regular polygons each concentric around the rotation axis 22.The bases of all the first tufts 23 are radially inward of the bases of all the second tufts 24 so that the polygon of the bases of the first tufts 23 lies within and concentric with the polygon of bases of the second tufts 24.There are nine tufts 23 and nine tufts 24, so the polygons are nonagons, but there may be more or less bases in each polygon.",
"It is also seen that outwardly leaning first tufts 23 are arranged radially inward from gaps between adjacent second tufts 24 so that an outwardly leaning tuft 23 leans outwardly between flanking pairs of second tufts 24, and similarly a second tuft 24 extends parallel to the axis 22 between flanking pairs of outwardly leaning first tufts 23.FIG.",
"12 shows an arrangement similar to FIGS.",
"8 and 9, showing a view in the direction of a tangent to a circle radially mid way between the polygons of tufts 23 and 24.It is seen that the distal ends of tufts 23 are further out radially than the distal ends of the tufts 24, and that as seen superimposed upon each other the tufts 23, 24 are seen to cross.",
"FIGS.",
"2-9 also show the presence of other, third, tufts of bristles 25.Although these are shown oriented parallel to the axis 22 they may alternatively lean inwardly toward or outwardly from the rotation axis 22.Such bristles 25 may be arranged at other points on the surface 21 to that shown, for example to increase the packing density of the bristle cluster.",
"In the arrangements shown these third tufts 25 form an innermost polygon of tufts arranged around the axis of rotation 22, and extend further from the surface 21 than do the first and second tufts 23, 24, which all have their distal ends at the same perpendicular distance from surface 25.All or some of the tufts of bristles 23, 24, 25 may also include a component of lean (not shown) in and/or against the direction of rotation of the holder 20 about axis 22.FIG.",
"13 shows another arrangement of first 23 and second 24 tufts, in which first tufts 23 project from surface 21 of the bristle holder 20, having their bases arranged in a polygon (not shown) around the rotation axis, leaning outwardly away from the rotation axis so that their distal end is further out from the rotation axis than their base.",
"In the arrangement second tufts 24 also lean outwardly away from the rotation axis so that their distal end is further out from the rotation axis than their base, and their base is further radially out from the rotation axis than the bases of the first tufts 23, but the tufts 24 have a lesser angle of lean relative to the rotation axis than the first tufts 23.The view of FIG.",
"13 is at a tangent to a circle radially mid-way between the bases of the tufts 23, 24 and as seen superimposed upon each other the tufts 23, 24 are seen to cross.",
"Referring to FIG.",
"14, a plurality of first tufts 23 project from the surface 21 of the bristle holder 20, and have their bases arranged in a polygon around the rotation axis 22 as in the embodiments illustrated above.",
"The first tufts 23 lean outwardly away from the rotation axis 22 so that their distal end is further out from the rotation axis 22 than their base.",
"There is also a plurality of second tufts 24 which also have their bases arranged in a polygon around the rotation axis 22 as in the embodiments illustrated above.",
"In FIG.",
"14 the second tufts 24 are oriented parallel to the rotation axis 22, but they could lean as in the other illustrated embodiments.",
"The first 23 and second 24 tufts have their bases at different radial distances R1, R2 from the rotation axis D—D, R1 being less than R2, with a difference R2 minus R1 being d1.The first 23 and second 24 tufts are oriented relative to the rotation axis 22 such that at a point, e.g.",
"P1, P2 along their base-distal end length the first 23 and second 24 tufts are respectively at radial distances from the rotation axis 22 with a difference d2 in their respective radial distances less than the difference d1 in the radial distances R1, R2 of their bases from the rotation axis 22.It is seen that the difference d2 is minimal at the distal ends on the tufts 23, 24.The same principle is seen to apply to the embodiments illustrated in FIGS.",
"10-13, and in FIGS.",
"11, 12 and 13 it is seen that d2 drops to zero at a point as the tufts 23, 24 are at the same radial distances from the rotation axis 22, but proceeding further in the base-distal direction d2 again increases as the tufts 23,24 diverge in their radial distance.",
"FIGS.",
"15-20 illustrate a brush part according to the second aspect of this invention.",
"Referring to FIG.",
"15, FIGS.",
"15A and 15B show perspective views of a bristle holder 20, on a surface 21 of which a bristle cluster 17 is mounted.",
"FIG.",
"15C shows a plan view of the cluster 17 and holder 20, looking down on surface 21 along the axis 22 about which holder 20 rotates with oscillating rotation.",
"As seen in FIGS.",
"15A, 15B and 15C, the cluster 17 comprises a plurality of tufts of bristles arranged in two regular polygons of tufts, viz.",
"a first, inner polygon 23 of twelve tufts and a second outer polygon 24 of eighteen tufts surrounding the inner polygon 23.Both the inner 23 and outer 24 polygons are arranged around a common centre.",
"In both polygons 23, 24 all of the tufts are mounted inclined at an angle such that the distal end 23A,24A of the tuft (furthest from the surface 21 of the bristle holder 20) is further around the polygon which contains it than the opposite base end 23B,24B of the tuft mounted in the surface 21, by the distance “d” shown in FIG.",
"15C.",
"In these inner 23 and outer 24 polygons their respective inclined tufts are inclined in opposite directions around the polygons 23, 24, such that the tufts in the inner polygon 23 are inclined in a clockwise direction, whilst the tuft(s) in the outer polygon 24 are inclined in an anti-clockwise direction.",
"The tufts in both polygons 23,24 are inclined at ca.",
"75° to the flat surface 21 of the bristle holder 20.The tufts 23,24 are also aligned outwardly angled relative to the centre of the polygon as viewed in plan, relative to the direction of an adjacent side of the polygon, by the angle A seen in FIG.",
"15C.",
"The cluster 17 also includes a polygon 25 of six third bristles having a common centre with the polygons 23, 24, situated within and surrounded by the inner polygon 23.The tufts of this third polygon 25 are substantially parallel to the axis of rotation 22 of the holder 20.At the centre of all three of the polygons 23, 24, 25 is a single third tuft 26, also aligned substantially parallel to the axis of rotation 22.As viewed in plan, the clusters 17 of FIGS.",
"15, 16, 17 and 18 are identical, but these clusters differ in the profile of the ends of the tufts remote from the surface 21 of the bristle holder 20.In FIGS.",
"15, 16 and 17 the ends of the two outer polygons 23, 24 are cut so as to lie in a plane parallel to the bristle surface 21.In FIG.",
"15 the ends of the tufts of the innermost polygon 25 and the central tuft 26 are profiled to form a peak about the centre of the cluster 17.The ends of the tufts of the innermost polygon 25 are cut so as to incline upwards toward the centre 22 of the polygon in a generally conical shape with its apex pointing away from the bristle surface 21.FIG.",
"16 shows a similar arrangement, but with the ends of the tufts of the innermost polygon 25 profiled into a generally hemispherical dome shape.",
"In FIG.",
"17 the ends of the tufts of the tufts of the innermost polygon 25 are profiled to incline downwards toward the centre 22 of the polygons 23,24,25 in a generally conical shape with its apex pointing toward the bristle surface 21.In FIG.",
"18 the ends of all of the tufts 23,24,25,26 are profiled in a concave cylindrical surface having the longitudinal axis of the cylinder C—C aligned perpendicular to the axis of rotation 22.Referring to FIG.",
"19, FIG.",
"19A shows a plan of the arrangement of tufts of bristles, and FIG.",
"19B shows a section through the cluster parallel to the axis of rotation.",
"As seen in FIG.",
"19A the cluster 17 comprises a plurality of tufts of bristles arranged in two regular polygons, viz.",
"a first, inner polygon 23 of eight tufts and a second outer polygon 24 of twelve tufts surrounding the inner polygon 23.Both the polygons 23,24 are arranged around a common centre being rotation axis 22.In both polygons 23, 24 all of the tufts are mounted inclined at an angle such that the distal end of the tufts are further around the polygon which contains them than the base end, the inclination of only one tuft 231, 241 in each of the polygons 23, 24 being shown in FIG.",
"19A which is a projection on a plane perpendicular to the axis of rotation 22.In these inner 23 and outer 24 polygons their respective inclined tufts are inclined in opposite directions around the polygons 23, 24, such that the tufts in the inner polygon 23 are inclined in a clockwise direction, whilst the tufts in the outer polygon 24 are inclined in an anti clockwise direction.",
"The tufts in polygons 23,24 are also aligned outwardly angled relative to the centre 22 of the polygon, relative to the direction of an adjacent side of the polygon.",
"The cluster 17 also includes an innermost polygon 25 of four third bristles in a regular square having a common centre with the polygons 23, 24, the tufts of third polygon 25 being substantially parallel to the axis of rotation 22 of the holder 20.As seen in the sectional view of FIG.",
"19B, being a sectional view through the bristle cluster 17 cut through rotation axis 22, the ends of the tufts 23,24,25 are profiled to form a peak about the centre of the cluster, the tufts of the innermost polygon 25 being the highest, the tufts of intermediate polygon 23 being lower than both the tufts of the outermost 24 and innermost 25 polygons, so that the height of the polygons dips between the outermost 24 and innermost 25 polygons.",
"Referring to FIG.",
"20, FIG.",
"20A shows a plan of the arrangement of tufts of bristles at the bristle surface 21.FIG.",
"20B shows a plan view of the cluster looking down the rotation axis 22 onto the surface 21.FIG.",
"20C shows a side view perpendicular to the axis of rotation 22.As seen in FIGS.",
"20A and 20B the cluster 17 comprises a plurality of tufts of bristles arranged in two regular polygons, a first inner regular polygon 23 of eight tufts and a second outer regular polygon 24 of twelve tufts surrounding the inner, polygon 23, both polygons 23,24 being arranged around a common centre.",
"As seen in FIG.",
"20B in both polygons 23, 24 all of the tufts are mounted inclined at an angle such that the distal end 23A, 24A of the tuft is further around the polygon which contains it than the opposite base end 23B,24B of the tuft, the respective inclined tufts being inclined in opposite directions around the respective polygons 23, 24.In two versions of the FIG.",
"20 embodiment the inclination of the tufts of the outer polygon is 8° or 10°, and the respective inclination of the tufts of the inner polygon is 4° or 5° in the opposite direction.",
"The tufts of the outer polygon 24 are also aligned outwardly at an angle 5° relative to the centre of the polygon as viewed in plan, relative to the direction of an adjacent side of the polygon, whilst the tufts 20 of the inner polygon 24 are aligned parallel to the direction of an adjacent side of the polygon.",
"The cluster 17 also includes an innermost polygon 25 of four third bristles in a regular square having a common centre with the polygons 23, 24, the tufts of which are substantially parallel to the axis of rotation 22 of the holder 20, and the distal ends of the which are higher than the tufts 23,24, the ends of which lie in a plane.",
"The height of the tufts 24 above surface 21 is 8 mm and of the tufts 23,24 is 6 mm."
]
] | Patent_10433850 |
[
[
"Package, particularly a cigarette package, and method and device for the production thereof",
"A cuboidal cigarette pack with a large-surface-area pack side, namely rear side (13), is provided with a blank (23), which serves as a printing carrier, in the region of this rear side (13).",
"Said blank is positioned between an outer wrapper (19) made of film and the pack (10).",
"A top region of the blank (23) is connected to a detachable top part of the outer wrapper, namely a film cap (22), and is also removed when the pack (10) is opened for the first time."
],
[
"1.-8.",
"(canceled) 9.A pack, in particular cigarette pack, having a large-surface-area front side (15) and a corresponding rear side (13) and also having an outer wrapper (19) made of film which has a tear-open strip (20) which runs all the way round and is intended for detaching a (top) film cap (22) of the outer wrapper (19), a separate blank (23) being positioned, as a printing carrier or coupon, between the outer wrapper (19) and the pack (10), in the region of a large-surface-area pack side (13, 15), and being connected to the inside of the outer wrapper, characterized by the following features: a) the blank (23) is connected to the outer wrapper (19), exclusively in the region of the film cap (22), by adhesive bonding or thermal sealing, b) the blank (23) is further connected to the pack in the region of the large-surface-area pack side (13, 15), to be precise by an easily releasable blob of glue (27).",
"10.The pack as claimed in claim 9, characterized by the following features: a) the blank (23) is arranged in the region of a rear side (13) of the pack, b) the blank (23) is connected to the detachable film cap (22) in the region of a cap rear wall (24), c) the blob of glue (27) for releasably connecting the blank (23) to the pack is arranged in the region of the rear side (13).",
"11.The pack as claimed in claim 9, characterized in that the blank (23) is connected to the outer wrapper (19) by spots of glue, in particular by two spots of glue (25, 26) which are spaced apart from one another on the inside of the outer wrapper (19) in the region of the film cap (22).",
"12.The pack as claimed in claim 9, characterized in that the blank (23) is of rectangular design with dimensions which are slightly smaller than the dimensions of the rear side (13).",
"13.The pack as claimed in claim 9, characterized in that the blank (23) is of multi-layered design, in particular in the form of a strip with concertina-like folding, such that an inner leg (28) of the folded blank (23), said leg being connected to the rear side (13), has a free end directed downward, and an outer leg (29), which is connected to the outer wrapper (19) and/or film cap (22), has a free end directed upward.",
"14.A method of producing packs as claimed in claim 9, characterized by the following features: a) a film web (30) is provided with spots of glue (25, 26), in particular with pairs of spots of glue (25, 26), at intervals from one another in the region of the film cap (22), b) a continuous tear-open strip (20) in the form of a strip web (36) is then fitted in a precisely positioned manner on the film web (30), c) in the region of a blank subassembly (32), blanks for the outer wrapper (19) are severed from the film web (30) and held ready in an upright plane, d) on a transversely directed, horizontal pack path (33), packs with a blank (23) fitted in a precisely positioned manner on the top side are fed to the blank subassembly (32), e) the outer wrapper (19) is folded around the pack (10) provided with the blank (23), the spots of glue (25, 26) being connected to the blank (23).",
"15.The method as claimed in claim 14, characterized by the following features: a) the packs (10) are transported along a pack path (33) with the rear side (13) oriented upward, b) a blob of glue (27) is applied to the rear side (13) of the pack (10), c) thereafter, the blank (23) is applied to the rear side (13) of the pack (10) in the region of the blob of glue (27), d) the outer wrapper (19) is then folded around the pack (10).",
"16.An apparatus for producing packs as claimed in claim 9, characterized by the following features: a) a film web (30) for producing blanks for the outer wrapper (19) can be conveyed through a glue station (34) for the purpose of applying spots of glue (25, 26), b) a continuous strip web (36) can be fitted on the film web (30) for the purpose of forming a tear-open strip (20), c) in the region of a blank subassembly (32), blanks for the outer wrapper (19) can be severed from the prepared film web (30) in a vertical plane, d) on a horizontal pack path (33), packs (10) are fed to the blank subassembly (32) for the purpose of receiving a prepared outer wrapper (19), e) arranged above the pack path (33), as seen in the conveying direction, are a glue subassembly (38) for applying a blob of glue (27) to the pack (10) and, following this, a blank supplier (39) for providing the blank (23)."
],
[
"The invention relates to a pack, in particular cigarette pack, having a large-surface-area front side and a corresponding rear side and also having an outer wrapper made of film which has a tear-open strip running all the way round, a separate blank being positioned, as a printing carrier, in particular a coupon, between the outer wrapper and the (cigarette) pack.",
"The invention also relates to a method of producing such packs and to an apparatus.",
"There are increasing requirements, in the fabrication of cigarette packs, for sheet-like inserts with printed information or advertising material to be fitted on the pack.",
"Various proposals are known for adding printing carriers made of thin paper or the like to the cigarette pack, to be precise even in the region between the conventional outer wrapper made of film and the pack itself.",
"The object of the invention is to propose a pack in the case of which the insert, namely the separate blank, is fitted straightforwardly in packaging terms and is presented in a user-friendly manner when the pack is opened for the first time, with the result that the advertising or information reaches the consumer.",
"In order to achieve this object, the pack according to the invention is characterized by the following features: a) the blank or printing carrier is fitted in the region between one of the large-surface-area pack side, on the one hand, and the outer wrapper, on the other hand, b) the blank or printing carrier is connected to the outer wrapper by adhesive bonding.",
"Positioning the printing carrier in particular on the large-surface-area rear side of the (cigarette) pack makes available a corresponding surface area for imparting information even with the pack closed, because the printing carrier can cover the entire side or rear side of the pack.",
"When the pack is opened for the first time, namely when the wrapper is removed, the fact that the printing carrier is connected to the wrapper additionally draws the consumer's attention thereto.",
"A cigarette pack according to the invention in the case of which the printing carrier is fitted on the rear side of the pack and is connected to a top outer-wrapper part which can be detached by tear-open strips—a film cap—is particularly advantageous.",
"When this pack is customarily opened for the first time, the film cap is detached from the outer wrapper.",
"In this case, the printing carrier, which is connected to said film cap, is drawn out of the position within the pack and presented to the consumer.",
"The amount of printing surface area available may be increased by a multi-layered, in particular concertina-like design of the printing carrier.",
"Moreover, it is a special feature that the printing carrier is connected to the pack or the rear side thereof, to be precise by releasable adhesive bonding.",
"A possible connecting means is a “stick/no stick” type of adhesive, that is to say an adhesive which automatically loses its effect after a certain period of time.",
"During production of a pack within the context of the invention, the procedure is such that a film web for producing the outer wrapper is provided with areas of glue in a precisely positioned manner, in the region of a film cap which is to be produced, for the attachment of the printing carrier.",
"Spots of glue are preferably applied to the film web in pairs at appropriate intervals from one another, a glue in the form of both a hot-melt and a long-term adhesive being advantageous.",
"Further details regarding the configuration and production of the packs according to the invention are explained in more detail hereinbelow with reference to the drawings, in which: FIG.",
"1 shows a perspective view of a cigarette pack, FIG.",
"2 shows, likewise in a perspective illustration, the pack according to FIG.",
"1 during a production phase, FIG.",
"3 shows the pack according to FIG.",
"2 without a printing carrier, FIG.",
"4 shows a side view of the pack according to FIG.",
"1, FIG.",
"5 shows an illustration corresponding to FIG.",
"4 of a different exemplary embodiment of the pack, FIG.",
"6 shows a perspective illustration of the pack according to FIG.",
"1 during the opening process, FIG.",
"7 shows, likewise in a perspective illustration, the pack according to FIG.",
"5 during the opening process, FIG.",
"8 shows a schematic side view of an apparatus for producing packs with printing carriers, and FIG.",
"9 shows a portion of a film web for an outer wrapper of a pack.",
"The drawings concern the configuration and production of cigarette packs, that is to say of cuboidal pack, which have at least two mutually opposite (large) surfaces.",
"This applies to the packs 10 of the hinge-lid-box type, which are illustrated as the preferred example.",
"This type of pack is constructed from a (bottom) box part 11 and a lid 12.The two parts are connected to one another such that they can be pivoted in the region of a transversely directed linear articulation 14 in the region of a large-surface-area rear side 13 of the pack 10.Accordingly, the rear side 13 of the pack 10 is in one piece throughout.",
"A correspondingly large-surface-area front side 15 is formed opposite.",
"Furthermore, the pack 10 is bounded by narrow, upright side surfaces 16 and by a base surface 17 and end surface 18.The resulting pack 10 is fully enclosed by an outer wrapper 19.The latter consists of a thin, transparent or clear film.",
"The outer wrapper 19 is removed, at least in part, when the pack 10 is opened for the first time.",
"For this purpose, connected to the outer wrapper 19, in a top region directed toward the end surface 18, is a tear-open strip 20 running all the way round.",
"A free, outer end of the tear-open strip 20 serves as a grip end 21.For the purpose of opening the outer wrapper 19, the latter is severed with the aid of the tear-open strip 20 such that a top sub-region of the outer wrapper 19 is completely detached from a bottom part.",
"The top part of the outer wrapper, a film cap 22, is removed.",
"The pack 10 is provided with a separate blank 23.The latter consists of thin material, in particular paper.",
"The blank 23 serves as a printing carrier, that is to say for receiving printed information and/or decoration, for example advertising and factual information, but also details for participation in prize draws.",
"The blank 23 is positioned in the region between the pack 10 and the outer wrapper 19, to be precise on one of the large pack surfaces.",
"It is particularly favorable for the blank 23 to be arranged in the region of the large-surface-area rear side 13, since the latter is designed in one piece throughout.",
"The blank 23 is dimensioned such that it extends more or less over the entire surface of the rear side 13.The outside of the blank 23 is clearly visible through the outer wrapper 19, with the result that appropriate printing and/or information may be provided in this region.",
"The blank 23 is dimensioned and positioned such that a sub-region of the same is located in that part which can be removed when the outer wrapper 19 is opened, that is to say in the region of the film cap 22.The blank 23 is connected to the outer wrapper 19 such that the blank 23 is removed with this outer wrapper 19.A special feature is the connection of the blank 23 to the detachable film cap 22 in the region of a cap rear wall 24.When the outer wrapper 19 is opened, the connection causes the blank 23 to be drawn out of position along with the film cap 22, to be precise without being destroyed or impaired in any other way (FIG.",
"6).",
"The blank 23 is connected to the outer wrapper 19 by adhesive bonding.",
"It is favorable for the blank 23 to be fastened on the cap rear wall 24 by a plurality of, in particular two, spots of glue 25, 26.These are spaced apart from one another adjacent to the end boundary of the blank 23, namely virtually at the top corners of the blank 23.The spots of glue 25, 26 may comprise a long-lasting adhesive, to be precise also in hot-melt form.",
"Provision is also made for the blank 23 to be connected to the pack 10 in a region which is located at the bottom or remote from the spots of glue 25, 26, namely connected to the rear side 13 of the pack in the region of the box part 11.This connection has to be easily releasable in order that the blank 23 can easily be withdrawn from the position on the rear side 13 when the film cap 22 is removed.",
"Provided for this purpose is a single, central blob of glue 27, which connects the blank 23 to the rear side 13.This connection has, in particular, the purpose of ensuring that the blank 23 is positioned precisely on the pack 10 during the production process and thereafter, at any rate before the pack is opened for the first time.",
"The blob of glue 27 may advantageously consist of the “stick/no stick” type of glue, that is to say glue with a decreasing adhesive action.",
"In order to increase the useful surface area, the blank 23 may be of multi-layered design.",
"According to FIG.",
"5, the blank 23 is folded in a concertina-like manner as an elongate strip, for example to the width of the rear side 13.An inner leg 28 butts against the rear side 13 and has a free end directed downward.",
"An outer leg 29 is directed toward the outer wrapper 19 and has a free end oriented upward.",
"The inner leg 28 is connected (in a releasable manner) to the pack 10 in the bottom region, via the blob of glue 27, and the outer leg 29 is connected to the outer wrapper 19 or the film cap 22 via preferably two spots of glue 25, 26.In the case of the concertina-like arrangement of the blank 23, folding lines are directed transversely at the top and bottom regions of the rear side 13.When the pack 10 is opened for the first time by virtue of the film cap 22 being lifted off, the latter causes the folded blank 23 first of all to be unfolded and then to be drawn out of the position on the rear side 13 (FIG.",
"7).",
"Accordingly, the blank 23 is not just drawn out of the position between the outer wrapper 19 and the pack 10, but is also unfolded in the process.",
"A further special feature is the production of packs 10 of the type described.",
"In the case of the apparatus shown schematically in FIG.",
"4, a film web 30 is withdrawn from a reel 31 and fed to a blank subassembly 32.In the region of the latter, blanks for the outer wrapper 19 are severed in the region of a vertical plane and transferred to packs 10, which can be fed along a horizontal pack path 33, likewise to the blank subassembly 32.In the region of a glue station 34, the film web 30 is provided with glue, preferably with the spots of glue 25, 26, for the purpose of fixing the blank 23.A glue subassembly 35 is provided for this purpose, and preferably applies the spots of glue by glue nozzles.",
"The tear-open strip 20 is then applied to the film web, to be precise as a strip web 36, which is likewise withdrawn from a reel 37.The blank for the outer wrapper 19, this blank being produced in the blank subassembly 32, thus already has the tear-open strip 20 and spots of glue 25, 26.As can be seen from FIG.",
"9, the latter are applied in a precisely positioned manner to the film web 30 as pairs of spots of glue 25, 26, corresponding to the position within the blank which is to be produced, to be precise alongside the tear-open strip 20 or the strip web 36.The packs 10, which are fed from a corresponding packer, are glued, namely provided with the blob of glue 27, on the upwardly oriented rear side 13 of the pack 10, in the region of the pack path 33, by a glue subassembly 38.The glue subassembly 38 has a glue nozzle which discharges an appropriate portion of glue from above.",
"Following the glue subassembly 38, a subassembly for applying the blanks 23 to the packs 10, that is to say a blank supplier 39, is positioned above the pack path 33.Said blank supplier transfers a blank 23 of the configuration according to FIG.",
"4 or 5, with precise relative positioning in each case, onto the rear side of the pack 10, the blank 23 being fixed by the blob of glue 27.Thereafter, in the region of the blank station 32, the blank of the outer wrapper 19 is folded in the customary manner around the pack 10, which has been prepared in respect of the blank 23.The measures described can also be used analogously for cigarette packs of the soft-carton type or capped-box type.",
"With corresponding fabrication, the blank 23 may alternatively be connected to the outer wrapper 19 and/or the pack 10 by thermal sealing.",
"LIST OF DESIGNATIONS 10 Pack 11 Box part 12 Lid 13 Rear side 14 Linear articulation 15 Front side 16 Side surface 17 Base surface 18 End surface 19 Outer wrapper 20 Tear-open strip 21 Grip end 22 Film cap 23 Blank 24 Cap rear wall 25 Spot of glue 26 Spot of glue 27 Blob of glue 28 Leg 29 Leg 30 Film web 31 Reel 32 Blank subassembly 33 Pack path 34 Glue station 35 Glue subassembly 36 Strip web 37 Reel 38 Glue subassembly 39 Blank supplier"
]
] | Patent_10433892 |
[
[
"Emergency release assembly and sliding door incorporating the same",
"An emergency release assembly for a door includes a latch mechanism to retain the door in a normally closed position yet releases to let the door move to an emergency open position.",
"A release mechanism triggers the latch mechanism.",
"The release mechanism has a reciprocating member that operates the latch mechanism.",
"A push bar moves from an extended position through an intermediate position to a depressed position.",
"A link member interconnects the push bar and the reciprocating member.",
"When the push bar moves from the intermediate to the depressed position, it operates the reciprocating member to trigger the latch mechanism.",
"A catch member operates to limit movement of the push bar between the intermediate and depressed positions but can be released to allow the push bar to move to the extended position.",
"When the push bar is in the intermediate position, it is flush with the door muntin."
],
[
"1.An emergency release assembly adapted for use in a door that is supported for motion in a framework when in a normally closed configuration yet movable in an emergency condition between the normally closed configuration to an emergency open configuration, said release assembly comprising: (A) a latch mechanism operative in a first state to retain the door in the normally closed position and actuable to a second state that allows the door to be moved to the emergency open configuration; and (B) a release mechanism operative to advance the latch mechanism from the first state to the second state, said release mechanism including (1) a reciprocating member coupled to said latch mechanism and supported by said door for sliding movement; (2) a push bar; (3) at least one link member interconnecting said push bar and said reciprocating member such that said push bar may be moved between a fully extended position and a depressed position through an intermediate extended position, said push bar when moved from the intermediate extended position to the depressed position being operative to move said reciprocating member such that said latch mechanism is advanced from the first state to the second state; and (4) a catch member operative in a retain state to limit movement of said push bar between the intermediate extended position and the depressed position yet releasable to an access state wherein said push bar can move from the intermediate extended position to the fully extended position.",
"2.An emergency release assembly according to claim 1 wherein said link member is defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another.",
"3.An emergency release assembly according to claim 2 wherein said first leg has a proximal end secured to said reciprocating member with a distal end opposite said proximal end and said second leg having a proximal end secured relative to said door with a distal end thereof secured to a medial portion of said first leg.",
"4.An emergency release assembly according to claim 3 including a mounting plate supported on the distal end of said first leg, said push bar being secured to said mounting plate.",
"5.An emergency release assembly according to claim 1 including at least two link members interconnecting said push bar and said reciprocating member, said link members being positioned in spaced-apart relation to one another.",
"6.An emergency release assembly according to claim 5 wherein each of said link members is defined by a scissors unit.",
"7.An emergency release assembly according to claim 1 wherein said latch mechanism includes a reciprocating latch actuator rod, said release mechanism including a rotatable drive element and a crank arm, said crank arm having a first crank arm end secured to said reciprocating member and a second crank arm end secured to said drive element whereby reciprocation of said reciprocating member acts to reciprocally rotate said drive element, said actuator rod having a first rod end secured to said drive element whereby reciprocal rotation of said drive element acts to reciprocate said actuator rod.",
"8.An emergency release assembly according to claim 7 wherein said first crank arm end is slideably secured to said reciprocating member such that said drive element is not rotated when said push bar moves from the intermediate extended position to the fully extended position.",
"9.An emergency release assembly according to claim 1 wherein said push bar is biased toward the fully extended position.",
"10.An emergency release assembly according to claim 1 wherein said release mechanism includes a base member adapted to be supported by said door, said link member interconnecting said base member and said reciprocating member.",
"11.An emergency release assembly according to claim 10 wherein said link member is defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another, said first leg having a proximal end pivotally secured to said reciprocating member by a first axle pin with a distal end opposite said proximal end and said second leg having a proximal end pivotally secured to said base member by a second axle pin with a distal end thereof secured to a medial portion of said first leg.",
"12.An emergency release assembly according to claim 11 wherein said catch member is defined by a lever pivotally secured to said base member by said second axle pin, said lever including an arm portion that abuts said first axle pin when in the retain state, said lever being pivotable so that said arm portion is pivoted out of the retain state whereby said push bar may move from the intermediate extended position to the fully extended position.",
"13.An emergency release assembly according to claim 12 wherein said push bar has access opening whereby said lever may be manually pivoted out of the retain state.",
"14.A release assembly adapted for use in a door that is movable between a closed position and an open position, the frame structure of said door including a centrally located muntin that has a muntin recess, said release mechanism comprising: (A) a latch mechanism disposed on said frame structure and operative in a first state to retain the sliding door in the normally closed position and actuable to a second state that allows the sliding door to be moved to the open position; and (B) a release mechanism adapted to be disposed in the muntin recess and operative to advance the latch mechanism from the first state to the second state, said release mechanism including (1) a control member coupled to said latch mechanism; (2) a push bar; (3) at least one link member interconnecting said push bar and said control member such that said push bar may be moved between a fully extended position and a depressed position through an intermediate extended position, said push bar when moved from the intermediate extended position to the depressed position being operative to move said control member such that said control member advances said latch mechanism from the first state to the second state; and (4) a catch member operative in a retain state to limit movement of said push bar between the intermediate extended position and the depressed position yet releasable to an access state wherein said push bar can move from the intermediate extended position to the fully extended position.",
"15.A release assembly according to claim 14 wherein said link member is defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another.",
"16.A release assembly according to claim 15 wherein said catch member engages said control member and is operative to in the retain state to limit movement thereof.",
"17.A release assembly according to claim 16 wherein said push bar has access opening whereby said catch member may be manually moved out of engagement with said control member thereby to permit said push bar to move from the intermediate extended position to the fully extended position.",
"18.A release assembly according to claim 15 including a mounting plate supported on the scissors unit, said push bar being secured to said mounting plate.",
"19.A release assembly according to claim 14 including at least two link members interconnecting said push bar and said reciprocating member, said link members being positioned in spaced-apart relation to one another.",
"20.A release assembly according to claim 19 wherein each of said link members is defined by a scissors unit.",
"21.A release assembly according to claim 14 wherein said latch mechanism includes a reciprocating latch actuator rod, said release mechanism including a rotatable drive element and a crank arm, said crank arm having a first crank arm end secured to said reciprocating member and a second crank arm end secured to said drive element whereby movement of said control member acts to rotate said drive element, said actuator rod having a first rod end secured to said drive element whereby rotation of said drive element acts to reciprocate said actuator rod.",
"22.A release assembly according to claim 21 wherein said first crank arm end is slideably secured to said control member such that said drive element is not rotated when said push bar moves from the intermediate extended position to the fully extended position.",
"23.A release assembly according to claim 14 wherein said push bar is biased toward the fully extended position.",
"24.A sliding door supported for sliding motion in a doorway when said sliding door is in a normally closed configuration yet which is movable in an emergency condition between the normally closed configuration to an emergency open configuration, comprising: (A) a framework including a muntin; (B) a latch mechanism disposed in said framework and operative in a first state to retain the sliding door in the normally closed configuration and actuable to a second state that allows the sliding door to be moved to the emergency open configuration; and (C) a release mechanism disposed in the muntin and operative to advance the latch mechanism from the first state to the second state, said release mechanism including (1) a control member coupled to said latch mechanism and supported for reciprocal sliding movement; (2) a push bar; (3) at least one link member interconnecting said push bar and said control member such that said push bar may be moved between a fully extended position and a depressed position through an intermediate extended position, said push bar when moved from the intermediate extended position to the depressed position being operative to move said reciprocating member such that said latch mechanism is advanced from the first state to the second state; and (4) a catch member operative in a retain state to limit movement of said push bar between the intermediate extended position and the depressed position yet releasable to an access state wherein said push bar can move from the intermediate extended position to the fully extended position.",
"25.An emergency release assembly according to claim 24 wherein said link member is defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another.",
"26.An emergency release assembly according to claim 25 including at least two link members interconnecting said push bar and said reciprocating member, each of said link members defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another and being positioned in spaced-apart relation to one another.",
"27.An emergency release assembly according to claim 26 wherein each said first leg has a proximal end secured to said control member with a distal end opposite said proximal end and said second leg having a proximal end secured relative to said door with a distal end thereof secured to a medial portion of a respective said first leg.",
"28.An emergency release assembly according to claim 27 including a mounting plate supported on the distal end of each said first leg, said push bar being secured to said mounting plate.",
"29.An emergency release assembly according to claim 24 wherein said latch mechanism includes a reciprocating latch actuator rod, said release mechanism including a rotatable drive element and a crank arm, said crank arm having a first crank arm end secured to said control member and a second crank arm end secured to said drive element whereby reciprocation of said control member acts to reciprocally rotate said drive element, said actuator rod having a first rod end secured to said drive element whereby reciprocal rotation of said drive element acts to reciprocate said actuator rod.",
"30.An emergency release assembly according to claim 29 wherein said first crank arm end is slideably secured to said control member such that said drive element is not rotated when said push bar moves from the intermediate extended position to the fully extended position.",
"31.An emergency release assembly according to claim 24 wherein said push bar is biased toward the fully extended position.",
"32.An emergency release assembly according to claim 24 wherein said release mechanism includes a base member adapted to be releasaby supported in said muntin, said link member interconnecting said base member and said control member.",
"33.An emergency release assembly according to claim 32 wherein said link member is defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another, said first leg having a proximal end pivotally secured to said control member by a first axle pin with a distal end opposite said proximal end and said second leg having a proximal end pivotally secured to said base member by a second axle pin with a distal end thereof secured to a medial portion of said first leg.",
"34.An emergency release assembly according to claim 33 wherein said catch member is defined by a lever pivotally secured to said base member by said second axle pin, said lever including an arm portion that abuts said first axle pin when in the retain state, said lever being pivotable so that said arm portion is pivoted out of the retain state whereby said push bar may move from the intermediate extended position to the fully extended position.",
"35.An emergency release assembly according to claim 34 wherein said push bar has access opening whereby said lever may be manually pivoted out of the retain state.",
"36.A sliding door supported for sliding motion in a doorway when said sliding door is in a normally closed configuration yet which is movable in an emergency condition between the normally closed configuration to an emergency open configuration, comprising: (A) a framework including a muntin that has a recess and peripheral frame portions that define a substantially planar outer muntin face; (B) a latch mechanism disposed in said framework and operative in a first state to retain the sliding door in the normally closed position and actuable to a second state that allows the sliding door to be moved to the emergency open configuration; and (C) a release mechanism disposed in the muntin recess and operative to advance the latch mechanism from the first state to the second state, said release mechanism including (1) a control member coupled to said latch mechanism and supported for reciprocal sliding movement; (2) a push bar having an outer surface; (3) at least one link member interconnecting said push bar and said control member such that said push bar may be moved between a fully extended position and a depressed position through an intermediate extended position, said push bar when moved from the intermediate extended position to the depressed position being operative to move said reciprocating member such that said latch mechanism is advanced from the first state to the second state, said push bar when in the intermediate extended position being located entirely within the recess and when in the fully extended position protruding out of the recess; and (4) a catch member operative in a retain state to limit movement of said push bar between the intermediate extended position and the depressed position yet releasable to an access state wherein said push bar can move from the intermediate extended position to the fully extended position.",
"37.An emergency release assembly according to claim 36 wherein said release mechanism includes a base member adapted to be supported by said door, said link member interconnecting said base member and said reciprocating member.",
"38.An emergency release assembly according to claim 37 wherein said link member is defined by a scissors unit formed by at least first and second legs pivotally interconnected to one another, said first leg having a proximal end secured to said control member with a distal end opposite said proximal end and said second leg having a proximal end secured to said base member with a distal end thereof secured to a medial portion of said first leg.",
"39.An emergency release assembly according to claim 38 wherein said first leg is formed by a pair of spaced-apart, substantially parallel first leg sections each having proximal and distal ends and wherein said second leg is formed by a pair of spaced-apart, substantially parallel second leg sections each having proximal and distal ends.",
"40.An emergency release assembly according to claim 39 wherein said base member has a pair of opposed ears projecting therefrom, the proximal end of each said second leg section pivotally secured to said ears by an axle pin.",
"41.An emergency release assembly according to claim 40 wherein the proximal end of each said first leg section is pivotally secured to said control member by a first axle pin and the proximal end of each said second leg section is pivotally secured to said base member by a second axle pin with a distal end of each second leg section secured to a medial portion of a respective said first leg section.",
"42.An emergency release assembly according to claim 41 wherein said catch member is defined by a lever pivotally secured to said base member by said second axle pin, said lever including one arm portion that abuts said first axle pin when in the retain state, said lever being pivotable so that said arm portion is pivoted out of the retain state whereby said push bar may move from the intermediate extended position to the fully extended position.",
"43.An emergency release assembly according to claim 42 wherein said catch member is U-shaped in configuration including a pair of spaced-apart arm portions joined by a web portion, each arm portion abutting said first axle pin when in the retain state yet pivotable out of the retain state whereby said push bar may move from the intermediate extended position to the fully extended position.",
"44.An emergency release assembly according to claim 37 wherein said muntin includes a pair of facing channel structures, said base member slideably received in and supported by said channel structures.",
"45.An emergency release assembly according to claim 36 wherein said latch mechanism includes a reciprocating latch actuator rod, said release mechanism including a rotatable drive element and a crank arm, said crank arm having a first crank arm end secured to said control member and a second crank arm end secured to said drive element whereby reciprocation of said control member acts to reciprocally rotate said drive element, said actuator rod having a first rod end secured to said drive element whereby reciprocal rotation of said drive element acts to reciprocate said actuator rod.",
"46.An emergency release assembly according to claim 45 wherein said first crank arm end is slideably secured to said control member such that said drive element is not rotated when said push bar moves from the intermediate extended position to the fully extended position.",
"47.A sliding door system with breakaway capability, comprising: (A) a fixed panel oriented in a first plane; (B) a guide structure extending parallel to the fixed panel; (C) a carriage piece disposed in said guide structure for sliding movement relative to said fixed panel, said carriage piece including a catch element; and (D) a sliding door secured to said carriage piece, said door (1) slidably movable with said carriage piece between (a) a slidable closed position relative to said fixed panel and (b) a slidable open position relative to said fixed panel, and (2) pivotally mounted with respect to said carriage piece at a location spaced from said catch element to be pivotally movably between (a) a normally closed configuration relative to said carriage piece and said fixed panel and (b) an emergency open configuration relative to said carriage piece and said fixed panel, (3) said door including (a) a frame extending around a perimeter portion of said door and a muntin that has a recess and peripheral muntin sections that have defining surfaces a substantially planar outer muntin face oriented in closely-spaced facing relationship with said fixed panel when in the slideable open position, (b) a latch mechanism disposed in said frame and operative in a first state to engage said catch thereby to retain said door in the pivotally closed state and in a second state to release said catch thereby to permit said door to pivot from the pivotally closed state to the pivotally open state, and (c) a release mechanism disposed in the muntin recess and mechanically coupled to said latching mechanism and actuable to advance said latching mechanism from the first state to the second state, said release mechanism including (i) a control member disposed in the recess and movable within the recess to selectively actuate said release mechanism, (ii) a push bar having an outer surface, (iii) at least one link member interconnecting said push bar and said control member such that said push bar may be moved between a fully extended position and a depressed position through an intermediate extended position, said push bar when moved from the intermediate extended position to the depressed position being operative to move said control member such that said latch mechanism is advanced from the first state to the second state, said push bar when in the intermediate extended position being located entirely within the recess and within a geometric projection of the planar outer mount in face and when in the fully extended position protruding out of the recess; and (iv) a catch member operative in a retain state to limit movement of said push bar between the intermediate extended position and the depressed position yet releasable to an access state wherein said push bar can move from the intermediate extended position to the fully extended position."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Various closure structures have been associated with the entryways of dwellings and buildings throughout history.",
"These closure structures range from simple draped fabrics to a standard hinged door and to more elaborate mechanical devices that are used in commercial applications.",
"Many simple closure structures do not include latch and/or locking capabilities.",
"On the other hand, more elaborate structures can include a variety of components which secure the door and position and control access to the entryway.",
"In modern commercial buildings, automatic doors have become commonplace.",
"Automatic doors are typically electronically operated so that, as a person approaches, the door moves from a closed position to an open position.",
"Automatic doors typically respond to an input signal that senses the presence of a person.",
"In the early days of automatic doors, a pressure pad was placed in front of the door on the walking surface.",
"When a person steps on the pressure pad, the weight of the person closes a switch that sends a signal to the door operating mechanism to cause that mechanism to open the door.",
"In more recent years, motion and proximity to detectors have been used to determine the presence of a person at the entryway.",
"When a person enters the sensor zone, a signal is produced that causes the door operating mechanism to open the door.",
"In earlier days, automated doors typically were swinging doors that were hinged, along a vertical edge and swung between the open and closed positions similarly to a standard household door.",
"In the event of a power outage, the door could forcefully be swung to an open condition to allow emergency entry or exit through the door.",
"This is because the mechanics of the door could be overcome by torque applied to the edge of the door opposite the vertical hinged location.",
"In more recent times, automated sliding doors have proliferated.",
"Automated sliding doors are typically constructed as a unit wherein a slide panel is slightly offset from but is parallel to a fixed panel.",
"The slide panel slides relative to the fixed panel to create a doorway opening for ingress and egress.",
"Often, a pair of such units are placed in end to end relation so that the slide panels abut one another in a common plane.",
"In such configurations, the presence of an operative signal causes both slide panels to slide away from one another presenting a doublewide entryway opening for ingress and egress.",
"While the closing force of a traditional automated swinging door can be manually overcome, it is more difficult to overcome the closing force of an automatic sliding door.",
"This could potentially cause a problem should power fail to be supplied to an automated door.",
"This can occur in situations such as power outages, fires and the like.",
"Since it is desirable to permit persons located within the building to exit the building under such circumstances, it is known to provide automatic sliding doors with a breakaway capability.",
"Here, in addition to its sliding capability, the sliding door is mounted on a hinge or pivot that defines a vertically oriented axis for swinging motion of the sliding door.",
"A latch mechanism is located along an opposite edge of the door with this latch mechanism normally retaining the sliding door in its normally closed position.",
"A manually actuable release mechanism is then associated with the door such that, upon activation, the release mechanism operates the latch mechanism to release the sliding door so that it may pivot along the vertical axis in a manner of a normal door.",
"This feature is called a “break-away capability”.",
"Traditional automatic slide doors with break-away capability suffer from a disadvantage, however.",
"In automatic sliding door configurations it is normally desirable that the slide panel be located exteriorly of the fixed panel.",
"However, if so mounted, the break-away activation mechanism on the slide panel can interfere with the ability of the slide panel to undergo reciprocal sliding movement do to the physical dimensions of the release mechanism.",
"For this reason, where an automatic slide door is provided with break-away capability, it is known to mount the slide panel in the less desirable interior location so that the release mechanism, located on the interior side of the side panel, does not interfere with the reciprocal sliding motion.",
"Accordingly, there has been a long felt need for an emergency release assembly and a sliding door assembly that incorporates the same, that permits the release assembly to be mounted on the interior side of the sliding panel yet which also is constructed so as to allow the slide panel to be positioned on the exterior side of the fixed panel of an automatic sliding door.",
"The present invention addresses this need."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a new and useful emergency release assembly adapted for use on a door structure as well as a door structure incorporating such an emergency release assembly.",
"Another object of the present invention is to provide a new and useful emergency release assembly adapted for use in a sliding door as well as a sliding door incorporating such an emergency release assembly.",
"It is another object of the present invention to provide an emergency release assembly that fits entirely within the geometric extension of the surface of a sliding door muntin during normal use.",
"It is a further object of the present invention to provide an emergency release mechanism for a sliding door that can be provided as original equipment for a sliding door or that may be retrofit into many types of existing sliding doors assemblies.",
"Yet another object of the present invention is to provide an emergency release mechanism and sliding door incorporating the same where the mechanism normally fits entirely within the recess of a muntin yet which may be manually tripped so that it may be expanded for ease of installation, maintenance and repair.",
"Still another object of the present invention is to provide an emergency release assembly and sliding door incorporating the same that is simple and reliable in construction and operation.",
"According to the present invention, then, an emergency release assembly is disclosed that is adapted for use with a door that is supported by a framework when in a normally closed configuration yet that is moveable in an emergency condition between the normally closed configuration and an emergency open configuration.",
"This emergency release assembly is especially incorporated, according to the present invention, in a sliding door system with break-away capability wherein the sliding door system includes a fixed panel oriented in the first plane a guide structure extending parallel to the fixed plane and a carriage piece disposed in the guide structure for sliding movement relative to the fixed panel.",
"A sliding door is secured to the carriage piece so that the door slidingly moves with the carriage piece between a slideable closed position relative to the fixed panel and a slideable open position relative to the fixed panel and wherein the sliding door is also pivotally mounted with respect to the carriage piece so that it may pivot between a normally closed configuration and an emergency open configuration.",
"The release assembly according to the present invention, whether for a swinging door or a sliding door, includes a latch mechanism that is operative in the first state to retain the door in the normally closed position and is actuable to a second state that allows the door to be moved to an open position.",
"The release assembly also includes a release mechanism that is operative to advance the latch mechanism from the first state to the second state.",
"The release mechanism includes a reciprocating control member coupled to the latch mechanism and supported by the door for reciprocal sliding movement.",
"A manually actuable push bar is provided and at least one link member interconnects the push bar and the reciprocating member in a manner such that the push bar may be moved between a fully extended position and a depressed position through an intermediate extended position.",
"When the push bar is moved from the intermediate extended position to the depressed position, it operates to move the reciprocating member such that the latch mechanism is advanced from the first state to the second state.",
"A catch member is provided that operates in a retained state to limit movement of the push bar between the intermediate extended position and the depressed position yet the catch member is releasable to an access state when the push bar can move from the intermediate position to the fully extended position.",
"This release mechanism can be used as a primary latch and release assembly for a door as an emergency or “one-way” release.",
"In the disclosed embodiment, the link member is defined by a scissors unit formed by first and second legs that are pivotally interconnected to one another.",
"Specifically, a pair of link members interconnect the push bar and the reciprocating members with these link members being positioned and spaced apart in relation to one another.",
"In each case, both link members are then defined by a scissors unit.",
"Whether using one or a plurality of scissor units, the first leg of each scissor unit has a proximal end secured to the reciprocating member and a distal end opposite the proximal end.",
"A second leg then has a proximal end secured relative to the door and a distal end secured to a medial portion of the first leg.",
"A mounting plate is supported on the distal end of the first leg and the push bar may be secured to the mounting plate.",
"Thus, depression of the push bar causes the scissor units to collapse and expand thereby moving the reciprocating member relative to the door.",
"In the disclosed embodiment, the latch mechanism includes a reciprocating latch actuator rod, and the release mechanism includes a rotatable drive element and a crank arm.",
"The crank arm has a first crank arm end that is secured to the reciprocating member and a second crank arm end secured to the drive element such that reciprocation of the reciprocating member acts to reciprocally rotate the drive element the actuator rod has a first rod end secured to the drive element whereby reciprocal rotation of the drive element acts to reciprocate the actuator rod.",
"The first crank arm end moreover may be slideably secured to the reciprocating member such that the drive element is not rotated when the push bar moves from the intermediate extended position to the fully extended position.",
"In the disclosed embodiment, the push bar is biased toward the fully extended position.",
"This is accomplished by means of a return spring mounted on the legs of the scissor unit(s) which biases them into an extended position.",
"In the disclosed embodiment, the release mechanism includes a base member that is adapted to be supported by the door, such as by slide channels formed within the muntin recess.",
"The link member or members interconnect the base member and the reciprocating member for reciprocal motion of the reciprocating member relative to the base member.",
"The catch member is defined by a lever that is pivotally secured to the base member with the lever including a lever arm section that interacts with the scissor unit to limit movement thereof so that is may not move from the intermediate extended position to the fully extended position when in the retained state.",
"This lever is pivotable out of the retained state so that the scissor assembly may expand to the fully extended position.",
"The push bar then has an access opening whereby the lever may be manually pivoted out of the retained state.",
"As noted, the release mechanism is adapted to be disposed in the muntin recess of a sliding door so that, when the push bar is in the intermediate extended position and in the depressed position, it is captured completely within the interior of the recess so that the push bar does not protrude outwardly therefrom.",
"The catch member retains the push bar within the muntin recess but, when moved to the access state, allows the push bar to extend out of the recess when in the fully extended position thereby to allow access for installation and maintenance.",
"The present invention also is directed to a sliding door incorporating a latch mechanism and a release mechanism, as described above.",
"Here, the sliding door includes a framework that has a muntin.",
"A latch mechanism is disposed in the framework and operates in the first state to retain the sliding door in the normally closed configuration yet which is actuable to a second state that allows the sliding door to be moved to the emergency open configuration.",
"A release mechanism is then disposed in the muntin and operates to advance the latch mechanism from the first state to the second state.",
"The release mechanism includes a control member coupled to the latch mechanism and supported for reciprocal sliding movement, a push bar at least one link member interconnecting the push bar and the control member and a catch member, all as described above.",
"These and other objects of the present invention will become more readily appreciated and understood from a consideration of the following detailed description of the exemplary embodiments of the present invention when taken together with the accompanying drawings, in which:"
],
[
"FIELD OF THE INVENTION The present invention broadly concerns latch and release mechanisms for doors.",
"The present invention particularly concerns latch and release mechanisms for doors, including glass panel doors and the like.",
"This invention specifically concerns an emergency release or break-away latch assembly for automatic sliding doors, but the invention also may apply to swinging doors.",
"BACKGROUND OF THE INVENTION Various closure structures have been associated with the entryways of dwellings and buildings throughout history.",
"These closure structures range from simple draped fabrics to a standard hinged door and to more elaborate mechanical devices that are used in commercial applications.",
"Many simple closure structures do not include latch and/or locking capabilities.",
"On the other hand, more elaborate structures can include a variety of components which secure the door and position and control access to the entryway.",
"In modern commercial buildings, automatic doors have become commonplace.",
"Automatic doors are typically electronically operated so that, as a person approaches, the door moves from a closed position to an open position.",
"Automatic doors typically respond to an input signal that senses the presence of a person.",
"In the early days of automatic doors, a pressure pad was placed in front of the door on the walking surface.",
"When a person steps on the pressure pad, the weight of the person closes a switch that sends a signal to the door operating mechanism to cause that mechanism to open the door.",
"In more recent years, motion and proximity to detectors have been used to determine the presence of a person at the entryway.",
"When a person enters the sensor zone, a signal is produced that causes the door operating mechanism to open the door.",
"In earlier days, automated doors typically were swinging doors that were hinged, along a vertical edge and swung between the open and closed positions similarly to a standard household door.",
"In the event of a power outage, the door could forcefully be swung to an open condition to allow emergency entry or exit through the door.",
"This is because the mechanics of the door could be overcome by torque applied to the edge of the door opposite the vertical hinged location.",
"In more recent times, automated sliding doors have proliferated.",
"Automated sliding doors are typically constructed as a unit wherein a slide panel is slightly offset from but is parallel to a fixed panel.",
"The slide panel slides relative to the fixed panel to create a doorway opening for ingress and egress.",
"Often, a pair of such units are placed in end to end relation so that the slide panels abut one another in a common plane.",
"In such configurations, the presence of an operative signal causes both slide panels to slide away from one another presenting a doublewide entryway opening for ingress and egress.",
"While the closing force of a traditional automated swinging door can be manually overcome, it is more difficult to overcome the closing force of an automatic sliding door.",
"This could potentially cause a problem should power fail to be supplied to an automated door.",
"This can occur in situations such as power outages, fires and the like.",
"Since it is desirable to permit persons located within the building to exit the building under such circumstances, it is known to provide automatic sliding doors with a breakaway capability.",
"Here, in addition to its sliding capability, the sliding door is mounted on a hinge or pivot that defines a vertically oriented axis for swinging motion of the sliding door.",
"A latch mechanism is located along an opposite edge of the door with this latch mechanism normally retaining the sliding door in its normally closed position.",
"A manually actuable release mechanism is then associated with the door such that, upon activation, the release mechanism operates the latch mechanism to release the sliding door so that it may pivot along the vertical axis in a manner of a normal door.",
"This feature is called a “break-away capability”.",
"Traditional automatic slide doors with break-away capability suffer from a disadvantage, however.",
"In automatic sliding door configurations it is normally desirable that the slide panel be located exteriorly of the fixed panel.",
"However, if so mounted, the break-away activation mechanism on the slide panel can interfere with the ability of the slide panel to undergo reciprocal sliding movement do to the physical dimensions of the release mechanism.",
"For this reason, where an automatic slide door is provided with break-away capability, it is known to mount the slide panel in the less desirable interior location so that the release mechanism, located on the interior side of the side panel, does not interfere with the reciprocal sliding motion.",
"Accordingly, there has been a long felt need for an emergency release assembly and a sliding door assembly that incorporates the same, that permits the release assembly to be mounted on the interior side of the sliding panel yet which also is constructed so as to allow the slide panel to be positioned on the exterior side of the fixed panel of an automatic sliding door.",
"The present invention addresses this need.",
"SUMMARY OF THE INVENTION It is an object of the present invention to provide a new and useful emergency release assembly adapted for use on a door structure as well as a door structure incorporating such an emergency release assembly.",
"Another object of the present invention is to provide a new and useful emergency release assembly adapted for use in a sliding door as well as a sliding door incorporating such an emergency release assembly.",
"It is another object of the present invention to provide an emergency release assembly that fits entirely within the geometric extension of the surface of a sliding door muntin during normal use.",
"It is a further object of the present invention to provide an emergency release mechanism for a sliding door that can be provided as original equipment for a sliding door or that may be retrofit into many types of existing sliding doors assemblies.",
"Yet another object of the present invention is to provide an emergency release mechanism and sliding door incorporating the same where the mechanism normally fits entirely within the recess of a muntin yet which may be manually tripped so that it may be expanded for ease of installation, maintenance and repair.",
"Still another object of the present invention is to provide an emergency release assembly and sliding door incorporating the same that is simple and reliable in construction and operation.",
"According to the present invention, then, an emergency release assembly is disclosed that is adapted for use with a door that is supported by a framework when in a normally closed configuration yet that is moveable in an emergency condition between the normally closed configuration and an emergency open configuration.",
"This emergency release assembly is especially incorporated, according to the present invention, in a sliding door system with break-away capability wherein the sliding door system includes a fixed panel oriented in the first plane a guide structure extending parallel to the fixed plane and a carriage piece disposed in the guide structure for sliding movement relative to the fixed panel.",
"A sliding door is secured to the carriage piece so that the door slidingly moves with the carriage piece between a slideable closed position relative to the fixed panel and a slideable open position relative to the fixed panel and wherein the sliding door is also pivotally mounted with respect to the carriage piece so that it may pivot between a normally closed configuration and an emergency open configuration.",
"The release assembly according to the present invention, whether for a swinging door or a sliding door, includes a latch mechanism that is operative in the first state to retain the door in the normally closed position and is actuable to a second state that allows the door to be moved to an open position.",
"The release assembly also includes a release mechanism that is operative to advance the latch mechanism from the first state to the second state.",
"The release mechanism includes a reciprocating control member coupled to the latch mechanism and supported by the door for reciprocal sliding movement.",
"A manually actuable push bar is provided and at least one link member interconnects the push bar and the reciprocating member in a manner such that the push bar may be moved between a fully extended position and a depressed position through an intermediate extended position.",
"When the push bar is moved from the intermediate extended position to the depressed position, it operates to move the reciprocating member such that the latch mechanism is advanced from the first state to the second state.",
"A catch member is provided that operates in a retained state to limit movement of the push bar between the intermediate extended position and the depressed position yet the catch member is releasable to an access state when the push bar can move from the intermediate position to the fully extended position.",
"This release mechanism can be used as a primary latch and release assembly for a door as an emergency or “one-way” release.",
"In the disclosed embodiment, the link member is defined by a scissors unit formed by first and second legs that are pivotally interconnected to one another.",
"Specifically, a pair of link members interconnect the push bar and the reciprocating members with these link members being positioned and spaced apart in relation to one another.",
"In each case, both link members are then defined by a scissors unit.",
"Whether using one or a plurality of scissor units, the first leg of each scissor unit has a proximal end secured to the reciprocating member and a distal end opposite the proximal end.",
"A second leg then has a proximal end secured relative to the door and a distal end secured to a medial portion of the first leg.",
"A mounting plate is supported on the distal end of the first leg and the push bar may be secured to the mounting plate.",
"Thus, depression of the push bar causes the scissor units to collapse and expand thereby moving the reciprocating member relative to the door.",
"In the disclosed embodiment, the latch mechanism includes a reciprocating latch actuator rod, and the release mechanism includes a rotatable drive element and a crank arm.",
"The crank arm has a first crank arm end that is secured to the reciprocating member and a second crank arm end secured to the drive element such that reciprocation of the reciprocating member acts to reciprocally rotate the drive element the actuator rod has a first rod end secured to the drive element whereby reciprocal rotation of the drive element acts to reciprocate the actuator rod.",
"The first crank arm end moreover may be slideably secured to the reciprocating member such that the drive element is not rotated when the push bar moves from the intermediate extended position to the fully extended position.",
"In the disclosed embodiment, the push bar is biased toward the fully extended position.",
"This is accomplished by means of a return spring mounted on the legs of the scissor unit(s) which biases them into an extended position.",
"In the disclosed embodiment, the release mechanism includes a base member that is adapted to be supported by the door, such as by slide channels formed within the muntin recess.",
"The link member or members interconnect the base member and the reciprocating member for reciprocal motion of the reciprocating member relative to the base member.",
"The catch member is defined by a lever that is pivotally secured to the base member with the lever including a lever arm section that interacts with the scissor unit to limit movement thereof so that is may not move from the intermediate extended position to the fully extended position when in the retained state.",
"This lever is pivotable out of the retained state so that the scissor assembly may expand to the fully extended position.",
"The push bar then has an access opening whereby the lever may be manually pivoted out of the retained state.",
"As noted, the release mechanism is adapted to be disposed in the muntin recess of a sliding door so that, when the push bar is in the intermediate extended position and in the depressed position, it is captured completely within the interior of the recess so that the push bar does not protrude outwardly therefrom.",
"The catch member retains the push bar within the muntin recess but, when moved to the access state, allows the push bar to extend out of the recess when in the fully extended position thereby to allow access for installation and maintenance.",
"The present invention also is directed to a sliding door incorporating a latch mechanism and a release mechanism, as described above.",
"Here, the sliding door includes a framework that has a muntin.",
"A latch mechanism is disposed in the framework and operates in the first state to retain the sliding door in the normally closed configuration yet which is actuable to a second state that allows the sliding door to be moved to the emergency open configuration.",
"A release mechanism is then disposed in the muntin and operates to advance the latch mechanism from the first state to the second state.",
"The release mechanism includes a control member coupled to the latch mechanism and supported for reciprocal sliding movement, a push bar at least one link member interconnecting the push bar and the control member and a catch member, all as described above.",
"These and other objects of the present invention will become more readily appreciated and understood from a consideration of the following detailed description of the exemplary embodiments of the present invention when taken together with the accompanying drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a diagrammatic front plan view of a sliding door assembly according to the present invention located within a representative entryway of a building and viewed from the interior of the building with a pair of sliding doors thereof shown in a normally closed condition and with the door assembly closing the entryway; FIG.",
"2 is a front view in elevation, similar to FIG.",
"1, but showing the pair of sliding doors opened to allow access to the entryway of the building; FIG.",
"3 is a top plan view taken about lines 3-3 of FIG.",
"1 showing a representative slide door unit with the slide door in the normally closed position; FIG.",
"4 is a top plan view, similar to FIG.",
"3, but showing the slide door in an emergency open or “break-away” configuration; FIG.",
"5 is a perspective view of the top portion of a slide door and break out arm according to the present invention; FIG.",
"6 is a perspective view of the top corner portion of a sliding door shown in FIG.",
"5 broken away to reveal the latch mechanism with the latch mechanism in a first state that retains the sliding door in the normally closed position; FIG.",
"7 is a perspective view, similar to FIG.",
"6, but showing the latch mechanism actuated to second state that allows the sliding door to be moved to the emergency open configuration; FIG.",
"8 is cross-sectional view showing the muntin of a sliding door; FIG.",
"9 is an exploded perspective view of the muntin and no stile of the present invention receiving the latch mechanism and the release mechanism according to a first exemplary embodiment of the present invention; FIG.",
"10 is an exploded perspective view showing the release mechanism according to the present invention; FIG.",
"11(a) is an exploded perspective of a scissor unit forming a representative link member; FIG.",
"11(b) is perspective view showing a link member in the form of a scissor unit according to the present invention; FIG.",
"12(a) shows a cross-sectional view of the push bar and release mechanism of the present invention installed in the muntin and illustrated in an intermediate extended position; FIG.",
"12(b) is a front view in elevation showing a portion of the release mechanism of the present invention in the intermediate extended position; FIG.",
"13(a) is an end view in elevation, similar to FIG.",
"12(a), but showing the push bar and release mechanism in the depressed position; FIG.",
"13(b) is a front view in elevation, similar to FIG.",
"12(b), but showing the release mechanism in the depressed position; FIG.",
"14(a) is a top plan view showing a link member in the form of a scissor unit and catch member with the catch member in the retain state and the scissor unit in the intermediate extended position; FIG.",
"14(b) is a top plan view, similar to FIG.",
"14(a), but showing the catch member in an access state such that the scissor unit and the push bar connected thereto is in the fully extended position; FIG.",
"15 is a side view in elevation showing the push bar and release mechanism in the fully extended position; FIG.",
"16 is a front view in elevation showing a second exemplary embodiment of the present invention employing the emergency release assembly with a swinging door; FIG.",
"17 is a perspective view of the top of the swinging door of FIG.",
"16 with the door panel in the normally closed position; FIG.",
"18 is a perspective view of the top of the swinging door, similar to FIG.",
"17, but showing the door panel in the emergency open position; FIG.",
"19 is a front view in elevation showing a third exemplary embodiment of the present invention employing the release assembly and latch mechanism as the primary latch for a swinging door shown in a closed position; and FIG.",
"20 is a side view in elevation showing the swinging door of FIG.",
"19 moving to an open position.",
"DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS The present invention broadly concerns door closures used with respect to the entryways of dwellings and other buildings.",
"The present invention, however, is specifically directed to a sliding door assembly such as that used in many commercial establishments.",
"It specifically concerns mechanisms, and doors incorporating those mechanisms, which are automated but which have an emergency break-away capability.",
"However, while the present invention is described with respect to an automatic sliding door assembly, it should be understood that this invention may be used in any application where an emergency break-away capability is desired, even on non-sliding doors.",
"Moreover, the present structure may also be used for the primary latching of a door without break-away capability.",
"It should be further understood that the present invention is contemplated to be manufactured as original equipment with such a sliding door, but also may be constructed for retrofit capability.",
"With these concepts in mind, a sliding door system having many features according to the prior art but also incorporating the structure of the exemplary embodiment of the present invention is introduced in FIGS.",
"1-4.In these figures, however, little, if any, of the detail of the new and useful emergency release assembly according to this invention is depicted.",
"However, the structure shown in these figures assists in the understanding of the operation of the present invention.",
"Turning first to FIGS.",
"1 and 2, a sliding door system 10 is shown mounted in a wall portion 12 of a representative structure, such as a commercial building, having a floor 16.Sliding door system 10 includes a pair of sliding door units 14 and 15.Sliding door unit 14 includes a fixed panel in the form of a glass pane 18 and a sliding door 20 which is mounted for sliding movement with respect to panel 18 on a rail 22.Likewise, sliding door unit 15 includes a fixed panel 19, again in the form of a glass panel, and a sliding door 21 that slides relative to panel 19 on rail 22.As is shown in FIG.",
"1, sliding doors 20 and 21 are in a normally closed position such that, when slid together abut one another generally in a common plane.",
"In FIG.",
"2, however, sliding doors 20 and 21 have been slid in the direction of arrows “A” (FIG.",
"1) to open thereby allowing access through an entryway 24.Generally speaking, each of fixed panels 18 and 19 as well as sliding doors 20 and 21 are mounted in a framework that includes guide rail 22 and side frame pieces 26 and 27, although this exact structure is not necessary for an appreciation of the present invention.",
"Turning to FIGS.",
"3 and 4, representative sliding door unit 14 is depicted, and it may be seen especially in FIG.",
"3 that fixed panel 18 is oriented generally in a first plane while sliding door 20, when in a normally closed configuration is oriented in a second or slide plane that is in closely spaced parallel relation to the plane of fixed panel 18.With reference next to FIG.",
"4, however, it may be seen that sliding door 20 can pivot into an emergency open configuration to allow emergency egress through entryway 24, if necessary.",
"To this end, sliding door 20 pivots about a hinge 28 that interconnects door 20 to a breakout arm 30, as is known in the art.",
"The construction of sliding door 15 is substantially identical to that of sliding door unit 14 except that the structure is symmetrical or forms a mirror image of that described with respect to FIGS.",
"3 and 4, so that the details of that structure are not again repeated.",
"As noted in the background of this invention, the typical sliding door unit with breakaway capability requires that the sliding door 20 be located interiorly of the building relative to the affixed panel.",
"In FIGS.",
"1-4, however, the present invention allows the sliding panel to be located exteriorly of the building by virtue of the emergency release assembly of the present invention.",
"To understand this emergency release assembly, reference is made to FIGS.",
"1 and 5-7 where it may be seen that representative sliding door 20 has a frame that includes a hinge stile 32 and a nose stile 34 that are interconnected by top and bottom frame pieces 36 and 38.A central, horizontal muntin 40 extends between nose stile 34 and hinge stile 38 midway between frame pieces 36 and 38 so as to support a pair of glass panels 42 as is known in the art.",
"With reference to FIGS.",
"5-7, it may be seen that nose stile 34 has a latch mechanism 44 disposed therein with this latch mechanism being adapted to engage a catch 46 that is part of breakout arm 30, as is known in the art.",
"Latch mechanism 44 is of a type described in U.S. Pat.",
"No.",
"4,368,905, the disclosure of which is incorporated herein by reference.",
"Broadly, latch mechanism 44 includes a spring loaded, U-shaped latch piece 48 that is pivotally mounted on shaft 50.When latch piece 48 engages catch 46, as is shown in FIG.",
"6, an end 54 of latch rod 52 abuts tongue 49 of latch piece 48 to maintain engagement with catch 46 so that the door 20 may not be moved into the emergency open position.",
"However, as is shown in FIG.",
"7, when latch rod 52 is moved downwardly a small distance in the direction of arrow “B” end 54 moves out of this blocking engagement so that latch piece 48 may flip to a release position thereby disengaging the catch 46 allowing door 20 to pivot into the emergency open position.",
"When the door 20 is moved from the emergency open position toward the normally closed position, the pressure of catch 46 against latch piece 48 again moves it into the position shown in FIG.",
"6 allowing rod 52 to move upwardly in the direction opposite arrow “B” to again secure the door in the normally closed position.",
"From this description, it should now be apparent to the ordinarily skilled artisan that, to release door 20 for pivoting motion, it is only necessary to toggle rod 52 a sufficient amount to move end 54 out of abutment with tongue 49.This is normally accomplished by a release mechanism that is mounted in the muntin 40 of the door.",
"However, the dimensions of such a release mechanism disposed in the muntin has here to for been too great to fit within the geometric projection of the face of the muntin, a problem resolved by the present invention.",
"Turning, then, to FIG.",
"8, it may be seen that a representative muntin 40 is in the form of an extrusion, such as aluminum, that includes a pair of opposed muntin sections 56 and 58 that are adjoined by a web piece 60 as an integral one piece extrusion.",
"Upper and lower muntin sections 56 and 58 define a recess 62 therein within which the release mechanism, described below, is to be mounted.",
"To this end, muntin 40 concludes a pair of spaced apart ribs 64 that form facing channels to receive the release mechanism as described below.",
"In any event, muntin sections 56 and 58 respectively have front surfaces 57 and 59 that define a substantially plainer outer muntin face “F”, as is shown in FIG.",
"8.In order for the sliding doors 20 and 21 to be mounted exteriorly of fixed panels 18 and 19, respectively, it is necessary that the release mechanism, during operation, fit entirely within recess 62 as bounded by web 60 and plane “F” that may also be termed the geometric projection of front surfaces 57 and 59 that define plane “F”.",
"The release mechanism 64 according to the exemplary embodiments of the present invention is introduced in FIG.",
"9 where it may be seen that release mechanism 64 includes a control member 66 in the form of a reciprocating piece, a push bar 68 and a pair of link members 70 that interconnect push bar 68 and reciprocating control member 66.In this exemplary embodiment, these pieces are mechanically secured to a base member 72 that is slideably engageable in the channels formed by ribs 63.In its more detailed construction, release mechanism 64 also includes a rotatable cam drive element 74 that is linked to control member 66 by means of a crank arm 76 slid into and supported in recess 62.The elements and construction of release mechanism 64 is shown in greater detail in FIG.",
"10.In this figure, it may be seen that base member 72 is in the form of a metallic strip or plate that has a first pair of ears 78 adjacent a first end thereof.",
"Ears 78 are stamped out of the material of base member 72 and are bent generally perpendicularly thereto to form a pair of opposed pin supports, as defined below.",
"Accordingly, each of ears 78 has a hole 79 formed therethrough.",
"A second pair of ears 80 having holes 81 are similarly formed at a second end portion of base member 72 to again form opposed supports for an axle pin, as described below.",
"Reciprocating control member 66 is in the form of a channel shaped piece that includes a bottom wall 82 and a pair of opposed sidewalls 84 that extend a majority of the length of bottom wall 82.Control member 86 has a pair of spaced apart ears 86 having holes 87 formed at a first end thereof to form axle mounts, again as described below.",
"Sidewalls 84 have a pair of spaced apart holes 86 formed at an end thereof that is opposite ears 86.Control member 66 is sized and configured to be placed in confronting relationship with base member 72 so that it may slide in a longitudinal direction between ears 78 and 80 alongside the surface of base member 72.A pair of link members in the form of scissor units 70 operate to link base member 70 to reciprocating control member 66.In FIG.",
"10, one such scissor unit 70 is shown in an assembled configuration while the other scissor unit 70 is shown in an exploded perspective.",
"The structure of a scissor unit 70 is depicted in greater detail, also, in FIGS.",
"11(a) and 11(b).",
"In either case, it may be seen that each scissor unit 70 is formed by a first leg 90 and a second leg 98.Each first leg 90 includes a pair of leg sections 91 that are in generally parallel spaced apart relation to one another and are interconnected by a web 92.A central hole 93 is located on each leg section 91 and opposite end holes 94 and 95 are located at opposite ends of each leg section 91.Second leg 98 includes a pair of leg sections 99 that are in generally parallel spaced relation to one another and are joined by a web 100.Each leg section 99 includes end holes 102 and 103.A mounting plate 104 is provided and includes ears, such as ears 105 having holes 106 having a hole 106 therethrough.",
"In addition, scissor unit 70 includes two axle pins 107 and 109 along with a torsion spring 110.Scissor unit 70 is shown in FIG.",
"11(b) in an assembled state.",
"Here, it may be seen that end holes 103 of second leg 88 have been co-axially aligned so that the distal end of each leg section 99, corresponding to end holes 103, are pivotally secured to a medial portion of first leg 90, and specifically, to a medial portion of leg sections 91 by means of axle pin 107.Here, also, it should be understood that axle pin axially receives torsion spring 110 thereon with torsion spring 110 being positioned between the leg sections 99.The ends of torsion spring 110 bear against webs 92 and 100 so as to bias legs 90 and 98 into an expanded or fully extended position.",
"Mounting plate 104 is secured to the distal end of first leg 90 by axially aligning holes 95 and 106 together with ears 105 being positioned between leg sections 91.Mounting plate 104 is then secured by means of axle pin 109.In this manner, legs 90 and 98 may pivot relative to one another about axle pin 107 while mounting plate 104 may pivot relative to first leg 90 by means of axle pin 109.Further construction of release mechanism 64 may be seen with reference again to FIG.",
"10.In interconnecting control member 66 to base member 72, it may now be appreciated that this is accomplished by means of a pair of axle pins 108 and 111.Specifically, after the assembly of a scissor unit 70, the proximal end of first leg 90 is secured to control member 66 by placing ears 86 between leg sections 91 and pivotally securing them by axle pin 111 passing through holes 87 and holes 94.The proximal end of second leg 98 is secured to base member 72 by placing the proximal end of second leg 98 between ears 78 and pivotally securing them together by means of axle pin 108 which passes through holes 79 and end holes 102.The second scissor unit 70 is secured in a similar manner with the proximal end of its first leg 90 being secured at the end of sidewalls 88 by connecting the proximal end portion of leg sections 91 with an axle pin 111 passing through holes 88 and end holes 94.The proximal end of the respective second leg 98 is secured to ears 80 by means of axle pin 108 which extends through holes 81 and end holes 102.With continued reference to FIG.",
"10, it should also be understood that a catch member 114 is provided and is generally U-shaped in configuration having a pair of spaced apart, generally parallel arm sections 115 joined by a web 116 at one end thereof.",
"Arm sections 115 are provided with medial holes 117 sized to receive a respective axle pin 111.Thus, catch member 114 is mounted for pivotal movement on a common axle pin 111 as is the proximal end of a respective second leg 98.Reciprocating control member 66 is used to operate actuator rod 52.To this end, a crank arm 118 is connected to an end of control member 66 by means of pin 120 passing through a slot 122 and being press fit into a hole 124 formed in the proximal end of crank arm 118 the distal end of crank arm 118 is formed as an angled foot 126 that has a hole 128.Base member 172 has a bracket 130 secured thereto by means of rivets 132.Bracket 130 supports a shaft 134 through a hole 136 formed in an offset lobe 131 of bracket 130.A cam disk 138 is then rotatably journaled relative to bracket 132 by being pressed fit on a rectangular head of shaft 134 by way of a central rectangular opening 139 formed in the center of disk 138, and it may be secured by clip 137.A pin 140 then attaches foot 126 of crank arm 118 to cam disk 138 by extending through a hole 142 and mating with hole 128.A post 144 is secured to disk 138 by means of a hole 146 in order to attach the latch actuator rod 52.This assembled unit may further be viewed in FIG.",
"12(b), as discussed below.",
"The operation of release mechanism 64 may now be more fully appreciated with reference to FIGS.",
"12(a), 12(b), 13(a) and 13(b).",
"In FIGS.",
"12(a) and 12(b), release mechanism 64 is shown in a condition wherein the push bar 68 and the scissor units are in an intermediate extended position.",
"In this position, push bar 68 has an outer face 69 that is substantially co-planer with muntin plane “F”.",
"Push bar 68 and scissor units 70 are held in this position since the ends of arm sections 115 of catch member 114 that are located oppositely web 16 are in abutment with axle pin 111.Catch member 114 is biased into a position wherein abutment occurs by means of a biasing spring 146 that extends around axle pin 108 which mounts the proximal end of the section leg section 98 to base member 72.However, when push bar 68 is depressed so that it moves into muntin recess 62, as is shown in FIG.",
"13(a), the scissor units 70 are compressed which results in a separation of the proximal ends of each of legs 90 and 98.When the proximal ends of legs 90 and 98 move apart, control member 66 is moved to the left, as is shown by arrow “C” in FIG.",
"13(b).",
"When this occurs, crank arm is drawn to the left, as is shown by arrow “D” since pin 120 is in the right hand location in slot 122.Movement of crank arm 118 to the left, in the direction of arrow “D” causes a rotation of disk 138 in the direction of arrow “R” as is shown in FIG.",
"13(b).",
"When this happens, actuator rod 152 moves downwardly in the direction of arrow “B”.",
"This action, as discussed with reference to FIGS.",
"6 and 7, acts to release latch mechanism 44.Releasing push bar 68 from this depressed position, as is shown in FIG.",
"13(a) causes it to move back to the intermediate extended position as shown in FIG.",
"12(a).",
"This is due to the action of return torsion springs 110 acting to expand scissor units 70 so as to move the proximal end portion of legs 90 and 98 closer together.",
"This action can occur as a result of either of two conditions.",
"First, if latch piece 48 has pivoted to the position shown in FIG.",
"6, control rod 52 can move upwardly which causes cam disk 138 to rotate.",
"Alternatively, due to the configuration of slot 122, even if latch piece 48 is in the position shown in FIG.",
"7, torsion springs 110 can move the scissor units to the intermediate expanded position.",
"The illustration of a representative scissor unit 70 in the intermediate extended position is also shown in FIG.",
"14(a).",
"As is shown in FIG.",
"14(a), end 117 of a representative arm section 115 is in abutment with axle pin 111.Thus, the portion of arm section 115 between pin 108 and pin 111 is in compression which prevents further movement of the proximal ends of legs 90 and 98 to move closer together.",
"This prevents further expansion of scissor units 70 to the fully extended position.",
"However, pivoting catch member 114 to move end portions 117 out of abutment with axle pin 11, as is shown in FIG.",
"14(b) allows scissor units 70 to move to the fully extended position.",
"When this occurs, push bar 68 can move to the fully extended position as is shown in FIG.",
"15.In this condition, push bar 68 moves and projects outwardly of recess 62 so that it is projected outwardly through muntin face plane “F”.",
"This position facilitates assembly and disassembly of release mechanism 64 for assembly and maintenance purposes.",
"In order, therefore, to pivot catch member 14, an access hole 150 is provided in face 69 of press bar 68 so that an instrument, such as a small prong or screwdriver, may be inserted through access opening 150 and pressed against web 16 to force catch member 114 to undergo this pivotal motion.",
"In assembling this device, reference may be made to FIGS.",
"8 and 9.Release mechanism 64 is inserted into muntin 40 by slideably positioning base member 72 so that it is engaged by ribs 63 and held in position.",
"Next, push bar 68 is mounted on each of mounting plates 104 by means of internal ribs 152 as is shown in FIGS.",
"12(a), 13(a) and 15.End caps 154 may then be attached to press bar 68, such as by screws (not shown).",
"If desired, a fill or cover plate 156 is to provide cover any region of recess 62 that is not covered by push bar 68.A second exemplary embodiment of the present invention is shown in FIGS.",
"16-18 wherein an emergency release assembly the type described above is shown incorporated into a swinging door.",
"It should be understood that this second embodiment is described with respect to a swinging glass door however other swinging door configurations, including wooden doors, are contemplated.",
"Indeed, it should be appreciated by the ordinarily skilled person in this field that framework structures other than that described in FIGS.",
"16-18 could be employed and that the skilled artisan could modify existing structures in order to incorporate the emergency release assembly of the present invention.",
"Thus, the swing door structure described herein is for illustrative purposes only.",
"With respect to FIG.",
"16, then, it may be seen that door 210 is shown in a normally closed position in an opening 212 formed in wall 214 above floor 216.Door 210 is hinged relative to wall 214 by a pair of hinges 218 so that it may swing from the normally closed position to the normally open position.",
"Door 210 includes a primary frame 220 and a subframe 230.Primary frame 220 includes a pair of vertical pieces 222 and 224 with vertical piece 222 connected to hinges 218.A breakout piece or arm 226 is rigidly connected between the upper end portions of vertical pieces 222 and 224.In FIG.",
"16, a lock 250 is provided and secures mainframe 220 in a locked condition relative to wall 214.Subframe 230 includes vertically extending hinge stile 232, a vertically extending nose stile 234, a top stile 236 and a bottom stile 238.A muntin 240 extends at a central location between hinge stile 232 and nose stile 234 equidistantly between top stile 236 and bottom stile 238.Muntin 240 is constructed similarly to muntin 40 described above.",
"A glass panel 242 is mounted between muntin 240 and top stile 236 and extends between hinge stile 232 and nose stile 234.Similarly, a second glass panel 244 extends between muntin 240 and bottom stile 238 and between hinge stile 232 and nose stile 234.It should be understood, then, that swinging door 210 includes both frame 220 and subframe 230 as well as muntin 240 and glass panels 242 and 244.With reference to FIGS.",
"17 and 18, it may be seen that door 210 may move from a normally closed position to an emergency open condition even when door 210 is, for example, is closed and in a locked state as is shown in FIG.",
"16.However, as is shown in FIGS.",
"17 and 18, subframe 230 may pivot on hinge stile 226 to provide an emergency breakaway capability.",
"Normally, sub-frame 230 is mounted co-extensively with mainframe 220.However, in an emergency release condition, push bar 268 (FIG.",
"16) may be depressed to actuate a release mechanism that is identical in structure to release mechanism 64 described with respect to the first exemplary embodiment.",
"Activation of release mechanism 64 operates latch mechanism 44 that is identical in structure to that described above so as to release sub-frame 230 from a catch (not shown) located at the junction of hinge stile 226 and vertical piece 224 that is substantially identical to that described with respect to hinge stile 32 and catch 46, above.",
"Upon activation, latch piece 48 releases so that subframe 230 may pivot out of mainframe 220 and thus allow egress even when door 210 is otherwise locked or prevented from opening as a result of electrical failure or otherwise.",
"A third exemplary embodiment of the present invention is shown in FIGS.",
"19 and 20.Here, it should be understood that the present invention is used not as an emergency breakaway release but rather as the primary latch and release mechanism for a door.",
"Thus, as is shown in FIG.",
"19, door 310 is shown mounted in a doorframe 312 that surrounds an opening 314 in walls 316 over a floor 318.Door 310 is mounted relative to frame 312 by means of a pair of hinges 320 so that it may swing between an open and closed state.",
"Door 310 includes a hinge stile 332 a nose stile 334 a top stile 336 and a bottom stile 338.A muntin 340 extends between hinge stile 332 and nose stile 334 midway between top stile 336 and bottom stile 338 and supports a pair of glass panes 342 and 344.With reference to FIGS.",
"19 and 20, it may be seen that header 350 mounts a catch 346 that is in the form of a post with an enlarged flat head, similar to catch 46 described above.",
"Catch 346 is mounted to header 350 by means of a plate 348.When in a normally closed condition, such as shown in FIG.",
"19, door 310 includes a latch piece 48 (FIG.",
"20) that is adapted to latch door 310 in the closed position.",
"Latch piece 48 is identical to that described with respect to the first and second exemplary embodiments of the present invention and is operated by a latch mechanism, such as latch mechanism 44 that is mounted in muntin 340 identically to that described with respect to latch mechanism 44 in muntin 40.To this end, as shown in FIG.",
"20, muntin 340 includes muntin sections 356 and 358 that have front surfaces 357 and 359, respectively.",
"A pushbar 368 is mounted in the muntin recess provided by muntin sections 356 and 358 and is flanked, on either side, by cover plates 376.It should be appreciated that the front surface 369 of pushbar 368 is co-planar with front surfaces 357 and 359 of muntin 340.Since all of this is identical to the structure described with respect to muntin 40, latch mechanism 44 and release mechanism 64, the detail does not need to be described again.",
"In any event, depression of pushbar 68, in a matter similar to that of pushbar 68, above, acts to release the latch mechanism 44 so that latch piece 48 may pivot to permit disengagement from catch 346 thereby allowing door 310 to pivot into an open position, as is shown in FIG.",
"20.When door 310 is again closed, latch piece 348 engages catch 346 and pivots and becomes latched by latch mechanism 44 so that door 20 is retained in the closed position until pushbar 368 is once again depressed.",
"From this description, it may be appreciated that the release mechanism and latch assembly of the present invention may be used for breakaway doors, but also it may be used as the primary latch and release mechanism for a typical swinging door of the type shown in 310 or any other structure, including a wood panel door.",
"This mechanism may be used generally on any such swinging door structure but is especially useful for emergency exit doors or other doors where one-way passage is desired.",
"In the latter case, a pushbar located on one side of the door allows a person on that side to egress, but does not allow ingress in the opposite direction without the provision of some other latch release mechanism.",
"Accordingly, the present invention has been described with some degree of particularity directed to the exemplary embodiments of the present invention.",
"It should be appreciated, though, that the present invention is defined by the following claims construed in light of the prior art so that modifications or changes may be made to the exemplary embodiment of the present invention without departing from the inventive concepts contained herein."
]
] | Patent_10433974 |
[
[
"Method for control of a diagnosis of a catalyst in the exhaust of an internal combustion engine",
"A control for diagnosing a catalytic converter in the exhaust gas of an internal combustion engine having means for determining the catalytic converter temperature is presented.",
"The diagnostic method is carried out in dependence upon the catalytic converter temperature.",
"In the diagnosis, only such results are used which have been determined below a threshold value for the catalytic converter temperature."
],
[
"1-5.cancel 6.A method for controlling the diagnosis of a catalytic converter in the exhaust gas of an internal combustion engine having means for determining the temperature of said catalytic converter, the method comprising the steps of: carrying out the diagnosis in dependence upon said temperature of said catalytic converter; and, utilizing only such results for said diagnosis which were determined below a threshold value (TMAX) for said temperature of said catalytic converter and for which results said temperature of said catalytic converter was below said threshold value (TMAX) for a predetermined waiting time.",
"7.The method of claim 6, wherein said temperature of said catalytic converter must lie within a temperature interval (TMAX, TMIN).",
"8.The method of claim 6, wherein the length of the predetermined waiting time is dependent upon the speed with which the catalytic converter temperature drops.",
"9.The method of claim 8, wherein the predetermined waiting time for higher temperature rates of change is selected longer than for lower temperature rates of change.",
"10.An arrangement for diagnosing a catalytic converter in the exhaust gas of an internal combustion engine, the arrangement comprising: means for determining the temperature of said catalytic converter; means for comparing said temperature of said catalytic converter to a predetermined threshold value (TMAX); decision means for utilizing only such results for said diagnosis which were determined below said threshold value (TMAX) for said temperature of said catalytic converter; and, said comparing means functions to use only said results for which said temperature of said catalytic converter was below said threshold value (TMAX) for a predetermined waiting time."
],
[
"The invention relates to a method for diagnosing catalytic converters which are used to convert toxic substances in the exhaust gas of internal combustion engines.",
"Statutory requirements provide an on-board diagnosis of toxic-emission relevant vehicle components such as catalytic converters.",
"Such an on-board diagnosis known, for example, from German patent publication 196 23 335.A correction of the conversion capability, which is determined with on-board means, for considering temperature influences on the diagnostic result is known from European patent publication 0,626,507.Here, a diagnosis takes place only above a minimum temperature threshold.",
"There is further an interest to increase the quality and therewith the reliability of the diagnostic statements.",
"The subject matter of the present claims increases the reliability of the diagnostic statements in that only such diagnostic results are evaluated where the temperature of the catalytic converter was below a highest temperature threshold for the determination of these diagnostic results.",
"This technical teaching is based on the observation that a deteriorated catalytic converter can effect the same toxic substance conversion at very high temperatures of, for example, 800° C. as can a new catalytic converter at 400° C. By limiting the diagnosis to catalytic converter temperatures below a highest temperature threshold, the situation is precluded that a deteriorated catalytic converter is evaluated as being operational based only on a high diagnostic temperature.",
"An advantageous configuration results in that, during the drop of the catalytic converter temperature below the highest temperature threshold, a waiting time must elapse before a catalytic converter diagnosis takes place or before results are determined which are evaluated in a diagnosis.",
"This waiting time can be dependent upon the speed with which the catalytic converter temperature drops.",
"The waiting time for higher rates of change of the catalytic converter temperature are selected longer than for lower rates of change.",
"In this way, the especially significant advantage results that a diagnosis for a steep drop of the catalytic converter temperature from a very high level is delayed longer than in a flat temperature course.",
"The longer delay with a steep course allows the catalytic converter more time for reaching a thermal steady state condition in the diagnostic temperature range between lowest and highest temperatures.",
"For a flat temperature course, a long wait is not required and the diagnosis can begin earlier.",
"In the following, embodiments of the invention are described with reference to the figures.",
"FIG.",
"1 shows the technical background of the invention.",
"FIG.",
"2 shows a flowchart as an embodiment of the method of the invention and FIG.",
"3 shows possible courses of the catalytic converter temperature as a function of time.",
"FIG.",
"1 shows in detail an internal combustion engine 1 having an intake manifold 2, load detecting means 3, fuel metering means 4, an rpm sensor 5, an exhaust-gas system 6, a catalytic converter 7, exhaust-gas probes 8 and 9, a control apparatus 10 and means 11 for displaying a fault.",
"The control apparatus 10 receives the signals ML of the load detecting means, (n) of the rpm sensor and the signals USVK and USHK of the two exhaust-gas probes and forms therefrom, inter alia, a fuel-metering signal ti, for example, an injection pulse width for driving injection valves 4 as fuel-metering means.",
"For this purpose, a base value t1 of the drive signal ti is determined for the fuel-metering means as a function of the inducted air mass ML and the engine speed (n).",
"This base value is multiplicatively corrected in a closed control loop by a control actuating quantity FR.",
"This control actuating quantity FR is generated in a manner known per se by applying a PI control strategy to the deviation of the probe signal USVK from a desired value.",
"In the formation of the fuel-metering signal, the signal USHK can be included in the computation additionally, for example, for forming the desired value.",
"A temperature sensor 12 supplies a signal as to the catalytic converter temperature TKAT to the control apparatus.",
"Alternatively, the catalytic converter temperature TKAT can also be determined in the control apparatus 10 from other measurement quantities, especially, from the air quantity ML and the rpm (n) utilizing a computer model.",
"One such temperature modeling is, for example, disclosed in U.S. Pat.",
"No.",
"5,590,521.In this technical background, the method of the invention can be carried in the embodiment shown in FIG.",
"2.Step 2.1 in FIG.",
"2a is reached from a higher-order engine control program and serves for determining the catalytic converter temperature TKAT.",
"In step 2.2, a comparison of the catalytic converter temperature TKAT to a predetermined maximum value TMAX takes place.",
"If the TMAX-value is exceeded, then no diagnosis takes place and the main program is continued.",
"If, in contrast, the catalytic converter temperature TKAT remains below the threshold value TMAX, then the diagnosis is permitted in step 2.3 and the main program is continued after the diagnosis.",
"In this way, only such results are used for diagnosis where these results were determined with the catalytic converter temperature lying below the threshold value TMAX.",
"When the diagnosis, for example, is based on a quotient of the signals of the exhaust-gas probes forward and rearward of the catalytic converter, then this can mean that the quotient is formed only for a permitted diagnosis.",
"Alternatively, the quotient can be formed continuously but the quotient can be evaluated only for permitted diagnoses.",
"This distinction applies in the same manner for other diagnostic methods.",
"FIG.",
"2b shows a supplement of FIG.",
"2a as an additional embodiment.",
"According to this embodiment, the determination of a waiting time tv comes after step 2.2 in a step 2.2.1 and the initialization of a count variable t (t=0) is made in step 2.2.2.The waiting time tv can be a fixed value.",
"Alternatively, tv can be dependent upon operating parameters of the engine or the exhaust-gas system.",
"A dependency of the waiting time tv on the speed of the change of the catalytic converter temperature when there is a drop below TMAX is especially advantageous.",
"The dependency tv=F(d/dt(TKAT)) is to be so designed that the waiting time tv is that much longer the steeper the course of TKAT is when passing the threshold value TMAX.",
"The steps 2.13 and 2.14 form a waiting loop which is run through so often until the waiting time has elapsed.",
"In this case, the program is continued with step 2.3 and the diagnosis is thereby enabled.",
"FIG.",
"3 shows the various waiting times in dependence upon the time derivative of the temperature TKAT when there is a drop below the threshold value TMAX."
]
] | Patent_10433982 |
[
[
"Sliding litter scoop",
"The present invention provides an animal refuse scoop comprising a scoop, and a handle attached to the scoop.",
"The scoop defines a top and bottom surface, and a plurality of openings therein.",
"The openings are defined to allow the passage of litter granules therethrough.",
"A cover, having a solid body and a trigger attached to the solid body, is slidably attached to the scoop.",
"The solid body defines a plurality of openings therein.",
"The solid body is adjacent to the scoop bottom surface and is adapted to matingly cover the scoop openings in a first position to prevent the passage of granules therethrough.",
"The cover solid body is adapted to slide to a second position by pulling of the trigger, wherein the cover solid body openings align with the scoop openings to allow the passage of granules therethrough.",
"The scoop can be modified to switch the first and second positions."
],
[
"1.An animal refuse scoop comprising: (i) a scoop; (ii) a handle attached to the scoop; (iii) the scoop defining a top surface and a bottom surface, and a plurality of openings therein, the openings being defined to allow granules to pass therethrough; (iv) a cover having a solid body and a trigger attached to said solid body, the solid body defining a plurality of openings therein, the solid body being slidably attached to the scoop and adjacent to the scoop bottom surface and adapted to matingly cover the scoop openings in a first position to prevent the passage of granules through the cover openings; and (v) the cover solid body being adapted to slide to a second position by pulling of the trigger, wherein the cover solid body openings align with the scoop openings to allow the passage of granules through the scoop and cover openings.",
"2.An animal refuse scoop comprising: (i) a scoop; (ii) a handle attached to the scoop; (iii) the scoop defining a top surface and a bottom surface, and a plurality of openings therein, the openings being defined to allow granules to pass therethrough; (iv) a cover having a solid body and a trigger attached to said solid body, the solid body defining a plurality of openings therein, the solid body being slidably attached to the scoop and adjacent to the scoop bottom surface and adapted to align with the scoop openings to allow the passage of granules through the scoop and cover openings in a first position; and (v) the cover solid body being adapted to slide to a second position by pulling of the trigger wherein the cover solid body matingly covers the scoop openings to prevent the passage of granules through the cover openings.",
"3.The animal refuse scoop as defined in claim 1 further comprising a biasing means attached to the scoop, the biasing means engaging the cover to bias said cover into the first position.",
"4.The animal refuse scoop as defined in claim 2 further comprising a biasing means attached to the scoop, the biasing means engaging the cover to bias said cover into the first position.",
"5.The animal refuse scoop as defined in claim 1 wherein the biasing means is a wound coil.",
"6.The animal refuse scoop as defined in claim 2 wherein the biasing means is a wound coil.",
"7.The litter scoop as defined in claim 1 further comprising a plurality of fingers attached to the cover solid body.",
"8.The litter scoop as defined in claim 2 further comprising a plurality of fingers attached to the cover solid body."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Domesticated animals such as cats are often trained to use litter boxes for defecation and urination.",
"These litter boxes are typically filled with some type of disposable litter, which is usually comprised of some sort of fine absorbent granules.",
"A particularly useful type of disposable litter is a clumping litter, such as the type disclosed in Canadian patent nos.",
"2,323,103; 2,233,406 and 2,225,328.Clumping litter allows a user to clean the litter box by removing solidified clumps of litter, that have been coagulated together by an animal's urine, or attached to wet/fresh faeces, to form a clumped mass.",
"To remove such clumped masses of litter, the user should, for hygienic reasons, preferably employ a scoop.",
"Numerous animal refuse scoops are disclosed in the prior art.",
"Recently, animal refuse scoops have been adapted to work particularly well with clumping litter in that these scoops provide openings to allow the non-clumped granules to fall through the scoop, and back in to the litter box.",
"For example, the scoops disclosed in U.S. Pat.",
"No.",
"6,312,029 and U.S. Des.",
"patent no.",
"D332,675 are particularly well suited for use with clumping litter.",
"These scoops operate as sifting scoops, which allow the user to preserve and conserve unused litter by keeping most of the unused litter in the litter box.",
"A problem with these sifting scoops results from the fact that very often, the user will have to travel some distance from the litter box to a place of disposal, such as a garbage can, to dispose of the clumped masses.",
"During transport, litter granules that are held loosely to the clumped mass will typically fall away from the clumped mass, travel through the openings in the sifting scoop, and wind up on the user's floor or carpet.",
"Cleaning these loose granules is a nuisance, and the granules having been in contact with urine and faeces, are unhygienic."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides an animal refuse scoop comprising a scoop, and a handle attached to the scoop.",
"The scoop defines a top and bottom surface, and a plurality of openings therein.",
"The openings are defined to allow the passage of litter granules therethrough.",
"A cover, having a solid body and a trigger attached to the solid body, is slidably attached to the scoop.",
"The solid body defines a plurality of openings therein.",
"The solid body is adjacent to the scoop bottom surface and is adapted to matingly cover the scoop openings in a first position to prevent the passage of granules therethrough.",
"The cover solid body is adapted to slide to a second position by pulling of the trigger, wherein the cover solid body openings align with the scoop openings to allow the passage of granules therethrough."
],
[
"FIELD OF INVENTION The present invention relates to animal refuse scoops, and more particularly to scoops for use with clumping animal litter.",
"BACKGROUND OF THE INVENTION Domesticated animals such as cats are often trained to use litter boxes for defecation and urination.",
"These litter boxes are typically filled with some type of disposable litter, which is usually comprised of some sort of fine absorbent granules.",
"A particularly useful type of disposable litter is a clumping litter, such as the type disclosed in Canadian patent nos.",
"2,323,103; 2,233,406 and 2,225,328.Clumping litter allows a user to clean the litter box by removing solidified clumps of litter, that have been coagulated together by an animal's urine, or attached to wet/fresh faeces, to form a clumped mass.",
"To remove such clumped masses of litter, the user should, for hygienic reasons, preferably employ a scoop.",
"Numerous animal refuse scoops are disclosed in the prior art.",
"Recently, animal refuse scoops have been adapted to work particularly well with clumping litter in that these scoops provide openings to allow the non-clumped granules to fall through the scoop, and back in to the litter box.",
"For example, the scoops disclosed in U.S. Pat.",
"No.",
"6,312,029 and U.S. Des.",
"patent no.",
"D332,675 are particularly well suited for use with clumping litter.",
"These scoops operate as sifting scoops, which allow the user to preserve and conserve unused litter by keeping most of the unused litter in the litter box.",
"A problem with these sifting scoops results from the fact that very often, the user will have to travel some distance from the litter box to a place of disposal, such as a garbage can, to dispose of the clumped masses.",
"During transport, litter granules that are held loosely to the clumped mass will typically fall away from the clumped mass, travel through the openings in the sifting scoop, and wind up on the user's floor or carpet.",
"Cleaning these loose granules is a nuisance, and the granules having been in contact with urine and faeces, are unhygienic.",
"SUMMARY OF THE INVENTION The present invention provides an animal refuse scoop comprising a scoop, and a handle attached to the scoop.",
"The scoop defines a top and bottom surface, and a plurality of openings therein.",
"The openings are defined to allow the passage of litter granules therethrough.",
"A cover, having a solid body and a trigger attached to the solid body, is slidably attached to the scoop.",
"The solid body defines a plurality of openings therein.",
"The solid body is adjacent to the scoop bottom surface and is adapted to matingly cover the scoop openings in a first position to prevent the passage of granules therethrough.",
"The cover solid body is adapted to slide to a second position by pulling of the trigger, wherein the cover solid body openings align with the scoop openings to allow the passage of granules therethrough.",
"DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a perspective view of a preferred embodiment of the present invention in a first position.",
"FIG.",
"1A is an exploded view of FIG.",
"1.FIG.",
"2 is a side view of FIG.",
"1.FIG.",
"3 is a cross-sectional view taken along the line 3-3 in FIG.",
"2.FIG.",
"3A is a cross-sectional view taken along the line 3A-3A in FIG.",
"2.FIG.",
"4 is a side view of the preferred embodiment of the present invention in a second position.",
"FIG.",
"5 is cross-sectional view taken along the line 5-5 in FIG.",
"1 in the first position.",
"FIG.",
"6 is a cross-sectional view similar to FIG.",
"5 in the second position.",
"FIG.",
"7 is a cross-sectional view similar to FIG.",
"6 showing an alternate second position.",
"FIG.",
"8 is a cross-sectional view similar to FIG.",
"5 showing an alternate first position.",
"DETAILED DESCRIPTION OF THE DRAWINGS In accordance with the present invention, FIG.",
"1 shows a cat litter scoop (10) having a scoop portion (14) and a handle (18) attached thereto.",
"The handle (18) is preferably hollow (see FIG.",
"3A).",
"The scoop (14) defines a top (50) and bottom (54) surface, and a plurality of openings (22) therein (the openings (22) are best seen in FIG.",
"1A).",
"The openings (22) are defined to allow the passage of litter granules (68) therethrough.",
"The openings (22) can be rectangular, circular, or any other shape or orientation so long as they permit the passage of litter granules (68) therethrough.",
"Similarly, the scoop (14) can be any shape so long as it is functional for dragging through a cat litter box (not shown).",
"A cover having a solid body (42) and a trigger (30) is slidably attached to the scoop (10), and is positioned adjacent to the scoop (14) bottom surface (54).",
"The cover body (42) defines a plurality of openings (26) to allow the passage of litter granules (68) therethrough.",
"The cover body (42) is preferably slidably attached to the scoop (14) by way of a corresponding groove (34) and tab (38) mating arrangement (best seen in FIG.",
"3).",
"The slidable attachment can also be modified to allow the cover (42) to be releasable or removable from the scoop (14) (to facilitate cleaning of the scoop (10)).",
"The groove (34) can be provided on the scoop (14) and the tab (38) can be provided on the cover body (42).",
"This arrangement can be reversed so that the groove (34) is provided on the cover body (42) and the tab (38) is provided on the scoop (14).",
"The cover body (42) can optionally define projecting fingers (58) (best seen in FIG.",
"2) for raking litter in a litter box (not shown), to give the litter a smooth surface.",
"When in its first position (FIG.",
"8) (i.e.",
"the “closed position”), the cover (42) is adjacent to the scoop (14) bottom surface (54) and adapted to matingly cover the scoop (14) openings (22).",
"In the closed position (FIGS.",
"6 and 8), free litter granules (68) cannot pass through the cover openings (26).",
"The cover (42) preferably corresponds in shape to the scoop (14) so that the cover (42) can matingly engage the scoop (14).",
"For design purposes, the cover (42) can be of a different shape and configuration from the scoop (14), so long as the cover (42) matingly covers the scoop (14) openings (22) when in the closed position, and permits litter granules (68) to fall through the openings (22) when in the open position.",
"The trigger (30) is attached to the cover (42) to permit a user (not shown) to, by squeezing the trigger (30), move the cover (42) between the first position and a second position (see FIG.",
"7).",
"In the second position the openings (26) of the cover body (42) align with the openings (22) of the scoop (14), so that litter granules (68) can freely pass therethrough.",
"The scoop (10) can optionally be fitted with a biasing means (46) which is preferably attached to the scoop (14) and engages the cover body (42).",
"The biasing means can bias the cover (42) into either the first or the second position, depending upon a user's preference.",
"In operation, the scoop (14) and cover (42) are inserted into a cat litter box (not shown) filled with clumping cat litter (not shown).",
"The scoop (14) is dragged across and through the cat litter.",
"While dragging the scoop (14) through the litter, the user can squeeze the trigger (30) to move the cover (42) from the closed position to the open position.",
"If the cover (42) is maintained in the closed position during dragging, the amount of cat litter that can pass through the scoop (14) is more limited than if the cover (42) is in the open position.",
"When the scoop (14) is lifted out of the litter box (not shown), the user squeezes the trigger (30), resulting in the cover (42) moving to the open position.",
"When the cover (42) is in the open position, free litter granules (68) can freely pass through both the scoop openings (22) as well as the cover openings (26).",
"Clumped masses of litter (64) are retained within the scoop (14) while the non-clumped litter granules (68) freely pass through the openings (22, 26) and back into the litter box.",
"Once the free litter granules (68) have passed through the scoop openings (22, 26), the user can release the trigger (30) allowing the cover to return to the closed position (FIG.",
"8).",
"The clumped masses (64) can then be transported to a site of garbage disposal without allowing the passage of free litter granules (68) through the scoop openings (22, 26).",
"The arrangement of the scoop (14) and cover (42) can be varied so that in the first position, the cover openings (26) are aligned with the scoop openings (22) (see FIG.",
"5).",
"In this arrangement, squeezing of the trigger (30) would result in the cover (42) moving to the second position (see FIG.",
"6) resulting in the openings (22, 26) no longer being aligned.",
"In the second position the cover (42) would matingly cover the scoop openings (22) so that litter granules (68) could not pass freely therethrough."
]
] | Patent_10438966 |
[
[
"Universal clutch puller and installer",
"The materials needed for making the universal clutch puller and installer (UCPI) consist of flat steel stock and metal pins.",
"After outlining, cutting, drilling, and bending you will end up with a device that makes the installation and removal of clutches easier on motorcycles and small engines with the three shoe clutch system.",
"It cut my install/removal time from 1 ½ hours to approximately fifteen minutes.",
"As you can see in the drawings this device is 14 ½″ inches long, 1 ⅜″ inches wide and ¼″ inch high."
],
[
"1.I claim the universal clutch puller and installer makes the removal and installation of the clutch easier and faster."
],
[
"<SOH> BACKGROUND <EOH>After trying different tools and many frustrating hours installing and removing clutches on motorcycles, I came up with the idea for the universal clutch puller and installer.",
"One tool that would work for both removing and installing clutches on motorcycles and even on some small engines."
],
[
"<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>I designed the universal clutch puller and installer to make the removal and installation of the clutch easier and faster.",
"This tool is designed to work on a three shoe clutch system that is generally found on motorcycles and small engines of various makes and models.",
"I have outlined how to use and the making of this tool in the Detailed Description Of The Invention document."
],
[
"DETAILED DESCRIPTION OF THE INVENTION Making of the Universal Clutch Puller and Installer (UCPI): To make the universal clutch puller and installer (UCPI) you will need flat steel stock (¼″×4″×14½″).",
"Place clutch on one end of flat stock and outline the outside of the clutch.",
"Do not forget to outline in between the clutch shoes.",
"This is where your three (3) pins will be located on your clutch tool (refer to left side of drawing 1 of 3).",
"On the other end of the flat stock measure 1 5/16″ from the top and 1 5/16″ from the bottom.",
"You should have 1 ⅜″ in the middle.",
"Draw your middle lines all the way down your flat stock until you run into your outline of the clutch.",
"From the end of your flat stock, cut one of your middle lines heading towards your outline of the clutch.",
"Continue to cut around the outside of your clutch outline until you get to the other middle line.",
"Then continue cutting the middle line to the end of the flat stock.",
"Next measure across your clutch outlines and find the center.",
"Drill the center to 1″ in diameter all the way through.",
"Then measure from the center out to your pins.",
"Your pins will go in between the clutch shoes and should measure 2 1/18″ from each other (refer to drawing 2 of 3).",
"Drill your three (3) pin holes 5/16″ all the way through.",
"Your metal dowel pins should be ⅞″ in length and 0.358 inches in diameter.",
"Then proceed to press your pins through so that one side is flush with that of the flat stock (refer to left side of drawing 3 of 3).",
"On the other end, measure 11/16″ from the top or bottom and make a mark and then measure from the end of the flat stock over ½″.",
"From that mark drill through the flat stock with a ¼″ end mill bit.",
"Using that bit, mill the flat stock ¾″ towards the clutch end.",
"At the end of that hole, measure a ¼″ and drill a hole using a 27/64″ drill bit.",
"Then tap that hole with a ½″ 13 n.c. (national coarse) tap.",
"From that center hole measure across a ¼″ and drill with a ¼″ end mill bit through the flat stock and milling that hole ¾″ across towards the clutch end of the flat stock (refer to right side of drawing 1 of 3).",
"Place the tool in the middle of a vise, clamping on the outside edges.",
"Measure from each end 3 ¼″ towards the middle.",
"On the end that is 1 ⅜″ across, heat metal with a torch and bend flat stock down 45 degrees.",
"On the other end heat with a torch and bend the opposite direction towards the sky (refer to drawing 3 of 3).",
"Now your tool is complete.",
"Using the Universal Clutch Puller and Installer (UCPI): First you will have to drain the oil from the motor.",
"Then you will remove the side cover to expose the clutch.",
"Insert the clutch puller into the clutch by inserting the three (3) pins on the clutch puller in between the clutch shoes.",
"You will then brace puller on top of foot peg if working on a motorcycle.",
"Insert the proper size socket onto center nut through the 1″ hole in the center of the puller.",
"Loosen nut counter clockwise until nut is completely off.",
"Turn puller around and insert the two proper size bolts through slotted holes in the clutch puller and into the clutch.",
"Hold the clutch puller firmly with one hand and tighten the center bolt on the clutch puller against the center stud in which the clutch sits on.",
"Tighten the bolt until the clutch pops off.",
"Complete any necessary repairs to the clutch and then reinstall the clutch.",
"To reinstall the clutch, insert the clutch onto the center stud.",
"Then install nut onto stud by turning it clockwise until snug.",
"Insert the three (3) studs of the clutch installer in between the clutch shoes.",
"Then insert socket through the 1″ hole in the center of the clutch installer.",
"Firmly hold the clutch installer with one hand and tighten the center nut to the recommended torque from the manufacturer.",
"Remove the socket and clutch installer from engine.",
"Finally reinstall the side cover and do not forget to add the correct amount of oil to the engine.",
"SPECIFICATION I designed the universal clutch puller and installer to make the removal and installation of the clutch easier and faster.",
"My tool is designed to work on a three shoe clutch system that is generally found on motorcycles and small engines of various makes and models.",
"BACKGROUND After trying different tools and many frustrating hours installing and removing clutches on motorcycles, I came up with the idea for the universal clutch puller and installer.",
"One tool that would work for both removing and installing clutches on motorcycles and even on some small engines.",
"BRIEF SUMMARY OF THE INVENTION I designed the universal clutch puller and installer to make the removal and installation of the clutch easier and faster.",
"This tool is designed to work on a three shoe clutch system that is generally found on motorcycles and small engines of various makes and models.",
"I have outlined how to use and the making of this tool in the Detailed Description Of The Invention document.",
"BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Picture 1 of 3: This drawing is a view looking down at the device.",
"This drawing also shows the complete layout of where all the holes should be cut and drilled on the device.",
"Picture 2 of 3: This drawing is a view of one end that shows the measurements of each hole.",
"Picture 3 of 3: This drawing is a drawing looking at the product from the side.",
"It shows the angle degree of metal and shows the height of the three (3) pins."
]
] | Patent_10442901 |
[
[
"Information processing apparatus and program",
"The present invention is associated with an information processing apparatus and a program adapted to provide content to a terminal via a server without otherwise notifying the terminal thereof.",
"A personal computer 1 transmits an image taken by a CCD video camera 33 for example to a server 6 along with the address of a terminal 6, which allows the personal computer 1 to request the server 5 for providing the transmitted image to the terminal 6.",
"In response to the request from the personal computer 1, the server 5 provides the image supplied from the personal computer 1 to the terminal 6."
],
[
"1.An information processing apparatus connected to a network with a server and a terminal to provide content to said terminal via said server, comprising: first input means for inputting said content; second input means for inputting an address of said terminal; and requesting means for transmitting said address of said terminal along with said content to said server to request said server for providing said content to said terminal.",
"2.The information processing apparatus according to claim 2, further comprising: third input means for inputting login information necessary for logging in said server; and wherein said second input means inputs said address of said terminal after said server has been authenticated by inputting said login information.",
"3.A program for an information processing apparatus connected to a network with a server and a terminal to provide content to said terminal via said server, said program causing a computer to execute: a first input controlling step for inputting said content; a second input controlling step for inputting an address of said terminal; and a request controlling step for transmitting said address of said terminal along with said content to said server to request said server for providing said content to said terminal."
],
[
"<SOH> BACKGROUND ART <EOH>For example, a content providing system has been developed in which content is supplied from a user terminal to a server via a network to be stored in the server for another user terminal to get the stored content.",
"However, such a content providing system presents a problem that another terminal to which the content is eventually supplied must be notified of getting the content from the server for example, thereby complicating a user procedure."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.",
"1 illustrates a usage example of a personal computer 1 to which the present invention is applied.",
"FIG.",
"2 illustrates an exemplary configuration of the external view of the personal computer of FIG.",
"1 .",
"FIG.",
"3 illustrates another exemplary configuration of the external view of the personal computer of FIG.",
"1 .",
"FIG.",
"4 illustrates still another exemplary configuration of the external view of the personal computer of FIG.",
"1 .",
"FIG.",
"5 illustrates yet another exemplary configuration of the external view of the personal computer of FIG.",
"1 .",
"FIG.",
"6 illustrates an exemplary configuration of the external view of a port replicator of FIG.",
"1 .",
"FIG.",
"7 is a block diagram illustrating an exemplary internal configuration of the personal computer of FIG.",
"1 .",
"FIG.",
"8 illustrates an example of capture mode screen.",
"FIG.",
"9 illustrates a positional relationship between jog dial and file list.",
"FIG.",
"10 illustrates an example of send mode screen.",
"FIG.",
"11 illustrates a method of operating a capture button and a send button.",
"FIG.",
"12 is a flowchart describing the operation of the CPU of a personal computer for displaying a manipulated presentation image.",
"FIG.",
"13 illustrates the processing of displaying a manipulated presentation image.",
"FIG.",
"14 is a flowchart describing the operation of a video controller of a personal computer for displaying a manipulated presentation image.",
"FIG.",
"15 illustrates the contents of the recording in a frame memory of a video decoder of a personal computer.",
"FIG.",
"16 illustrates a method of converting an image of 720×480 to an image of 640×480.FIG.",
"17 illustrates a pixel configuration.",
"FIG.",
"18 illustrates another example of capture mode screen.",
"FIG.",
"19 is a flowchart describing the operation of an I/O controller of a personal computer for displaying an image corresponding to an AV signal superimposed with a viewing disable signal.",
"FIG.",
"20 is a flowchart describing the operation of a video decoder of a personal computer for displaying an image corresponding to an AV signal superimposed with a viewing disable signal.",
"FIG.",
"21 is a flowchart describing the operation of a personal computer when a send image is transmitted to a server 5 .",
"FIG.",
"22 is a flowchart describing the operation of a server when a send image is transmitted to a terminal 6 .",
"FIG.",
"23 is a flowchart describing the operation of a terminal when it receives a send image from a server.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates generally to an information processing apparatus and a program and, more particularly, to an information processing apparatus and a program which are adapted to transmit addresses along with content to a server and request this server for the provision of content to terminals having these addresses.",
"BACKGROUND ART For example, a content providing system has been developed in which content is supplied from a user terminal to a server via a network to be stored in the server for another user terminal to get the stored content.",
"However, such a content providing system presents a problem that another terminal to which the content is eventually supplied must be notified of getting the content from the server for example, thereby complicating a user procedure.",
"DISCLOSURE OF INVENTION It is therefore an object of the present invention to transmit content with the addresses of terminals to a server from which the content is to be supplied to these terminals by requesting the server thereof.",
"An information processing apparatus according to the present invention is characterized in that it includes: first input means for inputting the content; second input means for inputting an address of the terminal; and requesting means for transmitting the address of the terminal along with the content to the server to request the server for providing the content to the terminal.",
"The information processing apparatus according to the present invention may further include third input means for inputting login information necessary for logging in the server, and the second input means may input the address of the terminal after the server has been authenticated by inputting the login information.",
"A program according to the present invention is characterized in that it causes a computer to execute: a first input controlling step for inputting the content; a second input controlling step for inputting an address of the terminal; and a request controlling step for transmitting the address of the terminal along with the content to the server to request the server for providing the content to the terminal.",
"In the information processing apparatus and the program according to the invention, content is inputted, the address of a terminal is inputted, and the content and the address are transmitted to a server together.",
"Consequently, the user can request the server for providing content to the terminal.",
"It should be noted that the present invention may also be practiced by the provision of an image processing method for the image processing apparatus according to the invention and a recording medium recording the program of the present invention.",
"BRIEF DESCRIPTION OF DRAWINGS FIG.",
"1 illustrates a usage example of a personal computer 1 to which the present invention is applied.",
"FIG.",
"2 illustrates an exemplary configuration of the external view of the personal computer of FIG.",
"1.FIG.",
"3 illustrates another exemplary configuration of the external view of the personal computer of FIG.",
"1.FIG.",
"4 illustrates still another exemplary configuration of the external view of the personal computer of FIG.",
"1.FIG.",
"5 illustrates yet another exemplary configuration of the external view of the personal computer of FIG.",
"1.FIG.",
"6 illustrates an exemplary configuration of the external view of a port replicator of FIG.",
"1.FIG.",
"7 is a block diagram illustrating an exemplary internal configuration of the personal computer of FIG.",
"1.FIG.",
"8 illustrates an example of capture mode screen.",
"FIG.",
"9 illustrates a positional relationship between jog dial and file list.",
"FIG.",
"10 illustrates an example of send mode screen.",
"FIG.",
"11 illustrates a method of operating a capture button and a send button.",
"FIG.",
"12 is a flowchart describing the operation of the CPU of a personal computer for displaying a manipulated presentation image.",
"FIG.",
"13 illustrates the processing of displaying a manipulated presentation image.",
"FIG.",
"14 is a flowchart describing the operation of a video controller of a personal computer for displaying a manipulated presentation image.",
"FIG.",
"15 illustrates the contents of the recording in a frame memory of a video decoder of a personal computer.",
"FIG.",
"16 illustrates a method of converting an image of 720×480 to an image of 640×480.FIG.",
"17 illustrates a pixel configuration.",
"FIG.",
"18 illustrates another example of capture mode screen.",
"FIG.",
"19 is a flowchart describing the operation of an I/O controller of a personal computer for displaying an image corresponding to an AV signal superimposed with a viewing disable signal.",
"FIG.",
"20 is a flowchart describing the operation of a video decoder of a personal computer for displaying an image corresponding to an AV signal superimposed with a viewing disable signal.",
"FIG.",
"21 is a flowchart describing the operation of a personal computer when a send image is transmitted to a server 5.FIG.",
"22 is a flowchart describing the operation of a server when a send image is transmitted to a terminal 6.FIG.",
"23 is a flowchart describing the operation of a terminal when it receives a send image from a server.",
"BEST MODE FOR CARRYING OUT THE INVENTION Now, referring to FIG.",
"1, there is shown a usage example of a personal computer 1 to which the present invention is applied.",
"A personal computer 1 may execute the capture mode processing in a predetermined image processing program to display, on a capture mode screen, an image (still image or moving image) taken by an incorporated CCD (Charge Coupled Device) video camera 33 or an image inputted from a video recorder 3 via a port replicator 2.It should be noted that, in what follows, an image taken by the CCD video camera 33 and an image inputted from the video recorder 3 for example, which are displayed on the capture mode screen, will be appropriately referred to as presentation images.",
"The personal computer 1 may execute the send mode processing in an image processing program to connect to a network 4 via the port replicator 2, thereby transmitting the images supplied from the CCD video camera 33 and the video recorder 3 to the server 5.At this moment, the user of the personal computer 1 displays images on the send mode screen to select the image to be transmitted to the server 5.It should be noted that, in what follows, each image to be transmitted to the server 5 will be appropriately referred to as a send image.",
"The server 5 manages the images transmitted from the personal computer 1 for example and provides (or transfers) any of these images to the terminal 6 for example which requests them.",
"The following outlines the functionality of the personal computer 1.The personal computer 1 displays a screen for the capture mode (hereafter referred to as a capture mode screen) (FIG.",
"8) during the execution of capture mode processing, which will be detailed later, and displays a screen for the sending mode (hereafter referred to as a send mode screen) (FIG.",
"10) during the execution of the send mode processing.",
"On the capture mode screen, an image taken by the CCD video camera 33 or an image inputted from the video recorder 3 is displayed (in a finder 206 shown in FIG.",
"8).",
"On the other hand, a send image is displayed on the send mode screen (in a view area 222 shown in FIG.",
"10) and a presentation image (displayed in the finder 206 shown in FIG.",
"8) is displayed on the send mode screen as manipulated to the extent that its contents are recognizable (in the finder 206 shown in FIG.",
"10) (in the case of FIG.",
"10, the presentation image is displayed in a transparent manner).",
"It should be noted that, in what follows, a presentation image before manipulation (the presentation image displayed on the capture mode screen) is appropriately referred to as a normal presentation image and a presentation image manipulated to the extent that its contents are recognizable (the presentation image displayed on the send mode screen) is appropriately referred to as a manipulated presentation image.",
"Namely, if the send mode processing is mainly performed (a send image is selected and transmitted), because of the difference between the display forms of presentation image and manipulated presentation image, the corresponding send image may easily be distinguished from the image corresponding to the capture mode processing not mainly performed (for example, the image being taken by the CCD video camera 33).",
"At the same time, the image being taken by the CCD video camera 33 may be grasped.",
"As described above, the personal computer 1 may display an image taken by the CCD video camera 33 in the capture mode as well as an image inputted from the video recorder 3 via the port replicator 2.Normally, the frame size of a video signal (digital) inputted form the CCD video camera 33 is 640 pixels×480 lines, but the frame size of a video signal (analog) inputted from the video recorder 3 is 720 pixels×480 lines, presenting a difference therebetween.",
"Therefore, the personal computer 1 eventually forms a frame of 640×480 from the video signal supplied from the video recorder 3 to display the image corresponding to the video signal from the video recorder 3 by the same size as that of the image corresponding to the video signal supplied from the CCD video camera 33.Also, as described above, the personal computer 1 may input the video signal from the video recorder 3 to display the image corresponding to this signal; if a signal for disabling viewing for the purpose of copyright protection (hereafter referred to as a viewing disable signal) is superimposed on this video signal, the personal computer 1 may display the image corresponding to the viewing disable signal by attaching a predetermined effect which disables viewing.",
"In addition, the personal computer 1 may send, along with a send image, the address of the terminal 6 for example to the server 5, thereby causing the server 5 to execute the processing of providing the send image to the terminal 6.The following describes the configurations of external views of the personal computer 1 and the port replicator 2 with reference to FIGS.",
"2 through 6.The personal computer 1 (FIG.",
"2) is basically configured by a main body 11 and a display section 12 attached to the main body 11 in a pivotal manner.",
"The main body 11 has, on the outside thereof, a plurality of operator keys 21 for entering various letters, symbols, and numerals, a stick-type pointing device (hereafter referred to simply as a stick) 22 for moving the mouse cursor, a left click button 23A and a right click button 23B equivalent to the left button and the right button of an ordinary mouse, a center button 23C for operating the scroll bar without pointing the mouse cursor to the scroll button, an incorporated speaker 24, and a shutter button 25 for the CCD video camera 33 arranged on the display section 12.The main body 11 also has a jog dial 26 on the upper right corner thereof as shown in the top view (FIG.",
"3) of the main body 11.The jog dial 26 is rotated in the arrow direction (up and down direction) as shown in FIG.",
"3 and pressed in the vertical direction.",
"It should be noted that the position of the jog dial 26 and the rotational direction in its rotational operation correspond to the position and operational direction of a file list 209 (to be described later) which is displayed in each of the capture mode screen and the send mode screen displayed on an LCD 31.The display section 12 has, on the front thereof, the LCD (Liquid Crystal Display) 31 and, on the front upper portion thereof, an image capture section 32 having the CCD video camera 33 in a pivotal manner relative to the display section 12.Namely, the image capture section 32 pivotally moves within an angular range of 180 degrees between the front direction and rear direction of the display section 12, thereby being positioned at any place within that range.",
"On the right side of the main body 11, a headphone connector 41, a microphone connector 42, an I/O connector 43, a USB (Universal Serial Bus) connector 44, a DC IN connector 45 which is connected to a power receptacle through an AC adaptor, and a modular jack 46 which is connected to the telephone line are arranged as shown in FIG.",
"4.In this example, the port replicator 2 is connected to the I/O connector 43.On the left side of the main body 11, an IEEE (Institute of Electrical and Electronics Engineers) 1394 connector 51 and a DC OUT connector 52 are arranged in a predetermined positional relation as shown in FIG.",
"5.Namely, the personal computer 1 may be connected to electronic equipment (not shown) having the IEEE 1394 connector and the DC IN connector arranged in the same positional relation as above with a predetermined cable having connecting portions corresponding to these connectors to perform communication based on the IEEE 1394 standard and supply DC power to the connected electronic equipment.",
"On the left side of the main body 11, a PC card slot 53 for PC (Personal Computer) cards having the PCMCIA (Personal Computer Memory Card International Association) standard and an air vent 54 are also arranged.",
"FIG.",
"6 shows a connector arrangement of the port replicator 2.The port replicator 2 has an AV IN 61 which is connected to the video recorder 3 for entering AV signals (audio signal and video signal) (analog) therefrom and an AV OUT connector 62 which is connected to a television receiver, a video deck, an active speaker, or an amplifier (none shown) for outputting AV signals (analog) therefrom.",
"The port replicator 2 also has a USB connector 63, a network connector 64 to be connected to a network compliant with 10BASE-T or 100BASE-T (both trademark), and a monitor connector 65 to be connected to an external display device.",
"In this example, the network connector 64 is connected to the network 4.FIG.",
"7 shows an exemplary internal configuration of the personal computer 1.In the main body 11 of the personal computer 1, a CPU (Central Processing Unit) 82 for totally controlling each function in the main body 11 is connected to a RAM (Random Access Memory) 92 via a memory bus 83 to execute processing corresponding to various programs (for example, image processing programs) loaded in the RAM 92 on the basis of the system clock provided by a clock generator 91 at a predetermined operating speed.",
"A cache memory 81 caches data for use by the CPU 82, thereby realizing high-speed access operations.",
"The CPU 82 is connected to a PCI (Peripheral Component Interconnect) bus 87.The PCI bus 87 is connected to a video decoder 84, a video controller 85, an IEEE 1394 interface 86, a PCI-ISA bridge 90, a video capture processing chip 93, and a PC card interface 94, in addition to the CPU 82.The video capture processing chip 93 is connected to an I2C bus 95 (generally referred to an SM (System Management) bus).",
"When the image data captured by the CCD video camera 33 are supplied via the I2C bus, the video capture processing chip 93 temporarily stores the received image data into an incorporated frame memory (not shown) and performs image compression processing on the image data on the basis of the JPEG (Joint Photographic Experts Group) standard or the MPEG (Moving Picture Experts Group) 1 or 2 standard to generate image data and then stores the generated image data into the frame memory (640 pixels×480 lines) again.",
"In response to a request from the CPU 82, the video capture processing chip 93 directly transfers the image data from the frame memory to the RAM 92 by use of the bus master function and then transfers the image data to a hard disk drive (HDD) 88 as still-image data or moving-image data.",
"A video signal (an analog signal) from the video recorder 3 for example which is inputted from the port replicator 2 connected to the I/O connector 43 is inputted in the video decoder 84 via an I/O controller 103 and the I2C bus 95.The video decoder 84 decodes the received video signal and temporarily stores the resultant video signal into a frame memory (not shown) (720 pixels×480 lines).",
"In response to a request from the CPU 82, the video decoder 84 directly transfers the image data from the frame memory to the RAM 92 by use of the bus master function and then transfers the image data to the HDD 88 as still-image data or moving-image data.",
"The video controller 85 performs predetermined graphics processing on the image data obtained as a result of the capturing by the CCD video camera 33, the image data from the video capture processing chip 93, or the image data from the video decoder 84, stores these data into an internal VRAM (Video Random Access Memory), reads the stored image data appropriately, and displays the read image data on the LCD 31.In addition, the video controller 85 is adapted to output the image data based on the processing (in the capture mode or the send mode) by the CPU 82 supplied appropriately to the LCD 31 to display a plurality of window screens.",
"The PC card interface 94 is adapted so that a PC card is appropriately loaded in a PC card slot 53 when adding optional capabilities and the PC card interface 94 is connected to external devices such as a CD-ROM drive and DVD drive via the loaded PC card.",
"The IEEE 1394 interface 86 is directly connected to the IEEE 1394 connector 51 to be connected to external devices such as another computer device and a digital video camera via the IEEE 1394 connector 51.The PCI bus 87 is connected to an ISA (Industrial Standard Architecture) bus 96 via a CPI-ISA bridge 90 to which the HDD 88, the Bluetooth 89, and the USB connector 44 are connected.",
"The PCI-ISA bridge 90 is configured by an IDE (Integrated Drive Electronics) interface, a configuration register, an RTC (Real-Time Clock) circuit and a USB interface to control the HDD 88 via the IDE interface on the basis of the system clock supplied from the clock generator 91.The hard disk of the HDD 88 stores an OS (Operating System) such as Windows (R) XP, an electronic mail program, an auto pilot program, a jog dial utility program, a jog dial driver, a capture software, an image processing program, and other various application software programs, which are appropriately transferred to the RAM 92 in the course of startup processing.",
"The PCI-ISA bridge 90 controls, via the USB interface, external devices such as a floppy (trademark) disk drive, a printer, and a USB mouse for example connected via the USB connector 44 and, at the same time, controls a modem 97 and a sound controller 98 connected to the ISA bus 96.The modem 97 is connected to an Internet service provider (hereafter referred to as a provider) via a public telephone line and the Internet (not shown) through the modular jack 46 to make access with the provider.",
"The sound controller 98 captures a voice signal from the microphone via the microphone connector 42 and supplies a voice signal to the incorporated speaker 24 and the headphone connector 41.The I/O (In/Out) controller 103 is connected to the ISA bus 96.The I/O controller 103 receives power from the DC IN connector 45 via a power supply/charge controller 100 to supply power to each circuit when a power switch 106 is turned on.",
"It should be noted that the I/O controller 103 operates on the system clock supplied from the clock generator 91 also.",
"The power supply/charge controller 100 is controlled by the I/O controller 103 to control the charging of a battery pack 99 connected to the battery connector and, at the same time, supplies power to external electronic equipment via the DC OUT connector 52.The I/O controller 103 is configured by a microcontroller, an I/O interface, a CPU, a ROM, a RAM, and so on and controls the data input/output operation between the OS and application software, and the various peripheral devices such as the LCD 31 and the HDD 88 on the basis of the BIOS (Basic Input/Output System) stored in a flash memory 102.In addition, the I/O controller 103 is connected to an infrared port 104 to execute infrared communication with another computer device for example.",
"Further, the I/O controller 103 is connected to a reverse switch 105, which is turned on when the image capture section 32 of the CCD video camera 33 is rotated 180 degrees in the rear direction of the LCD 31, notifying the CPU 82 thereof through the PCI-ISA bridge 90.Moreover, the I/O controller 103 is connected to a full-push/half-push switch 108, which is turned on when the shutter button 25 arranged on top of the main body 11 is half-pushed, notifying the CPU 82 thereof, and turned on when the shutter button 25 is fully pushed, notifying the CPU 82 thereof.",
"Namely, when the shutter button 25 is half-pushed by the user with the capture software loaded in the RAM 92 from the hard disk of the HDD 88, the CPU 82 enters the still-image mode and controls the CCD video camera 33 to execute the freezing of the still image; when the shutter button is fully pushed, the CPU 82 captures the frozen still image to send it to the video controller 85.On the contrary, when the shutter button 25 is fully pushed by the user with the capture software not loaded, the CPU 82 enters the moving-picture mode and captures the moving image to send it to the video controller 85.Meanwhile, the ROM of the I/O controller 103 stores various control programs including a wakeup program, a key input monitor program, a LED control program, and jog dial status monitor program.",
"The jog dial status monitor program is a program associated with a jog dial utility program stored in the hard disk of the HDD 88, which monitors whether or not the rotary encoder section of the jog dial 26 has been rotated or pushed.",
"The wakeup program is controlled by the CPU 82 such that, when the current time supplied from the RTC circuit of the PCI-ISA bridge 90 reaches a preset start time, the wakeup program executes predetermined processing.",
"The key input monitor program monitors the inputting of the operator keys 21 and other various key switches.",
"The LED control program controls the turning on/off of various lights constituted by LEDs (Light Emitting Diodes) 101 including a power light PL, a battery light BL, a message light ML.",
"The RAM of the I/O controller 103 has various program registers including a set time register for the wakeup program, a key input monitor register for the key input monitor program, a LED control register for the LED control program, and an I/O register for the jog dial status monitor program.",
"The set time register is adapted to store the time information about the start time set by the user as desired beforehand for use by the wakeup program.",
"Therefore, on the basis of the wakeup program, the I/O controller 103 determines whether or not the current time supplied by the RTC circuit has reached the start time set by the user as desired.",
"If the current time is found reaching the start time, the I/O controller 103 notifies the CPU 82 thereof.",
"Consequently, the CPU 82 starts up the predetermined preset application software at the start time, thereby executing the predetermined processing in accordance with this application software.",
"The key input monitor register is adapted to store operation key flags on the basis of the inputting from the operator keys 21, stick 22, left click button 23A, the right click button 23B, and the center button 23C.",
"Consequently, the I/O controller 103 determines on the basis of the key input monitor program and the state of the operation key flag whether or not a pointing operation by the stick 22 or a click operation by the left click button 23A, the right click button 23B or the center button 23C for example has been performed.",
"If a pointing operation or a click operation is found performed, the I/O controller 103 notifies the CPU 82 thereof.",
"A pointing operation herein denotes an operation for moving the mouse cursor to a desired position by pushing the stick 22 horizontally or vertically with the finger.",
"A click operation herein denotes an operation of depressing the left click button 23A or right click button 23B with the finger quickly, and then releasing the finger from the button.",
"In response to any of these operations, the CPU 82 executes the predetermined processing according to the click operation.",
"The LED control register is adapted to store the turn on/off flags indicative of the turn on/off of various lights formed by LEDs including the power light PL, the battery light BL, and the message light ML.",
"Consequently, when the CPU 82 loads the electronic mail program from the hard disk of the HDD 88 into the RAM 92 upon pushing of the jog dial 26, thereby receiving electronic mail in accordance with the electronic mail program, the I/O controller 103 stores the turn on/off flag and controls the LEDs 101 to turn on the message light ML on the basis of the stored turn on/off flag.",
"The I/O register for the jog dial status monitor program is adapted to store a rotary operation flag and a pushing operation flag for a rotary operation and a pushing operation performed with the jog dial 26 respectively.",
"Consequently, when a menu item desired by the user is selected from a plurality of menu items by a rotary operation and a pushing operation of the jog dial 26 via the rotation detector 107, the I/O controller 103 stores the rotary operation flag and the pushing operation flag into the I/O register and notifies the CPU 82 thereof.",
"In response, in accordance with the jog dial utility program loaded from the HDD 88 and starting up in the RAM 92, the CPU 82 starts up the application software corresponding to the menu item selected by the rotation and pushing operations of the jog dial 26, executing predetermined processing.",
"Here, if the OS is not started up with the power switch 106 being off, the I/O controller 103 is always operating by being supported by the jog dial status monitor program under the control of the power supply/charge controller 100, thereby starting up user-desired application software and script files upon pushing of the jog dial 26 without arranging a dedicated key in the power save state or power off state.",
"Meanwhile, the I/O controller 103 is connected to the I2C bus 95 and supplies, via the I2C bus 95, various setting parameters for the CCD video camera 33 set by the operator keys 21 and the jog dial 26, thereby controlling the camera power turn on/off in the CCD video camera 33 and adjusting the brightness and contrast in the CCD video camera 33.The following describes the details of the operation of the personal computer 1.Referring to FIG.",
"8, there is shown an example of a screen (a capture mode screen) which is displayed in the capture mode.",
"At the center of the screen, the finder 206 is arranged in which an image taken by the CCD video camera 33 or supplied from a device (in this example, the video recorder 3) connected to the AV IN connector 61 (FIG.",
"6) or a device connected to the USB connector 44 (FIG.",
"4) or the USB connector 63 (FIG.",
"6).",
"In an image information display section 201 shown in the upper left portion of the capture mode screen, the information associated with the image shown in the finder 206 is displayed.",
"Below the image information display section 201, an input device specification section 202 is arranged which is composed of buttons to be operated for specifying a device from which an image to be displayed in the finder 206 is supplied.",
"By specifying the CCD video camera 33 by operating a button 202A of the input device specification section 202, the user can display an image inputted from the CCD video camera 33.By operating a button 202B to specify a device (in this example, the video recorder 3) (in what follows, the device connected to the AV IN connector 61 is appropriately referred to as an analog AV input device) connected to the AV IN connector 61 (FIG.",
"6), the user can display an image inputted from the analog AV input device.",
"In addition, by operating a button 202C to specify a device connected to the IEEE 1394 connector 51 (FIG.",
"5) (in what follows, the device connected to the IEEE 1394 connector 51 is appropriately referred to as a digital AV input device), the user can display an image inputted from the digital AV input device.",
"Further, by operating a button 202D to specify a device to be connected to the USB connector 44 (FIG.",
"4) or the USB connector 63 (FIG.",
"6) (in what follows, the device connected to the USB connector 44 or the USB connector 63 is appropriately referred to as a USB device), the user can display an image inputted from the USB device.",
"In a capture mode specification section 203 below the input device specification section 202, a button for switching the capture mode of the CCD video camera 33, a button for setting the display mode of an image supplied from the device specified by the input device specification section 202, and so on are arranged.",
"A button 203A is operated to capture a still image by the CCD video camera 33.A button 203B is operated to display, in high quality (to display an image in movie for a long time), an image taken by the CCD video camera 33 or inputted from an analog AV input device.",
"A button 203C is operated to display an image taken by the CCD video camera 33, inputted from a digital AV input device, or inputted from a USB device in a quality lower than that of long-time movie but with a smaller data amount than that of long-time movie (namely, to display the above-mentioned image in network movie).",
"Below the capture mode specification section 203, an option button 204 and an effect button 205 are arranged.",
"The option button 204 is operated to perform a predetermined setting operation and the effect button 205 is operated to add an effect to an image to be displayed in the finder 206.Below the finder 206, a capture button 207 and a send button 208 each having a light are arranged.",
"It should be noted that, in the initial state of the capture mode screen, the light of the capture button 207 is on and the light of the send button 208 is off.",
"By operating the capture button 207 with its light being on, the user can start displaying an image in the finder 206.By operating the send button 208 with its light being off, the user can switch from the capture mode to the send mode.",
"It should be noted that, in the capture mode, pushing the jog dial 26 also switches from the capture mode to the send mode.",
"A file list 209 arranged on the right side of the capture mode screen displays, in a list form, the thumbnails of images recorded to the HDD 88 for example.",
"Each thumbnail also displays the information about the image.",
"The file list 209 is displayed at a position corresponding to the position of the jog dial 26 as shown in FIG.",
"9.The frame enclosing a thumbnail in the file list 209 may be moved along the file list 209 as the jog dial 26 is rotated.",
"To be more specific, when the jog dial 26 is rotated in the up-arrow direction in FIG.",
"9, the frame moves upward by the amount of rotation.",
"If the jog dial 26 is rotated further upward with the frame enclosing the top thumbnail, all displayed thumbnails are moved upward.",
"Namely, the top thumbnail disappears and a new thumbnail comes appearing at the bottom.",
"That is, the frame of the file list 209 moves in response to the rotary direction of the jog dial 26.FIG.",
"10 shows an exemplary screen (send mode screen) to be displayed in the send mode.",
"In this screen, a view area 222 smaller than the finder 206 is arranged in front of the finder 206.In addition, a send form specification section 221 instead of the input device specification section 202, capture mode specification section 203, option button 204, and the effect button 205 of the capture mode screen is arranged in this screen.",
"The image information display section 201, the capture button 207, the send button 208, and the file list 209 are displayed in the same manner as the capture mode screen.",
"The view area 222 displays the image of a thumbnail which corresponds to the frame of the file list 209.At this moment, the finder 206 displays an image (presentation image) currently inputted from a device specified by the input device specification section 202 such that it is manipulated to a degree that its contents are recognizable (manipulated presentation image (in this case, a transparent presentation image)).",
"In the send form specification section 221, buttons to be operated for specifying send modes in which the send image displayed in the view area 222 is transmitted.",
"A button 221A is operated to send mail attached with a still image or network movie.",
"A button 211B is operated to send a still image or a network movie to the server 5.A button 221C is operated to send a still image to another terminal (for example, the terminal 6) via the server 5.A button 221D is operated to send a still image to a terminal connected to the Bluetooth.",
"Button 221E is operated to edit a moving image (to be more specific, this button is operated to supply a moving image to an editing program).",
"It should be noted that the operation of the personal computer 1 to be executed when the button 221C is operated will be described later.",
"Below the view area 222, an operator section 223 is arranged in which various buttons to be operated when the image displayed in the view area 222 is manipulated.",
"Below the finder 206, the capture button 207 with its light off and the send button 208 with its light on are displayed.",
"By operating the send button 208 with its light on, the user can send the image displayed in the view area 222 in the form specified by the send form specification section 221.By operating the capture button 207 with its light off, the user can switch from the send mode to the capture mode.",
"It should be noted that, in the send mode, pushing the jog dial 26 may also switch from the send mode to the capture mode.",
"The following describes together the effects of the operations of the capture button 207 and the send button 208 in the capture mode and the send mode, with reference to FIG.",
"11.As shown in A of FIG.",
"11, when the send button 208 is operated in the capture mode (the light of the capture button 207 is on and the light of the send button 208 is off), the capture mode is switched to the send mode as shown in B of FIG.",
"11 (the light of the capture button 207 goes off and the light of the send button 208 goes on).",
"Namely, a manipulated presentation image is displayed instead of a normal presentation image in the finder 206 and the view area 222 is displayed with the thumbnail image enclosed by the frame in the file list 209 for example.",
"When the send button is further operated in the send mode (the light of the capture button 207 is off and the light of the send button 208 is on), the send image displayed in the view area 222 is transmitted in the form specified by the send form specification section 221 as shown in C of FIG.",
"11.The following describes the operations of the CPU 82 and the video controller 85 of the personal computer 1 to be executed when displaying a manipulated presentation image in the finder 206 when the capture mode is switched to the send mode.",
"First, the operation of the CPU 82 will be described with reference to the flowchart shown in FIG.",
"12.In the capture mode, when a switching signal generated by operating the send button 208 or pushing the jog dial 26 for switching from the capture mode to the send mode is inputted in step S1, the CPU 82 notifies the video controller 85 thereof in step S2 and, at the same time, draws a frame of 640 pixels×480 lines into the VRAM of the video controller 85 as shown in A of FIG.",
"13.Next, in step S3, the CPU 82 sets blue pixel values (RGB(32, 32, 126)) to the pixels of the even-numbered lines of the frame drawn in step S2 as shown in B of FIG.",
"13 and sets black pixel values (RGB(16, 0, 16)) to the pixels of the odd-numbered lines.",
"It should be noted that the halftone lines in B of FIG.",
"13 are even-numbered lines while the black lines are odd-numbered lines; for the brevity of description, the width and the number of these lines are not the precise reflection of the actual lines.",
"Then, the above-mentioned procedure comes to an end.",
"The following describes the operation of the video controller 85 with reference to the flowchart shown in FIG.",
"14.In step S11, receiving a notification that the switching to the send mode is requested by the CPU 82, then the video controller 85 executes the processing of displaying, in the view area 222, the image of the thumbnail enclosed by frame in the file list 209 and the processing of displaying other portions constituting the send mode screen in step S12.Next, the video controller 85 generates a manipulated presentation image and displays it in the finder 206.First, in step S13, the video controller 85 starts the processing of retrieving, from the video decoder 84 or the video capture processing chip 93, the image data for one field inputted from a device specified by the input device specification section 202.In step S14, the video controller 85 detects the pixels to which the black pixel values are set from the VRAM with the blue pixel values or the black pixel values set in step S3 shown in FIG.",
"12 and starts the processing of replacing the detected pixel values of the pixels by the pixel values of the pixels for one field retrieved in step S13 corresponding to the detected pixels.",
"In step S15, the video controller 85 starts the processing of outputting the one field with the pixel values replaced in step S14 to the LCD 31.Then, the above-mentioned procedure comes to an end.",
"Consequently, a manipulated presentation image formed by the even-numbered lines with blue displayed and the odd-numbered lines with a portion of the presentation image displayed is generated, so that, in the finder 206, a manipulated presentation image with the presentation image being transparent is displayed as shown in FIG.",
"10.Therefore, the user may easily distinguish a send image corresponding to the mainly performed send mode processing from the presentation image corresponding to the capture mode processing not being mainly performed and understand the contents of the presentation screen.",
"Then, the above-mentioned procedure comes to an end.",
"The following describes the operation of the video controller 85 of the personal computer 1 to be executed when converting an image displayed in 720×480 (for example, an image supplied from the video recorder 3) into an image displayed in 640×480 (an image corresponding to the size of the finder 206).",
"A signal inputted from the port replicator 2 connected to the I/O connector 43, for example a video signal (an analog signal) inputted from the video recorder 3, is inputted in the video decoder 84 via the I/O controller 103 and the I2C bus 95.Next, the video decoder 84 decodes the inputted video signal and stores the resultant image data into the incorporated frame memory (720 pixels×480 lines) (FIG.",
"15).",
"The video controller 85 takes 9 pixels which are arranged horizontally in FIG.",
"15 from the image data of 720×480 stored in the frame memory of the video decoder 84 and performs the processing of generating 8 pixels from these 9 pixels.",
"Because 720 and 640 (the number of pixels in the horizontal direction) are in 9 to 8 relationship, an image of 640×480 may eventually generated by generating 8 pixels for every 9 pixels arranged in the horizontal direction of the 720×480 image.",
"The following describes a more specific generating method with reference to FIG.",
"16.When any 9 pixels continuously arranged horizontally in an image of 720×480 are shown as with A of FIG.",
"16, a new pixel B1 (B of FIG.",
"16) forming an image of 640×480 is generated by computing color difference value Vb1, color difference value Ub1, and luminance value Yb1 by substituting color difference value Va1, color difference value Ua1, and luminance value Ya1 forming pixel A1 and color difference value Va1, color difference value Ua1, and luminance value Ya2 forming pixel A2 into the following equations.",
"It should be noted that one pixel value is formed by color difference value V, color difference value U, and luminance value Y; in an image of 720×480, color difference value V and color difference value U are shared by adjacent pixels as shown in FIG.",
"17.Color difference value Vb1=(color difference value Va1×8+color difference value Va1×1)/9 Color difference value Ub1=(color difference value Ua1×8+color difference value Ua1×1)/9 Luminance value Yb1=(luminance value Ya1×8+luminance value Ya2×1)/9 Color difference value Vb2, color difference value Ub2, and luminance value Yb2 forming pixel B2 are computed by substituting color difference Va1, color difference Ua1, and luminance Ya2 forming pixel A2 and color difference Va2, color difference Ua2, and luminance Ya3 forming pixel A3 into the following equations.",
"Color difference value Vb2=(color difference value Va1×7+color difference value Va2×2)/9 Color difference value Ub2=(color difference value Ua1×7+color difference value Ua2×2)/9 Luminance value Yb2=(luminance value Ya2×7+luminance value Ya3×2)/9 Color difference value Vb3, color difference value Ub3, and luminance value Yb3 forming pixel B3 are computed by substituting color difference value Va2, color difference value Ua2, and luminance value Ya3 forming pixel A3 and color difference value Va2, color difference value Ua2, and luminance value Ya4 forming pixel A4 into the following equations.",
"Color difference value Vb3=(color difference value Va2×6+color difference value Va2×3)/9 Color difference value Ub3=(color difference value Ua2×6+color difference value Ua2×3)/9 Luminance value Yb3=(luminance value Ya3×6+luminance value Ya4×3)/9 Color difference value Vb4, color difference value Ub4, and luminance value Yb4 forming pixel B4 are computed by substituting color difference value Va2, color difference value Ua2, and luminance value Ya4 forming pixel A4 and color difference value Va3, color difference value Ua3, and luminance value Ya5 forming pixel A5 into the following equations.",
"Color difference value Vb4=(color difference value Va2×5+color difference value Va3×4)/9 Color difference value Ub4=(color difference value Ua2×5+color difference value Ua3×4)/9 Luminance value Yb4=(luminance value Ya4×5+luminance value Ya5×4)/9 Color difference value Vb5, color difference value Ub5, and luminance value Yb5 forming pixel B5 are computed by substituting color difference value Va3, color difference value Ua3, and luminance value Ya5 forming pixel A5 and color difference value Va3, color difference value Ua3, and luminance value Ya6 forming pixel A6 into the following equations.",
"Color difference value Vb5=(color difference value Va3×4+color difference value Va3×5)/9 Color difference value Ub5=(color difference value Ua3×4+color difference value Ua3×5)/9 Luminance value Yb5=(luminance value Ya5×4+luminance value Ya6×5)/9 Color difference value Vb6, color difference value Ub6, and luminance value Yb6 forming pixel B6 are computed by substituting color difference value Va3, color difference value Ua3, and luminance value Ya6 forming pixel A6 and color difference value Va4, color difference value Ua4, and luminance value Ya7 forming pixel A7 into the following equations.",
"Color difference value Vb6=(color difference value Va3×3+color difference value Va4×6)/9 Color difference value Ub6=(color difference value Ua3×3+color difference value Ua4×6)/9 Luminance value Yb6=(luminance value Ya6×3+luminance value Ya7×6)/9 Color difference value Vb7, color difference value Ub7, and luminance value Yb7 forming pixel B7 are computed by substituting color difference value Va4, color difference value Ua4, and luminance value Ya7 forming pixel A7 and color difference value Va4, color difference value Ua4, and luminance value Ya8 forming pixel AB into the following equations.",
"Color difference value Vb7=(color difference value Va4×2+color difference value Va4×7)/9 Color difference value Ub7=(color difference value Ua4×2+color difference value Ua4×7)/9 Luminance value Yb7=(luminance value Ya7×2+luminance value Ya8×7)/9 Color difference value Vb8, color difference value Ub8, and luminance value Yb8 forming pixel B8 are computed by substituting color difference Va4, color difference Ua4, and luminance value Ya8 forming pixel A8 and color difference value Va5, color difference Ua5, and luminance value Ya9 forming pixel A9 into the following equations.",
"Color difference value Vb8=(color difference value Va4×1+color difference value Va5×8)/9 Color difference value Ub8=(color difference value Ua4×1+color difference value Ua5×8)/9 Luminance value Yb8=(luminance value Ya8×1+luminance value Ya9×8)/9 As described, an image of 720×480 is converted into an image of 640×480.If, for example, an image of 720×480 is displayed in the capture mode screen without being converted, the presentation image is displayed running over from the finder 206 as shown in FIG.",
"18.Consequently, if that image is used as it is, the image information display section 201 through the effect button 205 and the capture button 207 through the file list 209 must be rearranged in order to provide a suitable capture mode screen.",
"On the other hand, when converting an image of 720×480 into an image of 640×480 for display, this image is displayed in correspondence with the size of the finder 206, so that the image information display section 201 and so on need not be rearranged.",
"The following describes the operations of the I/O controller 103 and the video decoder 84 of the personal computer 1 to be executed when displaying, in corresponding to a viewing disable signal, an image corresponding to an AV signal (analog) inputted from the video recorder 3.First, the operation of the I/O controller 103 will be described with reference to the flowchart shown in FIG.",
"19.In step S21, the I/O controller 103 determines whether or not a viewing disable signal has been detected from the AV signal of the video recorder 3 connected to the port replicator 2 (the AV IN connector 61) inputted from the I/O connector 43.If the viewing disable signal is found not detected, the procedure goes to step S22, in which the value of the detection bit incorporated in the video decoder 84 is changed to value 0.On the other hand, if the viewing disable signal is found detected in step S21, then the procedure goes to step S23, in which the I/O controller 103 changes the value of the detection bit of the video decoder 84 to value 1.When the detection bit of the video decoder 84 has been changed to the predetermined value in step S22 or S23, the I/O controller 103 returns to step S21 to repeat the processing thereafter.",
"The following describes the operation of the video decoder 84 with reference to the flowchart shown in FIG.",
"20.In step S31, the video decoder 84 checks the value of the incorporated detection bit and determines in step S32 whether it is value 1 or value 0.If the detection bit is determined to be value 0 in step S32, then the procedure goes to step S33, in which the video decoder 84 decodes the inputted video signal as usual and stores the resultant image data into the frame memory.",
"Namely, in this case, the image (presentation image) inputted from the video recorder 3 is displayed in the finder 206 of the capture mode screen as it is.",
"On the other hand, if the detection bit is found to be value 1 in step S32, then the procedure goes to step S34, in which the video decoder 84 does not decode the inputted video signal but sets the black pixel values for example to the incorporated frame memory.",
"Namely, in this case, a black screen is displayed in the finder 206 of the capture mode screen.",
"Therefore, at this time, the user cannot view the image supplied from the video recorder 3.After the process of step S33 or S34, the video decoder 84 returns to step S31 to repeat the processing thereafter.",
"The following describes the operation of the personal computer 1 to transmit a send image to the server 5 to provide it from the server 5 to the terminal 6, with reference to the flowchart shown in FIG.",
"21.In step S51, when the button 221C (the button to be operated to transmit a still image to another terminal via the server 5) of the send form specification section 221 of the send mode screen (FIG.",
"10) is operated, then the CPU 82 of the personal computer 1 controls the video controller 85 to display a login screen (not shown) for inputting the information such as the user name necessary for login in step S52 and, upon inputting of the login information, the procedure goes to step S53.In step S53, the personal computer 1 transmits the login information inputted in step S52 to the server 5 via the port replicator 2 and the network 4 and, in step S54, receives the authentication result based on the login information from the server 5.Next, in step S55, the CPU 82 of the personal computer 1 determines on the basis of the authentication result received in step S54 whether or not the user of the personal computer 1 has been successfully authenticated.",
"If the user is found successfully authenticated, the procedure goes to step S56.In step S56, the CPU 82 displays a screen for creating the address of send destination (in this example, the address of the terminal 6) and the mail to be transmitted to the send destination.",
"Next, In step S57, when the send button 208 in the send mode screen is operated, the procedure goes to step S58, in which the CPU 82 transmits the address and so on inputted in the screen shown in step S56 and the image (the image of the thumbnail enclosed by the frame in the file list 209) displayed in the view area 222 when the send button 208 was operated to the server 5.If the user is found not successfully authenticated in step S55 or if the send image and address were transmitted to the server 5 in step S58, then the processing comes to an end.",
"Then, the above-mentioned processing comes to an end.",
"The following describes the operation of the server 5 which is executed in response to the above-mentioned operation of the personal computer 1 with reference to the flowchart shown in FIG.",
"22.In step S61, the server 5 receives the login information from the personal computer 1 (step S53 of FIG.",
"21) and performs authentication processing in step S62, transmitting an authentication result to the personal computer 1 in step S63.Next, in step S64, the server 5 determines on the basis of the received authentication result in step S62 whether or not the user of the personal computer 1 is registered at this system.",
"If the user is found registered, then the procedure goes to step S65 in which the server 5 waits until the send image and so on (step S58 of FIG.",
"21) transmitted from the personal computer 1 are received.",
"When the send image and so on are received from the personal computer 1 in step S65, the procedure goes to step S66, in which the server 5 determines whether or not the received data include the address of the send destination of the send image.",
"If the address is found included, then the procedure goes to step S67.In step S67, the server 5 determines whether or not the received data include mail (message).",
"If mail is found included, then the procedure goes to step S68 to determine whether or not the data amount of the received mail is greater than predetermined data amount L. If the mail is found not included in step S67 or the data amount of the received mail is found less than predetermined data amount L in step S68, then the procedure goes to step S69, in which the server 5 transmits the mail and URL (if no mail is included, the URL and mail including a predetermined message) received from the personal computer 1 to the address destination (in this example, the terminal 6).",
"Next, in step S70, in response to the URL and so on transmitted in step S69, the server 5 receives a signal from the terminal 6 (a signal indicative of the model of the terminal 6) to understand the type of the terminal 6.In step S71, the server 5 determines the image processing capacity of the terminal 6 from the obtained model and changes the size of the send image supplied from the personal computer 1 for example so that the send image can be processed within the determined image processing capacity.",
"In step S72, the server 5 transmits the send image with its size changed in step S71 to the terminal 6.If no address is found included in step S66 or if the data amount of the mail is found greater than predetermined data amount L in step S68, then the procedure goes to step S73, in which the server 5 notifies the personal computer 1 thereof.",
"If the user is found not registered in step S64 or if the send image was transmitted to the terminal 6 in step S72 or if the notification was made in step S73, the above-mentioned processing comes to an end.",
"The following describes the operation of the terminal 6 to be executed in response to the above-mentioned operation of the server 5 with reference to the flowchart shown in FIG.",
"23.In step S81, the terminal 6 receives the URL and so on supplied from the server 5 (step S69 in FIG.",
"22) and displays them in response to the operation performed by the user on the terminal 6.In step S82, when the URL of the server 5 is specified by the user, the terminal 6 accesses the server 5 to transmit a signal indicative of the terminal's model to the server 5.Next, in step S83, the terminal 6 receives the send image transmitted from the server 5 (step S72 of FIG.",
"22) and displays the image appropriately.",
"Then, the above-mentioned processing comes to an end.",
"It should be noted that the steps describing each program include not only the processes which are sequentially executed in a time-dependent manner, but also those processes which are executed in parallel to each other or discretely.",
"INDUSTRIAL APPLICABILITY As described and according to the information processing apparatus and program associated with the present invention, content is inputted, the address of a terminal is inputted, and the content and the address are transmitted to a server together.",
"Consequently, the user can request the server for providing content to the terminal."
]
] | Patent_10450041 |
[
[
"Relating to fabric care",
"The invention is concerned with improvements relating to fabric care and in particular to means by which the apparent ageing of clothes can be reduced or retarded.",
"Colour loss during laundering, as opposed to during wear, is a significant source of colour loss.",
"We have determined that this problem may be overcome by use of a lubricant during the laundering process, to prevent the visible appearance of local colour loss through mechanical damage by the laundering process."
],
[
"1.Use of a lubricant during the laundering process, to prevent the visible appearance of local colour loss through mechanical damage by the laundering process.",
"2.Use according to claim 1, wherein the lubricant is a nonionic or anionic, polymeric material.",
"3.Use according to claim 2, wherein the lubricant has an index of wet cotton lubrication as defined herein greater than 15 when measured with a solution containing 1 g dm−3 of the lubricant.",
"4.Use according to claim 2, wherein the lubricant is a material which when dissolved at a concentration of 1 g dm−3 in water at 25° C. gives a solution that has a viscosity greater than 0.05 Pa s when measured at a shear rate of 100 s−1.5.Use according to claim 2 wherein the lubricant has a molecular weight greater than 100,000 Dalton.",
"6.Use according to claim 5 wherein the lubricant is a polyacrylate, polyacrylic acid, polyacrylamide, poly vinyl pyrrolidone or a co-polymer thereof.",
"7.Use according to claim 2 wherein the lubricant is a polydimethyl siloxane.",
"8.Use according to claim 2 wherein the lubricant is an oxidised polyethylene wax.",
"9.Use according to claim 2 wherein the lubricant is present at a level of 0.5-5% wt.",
"10.Use according to claim 2 wherein the articles being laundered are garments."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>It is well known that clothes, particularly lower cost and quality clothes, lose dye when washed.",
"Almost everyone who washes a mix of clothes has at some time experienced the detrimental effects of washing a new dyed article with white articles.",
"In simple terms, the dye is transferred from the dyed article to the white articles causing a significant change in their colour.",
"The commonplace solution to this problem is to wash new clothes separately.",
"Articles which are not new also release dye, but at a reduced rate.",
"For this reason it has been proposed to incorporate various ‘dye fixatives’, ‘dye scavengers’ or ‘colour-safe’ bleaches for dyestuffs in laundry compositions.",
"These have been the subject of intensive and extensive research and variously fix the dye in place, prevent it re-depositing or chemically bleach it so as to overcome the problem of dye transfer in mixed washes.",
"It is well established that relatively small amounts of dye are released from older garments and that the bulk of dye release occurs in the first wash of a new garment.",
"As will be explained in further detail below, this initial dye loss is seldom noted by users except in incidents of dye transfer.",
"WO 01/53600 (P&G: 2000) relates to fabric dye protection which is specific to denim.",
"As noted in that specification and otherwise known, denim is a rather unique cloth.",
"First, it is woven from two forms of yarn, one of which is dyed and the other is not.",
"Second, the dyed yarn is typically dyed with indigo and has most of the dye located in the outer fibres of the yarn.",
"This form of dyeing is known as ‘ring-dyeing’ and results in a fabric where the dye is easily removed by abrasion during wear: a process known in the art as ‘crocking’.",
"Thus, during wear, the dye becomes detached from the fabric, particularly on the seat, knees and thighs and is then easily extracted during washing.",
"The characteristic colour loss from denim is thought by some to be a desirable feature.",
"Some may even go so far as to deliberately augment damage to denim goods to make the goods appear ‘older’.",
"Others, (according to the applicants of WO 01/53600) see such colour loss as a problem.",
"WO 01/53600 suggests that this rather specific problem can be overcome by the combined uses of a cationic polymer, a low molecular weight polyamine, crystal growth inhibitor and dye fixing agent to provide protection to denim fabric from dye loss which is primarily due to mechanical loss.",
"This mechanical loss occurs through the normal abrasive destruction of the fabric during wear.",
"The agents proposed to overcome this, apparently bind to the cloth and fix the colour in place and thereby prevent or reduce the appearance of ageing.",
"The characteristic colour loss of denim, which arises due to its peculiar structure, is somewhat unique.",
"Very few other garments are purposefully ring-dyed so as to encourage colour loss.",
"Ageing in garments which have not been ring-dyed still presents problems.",
"As noted above, a large part if not the bulk of, colour loss occurs in the first wash.",
"Despite this, clothes are not generally perceived as ‘old’ after the first wash.",
"Indeed, the first wash of an article generally produces no appreciable change in appearance to the normal observer.",
"While there may have been a significant change in hue or colour depth, this is simply not perceived without a suitable comparison.",
"Thus, prevention of actual colour loss (by the use of dye transfer inhibitors, dye fixatives etc) is not in itself sufficient to prevent apparent ageing of garments."
],
[
"<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>We have determined that the most significant cue for ageing in clothes and other articles is colour loss which is localised rather than general.",
"While an article may lose a significant proportion of its colour overall, this loss is not perceived by the casual spectator unless it is extreme.",
"Whereas any localised colour loss, for example on seams, acts as a strong indication that the article is ‘faded’.",
"In addition, we have determined that actual removal of dyestuff is not required for an apparent colour loss to be perceived.",
"On seams and the like, abrasion of the surface is sufficient to produce a modified scattering of light which gives the appearance of loss of dyestuff.",
"In reality, the dyestuff may still be present.",
"Moreover, we have determined that colour loss during laundering, as opposed to during wear, is a significant source of colour loss.",
"We have determined that this problem may be overcome by use of a lubricant during the laundering process, to prevent the visible appearance of local colour loss through mechanical damage by the laundering process.",
"By use of a lubricant in this manner, it is believed that localised abrasion of the articles being washed may be reduced or retarded.",
"Without wishing to be limited by reference to any theory of operation, it is believed that, when dyed textiles fade, this is, almost without exception, characterised by an increase in the luminance component of the colour.",
"It is known that human ability to perceive small differences in lightness is spatially dependent [M. D. Fairchild, ‘Colour Appearance models’, Addison Wesley Longman Publishing Co, New York (1998)].",
"The maximum in sensitivity corresponds to a spatial frequency in the range 2-15 cycles per degree.",
"It just so happens that when a garment (such as a pair of jeans) is viewed at a typical viewing distance, then the laundry-induced faded features tend to fall within this range of spatial frequencies.",
"Thus, relatively small changes in colour can strongly influence the perception of the garment provided that they are localised.",
"While generalised colour loss may still occur to a significant and easily measurable extent, the perceived effect of this colour loss is greatly reduced when the colour loss is even.",
"The effect of preventing local colour loss is to significantly reduce the degree to which the articles being washed appear visibly aged.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention is concerned with improvements relating to fabric care and in particular to means by which the apparent ageing of clothes can be reduced or retarded.",
"BACKGROUND OF THE INVENTION It is well known that clothes, particularly lower cost and quality clothes, lose dye when washed.",
"Almost everyone who washes a mix of clothes has at some time experienced the detrimental effects of washing a new dyed article with white articles.",
"In simple terms, the dye is transferred from the dyed article to the white articles causing a significant change in their colour.",
"The commonplace solution to this problem is to wash new clothes separately.",
"Articles which are not new also release dye, but at a reduced rate.",
"For this reason it has been proposed to incorporate various ‘dye fixatives’, ‘dye scavengers’ or ‘colour-safe’ bleaches for dyestuffs in laundry compositions.",
"These have been the subject of intensive and extensive research and variously fix the dye in place, prevent it re-depositing or chemically bleach it so as to overcome the problem of dye transfer in mixed washes.",
"It is well established that relatively small amounts of dye are released from older garments and that the bulk of dye release occurs in the first wash of a new garment.",
"As will be explained in further detail below, this initial dye loss is seldom noted by users except in incidents of dye transfer.",
"WO 01/53600 (P&G: 2000) relates to fabric dye protection which is specific to denim.",
"As noted in that specification and otherwise known, denim is a rather unique cloth.",
"First, it is woven from two forms of yarn, one of which is dyed and the other is not.",
"Second, the dyed yarn is typically dyed with indigo and has most of the dye located in the outer fibres of the yarn.",
"This form of dyeing is known as ‘ring-dyeing’ and results in a fabric where the dye is easily removed by abrasion during wear: a process known in the art as ‘crocking’.",
"Thus, during wear, the dye becomes detached from the fabric, particularly on the seat, knees and thighs and is then easily extracted during washing.",
"The characteristic colour loss from denim is thought by some to be a desirable feature.",
"Some may even go so far as to deliberately augment damage to denim goods to make the goods appear ‘older’.",
"Others, (according to the applicants of WO 01/53600) see such colour loss as a problem.",
"WO 01/53600 suggests that this rather specific problem can be overcome by the combined uses of a cationic polymer, a low molecular weight polyamine, crystal growth inhibitor and dye fixing agent to provide protection to denim fabric from dye loss which is primarily due to mechanical loss.",
"This mechanical loss occurs through the normal abrasive destruction of the fabric during wear.",
"The agents proposed to overcome this, apparently bind to the cloth and fix the colour in place and thereby prevent or reduce the appearance of ageing.",
"The characteristic colour loss of denim, which arises due to its peculiar structure, is somewhat unique.",
"Very few other garments are purposefully ring-dyed so as to encourage colour loss.",
"Ageing in garments which have not been ring-dyed still presents problems.",
"As noted above, a large part if not the bulk of, colour loss occurs in the first wash.",
"Despite this, clothes are not generally perceived as ‘old’ after the first wash.",
"Indeed, the first wash of an article generally produces no appreciable change in appearance to the normal observer.",
"While there may have been a significant change in hue or colour depth, this is simply not perceived without a suitable comparison.",
"Thus, prevention of actual colour loss (by the use of dye transfer inhibitors, dye fixatives etc) is not in itself sufficient to prevent apparent ageing of garments.",
"BRIEF DESCRIPTION OF THE INVENTION We have determined that the most significant cue for ageing in clothes and other articles is colour loss which is localised rather than general.",
"While an article may lose a significant proportion of its colour overall, this loss is not perceived by the casual spectator unless it is extreme.",
"Whereas any localised colour loss, for example on seams, acts as a strong indication that the article is ‘faded’.",
"In addition, we have determined that actual removal of dyestuff is not required for an apparent colour loss to be perceived.",
"On seams and the like, abrasion of the surface is sufficient to produce a modified scattering of light which gives the appearance of loss of dyestuff.",
"In reality, the dyestuff may still be present.",
"Moreover, we have determined that colour loss during laundering, as opposed to during wear, is a significant source of colour loss.",
"We have determined that this problem may be overcome by use of a lubricant during the laundering process, to prevent the visible appearance of local colour loss through mechanical damage by the laundering process.",
"By use of a lubricant in this manner, it is believed that localised abrasion of the articles being washed may be reduced or retarded.",
"Without wishing to be limited by reference to any theory of operation, it is believed that, when dyed textiles fade, this is, almost without exception, characterised by an increase in the luminance component of the colour.",
"It is known that human ability to perceive small differences in lightness is spatially dependent [M. D. Fairchild, ‘Colour Appearance models’, Addison Wesley Longman Publishing Co, New York (1998)].",
"The maximum in sensitivity corresponds to a spatial frequency in the range 2-15 cycles per degree.",
"It just so happens that when a garment (such as a pair of jeans) is viewed at a typical viewing distance, then the laundry-induced faded features tend to fall within this range of spatial frequencies.",
"Thus, relatively small changes in colour can strongly influence the perception of the garment provided that they are localised.",
"While generalised colour loss may still occur to a significant and easily measurable extent, the perceived effect of this colour loss is greatly reduced when the colour loss is even.",
"The effect of preventing local colour loss is to significantly reduce the degree to which the articles being washed appear visibly aged.",
"DETAILED DESCRIPTION OF THE INVENTION The extent of mechanical damage to articles being laundered will vary with the machine type, conditions and the stage of the wash being considered.",
"Use of low water levels and violent agitation are envisaged to promote damage whereas higher water levels and less violent agitation will reduce damage.",
"Unfortunately, mechanised washing machines and environmental concerns have led to increased agitation and lower water levels.",
"The degree of agitation and the level of water present vary during the laundry cycle and it is preferable that the lubricant is present in those parts of the cycle which involve low water and/or high agitation.",
"The lubricants which are especially preferred are polymeric materials.",
"A preferred feature of the present invention is that the in-wash protection is achieved by the use of a material which does not adsorb onto the fabrics being washed.",
"It is believed that this avoids adverse effects on the desirable properties of the fabric once it is in the post-wash dry state.",
"For example there is preferably no stiffening of the fabric, no increased tendency for the fabric to attract and retain soils or stains, no reduction in the breathability or water absorbency characteristics of the fabric.",
"It is therefore preferred that the lubricants are species which do not carry a cationic charge.",
"Nonionic and anionic species are preferred.",
"It is believed that anionic materials exhibit better performance due to their tendency to form an extended structure.",
"In order that the invention may be further understood it will be described hereinafter with reference to various preferred features.",
"Lubricants: It is possible to define suitable lubricants in several ways.",
"Preferred lubricants show a reduction of friction on wet cotton.",
"Preferred lubricants in the context of this invention are materials which, when measured using the frictional technique described below and with a solution containing 1 g dm−3 of the material, give an Iwcl (index of wet cotton lubrication) greater than 15.Preferably this index is greater than 50 more preferably greater than 75.Preferred lubricants exhibit particular viscosity properties.",
"A preferred lubricant in the context of this invention is a material which when dissolved at a concentration of 1 g dm−3 in water at 25° C. gives a solution that has a viscosity greater than 0.05 Pa s when measured at a shear rate of 100 s−1.Suitable lubricants include: polyacrylate salts with mol wt greater than 100 000 Dalton, preferably greater than 500,000 Dalton; polyacrylic acids with mol wt greater than 100,000 Dalton, preferably greater than 500,000 Dalton; polyacrylamides with mol wt greater than 100,000 Dalton preferably greater than 500,000 Dalton; or, co-polymers of these various acrylic materials.",
"Also suitable are dextrans, preferably with mol wt greater than 50,000 Dalton and preferably greater than 200,000 Dalton.",
"Other suitable materials are poly vinyl pyrrolidones with mol wt greater than 100,000 Dalton; polydimethyl siloxanes emulsified in nonionic surfactant; and, dispersions of lightly oxidise polyethylene wax e.g.",
"(Imacol C™ ex Clariant).",
"It is preferable that the lubricant material is presented together with a textile-compatible carrier.",
"In general, it is preferred that the carrier makes up the bulk of a product for use according to the present invention.",
"Levels for the polymeric lubricants in compositions for use according to the invention fall in the range 0.05% wt-20% wt, with levels of 0.5-5% wt being preferred.",
"Unless otherwise stated all % values given in this specification are % wt and all ratios are weight ratios.",
"As these levels of lubricants are relatively low, powders, granules, tablets, liquids and other forms of detergent product can be manufactured by conventional means and the lubricants included in the formulations as appropriate.",
"Surfactants: If the composition of the present invention is in the form of a typical detergent composition (such as a main wash composition), the textile-compatible carrier may be chosen from soap and non-soap anionic, cationic, nonionic, amphoteric and zwitterionic detergent active compounds, and mixtures thereof.",
"Many suitable detergent active compounds are available and are fully described in the literature, for example, in “Surface-Active Agents and Detergents”, Volumes I and II, by Schwartz, Perry and Berch.",
"The preferred textile-compatible carriers that can be used are soaps and synthetic non-soap anionic and nonionic surfactant compounds.",
"Anionic surfactants are well-known to those skilled in the art.",
"Commonly employed materials include alkylbenzene sulphonates, particularly linear alkylbenzene sulphonates having an alkyl chain length of C8-C15; primary and secondary alkyl sulphates, particularly C8-C15 primary alkyl sulphates; alkyl ether sulphates; olefin sulphonates; alkyl xylene sulphonates; dialkyl sulphosuccinates; and fatty acid ester sulphonates.",
"Sodium salts are generally preferred.",
"Nonionic surfactants that may be used include the primary and secondary alcohol ethoxylates, especially the C8-C20 aliphatic alcohols ethoxylated with an average of from 1 to 20 moles of ethylene oxide per mole of alcohol, and more especially the C10-C15 primary and secondary aliphatic alcohols ethoxylated with an average of from 1 to 10 moles of ethylene oxide per mole of alcohol.",
"Non-ethoxylated nonionic surfactants include alkylpolyglycosides, glycerol monoethers, and polyhydroxyamides (glucamide).",
"Cationic surfactants that may be used include quaternary ammonium salts of the general formula R1R2R3R4N+X− wherein the R groups are independently hydrocarbyl chains of C1-C22 length, typically alkyl, hydroxyalkyl or ethoxylated alkyl groups, and X is a solubilising anion (for example, compounds in which R1 is a C8-C22 alkyl group, preferably a C8-C10 or C12-C14 alkyl group, R2 is a methyl group, and R3 and R4, which may be the same or different, are methyl or hydroxyethyl groups); and cationic esters (for example, choline esters) and pyridinium salts.",
"The total quantity of detergent surfactant in the composition is suitably from 0.1 to 60 wt %, preferably 0.5-55 wt %, more preferably 5-50 wt %.",
"Preferably, the quantity of anionic surfactant (when present) is in the range of from 1 to 50% by weight of the total composition.",
"More preferably, the quantity of anionic surfactant is in the range of from 3 to 55% by weight, e.g.",
"5 to 30% by weight.",
"Preferably, the quantity of nonionic surfactant when present is in the range of from 2 to 25% by weight, more preferably from 5 to 20% by weight.",
"Amphoteric surfactants may also be used, for example amine oxides or betaines.",
"Detergency Builders: The compositions may suitably contain from 10 to 70%, preferably from 15 to 70% by weight, of detergency builder.",
"Preferably, the quantity of builder is in the range of from 15 to 50% by weight.",
"The detergent composition may contain as builder a crystalline aluminosilicate, preferably an alkali metal alumino-silicate, more preferably a sodium alumino-silicate.",
"The alumino-silicate may generally be incorporated in amounts of from 10 to 70% by weight (anhydrous basis), preferably from 25 to 50%.",
"Alumino-silicates are materials having the general formula: 0.8-1.5 M2O Al2O3.0.8-6 SiO2 where M is a monovalent cation, preferably sodium.",
"These materials contain some bound water and are required to have a calcium ion exchange capacity of at least 50 mg CaO/g.",
"The preferred sodium alumino-silicates contain 1.5-3.5 SiO2 units in the formula above.",
"They can be prepared readily by reaction between sodium silicate and sodium aluminate, as amply described in the literature.",
"Rinse and Conditioner Compositions: Compositions according to the invention may be in the form of a rinse composition, such as a fabric conditioner composition.",
"If the fabric care composition or the laundry rinse composition of the present invention is in the form of a fabric conditioner composition, the textile-compatible carrier will be a fabric softening and/or conditioning compound (hereinafter referred to as “fabric softening compound”), which may be a cationic or nonionic compound.",
"The softening and/or conditioning compounds may be water insoluble, non-polymeric, quaternary ammonium compounds.",
"The compounds may be present in amounts of up to 8% by weight (based on the total amount of the composition) in which case the compositions are considered dilute, or at levels from 8% to about 50% by weight, in which case the compositions are considered concentrates.",
"Compositions suitable for delivery during the rinse cycle may also be delivered to the fabric in the tumble dryer if used in a suitable form.",
"Thus, another suitable product form is a composition (for example, a paste) suitable for coating onto, and delivery from, a substrate e.g.",
"a flexible sheet or sponge or a suitable dispenser (such as a container having apertures therein, for example) during a tumble dryer cycle.",
"Suitable cationic fabric softening compounds are substantially water-insoluble quaternary ammonium materials comprising a single alkyl or alkenyl long chain having an average chain length greater than or equal to C20 or, more preferably, compounds comprising a polar head group and two alkyl or alkenyl chains having an average chain length greater than or equal to C14.Preferably the fabric softening compounds have two long chain alkyl or alkenyl chains each having an average chain length greater than or equal to C16.Most preferably at least 50% of the long chain alkyl or alkenyl groups have a chain length of C18 or above.",
"It is preferred if the long chain alkyl or alkenyl groups of the fabric softening compound are predominantly linear.",
"Quaternary ammonium compounds having two long-chain aliphatic groups, for example distearyldimethyl ammonium chloride and di (hardened tallow alkyl) dimethyl ammonium chloride, are widely used in commercially available rinse conditioner compositions.",
"Other examples of these cationic compounds are to be found in “Surface-Active Agents and Detergents”, Volumes I and II, by Schwartz, Perry and Berch.",
"Any of the conventional types of such compounds may be used in the compositions of the present invention.",
"The fabric softening compounds are preferably compounds that provide excellent softening, and are characterised by a chain melting Lβ to Lα transition temperature greater than 25° C., preferably greater than 35° C., most preferably greater than 45° C. This Lβ to Lα transition can be measured by DSC as defined in “Handbook of Lipid Bilayers”, D Marsh, CRC Press, Boca Raton, Fla., 1990 (pages 137 and 337).",
"Substantially water-insoluble fabric softening compounds are defined as fabric softening compounds having a solubility of less than 1×10−3 wt % in demineralised water at 20° C. Preferably the fabric softening compounds have a solubility of less than 1×10−4 wt %, more preferably less than 1×10−8 to 1×10−6 wt %.",
"Especially preferred are cationic fabric softening compounds that are water-insoluble quaternary ammonium materials having two C12-22 alkyl or alkenyl groups connected to the molecule via at least one ester link, preferably two ester links.",
"It is particularly advantageous if the quaternary ammonium material is biologically biodegradable.",
"Preferred materials of this class such as 1,2-bis (hardened tallowoyloxy)-3-trimethylammonium propane chloride and their methods of preparation are, for example, described in U.S. Pat.",
"No.",
"4,137,180 (Lever Brothers Co).",
"Preferably these materials comprise small amounts of the corresponding mono-ester as described in U.S. Pat.",
"No.",
"4,137,180, for example, 1-hardened tallowoyloxy-2-hydroxy-3-trimethylammonium propane chloride.",
"Other useful cationic softening agents are alkyl pyridinium salts and substituted imidazoline species.",
"Also useful are primary, secondary and tertiary amines.",
"The compositions may alternatively or additionally contain water-soluble cationic fabric softeners, as described in GB 2 039 556B (Unilever).",
"The compositions may comprise a cationic fabric softening compound and an oil, for example as disclosed in EP-A-0829531.The compositions may alternatively or additionally contain nonionic fabric softening agents such as lanolin and derivatives thereof.",
"Lecithins are also suitable softening compounds.",
"Nonionic softeners include Lβ phase forming sugar esters (as described in M Hato et al Langmuir 12, 1659, 1966, (1996)) and related materials such as glycerol mono-stearate or sorbitan esters.",
"Often these materials are used in conjunction with cationic materials to assist deposition (see, for example, GB 2 202 244).",
"Silicones are used in a similar way as a co-softener with a cationic softener in rinse treatments (see, for example, GB 1 549 180).",
"The compositions may also suitably contain a nonionic stabilising agent.",
"Suitable nonionic stabilising agents are linear C8 to C22 alcohols alkoxylated with 10 to 20 moles of alkylene oxide, C10 to C20 alcohols, or mixtures thereof.",
"Advantageously the nonionic stabilising agent is a linear C8 to C22 alcohol alkoxylated with 10 to 20 moles of alkylene oxide.",
"Preferably, the level of nonionic stabiliser is within the range from 0.1 to 10% by weight, more preferably from 0.5 to 5% by weight, most preferably from 1 to 4% by weight.",
"The mole ratio of the quaternary ammonium compound and/or other cationic softening agent to the nonionic stabilising agent is suitably within the range from 40:1 to about 1:1, preferably within the range from 18:1 to about 3:1.The composition can also contain fatty acids, for example C8 to C24 alkyl or alkenyl mono-carboxylic acids or polymers thereof.",
"Preferably saturated fatty acids are used, in particular hardened tallow C16 to C18 fatty acids.",
"Preferably the fatty acid is non-saponified, more preferably the fatty acid is free, for example oleic acid, lauric acid or tallow fatty acid.",
"The level of fatty acid material is preferably more than 0.1% by weight, more preferably more than 0.2% by weight.",
"Concentrated compositions may comprise from 0.5 to 20% by weight of fatty acid, more preferably 1% to 10% by weight.",
"The weight ratio of quaternary ammonium material or other cationic softening agent to fatty acid material is preferably from 10:1 to 1:10.The fabric conditioning compositions may include silicones, such as predominantly linear polydialkylsiloxanes, e.g.",
"polydimethylsiloxanes or aminosilicones containing amine-functionalised side chains; amphoteric surfactants; smectite type inorganic clays; zwitterionic quaternary ammonium compounds; and nonionic surfactants.",
"Preferably, the silicone component is a dimethylpolysiloxane with aminoalkyl groups.",
"The fabric conditioning compositions may also include an agent which produces a pearlescent appearance, e.g.",
"an organic pearlising compound such as ethylene glycol distearate, or inorganic pearlising pigments such as microfine mica or titanium dioxide (TiO2) coated mica.",
"The fabric conditioning compositions may be in the form of emulsions or emulsion precursors thereof.",
"Minor and Other Components: Other optional and minor ingredients include emulsifiers, electrolytes (for example, sodium chloride or calcium chloride) preferably in the range from 0.01 to 5% by weight, pH buffering agents, and perfumes (preferably from 0.1 to 5% by weight).",
"Further optional ingredients include one or more of non-aqueous solvent, perfume carriers, fluorescers, colourants, hydrotropes, antifoaming agents, enzymes, optical brightening agents, opacifiers, anti-shrinking agents, anti-wrinkle agents, anti-spotting agents, germicides, fungicides, anti-oxidants, UV absorbers (sunscreens), heavy metal sequestrants, chlorine scavengers, dye fixatives, anti-corrosion agents, drape imparting agents, antistatic agents and ironing aids.",
"This list is not intended to be exhaustive.",
"Product Form and Presentation: The compositions of the invention may be in the form of a liquid, solid (e.g.",
"powder or tablet), a gel or paste, spray, stick or a foam or mousse.",
"Examples include a pre-wash soaking product, a rinse treatment (e.g.",
"conditioner or finisher) or a main-wash product.",
"The composition may also be applied to a substrate (e.g.",
"a flexible sheet) or used in a dispenser which can be used in the wash cycle or the rinse cycle.",
"Embodiments of the invention which take the form of incorporation and use of lubricants in main wash products are preferred.",
"In such compositions the product form is generally a powder or other particulate form ranging in size from a granulate to a tablet, or a liquid.",
"Liquids according to the present invention may be structured or isotropic.",
"The fabrics which treated according to the present invention are generally in the form of garments and preferably comprise cellulosic fibres, preferably from 1% to 100% cellulosic fibres (more preferably 5% to 100% cellulosic fibres, most preferably 40% to 100% such as 75% to 100%).",
"When the fabric contains less than 100% cellulosic fibres, the balance comprises other fibres or blends of fibres suitable for use in garments such as polyester or polyamide, for example.",
"Preferably, the cellulosic fibres are of cotton or regenerated cellulose such as viscose.",
"As noted above, it is particularly preferred that the composition is used as part of a main wash formulation.",
"Typically the product will be packaged together with instructions for use in a main wash, in an automatic washing machine.",
"In order that the invention may be further understood it will be described hereinafter with reference to the following non-limiting examples.",
"EXAMPLES Example 1 Eye-Tracking Studies A pair of black cotton jeans were subjected to 10 repeat wash and dry cycles using the 40° C. cotton cycle in a Miele™ front loading automatic washing machine and a 1 hour dry cycle in a Miele™ tumble drier.",
"The jeans were not worn between washes.",
"These conditions generated a typical pattern of in-wash induced uneven colour fading: specifically a periodic light dark pattern along the double seam of the leg and increased colour fading at the seams of the pocket, fly and waistband.",
"Colour matched digital images were produced of the front and back of the jeans.",
"These images were used as stimulus material in the following experiment to determine how a human observer perceives that a garment has faded.",
"The subject was seated in front of a computer monitor screen.",
"Below the screen was position a small device which uses an infra red tracking system that, once calibrated, is able to determine in which direction the subject is looking.",
"This can then be transformed to calculate which part of the on-screen image the subject is fixating.",
"This information is recorded for the duration of the experiment so that it is possible to reconstruct the subject's eye movements and, in particular, determine the sequence, location and duration of their fixation at different parts of the on-screen image.",
"After initial calibration, the subject was informed of their task.",
"This was to look at the on screen image and determine which of the two garments shown had been washed the greater number of times.",
"Initially, they were presented with a blank screen apart from a coloured circle, the fixation point, which they were told to look at.",
"This was then replaced with the image of the front and back of the same pair of jeans.",
"From this point, the subject's eye movements were recorded until they voiced their decision.",
"Two images of the same garment were used to ensure that differences were not sufficiently large that the observers could decide instantly using peripheral vision alone.",
"The experiment was repeated using six different subjects.",
"The recorded eye-tracks showed that without exception, the great majority of fixations and fixation time was associated with the features of the garment showing uneven rather than even colour fading namely: the seams, fly, pockets and waistband.",
"Example 2 Human Sensitivity to Colour Changes The following experiment was performed to quantify the enhanced sensitivity of human colour change perception to uneven colour change compared to uniform colour change at a spatial frequency corresponding to abrasion induced faded features on washed garments.",
"Two sets of calibrated colour chips were produced using Adobe Photoshop™ in a calibrated printing set-up.",
"One set of chips consisted of a number of pairs of chips of uniform colour where the colour differed only in its lightness value.",
"The differences covered a range of values.",
"Six series of chips of different hue were used.",
"The second set of chips consisted of the same series except that this time the lighter of the two chips had the light colour superimposed as a band down the centre of the chip on top of the original colour.",
"The width of the band was such that, at the viewing distance, the spatial frequency was 2 cycles per degree.",
"24 panellists were asked to put each of the series in the two sets into order of increased fading.",
"Their performance was analysed using a Probit™ Statistical Analysis which then gave the ‘just noticeable difference’ (JND) value for colour fading perception.",
"The results are tabulated below (Table 1) in terms of CIELAB Delta-E values.",
"(Delta-E represents the magnitude of colour difference).",
"It is clear that while the absolute magnitudes vary with hue, there is a consistent ability of the panellist to discriminate lower levels of colour fading when presented with an unevenly faded sample compared to a uniformly faded sample.",
"TABLE 1 ‘Just noticeable difference’ values for even and uneven fading JND for even fading/ JND for uneven fading/ Hue CIELAB ΔE CIELAB ΔE Yellow 1.50 1.00 Magenta 1.10 0.50 Blue 0.45 0.25 Black 0.75 0.35 Example 3 Correlation Studies A number of black cotton ‘combat’ style trousers were washed and dried for various numbers of cycles (between 1 and 10) using a number of different wash protocols (different detergent, drying method, ironing or not).",
"The result of these various processes was to arrive at a wardrobe of clothes all showing different amounts of fading.",
"An area 10 cm×10 cm on the leg of the garments was identified which contained a double stitched seam showing enhanced colour fading and an area of uniform ‘background’ colour fading.",
"The reflectance spectrum of the background and the seam areas was measured using a Datacolor Spectraflash™ 500 spectrometer.",
"From these spectra, the CIELAB L* values were calculated for the seam and background areas of each garment.",
"The garments were then presented, one at a time to each of 20 different panellists.",
"The garments were presented in such a way that only the 10×10 cm area was visible.",
"The panellists were asked to score the level of fading on a 0 to 100 scale.",
"The average score was calculated for each garment.",
"From the data, two graphs were constructed: a plot of the L* value for the background area of the garment against the panel score; and a plot of the L* value of the seam against the panel score.",
"The correlation coefficient was calculated for each plot.",
"These are tabulated below in Table 2.It is clear from these values that the lightness of the unevenly faded seam correlates more closely to the panellists' visual assessment of fading than does the lightness value of the evenly faded background fabric.",
"TABLE 2 Correlation between measured lightness and visual assessment correlation coefficient L* (background) vs panel score 0.79 L* (seam) vs panel score 0.90 Example 4 Friction Measurements The coefficient of friction between two pieces of white cotton fabric immersed in a solution of the lubricant material was measured using an Eldredge™ tribometer that had been modified to permit the study of submerged materials.",
"In summary, the set up consisted of a cylindrical watertight trough, to the bottom of which was affixed a 30×8 cm strip of plain, woven, white, non-mercerised 100% cotton sheeting.",
"Above this was located a piece of similar fabric mounted a round a cylindrical holder 1 cm in diameter and 6 cm in length.",
"This was attached to a pivoted arm which could be driven in such a way that the fabric cylinder was placed in contact with the stationery strip with defined normal load and then driven at a controlled velocity along a 6.5 cm length of the strip.",
"Stain gauges on the arm permitted measurement of the frictional force opposing this motion.",
"From the frictional force and the normal load it is possible to calculate the coefficient of friction (μ).",
"This measurement can be made for a range of velocities; but for present purposes it is appropriate to choose a sliding velocity of 6 cm s−1.For the purposes of assessing efficacy of potential lubricant systems, the index of wet cotton lubrication (Iwcl) is defined as: I wcl = ( μ 0 - μ μ 0 ) × 100 where μ0 is the coefficient of friction measured by the above method using a solution containing none of the lubrication.",
"Examples 5-8 Formulations The examples given below are typical formulations according to the present invention.",
"The materials used in the examples are identified in table 3.TABLE 3 Raw material specification Component Specification LAS Linear Alkyl Benzene Sulphonic-acid, Marlon AS3 ™, ex Huls Na-PAS Primary Alkyl Benzene Sulphonic-acid, neutralised with NaOH Dobanol ™ 25-7 C12-15 ethoxylated alcohol, 7EO, ex Shell Zeolite Wassalith ™ P, ex Degussa STPP Sodium Tri Polyphosphate, Thermphos ™ NW, ex Hoechst Dequest ™ 2066 Metal chelating agent, ex Monsanto Lipolase Type 100L, ex Novo Savinase ™ 16L Protease, ex Novo Sokalan ™ CP5 Acrylic/Maleic Builder Polymer, ex BASF Deflocculating Polymer A-11 disclosed in EP-A-346 Polymer 995 SCMC Sodium Carboxymethyl Cellulose Minors Anti-redeposition polymers, transition-metal scavengers/bleach stabilisers, fluorescers, antifoam, dye-transfer-inhibition polymers, enzymes, perfume Example 5 Spray-Dried Powder Example 5 provides a spray dried powder according to the present invention.",
"The composition of the powder is given in Table 4.TABLE 4 Component % w/w Na PAS 11.5 Dobanol 25-7 6.3 Soap 2 Zeolite 24.1 SCMC 0.6 Na Citrate 10.6 Na Carbonate 23 sodium polyacrylate 0.7 (mol wt 1 300 000) Dequest 2066 0.4 Sokalan CP5 0.9 Savinase 16L 0.7 Lipolase 0.1 Perfume 0.4 Water/salts Up to 100% Example 6 Detergent Granulate Prepared by Non-Spray Drying Method Example 6 provides the formulation of a granulate manufactured according to the present invention by a non-tower route.",
"TABLE 5 Component % w/w Na PAS 13.5 Dobanol 25-7 2.5 STPP 45.3 Na Carbonate 4 Polyacrylamide 2.8 (mol wt 5-6 000 000) Na Silicate 10.1 Minors 1.5 Water Up to 100% Example 7 Isotropic Laundry Liquid Example 6 provides an example of an isotropic laundry liquid according to the present invention.",
"The formulation is given in Table 6: TABLE 6 Component % w/w Na Citrate 10.7 Propylene Glycol 7.5 Ethylene Glycol 4.5 Borax 3 Savinase 16L 0.3 Lipolase 0.1 Dextran (mol wt 800 000) 1.0 Monoethanolamine 0.5 Cocofatty acit 1.7 NaOH (50%) 2.2 LAS 10.3 Dobanol 25-7 6.3 LES 7.6 Minors (adjust pH to 7 white NaOH) 1.3 Water Up to 100% Example 8 Structured Laundry Liquids Example 8 provides an example of a structured laundry liquid according to the present invention.",
"The formulation of the product is given in Table 7.TABLE 7 Component % w/w LAS 16.5 Dobanol 25-7 9 Oleic acid (Priolene (6907)) 4.5 Zeolite 15 KOH, neutralisation of 8.5 acids and pH to Citric acid 8.2 Defloculating Polymer 1 Protease 0.38 Lipolase 0.2 Imacol C (ex Clariant) 2.0 Minors 0.4 Water Up to 100% Example 9 Powder Formulation Example 9 provides an example of a further powder formulation.",
"The composition of which is given in Table 8.TABLE 8 Component % w/w Na LAS 6.3 Nonionic 3EO branched 3.15 Nonionic 7EO branched 4.05 Soap 0.28 sodium tripolyphosphate 23.9 Sodium perborate 20.00 Sodiumsilicate 6.31 Tetra acetyl ethylene diamine 2.22 SCMC 0.23 Sodium sulphate 9.87 Na Carbonate 8.49 sodium polyacrylate 5.0 (mol wt 1 300 000) Dequest 2047 0.55 Sokalan CP5 1.13 Savinase 12 TXT 0.40 Lipolase 0.1 Perfume 0.24 Water/salts Up to 100%"
]
] | Patent_10450077 |
[
[
"Method for producing acrylate adhesive materials",
"A process for preparing pressure sensitive adhesives based on acrylate hotmelt, in which a monomer mixture including at least the following monomers (a) 70 to 100% by weight of compounds from the group of (meth)acrylic acid and the derivatives thereof corresponding to the following general formula is free-radically polymerized in solution wherein I. the polymerization is initiated using at least one dissociating photoinitiator and by irradiation with ultraviolet light, the photoinitiator being added to the monomer mixture before the beginning of the polymerization and/or to the reaction mixture in the course of the polymerization, II.",
"the polyacrylate is freed from the solvent, III.",
"the polyacrylate is processed further in the melt."
],
[
"1.A process for preparing pressure sensitive adhesives based on acrylate hotmelt, in which a monomer mixture including at least the following monomers (a) 70 to 100% by weight of compounds selected from the group consisting of (meth)acrylic acid and the derivatives thereof corresponding to the following formula with R1═H or CH3 and R2=an alkyl chain having 2 to 20 carbon atoms and also including (b) 0 to 30% by weight of olefinically unsaturated monomers containing functional groups and also including (c) if desired, optionally, further components is free-radically polymerized in solution to give a polyacrylate, wherein I. the polymerization is initiated using at least one dissociating photoinitiator and by irradiation with ultraviolet light, the photoinitiator being added to the monomer mixture before the beginning of the polymerization and/or to the reaction mixture in the course of the polymerization, II.",
"the polyacrylate is freed from the solvent, III.",
"the polyacrylate is processed further in the melt.",
"2.The process of claim 1, wherein the at least one photoinitiator is or are used in an amount of from 0.1 to 2% by weight, based on the weight of the monomer mixture.",
"3.The process of claim 1, wherein the irradiation with ultraviolet light is carried out in the form of two or more irradiation cycles and/or pulsed irradiation is carried out.",
"4.The process of claim 1, wherein the free-radical polymerization is conducted at least up to a conversion of 98% of the monomers.",
"5.The process of claim 1, wherein the removal of the solvent is carried out after the polymerization in a twin-screw extruder.",
"6.The process of claim 1, wherein crosslinkers are added to the monomer mixture, to the reaction mixture or to the polyacrylate.",
"7.The process of claim 1, wherein resins and/or additives are added to the monomer mixture, to the reaction mixture or to the polyacrylate.",
"8.The process of claim 1, further comprising the step of applying the pressure sensitive adhesive from the melt to a backing material.",
"9.An adhesive tape provided with a layer of a self-adhesive prepared by the process of claim 1 on one or both sides.",
"10.The process of claim 2, wherein said at least one photoinitiator is used in an amount of between 0.25 and 1% by weight, based on the weight of the monomer mixture.",
"11.The process of claim 6, wherein said crosslinkers are selected from the group consisting of difunctional or polyfunctional acrylates, difunctional or polyfunctional methacrylates, difunctional or polyfunctional isocyanates, difunctional or polyfunctional epoxides, or mixtures thereof.",
"12.The process of claim 7, wherein said resins and/or adhesives are selected from the group consisting of ageing inhibitors, light stabilizers, ozone protectants, fatty acids, plasticizers, nucleating agents, blowing agents, accelerators and fillers."
],
[
"The invention relates to a process for preparing acrylic hotmelts by free-radical addition polymerization and also to the use of such an acrylic hotmelt.",
"Within the field of pressure sensitive adhesives (PSAs), as a result of ongoing technological developments in the coating process, there exists a continuing demand for innovative developments.",
"In industry, hotmelt processes with solvent-free coating technology are of growing importance for the preparation of PSAs, since the environmental impositions are becoming ever greater and the prices of solvents continue to rise.",
"Consequently solvents are to be eliminated as far as possible from the manufacturing operation for PSA tapes.",
"As a result of the associated introduction of the hotmelt technology, the requirements imposed on the adhesives are becoming evermore stringent.",
"Acrylic PSAs in particular are being investigated very intensively with a view to their improvement.",
"For high-grade industrial applications there is a preference for polyacrylates, on account of their transparency and stability to weathering.",
"Besides these advantages, however, these acrylic PSAs must also meet exacting requirements in the areas of shear strength and of bond strength.",
"Acrylic PSAs can be prepared with great individuality.",
"They can be employed very variably, since a large number of different comonomers are available for polymerization and since the adhesive properties of the polyacrylate can be varied through the choice of the (co)monomer composition.",
"Comonomers used include as principal components commonly alkyl esters of acrylic and methacrylic acid, in smaller fractions usually (additionally) acrylic acid, methacrylic acid, acrylamides, maleic anhydride, hydroxy acrylates or itaconic acid.",
"These polyacrylates are prepared using free-radical addition polymerization in solution or in emulsion.",
"Both techniques are subject, among other things, to the problems depicted below, but are very cost-effective and have therefore long been carried out in production.",
"For processing as a hotmelt PSA it is necessary to remove the solvent.",
"For emulsion polymers this is a very energy-intensive operation, since water has a low vapor pressure and is therefore very difficult to evaporate.",
"Moreover, emulsifiers which remain in the polymer are disruptive, raising the sensitivity of the resultant PSA to water.",
"In the case of solution polymers it is desirable to lower the solvent fraction.",
"Here, however, the conventional methods are limited, since the polymerization is normally initiated thermally.",
"During the polymerization it is necessary to remove the developing heat of reaction by way of evaporative cooling.",
"Since the initiators generally used for the polymerization are azo initiators having decomposition half-lives of approximately 1 hour at T>60° C., moreover, fractions of initiator remain in the polymer and are then present even after the concentration operation as well.",
"These residual initiators may give rise to a variety of problems.",
"On the one hand it is possible, in the course of melt coating at very high temperatures, for a large fraction of the residual initiators to decompose.",
"The nitrogen gas which is liberated produces bubbles within the PSA, which are undesirable for the quality of the product.",
"On the other hand, polyfunctional acrylates are added in order to ensure that the acrylic PSA is crosslinkable by electron beams.",
"These acrylates are particularly essential for resin-blended acrylic PSAs.",
"In the course of coating not only is there generation of bubbles by the residual initiators but also the free radicals formed react with the polyfunctional acrylates and so bring about an additional, uncontrolled crosslinking of the acrylic PSA.",
"This gelling, as it is known, has an adverse influence on coating.",
"The partly gelled adhesive can no longer be applied as a coating, and, moreover, relatively solid areas with increased gelling may form in the adhesive, and adversely affect the adhesive properties of the PSA tape.",
"One solution is the UV polymerization of acrylates.",
"U.S. Pat.",
"No.",
"3,840,448 polymerized acrylates and crosslinked them by the irradiation of UV light.",
"The polymers prepared were not, however, used for PSAs.",
"U.S. Pat.",
"No.",
"4,181,752 prepares UV prepolymers which following application as a coating to the backing are polymerized to full conversion and crosslinked.",
"U.S. Pat.",
"No.",
"4,968,558 and U.S. Pat.",
"No.",
"5,183,833 likewise prepared PSAs from acrylates and vinyl compounds by way of the prepolymerization technique.",
"Prepolymerization was practised therein using UV-B lamps in conjunction with Irgacure 651™ (benzil dimethyl ketal, Ciba Geigy).",
"The prepolymerization technique is already very widespread but is also hampered by problems.",
"A high monomer content syrup has to be applied to the backing material; only on the backing material is polymerization carried out to high conversion.",
"Because of this operation, only very slow web speeds can be achieved; moreover, the residual monomer fraction in the adhesive is relatively high.",
"Furthermore, the incorporation of resins causes problems, since crosslinking is generally carried out using UV light and many resins of industrial importance absorb UV light and so act as regulators.",
"It is an object of the invention, therefore, to offer a process for preparing acrylic pressure sensitive adhesives which does not have the abovementioned disadvantages of the prior art.",
"In particular it ought to be possible to achieve a high conversion as early as during polymerization in solution without subsequent gelling processes occurring in the resulting acrylate composition.",
"The adhesive properties of the acrylate composition ought not to fall below a level which is necessary for its technical application.",
"This object is achieved, surprisingly and unforeseeably for the skilled worker, by a process as set out in the main claim.",
"The subclaims relate to advantageous developments of the invention and also to a use for the pressure sensitive adhesives prepared.",
"The invention accordingly provides a process for preparing pressure sensitive adhesives based on acrylate hotmelt, in which a monomer mixture including at least the following monomers (a) 70 to 100% by weight of compounds from the group of the (meth)acrylic esters corresponding to the following general formula with R1═H or CH3 and R2=an alkyl chain having 2 to 20 carbon atoms and also including (b) 0 to 30% by weight of olefinically unsaturated monomers containing functional groups and also including (c) if desired, further components is free-radically polymerized in solution to give a polyacrylate, and in which I. the polymerization is initiated using at least one dissociating photoinitiator and by irradiation with ultraviolet light, the photoinitiator being added to the monomer mixture before the beginning of the polymerization and/or to the reaction mixture in the course of the polymerization, II.",
"the polyacrylate is freed from the solvent.",
"III.",
"the polyacrylate is processed further in the melt.",
"The composition of the corresponding comonomers is preferably chosen so that the resultant PSAs possess pressure sensitive adhesive properties in accordance with D. Satas [Handbook of Pressure Sensitive Adhesive Technology, 1989, Verlag VAN NOSTRAND REINHOLD, New York].",
"The static glass transition temperature of the PSA is therefore below 25° C. For the inventive process for preparing the PSAs the comonomers used (component (b)) include olefinically unsaturated compounds which preferably contain functional groups, with a fraction of 0-30 percent by weight.",
"Examples of such olefinically unsaturated compounds are (meth)acrylic acid and the methyl esters thereof, methacrylic acid derivatives such as (meth)acrylamides, N-substituted (meth)acrylamides, dimethylacrylic acid, trichloroacrylic acid, hydroxyalkyl (meth)acrylate, amino-containing (meth)acrylates, hydroxy-containing (meth)acrylates, with particular preference 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl(meth)acrylate, and/or 4-hydroxybutyl(meth)acrylate, acrylonitrile, and also vinyl compounds such as vinyl esters, vinyl ethers, vinyl halides, vinylidene halides, and nitriles of ethylenically unsaturated hydrocarbons, vinyl compounds with aromatic rings and heterocycles in α position, particularly vinylacetic acid and vinyl acetate, N-vinylformamide, vinylpyridine, ethyl vinyl ether, vinyl chloride, vinylidene chloride, and also maleic anhydride, styrene, styrene compounds, β-acryloyloxypropionic acid, fumaric acid, crotonic acid, aconitic acid and/or itaconic acid; the above list is only exemplary and not conclusive.",
"As photoinitiators it is possible to use all Norrish type I photoinitiators (also referred to hereinbelow for short as type I photoinitiators).",
"The fraction of the photoinitiators, based on the monomers used, is advantageously between 0.1 and 2, preferably between 0.25 and 1 percent by weight.",
"With preference it is possible, for example, to use Irgacure 651™ or Irgacure 819™ (Ciba Geigy).",
"Photoinitiator mixtures as well are very suitable for initiation in the inventive sense.",
"Great preference is given to using photoinitiators with longwave absorption, since these possess a great depth of penetration and therefore penetrate the monomer/polymer mixture more readily.",
"For linear polymerization it is preferred to carry out initiation using a Norrish type I photoinitiator.",
"Norrish type II photoinitiators (type II photoinitiators) cause a greater level of grafting reactions (for the preparation of branched polyacrylates) and are therefore metered in preferably in the course of the UV polymerization.",
"Nevertheless, UV polymerizations can also be initiated using type II photoinitiators.",
"Norrish type I photoinitiators are those compounds which dissociate in accordance with a Norrish type I reaction when irradiated with light.",
"Such a reaction is, conventionally, the photofragmentation of a carbonyl compound in the course of which the bond to a carbon atom located a to the carbonyl group is free-radically cleaved (α cleavage), so forming an acyl radical and an alkyl radical.",
"In the inventive sense, the Norrish photoinitiators also include those in which instead of the carbonyl group another functional group is present and cleavage affects the bond between this group and an a carbon atom.",
"Norrish type II photoinitiators dissociate on irradiation with light in accordance with a Norrish type II reaction involving hydrogen abstraction, which is an intramolecular reaction.",
"In the case of aliphatic ketones it is possible in this instance for a hydrogen to be eliminated from the γ position to one corresponding to the above-depicted functional group.",
"Inventive examples of Norrish photoinitiators of both types are benzophenone, acetophenone, benzil, benzoin, hydroxyalkylphenone, phenyl cyclohexyl ketone, anthraquinone, thioxanthone, triazine or fluorenone derivatives, without wishing to impose any unnecessary restriction by giving this list.",
"The type I initiators include in particular aromatic carbonyl compounds, such as benzoin derivatives, benzil ketals, and acetophenone derivatives.",
"Type II photoinitiators are in particular aromatic ketones, such as benzophenone, benzil or thioxanthones, for example.",
"For further details see, for example, Römpp Lexikon Chemie-Version 2.0, Stuttgart/New York: Georg Thieme Verlag 1999.The polymerization for preparing the acrylic PSA can be conducted in polymerization reactors, which are generally provided with a stirrer, two or more feed vessels, a reflux condenser, heating and cooling and are equipped for operation under N2 atmosphere and superatmospheric pressure.",
"Moreover, a UV irradiation means should be integrated in the reactor.",
"The UV light is advantageously irradiated into the reactor from above.",
"For mixing it is preferred to use a propeller stirrer which ensures vertical mixing.",
"Effective distribution of the radicals formed in the reaction mixture promotes the polymerization, whereas if mixing is poor the polymerization results are likely to be unsatisfactory, owing to the limited depth of penetration of the UV light into the reaction mixture.",
"The polymerization proceeds favorably in accordance with a free-radical mechanism.",
"The monomer mixture is irradiated with UV light preferably under an inert gas atmosphere, such as nitrogen, helium or argon, for example.",
"The free-radical polymerization can be conducted in the presence of one or more organic solvents and/or in the presence of water or in bulk, with preference being given to polymerization in the presence of at least one organic solvent; in that case, it is advantageous to use as little solvent as possible.",
"The polymerization time—depending on conversion and temperature—is between 1 and 24 h. In the case of solution polymerization it is preferred as solvents to use esters of saturated carboxylic acids (such as ethyl acetate), aliphatic hydrocarbons (such as n-hexane or n-heptane), ketones (such as acetone or methyl ethyl ketone), special boiling point spirit or mixtures of these solvents.",
"For the polymerization it is also possible to use thiols, nitroxides, TEMPO derivatives (TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy pyrrolidinyloxyl)) or thioesters of any of a wide variety of kinds as further regulators for lowering molecular weight and reducing polydispersity.",
"Also effective as polymerization regulators, as they are known, are alcohols and ethers.",
"In one very favorable version of the inventive process the irradiation with ultraviolet light is carried out not continuously but instead in the form of one or more irradiation cycles.",
"By means of the irradiation cycles of the UV source it is possible to control the polymerization with regard to the products formed.",
"To control the polymerization it may be appropriate not to irradiate continuously.",
"In that case the sequence of irradiation times and dark times can be chosen advantageously.",
"By means of corresponding irradiation cycles it is possible to control the conversion and hence also the amount of heat produced.",
"The molecular weight distribution as well can be influenced by the irradiation cycle.",
"Particularly advantageous is the irradiation of the monomers to be polymerized with a defined pulse.",
"This method results in the formation only of a very few radicals which are able to react.",
"By means of the UV pulse, accordingly, it is possible to control the number of radicals produced and hence also to adjust the monomer/radical ratio.",
"With a constant ratio of monomer concentration to radical concentration it is possible using this technique to prepare polyacrylate PSAs with a narrow distribution.",
"On the other hand it is also possible very rapidly to generate a high concentration of radicals, which produce a high conversion.",
"UV-initiated polymerizations therefore proceed much more quickly and economically than conventional polymerizations using azobisisobutyronitrile (AIBN) as initiator.",
"A further advantage is the simple process technique.",
"Thermally initiated free radical polymerizations with AIBN, for example, generate heat in the course of the polymerization, as a result of which the dissociation of AIBN is accelerated and the number of free radicals formed increased.",
"In the critical case the polymerization may “run away”, i.e., become uncontrollable.",
"UV polymerization, in contrast, possesses the advantage that by not irradiating the monomer solution no new free radicals are formed and therefore the polymerization can be controlled more easily.",
"Depending on the solvent and the photoinitiator used the wavelength to be irradiated is chosen between 200 and 400 nm.",
"By way of example it is possible to use standard commercial high-pressure or medium-pressure mercury lamps with an output of, for example, 80 to 200 W/cm.",
"The free-radical polymerization is advantageously conducted at least up to a conversion of 98% of the monomers.",
"The polymer is subsequently freed from the solvent in a concentrating extruder.",
"It is preferred here to use a twin-screw extruder operated either counterrotatingly or corotatingly.",
"The volatile fraction remaining in the polymer should amount to less than 0.5 weight fraction.",
"The polymer is preferably concentrated under low shear.",
"Through the process of the invention it is also possible for the concentrating extruder to be operated at high temperatures without formation of free radicals as a result of thermal initiators dissociating.",
"To improve the adhesive properties it is preferred to admix resins to the acrylate hotmelt.",
"Examples of resins which can be used include terpene resins, terpene-phenolic resins, C5- and C9-hydrocarbon resins, pinene resins, indene resins, and rosins, alone and also in combination with one another.",
"In principle, however, it is possible to use any resins which are soluble in the corresponding polyacrylate; reference may be made in particular to all aliphatic, aromatic, and alkylaromatic hydrocarbon resins, hydrocarbon resins based on single monomers, hydrogenated hydrocarbon resins, functional hydrocarbon resins, and natural resins.",
"It is additionally possible to add various extenders (for example, carbon black, TiO2, solid or hollow beads of glass or other materials), nucleating agents, compounding agents, ageing inhibitors, light stabilizers, ozone protectants, fatty acids, plasticizers, nucleating agents, blowing agents, accelerators and/or fillers.",
"For some applications as a pressure sensitive adhesive it can be necessary to crosslink the polymer, particularly for the purpose of increasing the cohesion.",
"For the inventive process it is therefore very advantageous to add crosslinkers to the monomer mixture, to the reaction mixture or to the polyacrylate.",
"As crosslinkers it is possible to use any difunctional or polyfunctional compounds known to the skilled worker, whose functional groups are able to undergo linking reactions with the polyacrylates, particularly addition polymerization reactions, polycondensation reactions or polyaddition reactions.",
"Use is made in particular of difunctional or polyfunctional acrylates and/or methacrylates, difunctional or polyfunctional isocyanates or difunctional or polyfunctional epoxides.",
"For UV or EB curing, polyfunctional acrylates are preferred.",
"The crosslinkers are preferably metered into the acrylic hotmelt in the melt.",
"It is also possible to meter in, or to admix to the acrylate melt, substances which crosslink under UV radiation, such as UV photoinitiators, for example.",
"As photoinitiators it is possible to use benzophenone, acetophenone, benzil, benzoin, hydroxyalkylphenone, phenyl cyclohexyl ketone, anthraquinone, thioxanthone, triazine, or fluorenone derivatives, this list not being conclusive.",
"It is preferred to use type II photoinitiators.",
"It is also possible, furthermore, to admix any promoters known to the skilled worker to the acrylic hotmelt which might make UV crosslinking more efficient.",
"The pressure sensitive adhesive prepared by the process of the invention is suitable with particular advantage for the preparation of, for example, adhesive tapes.",
"For this purpose the acrylic hotmelt is applied from the melt to a backing in the form of the substance as it is or in a form modified as already described.",
"As backing materials, for adhesive tapes, for example, it is possible in this case to use the materials customary and familiar to the skilled worker, such as films (polyesters, PET, PE, PP, BOPP, PVC), nonwovens, foams, woven fabrics and woven films, and also release paper (glassine, HDPE, LDPE).",
"This list is not conclusive.",
"Following application, the crosslinking can then be carried out in the inventive sense, preferably directly on the backing material, preferably by means of UV radiation or by means of ionizing radiation, such as electron beams, for example.",
"In special circumstances it is also possible to carry out thermal crosslinking as well.",
"For UV crosslinking, irradiation takes place in a wavelength range from 200 to 400 nm using standard commercial high-pressure or medium-pressure mercury lamps having an output of, for example, 80 to 200 W/cm.",
"For UV crosslinking it may be appropriate to adapt the lamp output to the web speed or to carry out partial shading of the web in slow travel in order to reduce the thermal load thereon.",
"The irradiation time is guided by the construction and output of the respective lamps.",
"Moreover, it is preferred to carry out irradiation under an inert gas atmosphere.",
"The content of the invention further includes the use of the resultant pressure sensitive adhesive for an adhesive tape, it being possible for the adhesive tape to be provided with a self-adhesive layer on one or both sides.",
"EXAMPLES The invention is illustrated below by examples, without wishing to subject it to any unnecessary restriction as a result.",
"A selection is made of acrylic monomers and vinyl monomers depending on the desired adhesive properties of the acrylic hotmelts.",
"Test Methods The following test methods were employed to evaluate the adhesive properties of the PSAs prepared.",
"Shear Strength (Test A) A strip 13 mm wide of the adhesive tape was applied to a smooth steel surface which had been cleaned three times with acetone and once with isopropanol.",
"The area of application measured 20 mm×13 mm (length×width).",
"The adhesive tape was then pressed onto the steel backing four times with a weight of 2 kg.",
"At room temperature a weight of 1 kg was fastened to the adhesive tape, and the time taken for the weight to fall down was measured.",
"The shear stability times measured are indicated in minutes and correspond to the average of three measurements.",
"180° Bond Strength Test (Test B) A strip 20 mm wide of an acrylic PSA applied to a polyester was applied to steel plates which had been washed beforehand twice with acetone and once with isopropanol.",
"For the measurements on the PE substrate only new plates were used.",
"The PSA strip was pressed down twice onto the substrate using a 2 kg weight.",
"The adhesive tape was then removed immediately from the substrate at a speed of 300 mm/min and at an angle of 180°.",
"All measurements were conducted at room temperature under climatized conditions.",
"The results are reported in N/cm and are averaged from three measurements.",
"Determination of the Gel Fraction (Test C) The carefully dried solvent-free adhesive samples are welded into a pouch of polyethylene nonwoven (Tyvek web).",
"From the difference in the sample weights before and after extraction with toluene the gel value is determined, i.e., the weight fraction of the polymer that is not soluble in toluene.",
"Samples Investigated Commercially available substances used Substance Manufacturer Chemical composition Irgacure 819 Ciba Specialty Chemicals Bis(2,4,6- trimethylbenzoyl)phenyl- phosphine oxide Irgacure 651 Ciba Specialty Chemicals Benzyl dimethyl ketal Vazo 67 DuPont 2,2′-azobis (2-ethylpropiononitrile) Perkadox 16 Akzo Nobel Bis(4-tert-butylcyclohexyl) peroxydicarbonate Genomer 4212 Rahn Aliphatic polyurethane acrylate with a functionality of 2 PETIA UCB Pentaerythritol triacrylate SR 610 Sartomer Polyethylene glycol diacrylate with a degree of polymerization of 14 Norsolene M1080 Cray Valley Partially hydrogenated HC resin DT 110 DRT Terpene-phenolic resin Example 1 A mixture of 40 g of acrylic acid, 360 g of 2-ethylhexyl acrylate and 400 g of acetone was blended with 2 g of Irgacure 819™ (Ciba Geigy) and rendered inert with nitrogen gas for 45 minutes in a conventional 2 I reactor with a propeller stirrer.",
"The polymerization was initiated using a Bluepoint™ UV radiation source (340 nm wavelength, Spectrum).",
"Irradiation was carried out from above into the monomer solution.",
"The irradiation sequence was chosen in accordance with the following program: Time Duration Irradiation sequence Start to 20 min 19.5 min In each case 30 s irradiation, 60 s without irradiation up to 1 h 40 min In each case 30 s irradiation, 30 s without irradiation up to 2 h 60 min In each case 60 s irradiation, 30 s without irradiation The polymer was freed from the solvent in a drying cabinet under reduced pressure at a temperature of 50° C. Thereafter the polymer was coated at a rate of 50 g/m2 onto a Saran-primed PET film 23 μm thick through a slot die heated to 170° C. The PSA had a bubbly appearance.",
"The adhesive tape was subsequently crosslinked with electron beams (dose 15 kGy) with an acceleration voltage of 230 kV using an electron beam unit from Crosslinking.",
"The adhesive properties were analyzed by conducting test methods A and B.",
"Reference Example 2 A 2 I glass reactor conventional for free-radical polymerizations was charged with 40 g of acrylic acid, 360 g of 2-ethylhexyl acrylate and 300 g of acetone/isopropanol (97:3).",
"After nitrogen gas had been passed through the reactor for 45 minutes the reactor was heated to 58° C., with stirring using an anchor stirrer, and 0.2 g of Vazo 67™ (DuPont) was added.",
"The external heating bath was then heated to 75° C. and the reaction was carried out constantly at this external temperature.",
"After reaction times of 1 h and 1.5 h a further 0.2 g of Vazo 67™ was added.",
"After 3 h and 6 h dilution was carried out with in each case 150 g of acetone/isopropanol mixture (97:3).",
"After 16 h the residual initiators were reduced by dropwise addition of 0.4 g of Perkadox 16™ (Akzo Nobel) as a solution in 10 g of acetone.",
"The reaction was terminated after a time of 22 h and the product cooled to room temperature.",
"The polymer was freed from the solvent in a drying cabinet under reduced pressure at a temperature of 50° C. Thereafter the polymer was coated at a rate of 50 g/m2 onto a Saran-primed PET film 23 μm thick through a slot die heated to 170° C. The PSA had a smooth appearance.",
"The adhesive tape was subsequently crosslinked with electron beams (dose 15 kGy) with an acceleration voltage of 230 kV using an electron beam unit from Crosslinking.",
"The adhesive properties were analyzed by conducting test methods A and B.",
"Example 3 A mixture of 4 g of acrylic acid, 32 g of N-tert-butylacrylamide, 4 g of maleic anhydride, 180 g of 2-ethylhexyl acrylate, 180 g of n-butyl acrylate and 400 g of acetone was blended with 2 g of Irgacure 819™ (Ciba Geigy) and rendered inert with nitrogen gas for 45 minutes in a conventional 2 I reactor having a propeller stirrer.",
"The polymerization was initiated using a Bluepoin™ UV radiation source (340 nm wavelength, Spectrum).",
"Radiation was carried out from above into the monomer solution, proceeding in analogy to the irradiation program in Example 1.The polymer was blended in acetone with 30 weight fractions of DT 110™ (DRT) and 1 weight fraction of SR 610 (Sartomer) and was freed from the solvent in a drying cabinet under reduced pressure at a temperature of 50° C. Thereafter the polymer was coated at a rate of 100 g/m2 to a Saran-primed PET film 23 μm thick via a slot die heated to 170° C. The adhesive tape was subsequently crosslinked with electron beams (dose 50 kGy) at an acceleration voltage of 230 kV using an electron beam unit from Crosslinking.",
"The adhesive properties were analyzed by conducting test methods A and B.",
"In parallel thereto a specimen was stored in the drying cabinet at 140° C. for 8 hours and then test method C was carried out.",
"Reference Example 4 A 2 I glass reactor conventional for free-radical polymerizations was charged with 40 g of acrylic acid, 360 g of 2-ethylhexyl acrylate and 300 g of acetone/isopropanol (97:3).",
"After nitrogen gas had been passed through the reactor for 45 minutes the reactor was heated to 58° C., with stirring using an anchor stirrer, and 0.2 g of Vazo 67™ (DuPont) was added.",
"The external heating bath was then heated to 75° C. and the reaction was carried out constantly at this external temperature.",
"After reaction times of 1 h and 1.5 h a further 0.2 g of Vazo 67™ was added.",
"After 3 h and 6 h dilution was carried out with in each case 150 g of acetone/isopropanol mixture (97:3).",
"After 16 h the residual initiators were reduced by dropwise addition of 0.4 g of Perkadox 16™ (Akzo Nobel) as a solution in 10 g of acetone.",
"The reaction was terminated after a time of 22 h and the product cooled to room temperature.",
"The polymer was blended in acetone with 30 weight fractions of DT 110™ (DRT) and 1 weight fraction of SR 610 (Sartomer) and was freed from the solvent in a drying cabinet under reduced pressure at a temperature of 50° C. Thereafter the polymer was coated at a rate of 100 g/m2 onto a Saran-primed PET film 23 μm thick through a slot die heated to 170° C. The adhesive tape was subsequently crosslinked with electron beams (dose 15 kGy) with an acceleration voltage of 230 kV using an electron beam unit from Crosslinking.",
"The adhesive properties were analyzed by conducting test methods A and B.",
"In parallel thereto a specimen was stored in the drying cabinet at 140° C. for 8 hours and then test method C was carried out.",
"Example 5 The procedure of Example 3 was repeated.",
"The polymer prepared by UV polymerization was blended with 30 weight fractions of Norsolene M1080™ (Cray Valley) and 1 weight fraction of Genomer 4212™ (Rahn).",
"Reference Example 6 The procedure of Example 4 was repeated.",
"The polymer prepared by UV polymerization was blended with 30 weight fractions of Norsolene M1080™ (Cray Valley) and 1 weight fraction of Genomer 4212™ (Rahn).",
"Example 7 The procedure of Example 3 was repeated.",
"UV polymerization was carried out using 2% of acrylic acid, 49% of 2-ethylhexyl acrylate and 49% of n-butyl acrylate.",
"The polymer was blended with 30 weight fractions of Norsolene M1080™ (Cray Valley) and 1 weight fraction of PETIA™ (UCB).",
"Reference Example The procedure of Example 4 was repeated.",
"UV polymerization was carried out using 2% of acrylic acid, 49% of 2-ethylhexyl acrylate and 49% of n-butyl acrylate.",
"The polymer was blended with 30 weight fractions of Norsolene M1080™ (Cray Valley) and 1 weight fraction of PETIA™ (UCB).",
"Results Comparison of Examples 1 and 2 illustrates that residual initiators which remain in the polymer, such as Vazo 67™ from Du Pont, for example, produced constituents in the PSA which dissociate at high temperatures in the course of the coating operation and so give rise to nitrogen bubble inclusions.",
"Through UV-initiated polymerization this can be ruled out (see Example 1).",
"As a result of the UV-initiated polymerization, moreover, a different molecular weight and also a different molecular weight distribution are achieved.",
"This is reflected in the different adhesive properties.",
"Moreover, in Example 2, the adhesive properties are also adversely affected by the bubbly appearance (see Table 1).",
"TABLE 1 BS steel SST, 10 N EB dose Example [N/cm] [min] [kGy] 1 3.8 >10,000 15 2 3.9 6,795 15 50 g/m2 application rate SST: Shear stability time at RT BS: Bond strength Example 1 exhibits a much higher shear strength for the same crosslinking dose.",
"In Examples 3 to 8 the gelling behavior due to the residual initiators was investigated.",
"Investigations were carried out exclusively on polymers blended with resin.",
"Additionally, different polyfunctional acrylates were added in order to ensure electron beam crosslinkability.",
"For each example, as a reference, a polymer prepared conventionally by a thermally free-radical polymerization was executed.",
"All examples were first subjected to thermal storage, and then the gel value was measured.",
"The results are listed in Table 2.TABLE 2 Gel value Example [%] 3 0 4 5 5 0 6 8 7 0 8 14 Table 2 shows that for all of the UV-initiated polymerizations, independently of the resin used or of the crosslinker, no gel fraction could be detected.",
"The reference specimens prepared in comparison thereto, in contrast, had a gel fraction of 5 to 14%—a fraction which does not allow clean coating from the melt in the hotmelt operation.",
"Furthermore, the adhesive properties of the specimens prepared were compared with one another after the coating operation in Table 3.TABLE 3 BS steel SST, 10 N EB dose Example [N/cm] [min] [kGy] 3 11.4 5,145 50 4 12.1 3,320 50 5 9.4 4,765 50 6 10.1 2,920 50 7 10.2 3,005 40 8 10.6 1,855 40 100 g/m2 application rate SST: Shear stability time at RT BS: Bond strength There is hardly any variation in the adhesive properties of the examples depicted.",
"The specimens prepared by UV-initiated polymerization possess a somewhat higher shear strength.",
"On the other hand, the bond strength is situated on average at an only slightly lower level."
]
] | Patent_10450365 |
[
[
"Dual-pivot steering system and method",
"A system for guided travel over a surface between parallel guide rails includes a first frame including at least one rotatable element for travel over the surface; a first pin connected to the first frame, for rotation of the first frame thereon; a second frame including at least one rotatable element for travel over the surface; a second pin connected to the second frame, for rotation of the second frame thereon; and a base connected to the first pin and the second pin.",
"The first frame and the second frame each include guide wheels for tracking along the respective guide rails and between them.",
"The base can provide transit for people or products."
],
[
"1.A system for guided travel over a surface between parallel guide rails, comprising: a first frame including at least one rotatable element for travel over the surface; a first pin connected to the first frame, for rotation of the first frame thereon; a second frame including at least one rotatable element for travel over the surface; a second pin connected to the second frame, for rotation of the second frame thereon; and a base connected to the first pin and the second pin.",
"2.The system of claim 1, wherein the first frame and the second frame each travel retained between the guide rails.",
"3.The system of claim 1, wherein the guide rails are substantially perpendicular to the surface.",
"4.The system of claim 3, wherein the first frame and the second frame each travel retained between the guide rails.",
"5.The system of claim 4, further comprising a guide wheel for tracking along the guide rail, wherein the guide wheel is rotatingly attached to the first frame or the second frame.",
"6.A transport system for travel over a surface, comprising: a wheel for travel on the surface; a frame on which the wheel is rotatingly fixed; a guide fixed to the surface; and a roller, rotatingly fixed to the frame, for travel in close proximity to and along the guide.",
"7.The transport system of claim 6, further comprising: a second wheel for travel on the surface, rotatingly affixed to the frame.",
"8.The transport system of claim 7, further comprising: a second guide fixed to the surface in substantially parallel relationship to the first guide; and a second roller, rotatingly fixed to the frame for travel in close proximity to and along the second guide.",
"9.The system of claim 8, wherein the first roller and the second roller travel between the first and second guide rails.",
"10.The system of claim 9, further comprising a second frame, wherein the second frame includes a first and second roller, each rotatingly fixed to the second frame and traveling along respective ones of the first and second guide rails.",
"11.The system of claim 10, further comprising: a vehicle structure, connected independently to each of the first frame and the second frame.",
"12.The system of claim 11, further comprising: a first pin fixedly connected to the vehicle structure, and pivotally affixed to the first frame.",
"13.The system 12, further comprising: a second pin fixedly connected to the vehicle structure, and pivotally affixed to the second frame.",
"14.The system of claim 13, wherein the first pin and the second pin are centrally locatable between the first and second guides during transit.",
"15.A method of transit of a vehicle, comprising the steps of: providing a first guided wheel; providing a second guided wheel; and pivoting the first guided wheel and the second guided wheel with respect to the vehicle.",
"16.The method of claim 15, wherein the first guided wheel and the second guided wheel travel along substantially parallel to a guide.",
"17.A method of transit, comprising the steps of: providing a frame having a first pin and a second pin longitudinally displaced; providing a first guide, pivotally connected to the first pin; and providing a second guide pivotally connected to the second pin.",
"18.The method of claim 17, further comprising the steps of: providing a first wheel rotatingly connected to the first guide; and providing a second wheel rotatingly connected to the second guide.",
"19.The method of claim 18, wherein the first wheel and the second wheel travel across a surface during transit.",
"20.The method of claim 19, wherein the first wheel and the second wheel are retained between longitudinally parallel guides along a desired path of the transit; and wherein the first wheel and the second wheel each travel between the guides during transit."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention generally relates to transport vehicles and, more particularly, relates to guided mass transit vehicles having dual-pivot steering mechanisms.",
"Mass transit trains and other guided transit vehicles, such as subways and the like, operate in certain large cities and metropolitan areas in the U.S. and throughout the world.",
"Additional locations would, no doubt, be benefited by mass transport services.",
"Costs are quite substantial to acquire land, build tracks and infrastructure, and operate transport vehicles.",
"Thus, many locations forego, or do not have the ability and resources required to implement and provide, transit services.",
"Prior mass transit systems have typically been built in underground tunnels (e.g., as subways), as overhead railways, or as street-operated buses.",
"Each of these systems is problematic because of expense.",
"Moreover, each of the systems has certain requirements and peculiarities for construction and operations that make it physically and technically impractical, if not impossible in many cases.",
"Additionally, although most locations have railway and surface street facilities and access in place, the prior transit systems have only been capable of limited cross-use of the facilities and access.",
"For example, buses use city streets, however, the buses must typically conform to traffic flows of regular car and vehicle traffic.",
"Few, if any, buses operate in designated lanes and transit space.",
"The buses can be unwieldy and, in any event, further clog the normal vehicular traffic.",
"As another example, trains use rail systems.",
"These rail systems typically are limited to certain portions of cities or areas; therefore, the rails can not themselves provide general transit system access.",
"Additionally, as to tunnel and overhead transit systems, the transit vehicles typically operate only in dedicated lines or space.",
"However, these systems require special and costly infrastructure because of the dedicated usage, including land and space availability concerns.",
"It would be a significant improvement in the art and technology to provide systems and methods for mass transit that make best use of existing facilities and systems otherwise used for other purposes.",
"Additionally, it would be an improvement to provide such systems and methods operable among and between the conventional facilities and systems.",
"Moreover, it would be a significant improvement in the art and technology to limit costs, infrastructure, space usage, and other normal requirements for erection and operation of mass transit systems.",
"The present invention provides numerous advantages and improvements, including, for example, limited costs, use of existing infrastructures, minimization of land and space dedication and requirements, and other advantages.",
"The present invention further operates consistently, smoothly, and in superior respects to the conventional systems."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>An embodiment of the invention is a system for guided travel over a surface between parallel guide rails.",
"The system includes a first frame including at least one rotatable element for travel over the surface, a first pin connected to the first frame, for rotation of the first frame thereon, a second frame including at least one rotatable element for travel over the surface, a second pin connected to the second frame, for rotation of the second frame thereon, and a base connected to the first pin and the second pin Another embodiment of the invention is a transport system for travel over a surface.",
"The system includes a wheel for travel on the surface, a frame on which the wheel is rotatingly fixed, a guide fixed to the surface, and a roller, rotatingly fixed to the frame, for travel in close proximity to and along the guide.",
"Yet another embodiment of the invention is a method of transit of a vehicle.",
"The method includes providing a first guided wheel, providing a second guided wheel, and pivoting the first guided wheel and the second guided wheel with respect to the vehicle.",
"Another embodiment of the invention is a method of transit.",
"The method includes providing a frame having a first pin and a second pin longitudinally displaced, providing a first guide, pivotally connected to the first pin, and providing a second guide pivotally connected to the second pin."
],
[
"BACKGROUND OF THE INVENTION The present invention generally relates to transport vehicles and, more particularly, relates to guided mass transit vehicles having dual-pivot steering mechanisms.",
"Mass transit trains and other guided transit vehicles, such as subways and the like, operate in certain large cities and metropolitan areas in the U.S. and throughout the world.",
"Additional locations would, no doubt, be benefited by mass transport services.",
"Costs are quite substantial to acquire land, build tracks and infrastructure, and operate transport vehicles.",
"Thus, many locations forego, or do not have the ability and resources required to implement and provide, transit services.",
"Prior mass transit systems have typically been built in underground tunnels (e.g., as subways), as overhead railways, or as street-operated buses.",
"Each of these systems is problematic because of expense.",
"Moreover, each of the systems has certain requirements and peculiarities for construction and operations that make it physically and technically impractical, if not impossible in many cases.",
"Additionally, although most locations have railway and surface street facilities and access in place, the prior transit systems have only been capable of limited cross-use of the facilities and access.",
"For example, buses use city streets, however, the buses must typically conform to traffic flows of regular car and vehicle traffic.",
"Few, if any, buses operate in designated lanes and transit space.",
"The buses can be unwieldy and, in any event, further clog the normal vehicular traffic.",
"As another example, trains use rail systems.",
"These rail systems typically are limited to certain portions of cities or areas; therefore, the rails can not themselves provide general transit system access.",
"Additionally, as to tunnel and overhead transit systems, the transit vehicles typically operate only in dedicated lines or space.",
"However, these systems require special and costly infrastructure because of the dedicated usage, including land and space availability concerns.",
"It would be a significant improvement in the art and technology to provide systems and methods for mass transit that make best use of existing facilities and systems otherwise used for other purposes.",
"Additionally, it would be an improvement to provide such systems and methods operable among and between the conventional facilities and systems.",
"Moreover, it would be a significant improvement in the art and technology to limit costs, infrastructure, space usage, and other normal requirements for erection and operation of mass transit systems.",
"The present invention provides numerous advantages and improvements, including, for example, limited costs, use of existing infrastructures, minimization of land and space dedication and requirements, and other advantages.",
"The present invention further operates consistently, smoothly, and in superior respects to the conventional systems.",
"SUMMARY OF THE INVENTION An embodiment of the invention is a system for guided travel over a surface between parallel guide rails.",
"The system includes a first frame including at least one rotatable element for travel over the surface, a first pin connected to the first frame, for rotation of the first frame thereon, a second frame including at least one rotatable element for travel over the surface, a second pin connected to the second frame, for rotation of the second frame thereon, and a base connected to the first pin and the second pin Another embodiment of the invention is a transport system for travel over a surface.",
"The system includes a wheel for travel on the surface, a frame on which the wheel is rotatingly fixed, a guide fixed to the surface, and a roller, rotatingly fixed to the frame, for travel in close proximity to and along the guide.",
"Yet another embodiment of the invention is a method of transit of a vehicle.",
"The method includes providing a first guided wheel, providing a second guided wheel, and pivoting the first guided wheel and the second guided wheel with respect to the vehicle.",
"Another embodiment of the invention is a method of transit.",
"The method includes providing a frame having a first pin and a second pin longitudinally displaced, providing a first guide, pivotally connected to the first pin, and providing a second guide pivotally connected to the second pin.",
"BRIEF DESCRIPTION OF THE DRAWINGS The present invention is illustrated by way of example and not limitation in the accompanying figures, in which like references indicate similar elements, and in which: FIG.",
"1 illustrates a top view of a pivot steering mechanism for a transit system, according to certain embodiments of the invention; FIG.",
"2 illustrates an end view, along line A-A′ of FIG.",
"1, of the pivot steering mechanism for a transit system, according to certain embodiments of the invention; and FIG.",
"3 illustrates a top view of a transit vehicle foundation, including dual ones of the pivot steering mechanism for a transit system, according to certain embodiments of the invention.",
"DETAILED DESCRIPTION Referring to FIG.",
"1, a pivot steering mechanism 100 includes an axle 102 and two wheels 104a, 104b.",
"The wheels 104a, 104b are rotatingly fixed at respective ends of the axle 102.The axle 102 is maintained within an axle housing 106.The axle housing 106 includes a central, longitudinal void for holding the axle 102 in a freely rotating manner within the void.",
"Although the axle housing 106 is shown as a single element in FIG.",
"1, the axle housing 106 is and can include any device for affixing the axle 102 in rotational relationship to any structures fixed to the axle housing 106.The axle housing 106 is pivotally fixed to struts 108a, 108b at or near respective ends of the axle housing, close to the respective wheels 104a, 104b.",
"The struts 108a, 108b pivot with the axle housing 106 at the pivot points 110a, 110b, respectively.",
"The struts 108a, 108b have another end not fixed via the pivot points 110a, 110b to the axle housing 106.Each other end of each of the struts 108a, 108b is fixed at respective ends of a guide bar 112.The struts 108a, 108b pivot at the pivot points 114a, 114b, respectively, in relation to the guide bar 112.The guide bar 112 is fixed or incorporated with a center point pivot 120.The center point pivot 120 is a centrum for rotational movement of the entire combination of the wheels 104a, 104b, axle 102, axle housing 106, struts 108a, 108b, and guide bar 112 thereabout.",
"The guide bar 112 is fixed to the center point pivot 120 at a midway location of the length of the guide bar 112.The guide bar 112 is, thus, rotates around the point pivot 120 such that each half of the guide bar 112 is a spoke about the center point pivot 120.The wheels 104a, 104b, axle 102, axle housing 106, and struts 108a, on the other hand, are maintained in relative relation about the pivot points 110a, 110b and 114a, 114b and are circularly rotatable around the center point pivot 120 always some length away from the location of the center point pivot 120.In operations, the guide bar 112 rotates around its midway at the center point pivot 120.The struts 108a, 108b, in cooperation with the axle housing 106 and the axle 102 contained within the axle housing 106, causes the axle 102 to remain substantially perpendicular to the direction of travel of arrow X.",
"This operation causes the wheels 104a, 104b to roll on the axle 102 in substantially parallel direction to the arrow X of travel of the mechanism 100.Continuing to refer to FIG.",
"1, the axle housing 106 is fixedly connected to parallel bars 122a, 122b.",
"The parallel bars 122a, 122b are fixed in perpendicular relation to the axle housing 106.The parallel bars 122a, 122b extend along either side of the center point pivot 120.As the axle housing 106 moves rotationally around the center point pivot 120, the parallel bars 122a, 122b likewise rotate around the center point pivot 120 and maintain the perpendicular relation to the axle housing 106 and the parallel relation of the bars 122a, 122b across opposing sides to the center point pivot 120.The parallel bars 122a, 122b are substantially the same length, and extend sufficiently laterally beyond the wheels 104a, 104b.",
"At an end of the parallel bars 122a, 122b, extending beyond the axle housing 106 on a same side of the center point pivot 120, a front frame 130 is fixed to the parallel bars 122a, 122b.",
"The front frame 130 is connected to guide wheel housings 132a, 132b, at each end of the front frame 130.The guide wheel housings 132a, 132b fixed with the front frame 130 extend at least beyond a width of the wheels 104a, 104b in relation to the center point pivot 120.The guide wheel housings 132a, 132b each support a respective guide wheel 134a, 134b rotatingly affixed thereto.",
"Each guide wheel 134a, 134b is a substantially round wheel centered and mounted with a vertical (outward from the page) rotational axis 136a, 136b.",
"The guide wheels 134a, 134b each rotate around the respective rotational axis 136a, 136b.",
"Whereas the wheels 104a, 104b travel along a surface, such as the ground, the guide wheels 134a, 134b can travel along a guide (hereafter further detailed) perpendicularly to the surface on which the wheels 104a, 104b travel.",
"At another end of the parallel bars 122a, 122b, a rear frame 140 is fixed perpendicular to the parallel bars 122a, 122b.",
"The rear frame 140 is attached at its ends with respective guide wheel housings 142a, 142b.",
"Like the guide wheel housings 134a, 134b, the guide wheel housings 142a, 142b are each affixed with a rotation axis 144a, 144b, rising vertically (i.e., upward in the page of FIG.",
"1).",
"Each respective rotation axis 144a, 144b is fixed with a guide wheel 146a, 146b that rotates on the rotation axis 144a.",
"Like the guide wheels 134a, 134b, the guide wheels 146a, 146b can travel along a guide perpendicularly to the surface on which the wheels 104a, 104b travel.",
"Referring to FIG.",
"2, a rear view along line A-A′ of FIG.",
"1 shows the mechanism 100 and relative orientation of the wheels 104a, 104b and the guide wheels 146a, 146b.",
"Although not shown in detail in the Figures, a front view of the mechanism 100 would also show a substantially similar relative orientation of the wheels 104a, 104b and the guide wheels 134a, 134b.",
"The rear frame 140 is substantially horizontal with a surface on which the wheels 104a, 104b can roll.",
"The parallel bars 122a, 122b, and the respective guide wheel housings 142a, 142b fixed to the rear frame 140, each rotate around the center point pivot 120 for the mechanism 100.Referring to FIG.",
"3, a transit vehicle 300 includes two opposingly configured pivot steering mechanisms 100a, 100b.",
"Each pivot steering mechanism 100a, 100b is fixed at its center point pivot 120 to a vehicle base 302.The vehicle base 302 is a chassis or other foundation atop which can be fixed a vehicle housing (not shown in figure), such as a passenger compartment.",
"The vehicle base 302 extends a length beyond each mechanism 100a, 100b.",
"Each mechanism 100a, 100b is located under the vehicle base 302, and the vehicle base 302 (and any vehicle housing fixed to it) is fixed with each mechanism 100a, 100b via the respective center point pivot 120.Respective and parallel extending guides 304a,b form a guided path in which the entire structure of the transit vehicle 300 can travel.",
"The guides 304a,b extend upward from a travel surface on which the wheels 104a,b of each mechanism 100a, 100b can travel.",
"For example, if the wheels 104a, 104b travel along a relatively horizontal ground surface, the guides 304a,b are rails extending along the surface and forming an upwardly projecting side.",
"The adjacent and upwardly projecting sides of the guides 304a,b serve to retain the respective guide wheels 134a, 134b and 146a, 146b of each of the mechanisms 100a, 100b between the guides 304a,b.",
"In operation, the center point pivot 120 of each mechanism 100a,b is retained in approximately a center of the travel path formed by the guide rails 304a,b.",
"If and when the guide rails 304a,b vary from exact straight extension, the mechanisms 100a, b are maintained between the guide rails 304a,b.",
"Nonetheless, the vehicle base 302 need not be bendable or otherwise jointed in order to continue in the path formed between the guide rails 304a,b.",
"Moreover, the respective guide wheels 134a, 134b and 146a, 146b of each mechanism 100a, 100b can continue along the guide rails 304a, 304b of the path, because each mechanism 100a, 100b is able to rotate laterally in relation to the vehicle base 302, via the respective center pivot point 120.Furthermore as to operation, the entire transit vehicle 300 is automatically steered and guided in an appropriate path by the guide rails 304a, 304b forming the travel path.",
"The wheels 104a, 104b of each mechanism 100a,b are aligned to rotatingly travel along the appropriate path, because the wheels 104a, 104b are automatically aligned therein in travel, as the guide wheels 134a,b and 146a,b travel within and along the guide rails 304a,b.",
"The double pivot arrangement of the vehicle 300 permits travel along any path by the vehicle, corresponding to the appropriate and desired path formed via the guide rails 304a,b.",
"The steering provided by the foregoing will permit travel of the vehicle 300 along most any desired path, including along conventional mass transit roads and the like.",
"Moreover, the vehicle 300 is not restricted to travel on any railway or other particular surface.",
"The wheels 140a,b of the vehicle 300 can be regular tires or other round wheels.",
"If the vehicle 300 is incorporated with a manual or other steering assemblage, the vehicle can travel along as so steered.",
"In such instance, the guide rails 304a,b would not be limiting.",
"Moreover, the vehicle 300 can be equipped with a drive train and engine or motor.",
"In such instance, the vehicle 300 can automatically travel as driven, either along a path formed by guide rails 304a,b or along any other path provided there is some other steering assemblage or path.",
"In any event, the vehicle 300 is not limited to operations of travel via or within guide rails 304a,b or any other particular path.",
"On the other hand, the dual mechanism 100a,b arrangement and center point pivot 120 thereof, allows any type of vehicle base 302 (and vehicle housing) to effectively travel within a guide rail 304a,b path.",
"For other examples and alternatives, the Exhibit A hereto and incorporated herein includes additional features and concepts.",
"All such examples and alternatives, together with the features and concepts, are included herein and in the invention.",
"In the foregoing specification, the invention has been described with reference to specific embodiments.",
"However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the present invention as set forth in the claims below.",
"Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the present invention.",
"Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments.",
"However, the benefits, advantages, solutions to problems and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or element of any or all the claims.",
"As used herein, the terms “comprises, “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus."
]
] | Patent_10450376 |
[
[
"Dual polarisation antenna",
"A dual polarisation antenna comprising: one or more radiating elements (20); a pair of side walls (11,12) arranged on opposite sides of the radiating element(s); and one or more conductive isolating elements (44, 51, 70, 40, 41), each isolating element being at least partially supported by one or both of the side walls."
],
[
"1.A dual polarisation antenna comprising: one or more radiating elements; a pair of side walls arranged on opposite sides of the radiating element(s); and one or more conductive isolating elements, each isolating element being at least partially supported by one or both of the side walls.",
"2.An antenna according to claim 1 wherein at least one of the isolating elements is supported by one of the side walls only.",
"3.An antenna according to claim 2 wherein the isolation element is a substantially rectangular tab.",
"4.An antenna according to claim 2 wherein the isolation element subtends an angle of less than 180 degrees with the inner face of its supporting side wall.",
"5.An antenna according to claim 1 wherein at least one of the isolation elements is formed integrally with its supporting side wall(s).",
"6.An antenna according to claim 1 wherein at least one of the isolation elements subtends an angle between 80 and 100 degrees with the inner face of its supporting side wall.",
"7.An antenna according to claim 1 wherein at least one of the isolating elements is supported by both side walls.",
"8.An antenna according to claim 1 including an insulating element arranged between an isolating element and its supporting side wall.",
"9.An antenna according to claim 8 wherein the insulating element passes through a hole formed in the supporting side wall.",
"10.An antenna according to claim 1, comprising two or more radiating elements, each radiating element having first and second opposite sides, and third and fourth opposite sides; and three or more isolating elements, wherein the side walls and isolating elements are positioned such that each radiating element faces a side wall on its first side, a side wall on its second side, an isolating element on its third side, and an isolating element on its fourth side.",
"11.An antenna according to claim 1 wherein at least one of the isolating elements comprises a rod with a substantially circular cross-section.",
"12.An antenna according to claim 1 wherein at least one of the isolating elements is fully supported by one or both of the side walls.",
"13.An antenna according to claim 1 wherein the side walls and radiating elements are arranged in front of a planar reflector.",
"14.An antenna according to claim 1 wherein at least one of the isolating elements comprises a wall.",
"15.An antenna according to claim 14 wherein the wall is connected on a first side to the planar reflector, on a second side to one side wall, and on a third side to the other side wall.",
"16.An antenna according to claim 1 wherein each radiating element is a patch.",
"17.An antenna according to claim 1 wherein the side walls are substantially continuous.",
"18.An antenna according to claim 1 comprising a plurality of radiating elements.",
"19.An antenna according to claim 18 further comprising one or more phase shifters for generating a relative phase difference between two or more of the radiating elements.",
"20.An antenna according to claim 18 wherein at least one of the isolating elements is positioned between adjacent radiating elements.",
"21.An antenna according to claim 1 wherein at least one of the isolating elements is aligned with a respective radiating element.",
"22.An antenna according to claim 1 wherein at least one of the isolating elements is formed with a ridge or trough between the two side walls.",
"23.A dual polarisation antenna comprising: one or more radiating elements; a pair of side walls arranged on opposite sides of the radiating element(s); and one or more walls transverse to the side walls, wherein the or each transverse wall comprises a conductive sheet which is folded at each side to form a pair of connection walls, and wherein each connection wall is secured to a respective side wall."
],
[
"The present invention relates to a dual polarisation antenna.",
"A conventional dual polarisation antenna is described in U.S. Pat.",
"No.",
"6,072,439.A pair of side walls are arranged on opposite sides of a line of six crossed-dipole radiating elements.",
"In one embodiment the side walls are C-shaped in cross-section and have edges which create a diffraction pattern that increases the beamwidth by approximately ten degrees compared to similar antennas with no side walls.",
"In another embodiment the side walls are L-shaped in cross-section and narrow the 3 dB beamwidth of the antenna compared to similar antennas with no sidewalls.",
"A single parasitic isolation element is located midway along the length of the line of dipole radiating elements.",
"An alternative antenna arrangement is described in U.S. Pat.",
"No.",
"5,952,983.A line of three cross-dipole radiating elements is provided, with a parasitic element between two of the radiating elements.",
"In one arrangement, the parasitic element is inserted into a groove formed along the top edge of a non-conducting support which extends transversely across the array and is attached to the back plane.",
"In another arrangement, the parasitic element is supported and elevated by pairs of rod supports.",
"In U.S. Pat.",
"No.",
"5,940,044, isolation elements in the form of isolation plates of conductive material are disposed between each dipole sub-array.",
"The isolation plates are connected to the back plate by suitable fasteners.",
"Various different isolation devices are described in U.S. Pat.",
"No.",
"6,028,653, including isolation trees or bars arranged between bow tie radiating assemblies; isolation rails arranged alongside bow tie assemblies; rods or wires arranged in or on a radome that covers the bow tie assemblies; isolation strips arranged between a positive and negative arm of a dipole of a bow tie assembly; or a combination of one or more of the above.",
"An object of the invention is to provide an alternative antenna construction incorporating side walls and one or more isolating elements.",
"The present invention provides a dual polarisation antenna comprising: one or more radiating elements; a pair of side walls arranged on opposite sides of the radiating element(s); and one or more conductive isolating elements, each isolating element being supported by one or both of the side walls.",
"The invention provides an alternative method of supporting the isolating element(s).",
"In contrast to the prior art discussed above, which uses the back plane to support the isolating element(s), we use the side walls to fully or at least partially support the isolating element(s).",
"The side walls modify the beam width of the antenna (compared to a similar antenna with no side walls), and the isolating element(s) improve isolation between the two polarisation ports of the antenna.",
"By using the side walls to support the isolating element(s) we reduce the number of parts compared to U.S. Pat.",
"No.",
"5,952,983, which requires separate members to support the isolating element.",
"There may be a continuous conductive connection between the isolating element and the side wall(s).",
"For instance the isolating element may be soldered to one or both of the side walls, or the isolating element and its supporting side wall(s) may be formed integrally from a single piece of conductive material.",
"However a problem with a conductive connection is that in some circumstances, intermodulation distortion may occur at the joint between the element and its supporting side wall(s).",
"Therefore in some embodiments an insulating element is arranged between an isolating element and its supporting side wall.",
"The insulating element may be a strip of tape, or may be an element such as a rivet which passes through a hole in the supporting side wall.",
"Preferably, the antenna comprises: two or more radiating elements, each radiating element having first and second opposite sides, and third and fourth opposite sides; and three or more conductive isolating elements, wherein the side walls and isolating elements are positioned such that each radiating element faces a side wall on its first side, a side wall on its second side, an isolating element on its third side, and an isolating element on its fourth side.",
"This provides a more symmetrical arrangement than the arrangement in U.S. Pat.",
"No.",
"6,072,439.We have found that this added symmetry improves isolation between the two polarisation ports of the antenna.",
"At a minimum (that is, in the case where only two radiating elements are provided) only three isolating elements are required—one between the radiating elements and one at each end.",
"In the more general case, (that is, where n radiators are provided) n+1 isolating elements will typically be provided (although in some arrangements only n−1 may be required, with the isolating elements at each end omitted).",
"Typically the radiating elements are arranged in front of a planar reflector.",
"The isolating elements may be partially supported by the reflector (either directly or via an insulating element), or may be fully supported by one or both of the side walls.",
"The isolating elements may be supported by one of the side walls only.",
"In this case, the element is typically formed as a substantially rectangular tab.",
"The element is preferably directed inwardly: that is, the element subtends an angle of less than 180 degrees with the inner face of its supporting side wall.",
"Alternatively, the isolating elements(s) may be supported by both side walls.",
"In one embodiment at least one of the isolating element(s) comprises a rod with a substantially circular cross-section.",
"In another embodiment at least one of the isolating element(s) comprises a wall, which may be connected to the back reflector as well as the two side walls.",
"In another embodiment at least one of the isolating element(s) comprises a strip which is substantially rectangular in cross-section.",
"In one embodiment the isolating element is formed with a ridge or trough between the two side walls.",
"Preferably the ridge or trough is formed by bending a strip of metal.",
"The radiating elements may be dipoles, as in U.S. Pat.",
"No.",
"5,952,983.However, a problem with using dipoles is that they are relatively tall, and therefore the isolating element(s) need(s) to be mounted some distance away from the back reflector.",
"For this reason, in U.S. Pat.",
"No.",
"5,952,983 the isolating rods are mounted on rod supports.",
"Therefore preferably the radiating elements are patches.",
"Patches generally have a lower profile than dipoles, thus enabling the isolating element(s) to be supported by a side wall at a lower position.",
"Typically the side walls are substantially continuous (that is—with no slots or holes formed in them).",
"Typically the side walls are postioned to influence the azimuthal beamwidth of the antenna, for instance to provide an azimuthal beamwidth of 65 degrees.",
"Typically a plurality of radiating elements are provided, for instance eight.",
"One or more phase shifters may be provided to generate relative phase differences between the elements, for instance to control beam downtilt in a cellular communication system which communicates with mobile devices.",
"The isolating element(s) may be placed between adjacent radiating elements, and/or aligned with respective radiating elements.",
"Three embodiments of the invention will now be described with reference to the accompanying drawings, in which: FIG.",
"1 is an isometric exploded view of an antenna.",
"FIG.",
"2 is an isometric view of the patch tray assembly shown in FIG.",
"1.FIG.",
"2a is a plan view of the patch tray assembly.",
"FIG.",
"3 is an enlarged isometric view of one end of the patch tray assembly.",
"FIG.",
"4 is a plan view of three of the patches.",
"FIG.",
"5 is a cross-section taken along a line A-A in FIG.",
"4.FIG.",
"6 is an isometric view of an alternative patch tray assembly.",
"FIG.",
"7 is an enlarged isometric view of one end of the patch tray assembly of FIG.",
"6.FIG.",
"8 is plan view of two of the patches of FIG.",
"6.FIG.",
"9 is a cross-section taken along a line B-B in FIG.",
"8.FIG.",
"10 is an enlarged schematic cross-section through a corner of the patch tray assembly shown in FIG.",
"9.FIG.",
"11 is a cross-section through an alternative patch tray assembly showing a strip isolating element.",
"Referring to the first embodiment shown in FIGS.",
"1-5, an antenna assembly 1 is formed by a patch tray assembly 2 including a line of patch radiating elements (shown in detail in FIGS.",
"2-5); a secondary tray 3 which is fixed to the patch tray assembly 2 by screws or other fixing means; a radome 4 which is fixed to the secondary tray 3 by strips of double-sided adhesive tape 5,6: and a pair of end caps 7,8 which fit over the ends of the assembly.",
"The assembly 1 is mounted, when in use, on a mast with the line of radiating elements oriented vertically.",
"Referring to FIGS.",
"2-5, the patch tray assembly 2 includes a tray formed by folding a planar aluminium sheet to provide a back reflector 10, left side wall 11, right side wall 12 and end wall 13.Eight patch radiating elements are mounted in a single line on the reflector 10.The elements are identical and one is shown in cross-section in FIG.",
"5.A square top patch 20 (which may be brass or another conductive material) is attached to a block 21 of insulating foam material by a layer (not shown) of double-sided adhesive tape.",
"The block 21 is attached to a square brass bottom patch 22 by another layer (not shown) of double-sided adhesive tape.",
"A printed circuit board (PCB) 27 is attached to the back of the reflector 10 by adhesive (not shown).",
"The back reflector 10 has four holes 23-26 partially shown in plan view in FIG.",
"4 and the PCB 27 also has holes (not shown) lying in register with the holes 23-26.The bottom patch 22 has four feed probes which pass through the holes 23-26.Two of the feed probes are shown at 28,29 in FIG.",
"5.The probes have tabs which pass through to the rear of the PCB 27 (two of the tabs 30,31 being shown in FIG.",
"5) and are connected via solder (not shown) to feed lines (not shown) printed on the rear side of the PCB 27.The feed lines are also connected to a set of phase shifters which are partially shown in FIG.",
"1 mounted to the rear of the patch tray assembly.",
"The phase shifters introduce phase shifts between the signals provided to the radiating elements in order to control downtilt of the antenna beam.",
"The side walls 11,12 each support eight rectangular tabs which are each aligned with the centre of a respective patch radiating element.",
"Two of the tabs 40,41 are shown in cross-section in FIG.",
"5.The tabs 40,41 are formed by folding the same sheet of metal as the side walls 11,12 and act as isolating elements.",
"The tabs 40,41 subtend an angle of 90 degrees with the inner face of the side walls 11,12.Eight additional isolating elements are suspended between the side walls 11,12.One of the elements is shown in detail in FIG.",
"5.A pair of insulating plastic bushes 42,43 carry a cylindrical rod 44 formed of aluminium or another conductive material.",
"The bushes 42,43 each have stubs 45,46 which fit through holes in the side walls 11,12.As shown in FIG.",
"4, the rods bisect a line joining the centres of the adjacent patch radiating elements.",
"In an alternative embodiment (not shown) the cylindrical rods may be replaced by flat strips with a planar surface lying parallel with the reflector 10.These strips may be welded to the side walls 11,12 or insulated from the side walls by insulating elements.",
"An alternative patch tray assembly is shown in FIGS.",
"6-9.Integers which are equivalent to integers in FIGS.",
"1-5 are given the same reference numerals.",
"The rods are replaced by alternative isolating elements, one of which is shown in detail in FIGS.",
"7-9.The element is formed by folding a planar sheet of brass to form a wall 51 transverse to the reflector 10 and side walls 11,12, a pair of side walls 52,53 (labelled in FIG.",
"9) parallel with the side walls 11,12 and a rear wall 54 parallel with the back reflector 10.The element is secured to The tray by a plastic insulating rivet 55 passing through the rear wall 54, reflector 10 and PCB 27; a plastic insulating rivet 56 passing through the side wall 52 and side wall 11; and a plastic insulating rivet 57 passing through the side wall 53 and side wall 12.Prior to folding to form the reflector 10 and side walls 11,12, the aluminium sheet is powder coated with an electrically insulating layer 60 shown in FIG.",
"10 (which is not to scale).",
"Strips of single sided electrically insulating tape are also secured to the side walls 52,53 and back wall 54.Two of the strips 61,62 are shown in FIG.",
"10.The tape and powder layers prevent a direct electrical connection between the walls 52,53,54 and the tray.",
"In an alternative arrangement (not shown), powder coating of the reflector 10 and side walls 11,12 may not be necessary.",
"An alternative isolating element is shown in FIG.",
"11.A flat metallic strip 70 is bent upwardly to form a ridge 71 at its central point and attached at both ends to the side walls 11,12 by plastic rivets 72,73 passing through insulating clips 74,75.In an alternative arrangement (not shown), the strip 70 may be bent downwardly to form a trough at its central point.",
"The antenna is mounted vertically in use at a cellular telecommunication base station.",
"The patch radiating elements transmit and receive signals at +45 degrees and −45 degrees polarisation which are fed to/from the antenna via respective polarisation ports (not shown).",
"The −3 dB beamwidth of the antenna is reduced to approximately 65 degrees by the side walls 11,12.The antenna typically works in a cellular telecommunication band such as 1710-1880 MHz, 1750-1990 MHz or 1900-2170 MHz, but could be reasonably expected to work anywhere between 400 and 3000 MHz.",
"Isolation between the different polarisation ports is improved by the isolating elements positioned between the radiating elements and by the tabs mounted on the side walls.",
"It has also been found that isolation is improved in some cases by including isolating elements at the top and bottom of the array—that is, by including the rod 14 and end wall 13 shown in FIGS.",
"2 and 2a, or by including the wall 70 and end wall 13 shown in FIG.",
"6.As shown in FIGS.",
"4 and 8, each radiating element faces a side wall on its left and right side, and an isolation element on its upper and lower side.",
"The linearly polarised +45 degree and −45 degree electromagnetic waves transmitted by the array have horizontal components which are equal in amplitude.",
"The symmetrical construction is designed to maintain this equality, which results in improved isolation.",
"The symmetry is increased further by ensuring that the side walls 11,12 and isolation elements are all spaced equally from the centre of the radiating elements (resulting in a square configuration).",
"We have found that the gap between the side walls 52,53 of the isolating element 50 and the side walls 11,12 of the tray is particularly critical to the operation of the antenna.",
"This spacing can be accurately controlled by suitable selection of the thickness of the powder layer 60 and tape 61,62.We have found that the rods in the first embodiment do not need to be so close to the side walls, so can be spaced further away by the insulating bushes 42,43.The arrangement of FIGS.",
"1-6 does not require powder coating of the tray to prevent direct electrical connection with the rods.",
"Where in the foregoing description reference has been made to integers or components having known equivalents then such equivalents are herein incorporated as if individually set forth.",
"Although this invention has been described by way of example it is to be appreciated that improvements and/or modifications may be made thereto without departing from the scope of the invention as defined in the appended claims."
]
] | Patent_10450480 |
[
[
"Sound Reproducing Device",
"The sound-reproducing device according to the first embodiment of the invention comprises a front and back covers fastened together with sheets located therebetween, a sound message reproducing unit, a impact sensors unit and sheet position detecting unit the output of which is connected to an additional input of sound message reproducing unit.",
"This provides an automatic detection of the number of the open sheet taken into account when a sound message is selected.",
"Impact sensors unit comprises an activation point detection unit, N sensors, a commutator, and a pulse former.",
"The activation point is determined according to capacity changes between the cirresponding sensor and the common bus.",
"The sound-reproducing device according to the second embodiment of the invention further comprises a message number forming unit, the first and second inputs of which are connected to the outputs of sheet position detection unit and impact sensors unit, and the output is connected to the input of sound message reproducing unit.",
"Both embodiments provide a reliable operation of the device with a big number of book sheets."
],
[
"1.A sound-reproducing device comprising a front and back covers fastened together with sheets located therebetween, a sound message reproducing unit, an impact sensors unit, the output of which is connected to the input of said sound message reproducing unit, sheet position detection unit, the output of which is connected to an additional input of said sound message reproducing unit, wherein said impact sensors unit is located so that to be capable of detecting user actions, said sheet position detection unit is located so that to be capable of determining sheets positions, and said sound message reproducing unit is operative to reproduce sound messages depending on code numbers on its inputs.",
"2.The sound-reproducing device of claim 1, wherein said sound message reproducing unit is located in the space limited by surfaces of at least one of said front cover and said back cover.",
"3.The sound-reproducing device of claim 1, wherein said impact sensors unit comprises an activation point detection unit, N sensors, a commutator, and a pulse former, each of said sensors being located in said front or back cover or on the surface of one of said covers and being connected to the corresponding input of said commutator, the output of which via said pulse former is connected to the input of said activation point detection unit, the first output of which is the output of said impact sensors unit and the second output is connected to the control input of said commutator.",
"4.The sound-reproducing device of claim 3, wherein said activation point detection unit, commutator and pulse former are located in the space limited by surfaces of at least one of said front cover and said back cover.",
"5.The sound-reproducing device of claim 3, wherein at least one of said sensors is a capacity sensor.",
"6.The sound-reproducing device of claim 5, wherein said at least one sensor comprises a fragment of a metallized coating of the circuit board.",
"7.The sound-reproducing device of claim 5, wherein said at least one sensor comprises a fragment of a conducting coating on an insulating film.",
"8.The sound-reproducing device of claim 3, wherein said activation point detection unit is operative: to form on its second output cyclically repeating codes of numbers from 1 to N; to measure N pulse periods on its input corresponding to numbers from 1 to N; to store N pulse periods corresponding to numbers from 1 to N; and to form on its first output a code of number K so that the difference between the K-th measured period and the K-th earlier stored period is maximal for all measured periods from the first till the N-th and exceeds a preset threshold.",
"9.The sound-reproducing device of claim 3, wherein said pulse former comprises a threshold element, the input of which is the input of said pulse former and via a resistor is connected to a common bus, the output of said threshold element is the output of said pulse former, and wherein said commutator comprises an additional input connected to the output of said pulse former, a demultiplexer and a multiplexer, control inputs of which are connected to the input of said commutator, the output of which is connected to the output of said multiplexer, and an additional input is connected to the input of said demultiplexer, each out of at least part of outputs of which is connected to each out of at least part of inputs of said multiplexer via a consecutively connected resistor and diode, the united outputs of which are connected to a corresponding output of said commutator.",
"10.The sound-reproducing device of claim 1, wherein said sheet position detection unit comprises a sheets position sensor and an interrogation unit, the input of which is connected to the output of said sheets position sensor and the output being the output of said sheet position detection unit.",
"11.The sound-reproducing device of claim 10, wherein said interrogation unit is operative to cyclically form codes of numbers from 1 to L on its additional output connected to the input of said sheets position sensor, and wherein said sheets position sensor comprises L electrodes, an alternating voltage generator, a demultiplexer, a multiplexer, and a comparator, the output of which is the output of said sheets position sensor, and the input is connected to the output of said multiplexer, the control input of which is connected to the input of said sheet position sensor and is connected to the control input of said demultiplexer, the input of which is connected to the output of said alternating voltage generator, said electrodes being displaced on at least part of said sheets so that capacity connection appears between the electrodes on adjoining sheets, when said sheets adjoin each other, and each of said electrodes being connected at least to the corresponding output of said demultiplexer and to the corresponding input of said multiplexer.",
"12.The sound-reproducing device of claim 11, wherein said sheets position sensor comprises at least one of an additional electrode displaced on the inner side of said front cover and an additional electrode displaced on the inner side of said back cover, and wherein each of said additional electrodes is connected at least to the corresponding output of said demultiplexer and to the corresponding input of said multiplexer.",
"13.A sound-reproducing device comprising a front and back covers bound together with sheets displaced therebetween, a sound message reproducing unit, an impact sensors unit displaced so that to detect the user activation, a sheet position detection unit, and a message number forming unit, the first and second inputs of which are connected to the outputs of said sheet position detection unit and said impact sensors unit accordingly, and the output is connected to the input of said sound message reproducing unit, which is operative to reproduce sound messages depending on a number code on its input.",
"14.The sound-reproducing device of claim 13, wherein said sound message reproducing unit is displaced in the space limited by the surfaces of at least of said front cover and said back cover.",
"15.The sound-reproducing device of claim 13, wherein said sound message reproducing unit is displaced outside of the space limited by the surfaces of said front and back covers.",
"16.The sound-reproducing device of claim 13, wherein the connection between the output of said message number forming unit and the input of said sound message reproducing unit is an optical connection.",
"17.The sound-reproducing device of claim 13, wherein the connection between the output of said message number forming unit and the input of said sound message reproducing unit is a radio connection."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Russian Federation patent No 2141134 to McTaggart, 1993, discloses a sound-reproducing device comprising a front and back covers fastened together with sheets located between them and a audible message reproducing unit.",
"The above device is made in a form of a book, in which the sheets comprise sensors connected to the audible message reproducing unit.",
"When the user touches any of the sensors, an audible message is reproduced corresponding to the text and illustration on the sheet exactly at the point of the sensor location.",
"The drawback of the above device is its limited functionality.",
"The complicated design of the sheets with sensors and connecting wires does not allow to produce books comprising a big number of sheets.",
"The most close to the present invention device is the sound-reproducing device disclosed in EP 0686055 to Jessob, 1997.The device according to Jessob comprises a front and a back covers fastened together with sheets located between them, an audible message reproducing unit, and impact sensors unit located so that it detects the user actions, and the output of said unit is connected to the input of the audible message reproduction unit.",
"The above device is implemented in a form of a book with located in its covers capacity sensors that make up the impact sensors unit.",
"The point where the user touches the book sheet is determined by the capacity change between the capacity sensor located in the cover under the point where the sheet is touched and the common bus of the device.",
"The number of the opened sheet is determined by touching special markings on the sheet, under which there are additional capacity sensors in one of the book covers, said additional sensors being a part of the impact sensors unit.",
"The impact sensors unit transmits data about the user actions to the sound-reproducing unit.",
"The message for the reproduction is determined by the number of the open sheet and the point where the user touches this sheet.",
"The drawback of the above device is its limited functionality, because the sheet number cannot be determined automatically.",
"If the user turning over a new sheet forgets to touch the marking or fulfills this operation incorrectly, the wrong sound message will be reproduced.",
"This drawback makes it difficult to produce sound-reproducing books with a big number of sheets.",
"As the number of sheets increases, the number of markings increases accordingly and the input of the sheets number becomes complicated and time-taking.",
"The other limitation of the device according to Jessob is its comparative operation unreliability.",
"The capacity between the capacity sensor in the cover and the common bus of the device depends on the number of sheets located above this cover.",
"The change of said capacity taking place when many sheets are turned over, is comparable to the change of said capacity upon the user touch of the sheet that can result in mistakes in touch detection."
],
[
"<SOH> OBJECTS AND SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a sound-reproducing device simple in use due to automatic means and reliable having a big number of sheets.",
"A sound-reproducing device according to the first embodiment of the invention comprises a front and back covers fastened together with sheets located therebetween, a sound message reproducing unit, an impact sensors unit, the output of which is connected to the input of the sound message reproducing unit, sheet position detection unit, the output of which is connected to an additional input of said sound message reproducing unit, wherein said impact sensors unit is located so that to be capable of detecting user actions, said sheet position detection unit is located so that to be capable of determining sheets positions, and said sound message reproducing unit is operative to reproduce sound messages depending on code numbers on its inputs.",
"Further, the sound message reproducing unit is located in the space limited by surfaces of at least one of the front cover and back cover.",
"Further, the impact sensors unit comprises an activation point detection unit, N sensors, a commutator, and a pulse former, each of the sensors being located in the front or back cover or on the surface of one of the covers and being connected to the corresponding input of the commutator, the output of which via the pulse former is connected to the input of the activation point detection unit, the first output of which is the output of the impact sensors unit and the second output is connected to the control input of the commutator.",
"Further, the activation point detection unit, commutator and pulse former are located in the space limited by surfaces of at least one of the front cover and the back cover.",
"Further, at least one of said sensors is a capacity sensor.",
"Further, at least one sensor comprises a fragment of a metallized coating of the circuit board.",
"Further, at least one sensor comprises a fragment of a conducting coating on an insulating film.",
"Further, the activation point detection unit is operative to form on its second output cyclically repeating codes of numbers from 1 to N; to measure N pulse periods on its input corresponding to numbers from 1 to N; to store N pulse periods corresponding to numbers from 1 to N; and to form on its first output a code of number K so that the difference between the K-th measured period and the K-th earlier stored period is maximal for all measured periods from the first till the N-th and exceeds a preset threshold.",
"Further, the pulse former comprises a threshold element, the input of which is the input of the pulse former and via a resistor is connected to a common bus, the output of the threshold element is the output of the pulse former, and wherein the commutator comprises an additional input connected to the output of the pulse former, a demultiplexer and a multiplexer, control inputs of which are connected to the input of the commutator, the output of which is connected to the output of the multiplexer, and an additional input is connected to the input of the demultiplexer, each out of at least part of outputs of which is connected to each out of at least part of inputs of the multiplexer via a consecutively connected resistor and diode, the united outputs of which are connected to a corresponding output of the commutator.",
"Further, the sheet position detection unit comprises a sheets position sensor and an interrogation unit, the input of which is connected to the output of the sheets position sensor and the output being the output of the sheet position detection unit.",
"Further, the interrogation unit is operative to cyclically form codes of numbers from 1 to L on its additional output connected to the input of the sheets position sensor, and wherein the sheets position sensor comprises L electrodes, an alternating voltage generator, a demultiplexer, a multiplexer, and a comparator, the output of which is the output of the sheets position sensor, and the input is connected to the output of the multiplexer, the control input of which is connected to the input of the sheet position sensor and is connected to the control input of the demultiplexer, the input of which is connected to the output of the alternating voltage generator, the electrodes being displaced on at least part of the sheets so that capacity connection appears between the electrodes on adjoining sheets, when the sheets adjoin each other, and each of the electrodes being connected at least to the corresponding output of the demultiplexer and to the corresponding input of the multiplexer.",
"Further, the sheets position sensor comprises at least one of an additional electrode displaced on the inner side of the front cover and an additional electrode displaced on the inner side of the back cover, and wherein each of the additional electrodes is connected at least to the corresponding output of the demultiplexer and to the corresponding input of the multiplexer.",
"The sound-reproducing device according to the second embodiment comprises a front and back covers bound together with sheets displaced therebetween, a sound message reproducing unit, an impact sensors unit displaced so that to detect the user activation, a sheet position detection unit, and a message number forming unit, the first and second inputs of which are connected to the outputs of the sheet position detection unit and the impact sensors unit accordingly, and the output is connected to the input of the sound message reproducing unit, which is operative to reproduce sound messages depending on a number code on its input.",
"Further, the message reproducing unit in the device according to the second embodiment of the invention can be displaced in the space limited by the surfaces of at least the front cover and the back cover.",
"Further, the sound message reproducing unit can be displaced outside of the space limited by the surfaces of the front and back covers.",
"Further, the connection between the output of the message number forming unit and the input of the sound message reproducing unit is an optical connection.",
"Further, the connection between the output of the message number forming unit and the input of the sound message reproducing unit is a radio connection.",
"In the devices according to both embodiments of the invention, the introduction of the sheet position detection unit and the peculiarities of the sound message reproducing unit provide automatic determining of an open sheet number and taking it in the account, when a message for the reproduction is selected.",
"Besides, the peculiarities of impact sensors unit provide automatic tracing of changes in the capacity between each capacity sensor and the common bus of the device, when the sheets are turned over.",
"This ensures a reliable detection of the user touch when a big number of sheets are placed above capacity sensors."
],
[
"THE FIELD OF THE INVENTION The invention relates to radio-electronics and more particularly to devices for sound message reproduction and can be used in devices for educational, entertaining and advertising purposes, where sound messages accompany printed texts and graphic illustrations.",
"BACKGROUND OF THE INVENTION Russian Federation patent No 2141134 to McTaggart, 1993, discloses a sound-reproducing device comprising a front and back covers fastened together with sheets located between them and a audible message reproducing unit.",
"The above device is made in a form of a book, in which the sheets comprise sensors connected to the audible message reproducing unit.",
"When the user touches any of the sensors, an audible message is reproduced corresponding to the text and illustration on the sheet exactly at the point of the sensor location.",
"The drawback of the above device is its limited functionality.",
"The complicated design of the sheets with sensors and connecting wires does not allow to produce books comprising a big number of sheets.",
"The most close to the present invention device is the sound-reproducing device disclosed in EP 0686055 to Jessob, 1997.The device according to Jessob comprises a front and a back covers fastened together with sheets located between them, an audible message reproducing unit, and impact sensors unit located so that it detects the user actions, and the output of said unit is connected to the input of the audible message reproduction unit.",
"The above device is implemented in a form of a book with located in its covers capacity sensors that make up the impact sensors unit.",
"The point where the user touches the book sheet is determined by the capacity change between the capacity sensor located in the cover under the point where the sheet is touched and the common bus of the device.",
"The number of the opened sheet is determined by touching special markings on the sheet, under which there are additional capacity sensors in one of the book covers, said additional sensors being a part of the impact sensors unit.",
"The impact sensors unit transmits data about the user actions to the sound-reproducing unit.",
"The message for the reproduction is determined by the number of the open sheet and the point where the user touches this sheet.",
"The drawback of the above device is its limited functionality, because the sheet number cannot be determined automatically.",
"If the user turning over a new sheet forgets to touch the marking or fulfills this operation incorrectly, the wrong sound message will be reproduced.",
"This drawback makes it difficult to produce sound-reproducing books with a big number of sheets.",
"As the number of sheets increases, the number of markings increases accordingly and the input of the sheets number becomes complicated and time-taking.",
"The other limitation of the device according to Jessob is its comparative operation unreliability.",
"The capacity between the capacity sensor in the cover and the common bus of the device depends on the number of sheets located above this cover.",
"The change of said capacity taking place when many sheets are turned over, is comparable to the change of said capacity upon the user touch of the sheet that can result in mistakes in touch detection.",
"OBJECTS AND SUMMARY OF THE INVENTION It is an object of the present invention to provide a sound-reproducing device simple in use due to automatic means and reliable having a big number of sheets.",
"A sound-reproducing device according to the first embodiment of the invention comprises a front and back covers fastened together with sheets located therebetween, a sound message reproducing unit, an impact sensors unit, the output of which is connected to the input of the sound message reproducing unit, sheet position detection unit, the output of which is connected to an additional input of said sound message reproducing unit, wherein said impact sensors unit is located so that to be capable of detecting user actions, said sheet position detection unit is located so that to be capable of determining sheets positions, and said sound message reproducing unit is operative to reproduce sound messages depending on code numbers on its inputs.",
"Further, the sound message reproducing unit is located in the space limited by surfaces of at least one of the front cover and back cover.",
"Further, the impact sensors unit comprises an activation point detection unit, N sensors, a commutator, and a pulse former, each of the sensors being located in the front or back cover or on the surface of one of the covers and being connected to the corresponding input of the commutator, the output of which via the pulse former is connected to the input of the activation point detection unit, the first output of which is the output of the impact sensors unit and the second output is connected to the control input of the commutator.",
"Further, the activation point detection unit, commutator and pulse former are located in the space limited by surfaces of at least one of the front cover and the back cover.",
"Further, at least one of said sensors is a capacity sensor.",
"Further, at least one sensor comprises a fragment of a metallized coating of the circuit board.",
"Further, at least one sensor comprises a fragment of a conducting coating on an insulating film.",
"Further, the activation point detection unit is operative to form on its second output cyclically repeating codes of numbers from 1 to N; to measure N pulse periods on its input corresponding to numbers from 1 to N; to store N pulse periods corresponding to numbers from 1 to N; and to form on its first output a code of number K so that the difference between the K-th measured period and the K-th earlier stored period is maximal for all measured periods from the first till the N-th and exceeds a preset threshold.",
"Further, the pulse former comprises a threshold element, the input of which is the input of the pulse former and via a resistor is connected to a common bus, the output of the threshold element is the output of the pulse former, and wherein the commutator comprises an additional input connected to the output of the pulse former, a demultiplexer and a multiplexer, control inputs of which are connected to the input of the commutator, the output of which is connected to the output of the multiplexer, and an additional input is connected to the input of the demultiplexer, each out of at least part of outputs of which is connected to each out of at least part of inputs of the multiplexer via a consecutively connected resistor and diode, the united outputs of which are connected to a corresponding output of the commutator.",
"Further, the sheet position detection unit comprises a sheets position sensor and an interrogation unit, the input of which is connected to the output of the sheets position sensor and the output being the output of the sheet position detection unit.",
"Further, the interrogation unit is operative to cyclically form codes of numbers from 1 to L on its additional output connected to the input of the sheets position sensor, and wherein the sheets position sensor comprises L electrodes, an alternating voltage generator, a demultiplexer, a multiplexer, and a comparator, the output of which is the output of the sheets position sensor, and the input is connected to the output of the multiplexer, the control input of which is connected to the input of the sheet position sensor and is connected to the control input of the demultiplexer, the input of which is connected to the output of the alternating voltage generator, the electrodes being displaced on at least part of the sheets so that capacity connection appears between the electrodes on adjoining sheets, when the sheets adjoin each other, and each of the electrodes being connected at least to the corresponding output of the demultiplexer and to the corresponding input of the multiplexer.",
"Further, the sheets position sensor comprises at least one of an additional electrode displaced on the inner side of the front cover and an additional electrode displaced on the inner side of the back cover, and wherein each of the additional electrodes is connected at least to the corresponding output of the demultiplexer and to the corresponding input of the multiplexer.",
"The sound-reproducing device according to the second embodiment comprises a front and back covers bound together with sheets displaced therebetween, a sound message reproducing unit, an impact sensors unit displaced so that to detect the user activation, a sheet position detection unit, and a message number forming unit, the first and second inputs of which are connected to the outputs of the sheet position detection unit and the impact sensors unit accordingly, and the output is connected to the input of the sound message reproducing unit, which is operative to reproduce sound messages depending on a number code on its input.",
"Further, the message reproducing unit in the device according to the second embodiment of the invention can be displaced in the space limited by the surfaces of at least the front cover and the back cover.",
"Further, the sound message reproducing unit can be displaced outside of the space limited by the surfaces of the front and back covers.",
"Further, the connection between the output of the message number forming unit and the input of the sound message reproducing unit is an optical connection.",
"Further, the connection between the output of the message number forming unit and the input of the sound message reproducing unit is a radio connection.",
"In the devices according to both embodiments of the invention, the introduction of the sheet position detection unit and the peculiarities of the sound message reproducing unit provide automatic determining of an open sheet number and taking it in the account, when a message for the reproduction is selected.",
"Besides, the peculiarities of impact sensors unit provide automatic tracing of changes in the capacity between each capacity sensor and the common bus of the device, when the sheets are turned over.",
"This ensures a reliable detection of the user touch when a big number of sheets are placed above capacity sensors.",
"BRIEF DESCRIPTION OF THE DRAWINGS Further the invention will be illustrated by the accompanying drawings.",
"FIG.",
"1 shows the construction of the device according to the first embodiment of the invention; FIG.",
"2 shows a structural scheme of the sound-reproducing device according to the first embodiment of the invention; FIG.",
"3 shows a structural scheme of the sound-reproducing device according to the second embodiment of the present invention; FIG.",
"4 shows the construction of the sound-reproducing device according to the second embodiment of the present invention; FIG.",
"5 shows a structural scheme of an pulse former and a commutator for the both embodiments of the present invention; FIG.",
"6 shows a structural scheme of a sheet position sensor and an interrogation unit for both embodiments of the present invention; FIG.",
"7 shows sheet position sensors electrodes for the both embodiments of the present invention in enlarged scale; FIG.",
"8 shows a structural scheme of implementing the sound reproduction unit, interrogation unit, and the activation point detection unit in the device according to the first embodiment of the present invention; FIG.",
"9 shows a structural scheme for the sound reproduction unit, interrogation unit, the activation point detection unit, and the message number forming unit in the device according to the second embodiment of the present invention; FIG.",
"10 shows a flow-chart for the program run in the device according to the first embodiment of the invention; FIG.",
"11 shows a flow-chart for the program run in the device according to the second embodiment of the invention; FIG.",
"12 shows a flow-chart for the subroutine of determining the point of activation for the both embodiments of the invention; FIG.",
"13 shows a flow-chart for the subroutine of determining the open sheet number for the both embodiments of the present invention.",
"THE PREFERRED EMBODIMENT OF THE INVENTION The sound-reproducing device according to the first embodiment of the present invention (FIG.",
"1 and FIG.",
"2) comprises front cover 1 and back cover 2 fastened by bounding 3 with sheets 4.1 .",
".",
".",
"4.M located between them, sound message reproducing unit 5, sheet position detection unit 6, impact sensors unit 7.The outputs of impact sensors unit 7 and of sheet position detection unit 6 are accordingly connected to the input and the additional input of sound message reproducing unit 5 operative to reproduce sound messages depending on number codes on said inputs.",
"The device according to the first embodiment of the present invention is implemented in a form of a book shown open so that the sheets from 4.1 to 4.J are turned over, and the sheets from 4.",
"(J+1) to 4.M are not turned over.",
"On at least some of the sheets 4.1 .",
".",
".",
"4.M on one or both sides there are texts and/or illustrations made in some known way.",
"Sound message reproducing unit 5 can be located in the space limited by the surfaces of first cover 1 (that is inside the cover that has to have sufficient thickness) and/or in the space limited by the surfaces of back cover 2, as in FIG.",
"1, where it is shown relatively.",
"Sheet position detection unit 6 as a whole and impact sensors unit 7 as a whole are not shown in FIG.",
"1, as parts of these unit can be located in different parts of the device as it will be described further.",
"The device can be implemented in a form of a magazine, album, booklet, etc.",
"Besides, the device comprises a power supply, for example several batteries, the buses of which are connected to the supply outputs of device units (not shown in FIG.",
"1 and FIG.",
"2).The power can be switched on and off with the help of a regular switch located in an appropriate place of back cover 2.It is also possible that the power is switched on when the book is opened, and switched off when the book is closed with the use of a corresponding key-button.",
"Impact sensors unit 7 may comprise activation point detection unit 8, sensors 9.1 .",
".",
".",
"9.N, commutator 10, and pulse former 11.Each of sensors 9.1 .",
".",
".",
"9.N may be located in front cover 1, or in back cover 2, or on the surface of one of them, each sensor being connected to the corresponding input of commutator 10, the output of which via pulse former 11 is connected to the input of activation point detection unit 8, the first output of which is the output of impact sensors unit 7, and the second output is connected to the control input of commutator 10.In the example of the device structure shown in FIG.",
"1 sensors 9.1 .",
".",
".",
"9.",
"(Q−1) are located in front cover 1, sensors 9.Q .",
".",
".",
"9.N—in back cover 2.Activation point detection unit 8, commutator 10, and pulse former 11 may be located in the space limited by the surfaces of back cover 2 (In FIG.",
"2 shown relatively).",
"They may also be located inside front cover 1 or inside both said covers, or in other places of the device.",
"Sensors 9.1 .",
".",
".",
"9.N can be capacity sensors.",
"Each of them can comprise for example a fragment of a metallized coating of the circuit board forming the first plate of the capacitor.",
"The second plate of the capacitor is made up by the common bus of the device (“the ground”), that can be one of power supply buses.",
"Each of these sensors is separated from the others by strips with a remote metallized coating.",
"The capacity between sensor 9.n (n=1 .",
".",
".",
"N) and the common bus of the device changes, when the user touches the book sheets above said sensors or his/her hand just approaches this point.",
"Besides, each of sensors 9.1 .",
".",
".",
"9.N can comprise a strip of conducting coating on the insulating film that forms the first plate of the capacitor, the second plate of which is formed by the common bus of the device.",
"In this case, sensors 9.1 .",
".",
".",
"9.N can be produced according to the same technology as membrane keyboards.",
"Sensors 9.1 .",
".",
".",
"9.N may be implemented not only as capacity sensors but as for example inductive sensors.",
"In this case, the user has to touch the device not with his/her finger but with a special pointer comprising for example a piece of ferromagnetic material.",
"Activation point detection unit 8 can be operative to form on its second output cyclically repeating codes of numbers from 1 to N, to measure N values of pulse periods on its input corresponding to numbers from 1 to N, to store the values of pulse periods corresponding to numbers from 1 to N, and to form on its first output the code of number K out of the range from 1 to N, so that the difference between the K-th measured period value and the K-th earlier remembered period value is the highest among all period values from first to N-th and does not exceed the set threshold.",
"Impact sensors unit 7 can be based on other principles.",
"For example, it is possible to use the measurement of spreading times of an ultrasonic signal from the transmitter built into the pointer and activated upon pressing the button in the pointer to the receivers located in the corners of the covers.",
"Pointer coordinates can be calculates by the measured times.",
"Sheet position detection unit 6 can comprise sheets position sensor 12 and interrogation unit 13, the input of which is connected to the output of sheets position sensor 12, and the output is the output of sheet position detection unit 6.Sheets position sensor 12 in FIG.",
"1 is shown relatively and its implementation will be considered further.",
"Interrogation unit 13 can be located in back cover 2.The sound-reproducing device according to the second embodiment of the present invention (FIG.",
"3 and FIG.",
"4) comprises front cover 1 and back cover 2 fastened by binding 3 with 4.1 .",
".",
".",
"4.M sheets located between them, sound message reproducing unit 14, sheet position detection unit 6, impact sensors unit 7, and message number forming unit 15.The output of sheet position detection unit 6 and the output of impact sensors unit 7 are connected accordingly to the first and second inputs of message number forming unit 15, the output of which is connected to the input of sound message reproducing unit 14 operative to reproduce sound messages depending on the number code on its input.",
"The device according to the second embodiment of the present invention as well as the device according to the first embodiment can be implemented in a form of a book shown open so that the sheets from 4.1 to 4.J are turned over and the sheets from 4.",
"(J+1) to 4.M are not turned over.",
"Sound message reproducing unit 14 can be located in the space limited by surfaces of front cover 1 and/or in the space limited by the surfaces of back cover 2, or as shown in FIG.",
"4 outside covers 1 and 2 in a form of a constructively separate unit.",
"The output of message number forming unit 15 can be connected to the inputs of sound message reproducing unit 14 by wires or a radio-channel, or by for example infra-red radiation, or by other means known in the art.",
"Sheet position detection unit 6 and impact sensors unit 7 in the device according to the second embodiment of the present invention comprise the same units with the same specificities of construction and implementation as in the device according to the first embodiment of the invention.",
"Message number forming unit 15 can be located inside back cover 2 (in FIG.",
"4 shown relatively).",
"Besides, the device according to the second embodiment of the invention can comprise a power supply, for example several batteries the buses of which are connected to the supply inputs of the device units (not shown in FIG.",
"3 and FIG.",
"4 ).",
"If sound message reproducing unit 14 is made as a separate unit, it can have a separate power supply, for example of AC power.",
"In this case, it is possible that other device units located in the book receive power supply in sound message reproducing unit 14 via corresponding wires.",
"In devices according to the both embodiments of the invention, pulse former 11 comprises (FIG.",
"5) threshold element 16, the input of which is the input of pulse former 11 and via resistor 17 connected to the common bus, and the output is the output of pulse former 11.Commutator 10 is equipped with an additional input connected to the output of pulse former 11 (also shown in FIG.",
"2 and FIG.",
"3) and comprises demultiplexer 18, multiplexer 19, resistors 20.1 .",
".",
".",
"20.N and diodes 21.1 .",
".",
".",
"21.N.",
"The control inputs of demultiplexer 18 and multiplexer 19 are connected to the input of commutator 10, the additional input of which is the input of demultiplexer 18 and the output is the output of multiplexer 19.Demultiplexer 18 has NY outputs, and multiplexer 19 has NX inputs.",
"In the construction being described, NX×NY=N, but this condition generally speaking is not compulsory.",
"The K-th output of demultiplexer 18 (K=1 .",
".",
".",
"NY) is connected to the S-th input of multiplexer 19 (S=1 .",
".",
".",
"NX) via consecutively connected resistor 20 ((S−1)×NY+K) and diode 21.",
"((S−1)×NY+K), the united outputs of which are connected to the ((S−1)×NY+K)-th output of commutator 10.In the devices according to both embodiments of the present invention, sheets position sensor 12 may comprise (FIG.",
"6) electrodes 22.1 .",
".",
".",
"22.",
"(M+2), AC voltage generator 23, demultiplexer 24, multiplexer 25, and comparator 26, the output of which is the output of sheets position sensor 12, and the input is connected to the output of multiplexer 25, the control input of which is connected to the input of sheets position sensor 12 and connected to control input of demultiplexer 24, the input of which is connected to the output of AC voltage generator 23.The outputs of demultiplexer 24 from the first to the (M+1)-th are connected to electrodes 22.1 .",
".",
".",
"22.",
"(M+1), respectively.",
"The inputs of multiplexer 25 from the first to the (M+1)-th are connected to electrodes 22.2 .",
".",
".",
"22.(M+2).",
"The input of sheets position sensor 12 is connected to the additional output of interrogation unit 13 (also shown in FIG.",
"2 and FIG.",
"3), which can be made operative to cyclically form on its additional output codes of numbers from 1 to (M+1).",
"The sheets position sensor 12 can be also made without an input and the corresponding connection to the output of interrogation unit 13 (not shown in the drawings).",
"In this case, sheets position sensor 12 can comprise a clock generator connected to a pulse counter, the output of which is connected to control inputs of multiplexer 25 and demultiplexer 24 and to the output of sheets position sensor 12.AC voltage generator 23, demultiplexer 24, multiplexer 25, and comparator 26 can be located in front cover 1 and/or in back cover 2 (not shown in FIG.",
"2 and FIG.",
"3).",
"Upon supplying the code of number n to the control input of demultiplexer 24, the signal from its input goes to the (n+1)-th output and other outputs remain in the locked (high-Ohmed) position.",
"It is also possible that demultiplexers 18 and 24 are implemented as one demultiplexer integrated circuit (IC) and their functions are fulfilled by different groups of outputs of this integrated circuit.",
"Similarly, multiplexers 19 and 25 can be implemented as one multiplexer integrated circuit.",
"In this case, former 11 and AC voltage generator 23 are one generator.",
"Electrode 22.1 is located on the inner side of front cover 1 near binding 3.Electrodes 22.2 .",
".",
".",
"22.",
"(M+1) are located on sheets 4.1 .",
".",
".",
"4.M accordingly near binding 3.Electrode 22.",
"(M+2) is located on the inner side of back cover 2 near binding 3.In other embodiments of the device, electrodes can be placed not on every sheet and/or can be absent from the covers.",
"In this case, the range of variation of the number formed at the additional output of interrogation unit decreases.",
"Each of electrodes 22.1 .",
".",
".",
"22.",
"(M+2) can be implemented in a form of a foil plate fastened to one of the sides of the sheet (or a cover) and covered by some insulating material.",
"Sheets 4.1 .",
".",
".",
"4.M can be implemented as double sheets (FIG.",
"7) by gluing together of two sheets of paper.",
"In this case, electrodes 22.2 .",
".",
".",
"22.",
"(M+1) can be inserted between the sheets and the insulating material is not required.",
"FIG.",
"7 shows the location of electrodes on sheets 4.",
"(J−1) .",
".",
".",
"4.",
"(J+2) shown partially and in enlarged scale.",
"The last turned over sheet 4.J and the preceding list 4.",
"(J−1) touch each other.",
"In the results there is a capacity connection between electrodes 22.",
"(J+1) and 22.J located on these sheets.",
"Similarly, there is capacity connection between electrode 22.",
"(J+2) on sheet 4.",
"(J+1) and electrode 22.",
"(J+3) on sheet 4.(J+2).",
"At the same time, between electrode 22.",
"(J+1) on sheet 4.J and electrode 22.",
"(J+2) on sheet 4.",
"(J+1) there is almost no capacity connection, because the first of said sheets is turned over and the second is not.",
"The structural scheme of implementing sound message reproducing unit 5, interrogation unit 13 and activation point detection unit 8 in the device according to the first embodiment of the present invention (FIG.",
"8) shows controller 27 comprising memory 28 and connected to it message memory 29, and consecutively connected to digital-to-analog converter (DAC) 30, amplifier 31, and loudspeaker 32.Controller 27 can be implemented on for example microprocessor Atmel Inc., with memory 28 comprising non-volatile memory of 8 Kbyte for storing programs run by microprocessor, and operating memory of 256 byte for storing variables during the fulfilling of programs.",
"The functions of the input and the second output of activation point detection unit 8, input and additional output of interrogation unit 13 are fulfilled by designated for this purposes bits of input/output ports of microcontroller 27.The connections of the output of interrogation unit 13 and the first output of activation point detection unit 8 with the first and second inputs of sound message reproducing unit 5 accordingly are done by program means.",
"Message memory 29 can be based on an integrated circuit of non-volatile electric erasable memory, for example AT45D161 by Atmel Inc., USA.",
"The number of memory ICs used depends on information volume in recorded messages.",
"Message memory 29 can also be based on a miniature laser disc reproducing device or a miniature storage device on a hard disc, that allows to significantly increase the volume of sound information in the devices according to the present invention.",
"Sound messages are recorded during the manufacturing of the device into message memory 29 in a form of sequences of sound signals and can comprise speech messages, music fragments, various sound effects, etc.",
"Each sound message is designated by number NM.",
"The formula for calculating this number will be considered further.",
"Message memory 29 comprises address table, in which for each of numbers NM value the initial address of the message with this number and the number of bites with information in this message are recorded.",
"If no message is designated for some of number NM, the number of bites with information equal to zero is recorded.",
"To increase the volume of sound information recorded into message memory 29 any known method of sound compression can be used.",
"Other not considered herein variants for the device are possible, in which messages can be recorded by the user into message memory 29.The structural scheme of sound message reproducing unit 14, interrogation unit 13, activation point detection unit 8, and message number forming unit 15 in the device according to the second embodiment (FIG.",
"9) shows controller 33 fulfilling the functions of interrogation unit 13, activation point detection unit 8, and message number forming unit 15, and comprising memory 34, controller 35 used in sound message reproducing unit 14 and comprising memory 36, connected to it message memory 37 and consecutively connected DAC 38, amplifier 39, and loudspeaker 40.Controllers 33 and 35 can be implemented similarly to controller 27, and message memory 37—similarly to message memory 29 in the device according to the first embodiment of the invention.",
"The functions of the input and the second output of activation point detection unit 8, the input and additional output of interrogation unit 13, and the output of message number forming unit 15 is fulfilled by the designated for this purpose bits of input/output ports of microcontroller 33.The connections of the output of interrogation unit 13 and the first output of activation point detection unit 8 with the first and second inputs of message number forming unit 15 accordingly are made by program means.",
"If sound message reproducing unit 14 is implemented as a separate unit, it can have different constructions, for example in a form of a talking toy.",
"The connection of the output of message number forming unit 15 and the input of sound message reproducing unit 14 in this case can be established with wires, radio-waves, infra-red rays, etc.",
"Sound message reproducing unit 14 can be also made on the basis of a separate PC, the storage device on its hard disc serving as message memory 37, and sound card as DAC 38 and amplifier 39.The output of message number forming unit 15 and the input of sound message reproducing unit 14 in this case are connected with the use of some standard interface, for example RS-232.The flow-chart of the program run in the device according to the first embodiment of the present invention (FIG.",
"10) comprises program blocks 41 and 42, subroutine 43, program block 44, subroutines 45, program blocks 46 and 47.The flow-chart of the program run in the device according to the second embodiment of the present invention (FIG.",
"11) comprises program blocks 48 and 49, subroutine 50, program block 51, subroutine 52, program blocks 53 and 54.The flow-chart of the subroutine for determining the point of activation (FIG.",
"12) comprises program blocks 55 .",
".",
".",
"71.The flow-chart of subroutine for determining the number of the open sheet (FIG.",
"13) comprises program blocks 72 .",
".",
".",
"80.Operation of the Device The operation of the sound-reproducing device according to both embodiments of the present invention is based on detecting the user touch of one of the open book pages with the help of impact sensors unit 7, determining the number of the open sheet with the help of sheet position detection unit 6 and reproducing a sound message corresponding to the point of touch and the number of the open sheet.",
"The user opens the book so that some of sheets (from 4.1 to 4.J in FIG.",
"7) are turned over together with front cover 1, and the rest of the sheets (from 4.",
"(J+1) to 4.M) are not turned over together with back cover 2.The capacity connection between electrode 22.",
"(J+1) on sheet 4.J and electrode 22.",
"(J+2) on sheet 4.",
"(J+1) is unlocked.",
"Interrogation unit 13, the functions of which in the considered embodiments are fulfilled by controller 27 (FIG.",
"8) and controller 33 (FIG.",
"9), in turn records into demultiplexer 24 and multiplexer 25 (FIG.",
"6) codes of numbers from 1 to M+1.When a code of some number n is being recorded, the output of AC voltage generator 23 gets connected via demultiplexer 24 to electrode 22.n, and the input of comparator 26 via multiplexer 25 gets connected via multiplexer 25 to electrode 22 (n+1).",
"The interrogation of sheets position sensor is repeated periodically.",
"If electrodes 22.n and 22.",
"(n+1) adjoin each other, then there is a capacity connection between them.",
"AC voltage goes to the input of comparator 26, on the output of which the level of the voltage is equal to 1.If there is a sufficient distance between electrodes 22.n and 22.",
"(n+1) corresponding to n=(J+1) (FIG.",
"7), the capacity connection between them is unlocked, AC voltage is not applied to the input of comparator 26, on the output of which the level of voltage is equal to 0.In interrogation unit 13 the value of n=NP is determined, when at the output of sheets position sensor 12 the value of 0 appears.",
"The program fulfilling this function will be described further.",
"In the described embodiment of the invention, the number of the last turned over sheet is equal to NP−1, and the number of the first not turned over sheet is equal to NP.",
"If only front cover 1 is turned over, then NP=1.If all the sheets are turned over and back cover 2 is not, then NP=M+1.Determining of the point of touch is based on detecting the capacity increase between the common bus of the device and sensor 9.n (where n=1 .",
".",
".",
"N), located under the part of the open sheet touched or to which the user finger has approached close enough.",
"The increase of said capacity is determined by the increase of periods of pulses formed by pulse former 11, when a corresponding sensor is connected to its feedback network.",
"Activation point detection unit 8, the functions of which in the considered embodiments of the invention are fulfilled by controller 27 (FIG.",
"8) or controller 33 (FIG.",
"9), records in demultiplexer 18 and in multiplexer 19 (FIG.",
"5) number codes providing turn by turn connection via demultiplexer 18 of one of the pins of resistor 20.n to the output of pulse former 11, and via multiplexer 19 the cathode of corresponding diode 21.n to the input of pulse former 11.Sensor 9.n is connected to the point of connection of said resistor and diode, and the capacity between this sensor and the common bus determines the period of pulses formed by pulse former 11.The value of pulse periods is measured and stored in activation point detection unit 8.The described procedure is fulfilled for all values n=1 .",
".",
".",
"N. The loop of interrogation of sensors 9.1 .",
".",
".",
"9.N repeats periodically.",
"In the interrogation loop, the measured pulse period for each sensor 9.n is compared to the pulse period for the same sensor stored in the previous interrogation loop.",
"In the result, such number NT is determined that for sensor 9.NT the increase of pulse period compared to the previous interrogation loop is higher than for all other capacity sensors and it exceed the set threshold.",
"The program fulfilling the above functions will be considered later.",
"In the device according to the first embodiment of the invention (FIG.",
"2), the code of number NP indicating the number of the last turned over sheet and the code of number NT indicating the number of sensor 9.NT for which the touch has been detected arrive at the first and second inputs of sound message reproducing unit 5 accordingly.",
"In the result, a sound message is reproduced corresponding to the selected part on the selected page.",
"Sound signal counts are consecutively read from message memory 29 (FIG.",
"8) and are forwarded to DAC 30, where they are converted into the analog form.",
"The sound signal is amplified in amplifier 31 and reproduced via loudspeaker 32.In the device according to the second embodiment of the invention (FIG.",
"4), codes of numbers NP and NT arrive at the first and second inputs of message number forming unit 15, accordingly, in which the code number for the message arriving at sound message reproducing unit 5 is formed.",
"In the result, a sound message is reproduced corresponding to the selected part on the selected page.",
"In the described below flow-charts of the programs fulfilled by controllers 27 and 33 in the devices according to the first and second embodiments of the invention accordingly, the following designations are used: T is a array of N numbers, in which the measured values of pulse periods for sensors 9.1 .",
".",
".",
"9.N are stored, G is an array of N numbers, in which values of pulse period remembered for the use in the next loop are stored, nm is a number of the sensor for which the difference between the measured and the stored values of pulse periods is maximal, DTm is a value of said maximal difference between the measured and stored values of pulse periods, Thr is a threshold value of the difference between the measured and stored values of pulse periods, NT is a number of the sensor for which the touch is detected, U is a measured logical voltage level at the output of comparator 26 in sheets position sensor 12, NP is a number of the first not turned over sheet, NM is a number of a reproduced sound message, n is a counting variable.",
"The run of the program in the device according to the first embodiment of the invention (FIG.",
"10) begins when the power is switched on (block 41).",
"Then the initialization takes place (block 42), in the process of which the values of pulse periods for all sensors 9.1 .",
".",
".",
"9.N are measured and the received values are recorded in the corresponding cells T[1] .",
".",
".",
"T[N].",
"The procedure of measuring pulse periods is similar to the one used in subroutine 43 of determining the point of touch that will be described later.",
"Then the program goes into the loop comprising subroutine 43, program block 44, subroutine 45, and program blocks 46, 47.This loop continues till the power is switched off.",
"In the result of running subroutine 43 of determining the point of activation, number NT is found that shows the number of sensor 9.NT, for which the touch is detected.",
"Subroutine 43 fulfils the functions of activation point detection unit 8.Then it is checked, if there is a touch (block 44).",
"If NT=0, there is not touch and the program returns to the beginning of the loop to subroutine 43.In the opposite case, subroutine 45 of determining the number of the open sheet is fulfilled, in the result of which NP is found showing the number of the first not turned over sheet.",
"Subroutine 45 fulfils the functions of interrogation unit 13.Then NM number of the message for reproduction is determined (block 46) according to the formula NM=(NP−1)*N+NT.",
"Then the sound message with number NM is reproduced (block 47).",
"The values of sound signal counts are consecutively read from message memory 29 (FIG.",
"8) beginning with the address determined by number NM and are forwarded to DAC 30.These operations repeat till all the sound signal counts are reproduced.",
"If some method of sound compression was used during the recording of sound messages into message memory 29, then the corresponding decoding is fulfilled during the message reproduction.",
"The program fulfilling these functions can be similar to those used in the known devices for recording and reproduction or for transmission of sound information.",
"Thus, program blocks 46 and 47 fulfil the functions of sound message reproducing unit 5 in the device according to the first embodiment of the present invention.",
"Then the program returns to the beginning of the loop at subroutine 43.The program run in the device according to the second embodiment of the invention (FIG.",
"11) also begins when the power is switched on (block 48).",
"Initialization (block 49), subroutine 50 for determining the point of activation (block 51), subroutine 52 of determining the number of the open sheet, determining of the message number (block 53) are similar to the corresponding blocks of the program and to the subroutines in the program of the device according to the first embodiment of the invention.",
"Running of the loop beginning with subroutine 50 and ending in program block 54 continues till the power is switched off.",
"The found number NM of a message for reproduction is sent to sound message reproducing unit 14 (block 54).",
"Then the program returns to the beginning of the beginning of the loop at subroutine 50.After receiving number NM, controller 35 in sound message reproducing unit 14 (FIG.",
"9) fulfils the program of reproducing sound messages.",
"Sound signal counts are read beginning with the address determined by number NM from message memory 37 and are forwarded to DAC 38.If necessary, compressed sound information is decoded.",
"After getting into subroutine 43 of determining the point of touch (block 55 in FIG.",
"12) counting variable n receives the value of 1, and variable NT—the value of 0 (block 56).",
"Then the loop of blocks 57 .",
".",
".",
"60 is repeated N times.",
"In each run of the loop, the program measures periods of pulses formed by pulse former 11 upon connecting sensor 9.n, (n=1 .",
".",
".",
"N) into its feedback.",
"At the beginning of each loop, the following is calculated: the value of number n1 sent to multiplexer 19 and showing the number of its input used (FIG.",
"5), the value of number n2 sent to demultiplexer 18 and showing the number of its output used is (block 57).",
"The value of n1 is found by formula n1=Trunc(n/NY), where NY is a number of outputs of demultiplexer 18, Trunc(x) is a function returning the whole part of argument x.",
"Value n1 changes in the range from 0 to NX−1, where NX=N/NY is a number of multiplexer 19 inputs.",
"Value n1=0 corresponds to the first output of demultiplexer 18.Value n2 is found by formula n2=n−n1×NY and changes in the range from 0 to NY−1.Value n2=0 corresponds to the first input of multiplexer 19.In the result, sensor 9.n, where n=n1×NY+n2, is included into the feedback of pulse former 11.Pulse former 11 forms pulses, the period of which is determined by the capacity between said sensor and the common bus.",
"Further, the pulse period is measured (block 58).",
"This operation can be for example the operation of counting cycles in controller 27 (or 33) during the interval between two neighboring pulse fronts at the output of pulse former 11.The value received is stored in variable T[n].",
"Program blocks 59 and 60 provide the transition to the next run of the loop and its termination after the N-th run.",
"Then the subroutine calculates the difference between measured periods T[n] and stored after the previous run of the loop of the main program values of G[n] for all n=.",
".",
".",
"N (block 61).",
"In the result of the calculations, values of nm are determined, that is of the sensor number for which the difference between the measured and stored pulse period values is maximal; and DTm is determined, the value of said maximal difference between the measured and stored values of pulse period values.",
"Further, it is checked if there is a touch (block 62).",
"For this purpose, DTm is compared to constant Thr, the threshold value for the difference between the measured and stored pulse period values.",
"If DTm>Thr, then the touch for sensor 9.nm is detected.",
"All stored for the use in the next loop pulse period values G[n] (n=1 .",
".",
".",
"N) receive values of corresponding measured periods T[n] (block 63).",
"Variable NT comprising sensor number, for which the touch has been detected, receives value nm (block 64).",
"After this, the subroutine terminates in block 71.In case if Dtm does not exceed the threshold, that is no touch is detected, counting variable n receives the value of 1 (block 65).",
"After this, the loop of program blocks 66 .",
".",
".",
"70 is fulfilled, in which new values of G[n] (n=1 .",
".",
".",
"N) are determined.",
"In each run of the loop, the measured period T[n] is compared to an earlier stored value of G[n] (block 66).",
"If T[n]<G[n], then variable G[n] receives the value of T[n] (block 67).",
"In the opposite case, the new value of G[n] is found by formula G[n]=(3×G[n]+T[n])/4 (block 68).",
"Thus, if period T[n] has increases but has not exceeded threshold Thr, value G[n] grows slowly that increases the reliability of the device.",
"The formula for calculating G[n] can be different, but it has to ensure the above peculiarities for changes of the above values in time.",
"Then the value of variable n increases by 1 (block 69), and the condition for the loop termination is checked (block 70), by fulfilling which the subroutine terminates (block 71).",
"Variable NT remains equal to 0 showing that no touch has been detected.",
"The described subroutine 43 of determining the point of touch provides the protection against a repeated activation, when the user keeps the finger on the same part of the sheet for a long time.",
"Besides, the subroutine smoothly follows the gradual increase or decrease in the capacity between sensors 9.1 .",
".",
".",
"9.N and the common bus taking place, when book sheets 4.1 .",
".",
".",
"4.M are turned over.",
"As the values for the periods of each sensor 9.1 .",
".",
".",
"9.N are stored and analyzed separately, the influence of capacity range on said sensors is excluded.",
"Subroutine 50 of determining the point of touch in the device according to the second embodiment of the invention is similar to the described one.",
"After entering subroutine 45 for determining the number of the open sheet in block 72 (FIG.",
"13), counting variable n receives the value of 1, and variable NP showing the number of the first sheet that is not turned over receives the value of 0 (block 73).",
"Then the loop consisting of program blocks 74 .",
".",
".",
"79 is fulfilled.",
"In each run of the loop one pair of electrodes 22n.",
"and 22.",
"(n+1) is checked (FIG.",
"6).",
"In the beginning of each run of this loop, value n is sent to demultiplexer 24 and multiplexer 25 (block 74).",
"Then the logical voltage level received from the output of comparator 26 is stored in variable U (block 75).",
"Further, the value of variable U is compared to logical 0 (block 76).",
"If the result of the comparison is negative (U corresponds to logical 1), that corresponds to the absence of the gap between electrodes 22n.",
"and 22 (n+1), the value of variable n increases by 1 (block 78) and the condition for termination of the loop is checked (block 79).",
"If this condition is not met, the program returns to the beginning of the loop in block 74.In the opposite case, that is if n>M+1, the subroutine terminates (block 80) NP retaining the value of 0.The similar situation takes place, if the book is closed.",
"If in program block 76 it is found out that U corresponds to the logical 0, that is electrodes 22n.",
"and 22.n+1 are far enough from each other, variable NP receives the value of n (block 77).",
"After this, the subroutine terminates in block 80.Subroutine 52 of determining the number of the open sheet fulfilled in the device according to the second embodiment of the invention is similar to the described above.",
"Conclusion, Ramifications and Scope As it is clear from the description of the present invention, the invention provides sound-reproducing devices that have advantages over the devices known before.",
"The devices according to the present invention are operative to automatically determine the number of the open sheet and to reproduce a sound message in accordance with said number and the point of the user activation of one of the open sheets.",
"The change in the capacity between sensors and the common bus occurring when sheets are turned over is traced automatically that provides a precise determination of the sheet part touched even if a big number of sheets is placed above a capacity sensor.",
"Thus, a higher reliability of the device is achieved.",
"The sound-reproducing devices according to the present invention can be used as educational books, toys, advertising magazines, guide books, etc.",
"Automatic determining of the open sheet simplifies the use of the device and is advantageous for the user.",
"Another advantage of the present invention devices is the possibility to use a big number of sheets in the device that increases the amount of text and graphic information while the corresponding sound information can be stored on a memory IC, on a magnet or laser disc.",
"The sound-reproducing unit can be displaced both inside and outside of the present invention device.",
"For example, a PC can be used or a regular sound-reproducing center allowing to increase volume and the sound quality and in same cases can be more convenient for the user.",
"The above and other advantages of the devices according to the present invention open wide possibilities for their commercial use.",
"Having described the preferred embodiments of the invention with the reference to the accompanying drawings, it is to be understood that the invention is not limited to these precise embodiments, and that various changes and modifications may be effective therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims"
]
] | Patent_10450552 |
[
[
"Considerations, evaluations, investigations and searching",
"Methods of considering DNA based links between two or more situations are provided.",
"Amongst the methods is a method including obtaining a plurality of test results, each test result relating to a situation, each test result including information on the DNA from that situation, the plurality of test results providing a group of test results; selecting a plurality of test results from the group of test results, the plurality of results for the combination of test results; considering a genotype as possibly giving rise to each of the test results of the combination, evaluating the support for that genotype giving rise to all of the test results of the combination; considering the genotype as a DNA based link between the situations for the test results in a combination if the support meets the defined criteria.",
"The methods provide new techniques for considering preexisting DNA test results and/or new DNA test results so as to establish links between them in terms of the genotypes which are supported as contributing to them.",
"The methods enable the evaluation of the support for genotypes given the test results in relation to various combinations of test results."
],
[
"1.A method of considering DNA based links between two or more situations, the method including: obtaining a plurality of test results, each test result relating to a situation, each test result including information on the DNA from that situation, the plurality of test results providing a group of test results; selecting a plurality of test results from the group of test results, the plurality of results forming a combination of test results; considering a genotype as possibly giving rise to each of the test results of the combination, evaluating the support for that genotype giving rise to all of the test results of the combination; considering the genotype as a DNA based link between the situations for the test results in the combination if the support meets defined criteria.",
"2.A method according to claim 1 in which the method includes the further step of comparing a genotype which is considered a DNA based link against records of genotypes to identify matching genotypes in the records.",
"3.A method according to claim 1 or claim 2 in which the evaluation involves a direct evaluation of the support for a genotype giving the combination of test results or involves an evaluation of the support for a given genotype giving each of the test results, the individual evaluations being combined to give the overall evaluation.",
"4.A method according to any preceding claim in which the support meets the defined criteria when the probability that the genotype could have given rise to the test results of the combination is above a given level and/or when an expression of the support that the genotype could have given rise to the test results of the combination is below a given level.",
"5.A method according to any preceding claim in which the evaluation of the support for a genotype giving rise to the combination of test results includes a consideration of the probability of the test results arising given that genotype and the probability of occurrence of that genotype.",
"6.A method according to any preceding claim in which the evaluation of the support for a genotype giving rise to the combination of test results is defined by Pr ( G l | D… ) = ∏ ijk Pr ( D ijk | G l ) Pr ( G l ) ∑ i ∏ ijk Pr ( D ijk | G l ) Pr ( G l ) for all l .",
"where G1 represents the particular genotype, D represents the combination of test results, potentially including test results due to various scenes and/or samples from scenes and/or replicates of samples from scenes, i represents the range of replicates, j the range of samples, k the range of scenes and l the range of genotypes under consideration.",
"7.A method according to any preceding claim in which one or more limits are applied to the genotypes which are considered from amongst the full set of possible genotypes, the limits being based on one or more rules as to genotypes which could not practically give one or more of the results in the combination being considered.",
"8.A method according to any preceding claim in which the evaluation of the support for a genotype giving rise to the group of test results includes a consideration of the effect of one or more of contamination of the test results and/or allele drop out from the results and/or stutter in the results and/or preferential amplification of the results.",
"9.A method according to any preceding claim in which a genotype which is considered as a DNA based link between the situations of the combination is used in a further consideration, the further consideration including the review of possible matches between the genotype and a collection of genotype records.",
"10.A method of considering DNA based links between two or more situations, preferably according to claim 1, the method including: obtaining a first test result for a first situation, the first test result including information on the DNA from the first situation; considering a genotype as possibly giving rise to the first test result and evaluating the support that the genotype gave rise to the first test result, repeating the evaluation of the support for a plurality of other genotypes, generating a set of possible genotypes based on the evaluation with respect to the first test result; obtaining a second test result for a second situation, the second test result including information on the DNA from the second situation; considering a genotype as possibly giving rise to the second test result and evaluating the support that this genotype gave rise to the second test result, repeating the evaluation of the support for a plurality of other genotypes, generating a set of possible genotypes based on the evaluation with respect to the second test result; combining the set of possible genotypes for the first test result and the set of possible genotypes for the second test result, genotypes present in the first set and the second set being given a higher ranking in the combined set than genotypes not present in one or more of the sets; considering one or more of the higher ranked genotypes in the combined set as a DNA based link between the first situation and the second situation.",
"11.A method according to claim 10 in which the evaluation of the support involves a determination of the mixture proportions contributed by different individuals.",
"12.A method according to claim 10 or claim 11 in which the evaluation of the support is used to rank the set.",
"13.A method according to any of claims 10 to 12 in which the evaluation produces a list of possible genotypes for the first and second test results.",
"14.A method according to any of claims 10 to 13 in which the combining of the set for the first test result and the set for the second test result includes, for genotypes present in the first set and in the second set, adding the support for that genotype for the first set to the support for that genotype for the second set.",
"15.A method according to any of claims 10 to 14 in which the combining of the set or ranked evaluation for the first test result and the set or ranked evaluation for the second test result includes combining the support for that genotype for the set or ranked evaluation it is present in with a dummy support for the set or ranked evaluation it is absent from.",
"16.A method according to any of claims 10 to 15 in which the genotypes are ranked within the combined set or combined evaluation, genotypes present in each of the sets or ranked evaluations receiving a high ranking in the combined set or ranked evaluation and/or genotypes absent from one or more of the sets of ranked evaluations receiving a low ranking in the combined set or ranked evaluation."
],
[
"This invention concerns improvements in and relating to considerations, evaluations and investigations, particularly but not exclusively in relation to highlighting genotypes of interest, and to searching, particularly but not exclusively in relation to searching of databases of genotypes.",
"Substantial numbers of DNA test results exist from various scenes, samples taken from scenes and replicates of samples.",
"These relate to both solved and unsolved incidents.",
"It is desirable to be able to obtain as much information as possible from the consideration or evaluation of these test results.",
"The present invention seeks to provide a significantly more powerful tool for this purpose than presently exists.",
"A substantial number of genotypes are also recorded, for instance in The National DNA Database (UK Registered Trade Mark) of genotypes in the UK, and these provide a potential source of information to be considered.",
"The present invention seeks to provide a significantly more powerful tool for considering the information in such databases.",
"The present invention has amongst its aims to provide new techniques which enable useful information to be obtained by considering pre-existing DNA test results alone or in combination with new DNA test results so as to establish links between some of those test results in terms of genotypes which are supported as contributing to them.",
"The present invention has amongst its aims to evaluate the more supported genotypes given the test results in relation to various combinations of test results.",
"According to a first aspect of the invention we provide a method of considering DNA based links between two or more situations, the method including: obtaining a plurality of test results, each test result relating to a situation, each test result including information on the DNA from that situation, the plurality of test results providing a group of test results; selecting a plurality of test results from the group of test results, the plurality of results forming a combination of test results; considering a genotype as possibly giving rise to each of the test results of the combination, evaluating the support for that genotype giving rise to all of the test results of the combination; considering the genotype as a DNA based link between the situations for the test results in the combination if the support meets defined criteria.",
"According to a second aspect of the invention we provide a method of considering DNA based links between two or more situations, the method including: obtaining a plurality of test results, each test result relating to a situation, each test result including information on the DNA from that situation, the plurality of test results providing a group of test results; selecting a plurality of test results from the group of test results, the plurality of results forming a combination of test results; considering a genotype as possibly giving rise to each of the test results of the combination, evaluating the support for that genotype giving rise to all of the test result of the combination; considering the genotype as a DNA based link between the situations for the test results in the combination if the support meets defined criteria; comparing a genotype which is considered a DNA based link against records of genotypes to identify matching genotypes in the records.",
"The second and/or third aspects of the invention may include any of the features, options or possibilities set out elsewhere in this document, including the third and fourth aspects of the invention.",
"The situations may be test results for different scenes and/or different samples from the same or different scenes and/or replicates from the same samples or from different samples.",
"The situations may include test results for one or more known individuals.",
"The test results may relate to mixtures and/or single contributor cases.",
"The test results may relate to complete and/or partial profiles.",
"The information on the DNA may be the allele present at a loci, ideally for at least 6 different loci.",
"The information may include the peak height and/or peak area for those alleles.",
"Complete or partial profiles may be accepted as test results for application of the method.",
"The group of test results may be stored as a group or may be pulled together from discrete sources.",
"The discrete sources may be pulled together to form a group.",
"Alternatively one or more test results from such sources may be pulled together to form a group for the purposes of the application of the method.",
"All possible combinations of test results for the test results in the group may be considered.",
"Pairs, triplets and quadruplets of test results may particularly be considered.",
"The evaluation a direct evaluation of the support for a genotype giving the combination of test results or may involve an evaluation of the support for a given genotype giving each of the test results, the individual evaluations being combined to give the overall evaluation.",
"The support may meet the defined criteria when the probability that the genotype could have given rise to the test results of the combination is above a given level.",
"The level may be predetermined.",
"The support may meet the defined criteria when an expression of the support that the genotype could have given rise to the test results of the combination is below a given level.",
"That level may be predetermined.",
"The method may be used to establish DNA based links between different scenes.",
"The method may be used to establish DNA based links between different samples taken from different parts of the same scene.",
"The method may be used to establish DNA based links between replicates of the same sample.",
"The method may be used to establish DNA based links between combinations of such situations.",
"The method may be used to establish a DNA based link between a person whose genotype is known and one or more situations.",
"The method may be used to establish DNA based links between test results which are ambiguous or which could be suggested to be ambiguous.",
"Such test results might be one or more of test results which are or could be mixtures and/or test results which are or could involve low levels of DNA in the sample for one or more persons (low levels could be though of as less than 500 pg or even less than 100 pg of DNA) and/or test results which do or could involve effects due to stutter and/or allele drop out and/or allele contamination and/or preferential amplification.",
"The method may be used to establish a genotype or genotypes which are DNA based links between situations and which are then matched to an existing genotype record, ideally for a known person.",
"According to a third aspect of the invention we provide a method of considering DNA based links between two or more situations, the method including: obtaining a plurality of test results, each test result relating to a situation, the test results including information on the DNA from the corresponding situation, the plurality of test results providing a group of test results; selecting a combination of test results which includes a plurality of test results from the group of test results; considering one or more genotypes as possibly giving rise to the combination of test results and evaluating for each of those genotypes the support that the genotype gave rise to the combination of test results; considering a genotype whose support meets defined criteria as a DNA based link between the situations of the combination.",
"The third aspect of the invention may include any of the features, options or possibilities set out in this document, but particularly from amongst the following.",
"Preferably the evaluation of the support for a genotype giving rise to the combination of test results includes a consideration of the probability of the test results arising given that genotype and the probability of occurrence of that genotype.",
"Preferably the evaluation of the support for the genotype giving rise to the combination of test results includes a consideration of the overall probability of the test results arising given that genotype and the probability of occurrence of that genotype, for all possible genotypes.",
"Preferably the evaluation of the support for a genotype giving rise to the combination of test results includes a consideration of the probability of the test results arising given that genotype and the probability of occurrence of that genotype against a consideration of the overall probability of the test results arising given that genotype and the probability of occurrence of that genotype, for all possible genotypes.",
"Preferably the evaluation of the support for a genotype giving rise to the combination of test results is defined by Pr ( G l | D… ) = ∏ ijk Pr ( D ijk | G l ) Pr ( G l ) ∑ i ∏ ijk Pr ( D ijk | G l ) Pr ( G l ) for all l .",
"where G1 represents the particular genotype, D represents the combination of test results, potentially including test results due to various scenes and/or samples from scenes and/or replicates of samples from scenes, i represents the range of replicates, j the range of samples, k the range of scenes and l the range of genotypes under consideration.",
"Preferably the method is applied to a plurality of combinations selected from the group of test results.",
"Each possible combination of test results from amongst the group of test results may be considered.",
"Combinations may include two, three, four or more test results from the group.",
"A combination may include one or more test results for a situation which is from a different scene to one or more other situations represented by test results in the group.",
"A combination may include one or more test results for a situation which is from a different sample to one or more other situations represented by test results in the group.",
"A combination may include one or more test results for a situation which is from a different replicate to one or more other situations represented by test results in the group.",
"Each possible genotype may be considered as giving rise to the combination of test results.",
"One or more limits may be applied to the genotypes which are considered from amongst the full set of possible genotypes.",
"The limits may be based on one or more rules as to genotypes which could not practically give one or more of the results in the combination being considered.",
"The evaluation may be expressed as a posterior probability.",
"Preferably the evaluation of the support for a genotype giving rise to the group of test results includes a consideration of the effect of one or more of contamination of the test results and/or allele drop out from the results and/or stutter in the results and/or preferential amplification of the results.",
"The method may be used to consider one or more test results which are mixtures.",
"The method may be used to consider one or more test results which contain low levels of DNA from one or more persons.",
"The method may be used to consider one or more test results where there is ambiguity or suggested ambiguity as to the contributors and/or the genotypes of the contributors.",
"The method may be used to consider one or more test results to which there is only one contributor and/or for which the genotype is known.",
"A DNA based link may be present where the support that a genotype could give rise to the test result meets criteria for each of the test results for which a link is determined.",
"The criteria may be a predetermined support level.",
"The defined criteria may be a genotype whose support for giving rise to the test results in the combination is above a defined level.",
"The level may be predefined.",
"A DNA based link may be used to suggest or confirm a link between one or more situations.",
"The link may support other links for those situations based on other evidence types.",
"The link may suggest links for which links had not previously been suggested.",
"The link may be used to direct subsequent investigation of the situations and/or events which gave rise to those situations and/or individuals behind those situations by law enforcement agencies.",
"The DNA based link may be used as evidence in legal proceedings.",
"A genotype which is considered as a DNA based link between the situations of the combination may be used in a further consideration.",
"The further consideration may include the review of possible matches between the genotype and a collection of genotype records.",
"The existence of a match may be deemed to occur where correspondence at or above a given level of correspondence occurs.",
"The given level may be at least 80%, and ideally at least 90%, of alleles in common between the genotypes.",
"The recorded genotypes may be genotypes of known individuals.",
"The further consideration may link the genotype to an individual.",
"The further consideration may link the situations of the combination of test results to an individual.",
"The test result may be obtained, or have been obtained, by PCR based amplification of DNA collected from the situation.",
"The test result may be obtained, or have been obtained, by establishing allele identities for one or more loci of the DNA.",
"The peak area and/or peak height for the alleles may be obtained.",
"The test results may be obtained for use in the method and/or may have previously been obtained for other purposes.",
"The test results may be reused in the method after use in other analysis and/or consideration methods.",
"The situations may be test results for different scenes and/or different samples from the same or different scenes and/or replicates from the same samples or from different samples.",
"The situations may include test results for one or more known individuals.",
"The test results may relate to mixtures and/or single contributor cases.",
"The test results may relate to complete and/or partial profiles.",
"The number of test results obtained may be more than 10, more than 100 or even more than 1000.The information on the DNA may be the identity of one or more alleles at one or more loci.",
"The identity and peak height and/or peak area may be obtained.",
"The group of test results may be in a formal group or may be test results stored in a variety of locations.",
"The group of test results may include one or more test results from the test results for an investigation and/or one or more test results from the test results for another, (potentially at the time of combinations selection, unrelated) investigation and/or one or more test results from a centralised store, such as The National DNA Database.",
"The group of test results may include all available test results.",
"According to a fourth aspect of the invention we provide a method of considering DNA based links between two or more situations, the method including: obtaining a first test result for a first situation, the first test result including information on the DNA from the first situation; considering a genotype as possibly giving rise to the first test result and evaluating the support that the genotype gave rise to the first test result, repeating the evaluation of the support for a plurality of other genotypes, generating a set of possible genotypes based on the evaluation with respect to the first test result; obtaining a second test result for a second situation, the second test result including information on the DNA from the second situation; considering a genotype as possibly giving rise to the second test result and evaluating the support that this genotype gave rise to the second test result, repeating the evaluation of the support for a plurality of other genotypes, generating a set of possible genotypes based on the evaluation with respect to the second test result; combining the set of possible genotypes for the first test result and the set of possible genotypes for the second test result, genotypes present in the first set and the second set being given a higher ranking in the combined set than genotypes not present in one or more of the sets; considering one or more of the higher ranked genotypes in the combined set as a DNA based link between the first situation and the second situation.",
"The fourth aspect of the invention may include any of the features, options or possibilities set out elsewhere in this document.",
"Each possible genotype may be considered as giving rise to a test result.",
"One or more limits may be applied to the genotypes which are considered from amongst the full set of possible genotypes.",
"The limits may be based on one or more rules as to genotypes which could not practically give the result being considered.",
"The same or different genotypes may be considered for the different test results.",
"Different, but preferably the same rules may be used as limits in considering each test result.",
"The evaluation of the support may involve a determination of the mixture proportions contributed by different individuals.",
"The determination may involve the comparison of the observed and expected peak height and/or peak area results at one or more loci.",
"The peak area expected may be subtracted from the peak area observed for a locus, squared, and then summed with the values for the other loci to give a residual.",
"Account may be taken of errors in mixture proportion and/or peak area determinations.",
"The evaluation of the support may be used to rank the set.",
"Preferably the evaluation of the support includes a least squares based evaluation.",
"The lower the value of the residual a genotype has, the higher ranking it may be given in the ranked evaluation.",
"Preferably the same evaluation is used for each genotype considered and/or for each test result considered.",
"The evaluation may produce a list of possible genotypes for the first and second test results.",
"The list may be ranked.",
"The set may be in the form of a list.",
"The set or ranked evaluation or evaluations may include a pre-determined number of genotypes.",
"The number may be at least 200 or even at least 400.The number may be less than 1000.The set or ranked evaluation may include all genotypes with a support above a pre-determined threshold.",
"Preferably the genotypes included in a set or ranked evaluation are ranked within that evaluation.",
"Preferably the combining of the set for the first test result and the set for the second test result includes, for genotypes present in the first set and in the second set, adding the support for that genotype for the first set to the support for that genotype for the second set.",
"The residual value for a genotype in the first set may be added to the residual value for the same genotype in the second set.",
"Preferably the combining of the set or ranked evaluation for the first test result and the set or ranked evaluation for the second test result includes combining the support for that genotype for the set or ranked evaluation it is present in with a dummy support for the set or ranked evaluation it is absent from.",
"The dummy may be a pre-set support value.",
"The dummy may be a multiple of the support of the least likely genotype in the set or ranked evaluation from which the genotype was absent.",
"The multiple is preferably greater than 1, such as 2.Preferably the genotypes are ranked within the combined set or combined evaluation.",
"Preferably genotypes present in each of the sets or ranked evaluations receive a high ranking in the combined set or ranked evaluation.",
"Preferably genotypes absent from one or more of the sets of ranked evaluations receive a low ranking in the combined set or ranked evaluation.",
"The first and second test results may be from situations which differ in terms of the scene and/or the sample and/or the replicate in question.",
"The method may be applied to three, four or more test results.",
"The method may be applied to test results in existing records.",
"Each pair of test results and/or triple of test results and/or quadruple of test results and/or higher combinations could be considered according to the method.",
"The method may be used to consider one or more test results which are mixtures.",
"The method may be used to consider one or more test results which contain low levels of DNA from one or more persons.",
"The method may be used to consider one or more test results where there is ambiguity or suggested ambiguity as to the contributors and/or the genotypes of the contributors.",
"The method may be used to consider one or more test results to which there is only one contributor and/or for which the genotype is known.",
"A genotype which is considered as a DNA based link between the situations may be used in a further consideration.",
"The further consideration may include the review of possible matches between the genotype and a collection of genotype records.",
"The existence of a match may be deemed to occur where correspondence at or above a given level of correspondence occurs.",
"The given level may be at least 80%, and ideally at least 90%, of alleles in common between the genotypes.",
"The recorded genotypes may be genotypes of known individuals.",
"The further consideration may link the genotype to an individual.",
"The further consideration may link the situations of the combination of test results to an individual.",
"The test results may be obtained, or have been obtained, by PCR based amplification of DNA collected from the situations.",
"The test results may be obtained, or have been obtained, by establishing allele identities for one or more loci of the DNA.",
"The peak area and/or peak height for the alleles may be obtained.",
"The test results may be obtained for use in the method and/or may have previously been obtained for other purposes.",
"The test results may be reused in the method after use in other analysis and/or consideration methods.",
"The situations may be test results for different scenes and/or different samples from the same or different scenes and/or replicates from the same samples or from different samples.",
"The situations may include test results for one or more known individuals.",
"The test results may relate to mixtures and/or single contributor cases.",
"The test results may relate to complete and/or partial profiles.",
"The number of test results considered may be more than 10, more than 100 or even more than 1000.The number of test results for which sets of possible genotypes are combined may be two, three, four, five or more.",
"A DNA based link may be used to suggest or confirm a link between one or more situations.",
"The link may support other links for those situations based on other evidence types.",
"The link may suggest links for which links had not previously been suggested.",
"The link may be used to direct subsequent investigation of the situations and/or events which gave rise to those situations and/or individuals behind those situations by law enforcement agencies.",
"The DNA based link may be used as evidence in legal proceedings.",
"The test results may be selected from a formal group of results or may be test results stored in a variety of locations.",
"The test results may include one or more test results from the test results for an investigation and/or one or more test results from the test results for another, (potentially at the time of combinations selection, unrelated) investigation and/or one or more test results from a centralised store, such as The National DNA Database.",
"The method may be applied to all available test results.",
"Various embodiments of the invention will now be described, by way of example only.",
"The present invention is aimed at establishing links between DNA test results obtained from a series of situations.",
"In general the DNA will be collected and analysed using conventional techniques, such as PCR based amplification and gel electrophoresis to identify the alleles occurring at various loci for the DNA.",
"The situations being considered could be multiple scenes from which DNA is collected and/or multiple samples of DNA from different parts of a single scene and/or even multiple replicates of DNA from a single sample which are analysed and generate test results.",
"The DNA test results considered could, in one or more cases, have arisen from samples taken from known persons in controlled circumstances, such as the genotype profiles stored on The National DNA Database (Registered Trade Mark).",
"The aim is to establish whether there are any well supported genotypes (those offering a sufficiently high probability), given the various separate test results obtained, for a particular combination of situations.",
"Taking the example of a serial offender there may be scenes which include DNA of that offender and scenes which do not, samples from a given scene which include DNA of that offender and samples which do not and even replicates of a sample which include a report of the DNA of that offender and other replicates which do not, despite those replicates arising from the same sample.",
"Furthermore some of the samples may be single contributor samples and others may be mixtures involving DNA contributed from a plurality of individuals.",
"Determining links between such scenes and/or samples and/or replicates is not an easy task on an effective timescale.",
"The present invention provides an intelligence tool in which a DNA test result from a situation is considered in combination with one or more test results from other situations.",
"The tool seeks to establish genotypes which are more supported to arise from the test results in that particular combination of such situations and thereby provide a DNA based link between those situations.",
"Of course the tool can be used to establish that there is no likely link between the test results in that combination and still provide some useful information.",
"The combination of test results considered may particularly include test samples from two or more different scenes, but is useful even where the test results are from different samples at the same scene, and even for different replicates of the same sample.",
"The tool can also consider test result combinations which include test results from different types of situation.",
"The technique can be used to compare test results having one or more different timings, including: the test results from the analysis of present situations, for instance, recently occurred events under active law enforcement agency consideration for which test results are newly available; the test results from the analysis of past situations, for instance, past events no longer under active consideration for which test results were generated at the time or have now been generated; the test results from analysis of known situations, for instance test results obtained from known persons under controlled circumstances (such as those used to generate The National DNA Database.",
"The tool may be used to investigate speculated or informed links between situations, which links are arrived at through other processes, by indicating a high level of support for one or more genotypes linking those situations given the test results for them.",
"The tool may be used to investigate in a non-premeditated manner a body of test results from situations with a view to generating suggested links.",
"The tool may thus generated suggested links between situations not previously considered in conjunction with one another.",
"The tool can also be used to suggest links between one or more situations associated with a crime or scene and a stored test result, previously obtained from a known individual.",
"Two main embodiments of the invention are now described with similar intents behind the process they facilitate and the uses they can be put to.",
"In each case the aim is to identify one or more genotypes which is supported given the particular combination of test results (referred to as a partition) and hence the situations behind them.",
"This provides information on links between crimes, locations and the like.",
"As a further part of the process the genotypes identified in this way can then be compared with the genotype for a known situation to identify any matches between that genotype and one of the particular supported genotypes.",
"A link between the set of scenes and a known individual can thus be obtained.",
"In a first technique there is a general consideration of the probability of a genotype given the combination of test results/partition, using a consideration of the probability of the test results arising given a specific genotype (from amongst the many possibilities for the genotype) and the probability of occurrence of that specific genotype, compared with a consideration of the overall probability of the test results arising given a specific genotype and the probability of that genotype, for all the possible genotypes.",
"The consideration can be represented as Pr ( G l | D… ) = ∏ ijk Pr ( D ijk | G l ) Pr ( G l ) ∑ i ∏ ijk Pr ( D ijk | G l ) Pr ( G l ) for all l .",
"where i represents the range of replicates, j the range of samples, k the range of scenes and 1 the range of genotypes under consideration.",
"This general consideration can be applied to one or more combinations/partitions of test results from amongst the test results available.",
"Indeed all such combinations/partitions can be considered in obtaining suggestions as to those which are linked and/or the level of support for the linking supported genotype.",
"A combination of situations or a partition is a pair, triplet, quadruplet or higher number of test results each from a different situation (such as replicates and/or samples and/or scenes).",
"By considering each of the possible combinations/partitions an indication will be provided of those combinations/partitions which are linked by DNA reported in the test result for each of the situations behind it, due to the high posterior probability obtained from the consideration.",
"A very large number of the combinations/partitions will of course not be linked.",
"The outcome is one or more supported genotypes, each of the supported genotypes being the link for one or more of the combinations/partitions.",
"Thus a supported genotype X may be suggested as being involved in a partition consisting of test results from five particular scenes; a separate but supported genotype Y may be suggested as involved in four scenes and so on.",
"This linking of situations, particularly between scenes can be of great use in its own right.",
"For instance, it may allow other evidence from a number of scenes to be considered in combination when previously there was no such suggestion of a link.",
"The other evidence in combination may lead to the solving of the crime.",
"Once the supported genotypes with a high posterior probability given the test results for that combination of situations or partition are obtained these supported genotypes can be used in further considerations.",
"For instance each of those supported genotypes can be considered against records of genotypes for matches or near matches (90%+of the alleles in common, for example) so as to link the supported genotype to other situations, for instance scenes, samples or replicates.",
"This information can be used to confirm other evidence and/or to direct future enquiries and/or to open up new lines of enquiry too.",
"In particular a link to an individual may be produced.",
"As a substantial number of the possible genotypes cannot arise given the test results, the consideration is limited down quite significantly from having to consider all the approximately 1021 genotypes possible in total.",
"With the processing side performed by a computer operating the defined consideration, the large amount of processing involved can be realistically performed.",
"The manner in which the probability of the specific genotype given the test results is considered can be as simply or as sophisticatedly considered as is desired.",
"Obviously more sophisticated considerations can give greater confidence and worth to any combinations of situations or partitions for which links are suggested.",
"Thus it is desirable to include within that probability consideration one or more functions or models which can account for one or more factors such as genotype dropout, Pr(GD), allele dropout, Pr(AD), stutter, Pr(S), preferential amplification, Pr(PA) and others.",
"These issues can all have an effect on the test result from a situation compared with the actual genotype behind the DNA in that situation.",
"Accounting for them makes the consideration of the extent of support for the supported genotype more robust.",
"To this end models for one or more of these factors can be included.",
"An illustration of the way in which these factors can be modelled is provided in “An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA.” Gill et al., Forensic Science International 112 (2000) 17-40.Models to account for laboratory introduced contamination, allele drop out and stutter in particular are provided.",
"The paper is concerned with accounting for such factors in the analysis of a single test result, however, and is not concerned with the between result considerations involved in this invention.",
"The models are none the less useful in assisting.",
"Other models can be used, however, and other factors can be modelled.",
"In a second technique a test result for a situation is obtained and is submitted to an evaluation of the likelihood of a genotype arising given that test result.",
"A heuristic approach is taken; the general aim being to list more supported genotypes ranked by residual of Euclidean distance.",
"The evaluation may use a technique such the Pendulum™ technique detailed in Gill et al, Forensic Science International 91 (1998) 41-53 to generate the starting information.",
"That paper describes the consideration of the relative contribution of different individuals to a mixture of DNA, followed by a consideration of the likelihood of the possible genotypes as having been behind the test result obtained.",
"The contents of that paper and in particular the disclosure of the manner in which the proportions of a mixture and the ranking of likelihood is performed is incorporated herein by reference.",
"The technique involves, for a single test result, the consideration of a number of loci simultaneously when establishing the likely mixing proportions and the likely genotypes, but is only concerned with the analysis of an individual situation.",
"No between situation/test consideration is involved.",
"Other techniques for detailing likely genotypes behind an individual test result can be used in a similar way to provide the starting information.",
"In the preferred embodiment, the starting information is provided by the Pendlum™ technique and the output of this evaluation is a list of genotypes which give the lowest residual value from the evaluation used.",
"In effect the most supported genotypes are assumed to be those with the lowest residuals.",
"These are ranked from the lowest residual up to a cut off point, which could be a residual level, but is normally a number of genotypes (frequently 500).",
"An output listing for the test result of a situation A is provided below in Table A; the Genotype designation letters and residual values are schematic illustrations only.",
"TABLE A Number in list Genotype Residual value 1 Genotype AA 1.002 × 106 2 Genotype GR 1.058 × 106 3 Genotype LD 1.078 × 106 4 Genotype HU 1.092 × 106 — — — 499 Genotype KW 2.002 × 106 500 Genotype LM 2.097 × 106 By repeating the evaluation for another test result, test result B, a further output listing is obtained, as set out in Table B.",
"This output listing is the test result from another situation, such as a sample obtained from another scene, a sample taken from a different part of the same scene or the like.",
"TABLE B Number in list Genotype Residual value 1 Genotype JE 1.011 × 106 2 Genotype QP 1.023 × 106 3 Genotype LW 1.085 × 106 4 Genotype AA 1.101 × 106 — — — 499 Genotype AS 1.989 × 106 500 Genotype YR 2.001 × 106 If the output listings are then added to one another in a prescribed manner a combined output listing can be obtained.",
"This listing then provides indications as to those, if any, genotypes which are possibly involved in the situations behind both the test results.",
"The same concept applies even if a combination of more than two situations is being considered.",
"In this embodiment the prescribed manner of adding the output listings to give the combined output listing involves the following rules.",
"Where the same genotype is present in each of the output listings considered then the residuals for that genotype in each of the output listings are added together.",
"Where a genotype is absent from an output listing, a dummy residual for each output listing the genotype is absent from is provided and the residuals for the output listings the genotypes is present in are added to that dummy residual.",
"The dummy residual in this embodiment, in each case, is the largest residual of that output listing the genotype is absent from multiplied by a factor (two in the illustrated example).",
"The genotype can alternatively by rejected entirely.",
"The combined output listing presents the genotypes in order based on the residual level they have.",
"Considering the output listings of tables A and B it turns out that Genotype AA is present in both output listings, as is a Genotype DS which had position 206 in Table A and position 56 in Table B.",
"None of the other genotypes were present in both output listings.",
"The combined output listings is represented in Table C. TABLE C Number in list Genotype Combined residual value 1 Genotype AA 2.103 × 106 2 Genotype DS 3.103 × 106 3 Genotype GR 5.060 × 106 4 Genotype LD 5.080 × 106 5 Genotype HU 5.094 × 106 6 Genotype JE 5.205 × 106 7 Genotype QP 5.217 × 106 8 Genotype LW 5.279 × 106 — — — 995 Genotype KW 6.004 × 106 996 Genotype LM 6.099 × 106 997 Genotype AS 6.183 × 106 998 Genotype YR 6.195 × 106 As can be seen the two supported genotypes which are considered possibilities in each of the two output listings stand out in the combined output listing when compared with the other genotypes and their residual values.",
"This accurately reflects the status of these supported genotypes as being more supported candidates from both output listings and the test results behind them, given the respective test results.",
"Genotype AA ranks higher than Genotype DS in view of its higher ranking in each of the output listings.",
"Whilst the technique is illustrated above in relation to the combining of two output listings, which in turn represent two test results, the technique can be applied to a larger number of output listings and their underlying test results in the same manner.",
"In practice it is envisaged that the technique, in any of its embodiments, could be applied to a database of existing test results.",
"Each pair of test results, each triplet of test results, each quadruplet of test results and so on could be considered; and as a result the situations behind them.",
"In this way any links between situations can be considered and highlighted in the combined output listing, without speculating on links or suggesting links for consideration first.",
"This is useful in approaching a body of unsolved or unlinked situations with a view to generating fresh avenues for investigation.",
"The technique could also be employed when a new test result or series of test results are obtained from a new situation, such as a new scene.",
"These test results can be considered in the same way in all possible combinations of situations, or partitions, with the pre-existing test results to see whether this situation is linked to another.",
"The technique can also be used to test speculative or implied links between situations, such as scenes and/or samples there from, based on other information or evidence.",
"As well as applying the technique in this way it is possible to use a limited number of test results from a plurality of situations to produce a list of supported genotypes given those test results.",
"These likely genotypes can then form the basis of further searches or investigations.",
"A given supported genotype can be compared with records of genotypes determined or suggested for other situations, such as from other samples, for other scenes or from the testing of individuals to determine their genotype.",
"A match gives a link between them.",
"The search can include not only direct matches but also take into account the fact that not all the prior genotypes may be complete.",
"A match, therefore, may be deemed to extend to genotypes where the two are close to one another, for instance where they share 20 or 21 out of the 22 alleles considered in the determination of a test result.",
"Again the genotype links suggested can link a variety of situations, such as scenes and/or samples and/or individuals and/or events in a useful and informative way."
]
] | Patent_10450597 |
[
[
"Microspray column, mass spectrometer, and mass spectrometry",
"A problem is to provide a microspray column capable of increasing the ionization efficiency, and a high sensitive mass spectrometer and a mass spectrometric method using such a microspray column.",
"For solving this problem, we provide a column for introducing a sample in an ionization source of a mass spectrometer (ESI/MS) being designed to ionize a sample molecule with electrospray and introduce it into an analyzer, has structural features of: (1) 0.5 μm or less in an inner diameter of a tip opening of the column; (2) 0.5 μm or more and 5 μm or less in a particle size of a column filler; and (3) fritless, a microspray column having the above (1) to (3), a mass spectrometer having such a microspray column in an ionization source, and a mass spectrometric method capable of performing a nonoflow electrospray with the microspray column."
],
[
"1.A column for introducing a sample in an ionization source of a mass spectrometer (ESI/MS) being designed to ionize a sample molecule with electrospray and introduce it into an analyzer, comprising structural features of: (1) 0.5 μm or less in an inner diameter of a tip opening of the column; (2) 0.5 μm or more and 5 μm or less in a particle size of a column filler; and (3) fritless.",
"2.A mass spectrometer, comprising: an ionization source which is constructed such that a sample molecule in liquid droplets atomized from the microspray column described in claim 1 is ionized.",
"3.A mass spectrometric method, wherein a nonoflow electrospray is performed using the microspray column described in claim 1."
],
[
"<SOH> BACKGROUND ART <EOH>A mass spectrometer (hereinafter, referred to as “MS” (Mass Spectrometer)) is roughly composed of: an “ionization source” for ionizing a sample; an “analyzer” for separating ions according to the ratio of mass/charge, represented by m/ze (wherein m: mass, z: charge number, and e: unit charge); and a “detection and recording part” of ions being separated.",
"The electrospray ionization technique generally referred to “ESI” (the abbreviation for Electrospray Ionization) is known as one of the methods of ionizing and introducing a sample molecule to an analyzer of MS.",
"In this electrospray ionization technique, a spray is carried out by applying the high voltage on a sample molecule being brought in ionic state with acid or the like in a solvent.",
"This electrospray ionization technique is a technique for spraying a sample molecule, which is brought into an ionic state by acid or the like in the solution, by applying high voltage; forming liquid droplets (mist) in micron order, in which many solvent molecules are combined with multi-protonated molecules; and spraying nitrogen to dry and remove the solvent to ionize the sample molecule, followed by subjecting to the above analyzer.",
"As the charge number of ions being generated becomes large in this technique, it may be particularly useful in the measurements of peptide and proteins, respectively.",
"Here, the electrospray ionization of the sample molecule in the above ionization source of MS is performed by discharging and atomizing (spraying) the sample molecule in small quantities from a column formed of an elongated silica glass generally having an opening with a minute aperture.",
"This column will be referred to as a “microspray column” below.",
"FIG.",
"2 is a diagram that simplifies and expresses the configuration of the conventional electrospray ionization technique.",
"The reference numeral 10 denotes a conventional typical microspray column.",
"On the microspray column 10 being formed such that the tip portion thereof has a cusp form, a large number of fillers 10 a such as chemical bond type silica gels or the like having a particle size of about 50 μm is formed.",
"In addition, the inner diameter d of the tip portion of the column is about 10 to 15 μm.",
"Furthermore, the outermost tip portion of the microspray column 10 is loaded with a large-sized bead 10 b for preventing the discharge of beads, which are also referred to as a flit.",
"This microspray column 10 is a constituent member of the ionization source of a mass spectrometer 11 and is arranged such that it extends to the front of a pre-column 12 on which a high voltage is loaded.",
"It is configured that fine droplets 14 containing the sample molecule are atomized from the tip portion of the microspray column 10 to the analyzer 13 of the mass spectrometer 11 .",
"However, in the conventional microspray column, the separation efficiency of a chromatograph was insufficient since the particle diameter of the filler in the column was large (generally about 50 μm).",
"In addition that the particle size of the filler was large, the inner diameter of the tip opening of the above microspray column was also large.",
"Therefore, the discharge amount of the sample increased and the particle size of charged liquid droplets formed by the spray was also large.",
"As a result, in the process in which the solvent was dried and vaporized, the efficiency of transferring charged electrons to the sample molecule in the solvent was not sufficient.",
"In other words, the ionization efficiency of the sample molecule was insufficient.",
"Furthermore, the microspray column was configured such that many areas without filling with the filler were formed near the tip opening, resulting in a large discharge amount of the sample and a large particle size of the charged droplet formed with the spray.",
"Therefore, an object of the present invention is to provide a microspray column capable of improving the ionization efficiency, and a high-sensitive mass spectrometer and a mass spectrometric method using this microspray column."
],
[
"TECHNICAL FIELD The present invention relates to an improved technique in mass spectrometry.",
"In particular, the present invention relates to an improved technique of a mass spectrometer (ESI/MS) being designed to allow the introduction of a sample molecule with electrospray ionization, and a mass spectrometric method.",
"BACKGROUND ART A mass spectrometer (hereinafter, referred to as “MS” (Mass Spectrometer)) is roughly composed of: an “ionization source” for ionizing a sample; an “analyzer” for separating ions according to the ratio of mass/charge, represented by m/ze (wherein m: mass, z: charge number, and e: unit charge); and a “detection and recording part” of ions being separated.",
"The electrospray ionization technique generally referred to “ESI” (the abbreviation for Electrospray Ionization) is known as one of the methods of ionizing and introducing a sample molecule to an analyzer of MS.",
"In this electrospray ionization technique, a spray is carried out by applying the high voltage on a sample molecule being brought in ionic state with acid or the like in a solvent.",
"This electrospray ionization technique is a technique for spraying a sample molecule, which is brought into an ionic state by acid or the like in the solution, by applying high voltage; forming liquid droplets (mist) in micron order, in which many solvent molecules are combined with multi-protonated molecules; and spraying nitrogen to dry and remove the solvent to ionize the sample molecule, followed by subjecting to the above analyzer.",
"As the charge number of ions being generated becomes large in this technique, it may be particularly useful in the measurements of peptide and proteins, respectively.",
"Here, the electrospray ionization of the sample molecule in the above ionization source of MS is performed by discharging and atomizing (spraying) the sample molecule in small quantities from a column formed of an elongated silica glass generally having an opening with a minute aperture.",
"This column will be referred to as a “microspray column” below.",
"FIG.",
"2 is a diagram that simplifies and expresses the configuration of the conventional electrospray ionization technique.",
"The reference numeral 10 denotes a conventional typical microspray column.",
"On the microspray column 10 being formed such that the tip portion thereof has a cusp form, a large number of fillers 10a such as chemical bond type silica gels or the like having a particle size of about 50 μm is formed.",
"In addition, the inner diameter d of the tip portion of the column is about 10 to 15 μm.",
"Furthermore, the outermost tip portion of the microspray column 10 is loaded with a large-sized bead 10b for preventing the discharge of beads, which are also referred to as a flit.",
"This microspray column 10 is a constituent member of the ionization source of a mass spectrometer 11 and is arranged such that it extends to the front of a pre-column 12 on which a high voltage is loaded.",
"It is configured that fine droplets 14 containing the sample molecule are atomized from the tip portion of the microspray column 10 to the analyzer 13 of the mass spectrometer 11.However, in the conventional microspray column, the separation efficiency of a chromatograph was insufficient since the particle diameter of the filler in the column was large (generally about 50 μm).",
"In addition that the particle size of the filler was large, the inner diameter of the tip opening of the above microspray column was also large.",
"Therefore, the discharge amount of the sample increased and the particle size of charged liquid droplets formed by the spray was also large.",
"As a result, in the process in which the solvent was dried and vaporized, the efficiency of transferring charged electrons to the sample molecule in the solvent was not sufficient.",
"In other words, the ionization efficiency of the sample molecule was insufficient.",
"Furthermore, the microspray column was configured such that many areas without filling with the filler were formed near the tip opening, resulting in a large discharge amount of the sample and a large particle size of the charged droplet formed with the spray.",
"Therefore, an object of the present invention is to provide a microspray column capable of improving the ionization efficiency, and a high-sensitive mass spectrometer and a mass spectrometric method using this microspray column.",
"DISCLOSURE OF THE INVENTION In order to solve the above-mentioned technical subject, the following means are adopted in this invention.",
"At first, the microspray column for introducing a sample into an ionization source of a mass spectrometer (ESI/MS) configured to perform an electrospray ionization of the sample molecule and introduce the sample molecule into an analyzer was designed such that (1) the inner diameter of a tip opening of the column is 0.5 μm or less, (2) a particle size of the filler in the column is larger than 0.5 μm but not more than 5 μm, and (3) there is no frit at all (fritless) .",
"Here, “frit” means the member of a major-diameter bead and other members for blocking the bead loaded in the tip portion of the column.",
"On the other hand, “fritless” means the configuration in which only the filler is filled in the column without using the frit.",
"The particle size of the filler is a minimum diameter as much as it cannot be conventionally conceived.",
"In addition, the inner diameter of the tip opening of the microspray column is small.",
"Therefore, the discharge amount of the sample can be substantially made small, compared with the conventional one.",
"For this reason, the particle diameter of the charged droplet formed by the spray can be miniaturized.",
"As a result, it becomes possible to increase the efficiency of transferring the charged electron in the solvent to the sample molecule.",
"Therefore, an increase in ionization efficiency of the sample molecule becomes possible.",
"It is preferable to shape the tip portion of the microspray column into tapering form as much as possible.",
"In addition, it is preferable to minimize the area of the tip portion of the microspray as much as possible.",
"This is because, when the area of the tip portion of the column is too large, there is a phenomenon in which a large droplet is formed while the solution discharged from the column is being adhered on the tip.",
"Therefore, the liquid droplets can be prevented from becoming fine.",
"It is preferable that the inner diameter of the tip opening of the column is 0.1 μm or more.",
"This is because a high pressure is needed for the discharge of a solution when the inner diameter of the tip opening is less than 0.1 μm.",
"In the present invention, furthermore, the present invention offers a mass spectrometer characterized by comprising an ionization source constructed such that a sample molecule contained in charged droplets atomized from the above microspray column is ionized, and a mass spectrometric method for performing an nanoflow electrospray using the above microspray column.",
"Here, the term “nanoflow electrospray” means a technique capable of performing a stable electrospray ionization on a solution that contains a sample molecule to be fed at a flow rate in the order of nano liters (nL/min) and introducing into an analyzer of a mass spectrometer.",
"With this means, it is possible to provide a mass spectrometer having high detection sensitivity and excellent ionization efficiency and a mass spectrometric method.",
"As described above, the present invention is capable of improving the ionization efficiency of a sample molecule and perfectly performing a nanoflow electrospray by making fine charged liquid particles discharged from the microspray column.",
"This technique has a technical meaning that the high sensitivity measurement of a high molecular weight compound such as peptide or protein becomes possible because of an increase in the charge number of the ion to be generated.",
"BRIEF EXPLANATION OF THE DRAWINGS FIG.",
"1 is a diagram illustrating an extended view around the tip portion of the microspray column of the present invention; FIG.",
"2 is a diagram illustrating a simplified configuration of the conventional electrospray column technique; FIG.",
"3(A) is a schematic diagram of the chromatograph of the mass spectrometer when the conventional microspray column is used, and FIG.",
"3(B) is a schematic diagram of the chromatograph of the mass spectrometer when the microspray column of the present invention is used; FIG.",
"4 is a chromatograph of the mass spectrometer obtained in the example; and FIG.",
"5 is a table of the whole amino acid sequence (609 amino acids) which is the digestive product of trypsin enzyme of human serum albumin.",
"BEST MODE FOR CARRYING OUT THE INVENTION Preferable embodiments of the present invention will be described with reference to the attached drawings.",
"FIG.",
"1 is an enlarged view of around the tip portion of the microspray column of the present invention.",
"In FIG.",
"1, the reference numeral 1 denotes a microspray column (hereinafter, simply referred to as “column”) made of silica grass, which is shaped like an elongated cylinder with a hollow formed therein.",
"This column 1 functions as a sample-installation column which constitutes an ionization source of a mass spectrometer (ESI/MS) designed to introduce a sample molecule into the analyzer after ionizing the sample compound with electrospray.",
"As shown in FIG.",
"1, the tip portion 1a of the column 1 has a cusp form which tapers off gradually, and a tip opening 1b having a specific diameter for discharging a sample solution and atomizing the sample solution toward an analyzer of a mass spectrometer not shown in the figure is formed in the outermost tip portion.",
"Furthermore, in the inside of the column 1, a filler 2 functioned as a sorbent at the time of separating a sample is filled up with uniform density.",
"In the tip opening 1b, it is configured such that a frit is not loaded.",
"That is, the column 1 of the present invention is a fritless column.",
"Here, in the present invention, the inner diameter D1 of the tip opening 1b of the column 1 is 0.5 μm or less, preferably 0.1 μm or more and 0.5 μm or less.",
"The particle size D2 of the above filler 2 is more than 0.5 μm and 5 μm or less.",
"The reason of setting the inner diameter D1 of the tip opening 1b to 0.5 μm or less (D1≦0.5 μm) is that a charged droplet containing a sample molecule separated from the column 1 and atomized from the tip opening 1b is sufficiently made smaller to increase the ionization efficiency of the sample molecule with certainty.",
"Specifically, the inner aperture D1 of tip opening 1b is set to 0.5 μm or less to allow the mass spectrometer to generate a large charge number of ions in the sample molecule enough to realize the high-sensitive measurement of a high molecular weight compound such as peptide or protein using a mass spectrometer.",
"In addition, the reason of defining the particle size D2 of the filler 2 to more than 0.5 μm and 5 μm or less (0.5 μm<D2≦5 μm) is that for preventing the filer 2 from being discharged through the tip opening 1b of 0.5 μm or less in inner diameter.",
"The particle size D2 of the filler is made larger than the inner diameter D1 of the above tip opening 1 (D1<D2), while the particle size D2 of the filler 2 is made smaller than 5 μm or less to increase the total surface area of the whole filler 2.Furthermore, as the particle size D2 of the filler 2 is defined as 5 μm or less, the results can be obtained within a short time with a small amount of an eluent at a nanoflow level.",
"In addition, a chemical bond type silica gel (e.g., C18 having a large absorbency) may be used as the filler 2.Here, FIG.",
"3 is a schematic diagram for making a comparison between the chromatograph (A) of the mass spectrometer at the time of using the conventional microspray column and the chromatograph (B) of the mass spectrometer at the time of using the microspray column of the present invention.",
"As shown in FIG.",
"3, the microspray column of the present invention shows a sharp peak in the chromatograph of the mass spectrometer.",
"More concretely, reading of the rising peak and also no tailing in the second half of a peak cannot be , and, more specifically, the tendency of generating a sharp peak that exceeds a detection limit can be notably appeared.",
"The sensitivity of the mass spectrometer is concentration-dependent, so that the signal strength increases as the peak height increases.",
"Therefore, using the microspray column of this invention, the sensibility and the resolution of a mass spectrometer become high.",
"In addition, it becomes possible to raise the rate of decoding an amino acid sequence (sequence coverage), exponentially.",
"EXAMPLE A solution including a digestive product of a trypsin enzyme derived from human serum albumin (a molecular weight of 65,000 to 70,000) at a concentration of 50 femto mole or less was atomized from a microspray column of the present invention under the conditions in which the tip diameter of the column was 0.5 μm or less, fritless, and the particle size of the filler was 1.0 μm.",
"The chromatograph of the mass spectrometer obtained in the present example is shown in FIG.",
"4.As shown in the schematic diagram of FIG.",
"3, the sharp peak without reading or tailing was also obtained by the actual experimental findings as shown in the schematic diagram of FIG.",
"3, so that the decipherment of an amino acid sequence could be raised exponentially.",
"FIG.",
"5 shows a total amino acid sequence table (609 amino acids) of the above digestive product.",
"In this example, the 573 amino acid sequences except of 36 amino-acid portion surrounded by a square enclosure in FIG.",
"5 were deciphered.",
"The rate of a decipherment was dramatically as high as 94% (573/609).",
"Industrial Applicability According to the microspray column of the present invention, the particle size of the filler is a minimum diameter in addition to make the inner diameter of the microspray column equal to a predetermined diameter or less.",
"Therefore, the discharge amount of a sample solution per unit time can be sharply lessened as compared with the conventional one, so that the particle size of charged droplets formed by spraying can be made finer.",
"As a result, the efficiency of transferring the electrons charged in the solvent to the sample molecule can be extensively improved and the ionization efficiency of the sample molecule can be increased.",
"Furthermore, in the present invention, it is characterized by comprising an ionization source designed to ionize the sample molecule to be contained in the charged droplets atomized from the above microspray, so that a mass spectrometer having excellent ionization efficiency and high detection sensitivity can be provided.",
"Furthermore, according to the present invention, a mass spectrometric method that surely performs a nanoflow electrospray and nano-LC gradient analysis by the above microspray column, so that a high sensitive measurement of high molecular weight compound such as peptide and protein can be performed, positively.",
"Consequently, an extensive improvement in the rate of an amino acid decipherment of peptide or protein can be attained."
]
] | Patent_10450608 |
[
[
"Imaging head with laser diode array and a beam-shaping micro light-pipe array",
"An optical imaging heads that produce a plurality of light spots on light sensitive media such as photographic film or printing plate.",
"The optical head incorporates an array of multi-mode laser diodes as a light source, a Micro Light-Pipe Array (MLPA) as a beam-shaping element, means for reducing the divergence of the laser diode beam in the fast axis direction and means for imaging the laser diode emitters on a surface close to the micro light-pipe entrance aperture."
],
[
"1.An optical imaging head comprising: an array of multi-mode laser diode emitters; an array of micro light-pipes (MLPs), each of said individual micro light-pipes being associated with one of said laser diode emitters; and means for imaging the exit aperture of each of said micro light-pipes on a photosensitive medium.",
"2.An optical imaging head according to claim 1, wherein said means for imaging comprises a telecentric lens.",
"3.The optical imaging head of claim 1, additionally comprising means for reducing the divergence of the laser diode beam in the fast axis direction.",
"4.The optical imaging head of claim 3, wherein said means for reducing the divergence is an anamorphic lens.",
"5.The optical imaging head of claim 4, wherein said anamorphic lens is cylindrical.",
"6.The optical imaging head of claim 3, wherein said means for reducing the divergence is common for all said laser diode emitters.",
"7.The optical imaging head of claim 3, wherein said means for reducing the divergence is separate for each of said laser diode emitters.",
"8.The optical imaging head of claim 7, wherein said means for reducing the divergence is a micro-lens array.",
"9.The optical imaging head of claim 1, additionally comprising means for imaging the laser diode emitters on a surface close to the micro light-pipe entrance aperture.",
"10.The optical imaging head of claim 1 wherein the micro light-pipes are tapered.",
"11.The optical imaging head of claim 3 wherein the micro light-pipes are of funnel type.",
"12.An external-drum electro-optical plotter comprising the optical head of claim 1.13.A flatbed electro-optical plotter comprising the optical head of claim 1.14.A method of producing a plurality of writing spots on a photosensitive medium, comprising the steps of: providing an array of multi-mode laser diode emitters; providing an array of micro light-pipes, each of said individual micro light-pipes being associated with one of said laser diode emitter; and imaging the exit aperture of each of said micro light-pipes on said photosensitive medium.",
"15.The method of claim 14, additionally comprising, before said step of imaging, the step of providing means for reducing the divergence of the laser diode beam in the foist axis direction.",
"16.The method of clam 15, additionally comprising, after said step of providing means for reducing the divergence of the laser diode beam in the fast axis direction, the step of imaging the laser diode emitter in a surface close to the micro light-pipe entrance aperture."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Optical heads for imaging a plurality of light spots on a light sensitive media may incorporate an array of laser diodes as a light source.",
"The laser diodes array may be configured as an ordered plurality of individual laser diodes mounted on a common carriage, or as a plurality of laser emitters manufactured on a single-piece semiconductor material (such as GaAs).",
"For brevity, the light source (whether configured out of individual laser diodes or manufactured on a single semiconductor chip) will be referred to hereunder as Individually Addressed Laser Diode Array (IALDA).",
"The imaging speed in electro-optical plotters is generally limited by the power delivered to the medium by the laser beam(s).",
"This is especially true when the imaged medium is a thermal or ablative printing plate, or laser-transfer material, where the sensitivity is typically of the order of several hundreds mJ/cm 2 .",
"In order to achieve the required power, the IALDA has to be built of powerful multi-mode laser diodes (LD).",
"Multimode LDs are characterized by the light-emitting region having a very elongated shape, typically 1 micron across and 50 to 200 microns along the array axis, with the beam divergence in the cross-emitter direction high, typically 50-60 degrees FWHM, and the beam divergence in the length direction relatively low, typically 10 degrees FWHM.",
"For brevity, the cross-emitter direction will be referred to as the ‘fast axis’ and the emitter's length direction will be referred to as the ‘slow axis’ The near field emission pattern of multi-mode LDs is substantially rectangular.",
"An important characteristic of multi-mode LDs is that the energy distribution of the near field in the slow axis direction is non-uniform and changes with the LD's junction temperature, as well as with the data current driving the diode.",
"This effect is often displayed as a “hot spot” moving along the emitter's length.",
"When the image on the photosensitive medium is formed by imaging the near field of the LD, the non-uniform and frequently changing energy distribution of its pattern leads to undesired effects, such as image density irregularities.",
"A method and apparatus for overcoming these shortcomings of multi-mode LDs by using optical diffusers is disclosed in EP 0 992 343 A1 to Sousa U.S. Pat.",
"No.",
"6,208,371 to Takeshi et al, describes an optical beam-shaping system imaging the near field of a LD.",
"The present invention successfully solves the above mentioned shortcomings of imaging the multi-mode LD near field, by using a Micro-Light-Pipe Array (MLPA) for achieving spots with evenly distributed energy on the photosensitive medium, not depending on the LD's working conditions."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a multiple laser-beam recording apparatus producing a plurality of high-degree identical optical spots with uniform energy distribution.",
"Another object of the present invention is to provide a multiple laser-beam recording apparatus, which is free of image density irregularities due to non-uniform energy distribution of the LD near field.",
"Still another object of the present invention is to provide a high energy-efficient multiple laser-beam recording apparatus free of image density irregularities due to non-uniform energy distribution on the LD near field."
],
[
"FIELD OF THE INVENTION The present invention relates to optical imaging heads that produce a plurality of light spots on light sensitive media such as photographic film or printing plate.",
"The optical head incorporates an array of laser diodes as light source and a Micro Light-Pipe Array (MLPA) as a beam-shaping element.",
"BACKGROUND OF THE INVENTION Optical heads for imaging a plurality of light spots on a light sensitive media may incorporate an array of laser diodes as a light source.",
"The laser diodes array may be configured as an ordered plurality of individual laser diodes mounted on a common carriage, or as a plurality of laser emitters manufactured on a single-piece semiconductor material (such as GaAs).",
"For brevity, the light source (whether configured out of individual laser diodes or manufactured on a single semiconductor chip) will be referred to hereunder as Individually Addressed Laser Diode Array (IALDA).",
"The imaging speed in electro-optical plotters is generally limited by the power delivered to the medium by the laser beam(s).",
"This is especially true when the imaged medium is a thermal or ablative printing plate, or laser-transfer material, where the sensitivity is typically of the order of several hundreds mJ/cm2.In order to achieve the required power, the IALDA has to be built of powerful multi-mode laser diodes (LD).",
"Multimode LDs are characterized by the light-emitting region having a very elongated shape, typically 1 micron across and 50 to 200 microns along the array axis, with the beam divergence in the cross-emitter direction high, typically 50-60 degrees FWHM, and the beam divergence in the length direction relatively low, typically 10 degrees FWHM.",
"For brevity, the cross-emitter direction will be referred to as the ‘fast axis’ and the emitter's length direction will be referred to as the ‘slow axis’ The near field emission pattern of multi-mode LDs is substantially rectangular.",
"An important characteristic of multi-mode LDs is that the energy distribution of the near field in the slow axis direction is non-uniform and changes with the LD's junction temperature, as well as with the data current driving the diode.",
"This effect is often displayed as a “hot spot” moving along the emitter's length.",
"When the image on the photosensitive medium is formed by imaging the near field of the LD, the non-uniform and frequently changing energy distribution of its pattern leads to undesired effects, such as image density irregularities.",
"A method and apparatus for overcoming these shortcomings of multi-mode LDs by using optical diffusers is disclosed in EP 0 992 343 A1 to Sousa U.S. Pat.",
"No.",
"6,208,371 to Takeshi et al, describes an optical beam-shaping system imaging the near field of a LD.",
"The present invention successfully solves the above mentioned shortcomings of imaging the multi-mode LD near field, by using a Micro-Light-Pipe Array (MLPA) for achieving spots with evenly distributed energy on the photosensitive medium, not depending on the LD's working conditions.",
"SUMMARY OF THE INVENTION It is an object of the present invention to provide a multiple laser-beam recording apparatus producing a plurality of high-degree identical optical spots with uniform energy distribution.",
"Another object of the present invention is to provide a multiple laser-beam recording apparatus, which is free of image density irregularities due to non-uniform energy distribution of the LD near field.",
"Still another object of the present invention is to provide a high energy-efficient multiple laser-beam recording apparatus free of image density irregularities due to non-uniform energy distribution on the LD near field.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1a is a schematic isometric view of an IALDA and a beam-shaping MLPA according to the present invention; FIGS.",
"1b and 1e schematically illustrate an exemplary optical imaging head incorporating the IALDA and beam-shaping MLPA of FIG.",
"1a; FIG.",
"2a is a schematic isometric view of an IALDA with an anamorphic correcting lens and a beam-shaping MLPA according to the present invention; FIGS.",
"2b and 2c schematically illustrate an exemplary optical imaging head incorporating the IALDA with anamorphic correcting lens and beam-shaping MLPA of FIG.",
"2a; FIG.",
"3a is a schematic isometric view of an IALDA with a correcting and imaging lens system with virtual emitter image and a beam-shaping MLPA according to the present invention; FIGS.",
"3b and 3c schematically illustrate an optical imaging head incorporating the IALDA with correcting and imaging lens system with virtual emitter image and beam-shaping MLPA of FIG.",
"3a; FIG.",
"4a is a schematic isometric view of an IALDA with a correcting and imaging lens system and a beam-shaping MLPA according to the present invention; FIGS.",
"4b and 4c schematically illustrate an optical imaging head incorporating the IALDA with correcting and imaging lens system and beam-shaping MLPA of FIG.",
"4a; FIGS.",
"5a and 5b show the energy distribution of light in the entrance and exit apertures of a micro light-pipe respectively; FIG.",
"6a is a schematic isometric view of an IALDA and a tapered beam-shaping MLPA according to the present invention; FIGS.",
"6b and 6c schematically illustrate an exemplary optical imaging head incorporating the IALDA and tapered beam-shaping MLPA of FIG.",
"6a; FIG.",
"7a is a schematic isometric view of an IALDA with an anamorphic correcting lens and a funnel-type beam-shaping MLPA according to the present invention; FIGS.",
"7b and 7c schematically illustrate an exemplary optical imaging head incorporating the IALDA with anamorphic correcting lens and funnel-type beam-shaping MLPA of FIG.",
"7a; FIG.",
"8 is an exploded isometric view of a micro-machined MLPA; FIGS.",
"9a to 9d illustrate different channel shapes in micro-machined MLPA according to the present invention; FIG.",
"10 is a schematic isometric view of an IALDA and a bulk-type beam-shaping MLPA according to the present invention; FIG.",
"11 is a schematic isometric view of an external-drum-type electro-optical plotter with optical imaging head incorporating an IALDA and a beam-shaping MLPA according to the present invention; and FIG.",
"12 is a schematic isometric view of a flatbed-type electro-optical plotter with an optical imaging head incorporating an IALDA and a beam-shaping MLPA according to the present invention.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS There are generally two types of light-pipes: Bulk and Hollow.",
"The bulk-type light-pipe is a rod of transparent material with a polygonal cross-section (triangular, rectangular, etc.).",
"The index of refraction of the material forming the light-pipe is higher than the index of refraction of the surrounding material.",
"A typical example is glass rod in air.",
"This type of light-pipes employ the principle of Total Internal Reflection (TIR) on the interface of the two materials—in the above example on the interface glass—air.",
"The hollow light-pipes are tubes with a polygonal cross section (triangular, rectangular, etc.",
"), made of transparent or nontransparent material, with their internal walls coated with a highly reflective coating.",
"This type of light-pipes work on reflection from the reflective coating.",
"In all preferred embodiments described below, a hollow light-pipe is taken as an example.",
"It will be, however, appreciated by any person skilled in the art, that same performance can be achieved by using bulk-type light-pipes.",
"FIG.",
"1a shows an IALDA light source 20 and an MLPA 10, aligned in parallel with the array of laser emitters 21.The number of the channels 12 in the MLPA corresponds to the number of the laser diodes 21 in the IALDA and the MLPA is placed in close proximity to the IALDA, in order to avoid optical crosstalk between the channels.",
"The Micro Light-Pipes (MLP) 12 are hollow and their internal surface is coated with a highly reflecting coating, such as Au, enhanced Al or dielectric, depending on the base material and the wavelength of the light.",
"The light emitted from each diode 21 enters the corresponding MLP 12 trough its entrance aperture 13.Inside the MLP 12 each beam experiences a number of bounces from its walls before it exits from the opposite side through the exit aperture 14.Due to these multiple reflections, the illumination of the MLP exit aperture is relatively uniform The uniformity, defined as Edge Illumination Center Illumination , depends on the value L n = L · NA i n · A , called the MLP normalized-length, where L is the light-pipe length, NAi is the numerical aperture of the input beam n is the index of refraction of the MLP (n=1 for a hollow MLP), and A is the cross-sectional area of the MLP.",
"There is no precise theory of light pipes.",
"The scrambling efficiency is usually checked experimentally, or by non-sequential ray tracing.",
"It is, however, an empirical fact that when Ln≧4, the illumination uniformity at the MLP exit can be expected to be better than 90%.",
"FIGS.",
"1b and 1c schematically show an optical imaging head 100 incorporating an IALDA 20, represented by a limited number of emitters 21, and an MLPA 10.FIG.",
"1b illustrates the beams propagation in a plane coinciding with the emitters' fast axis, while FIG.",
"1c illustrates the beams propagation in a plane coinciding with emitters' slow axis.",
"The exit aperture 14 of the MLPs 12 is imaged by means of imaging lens 70, preferably telecentric, on the photosensitive medium 50, i.e.",
"the exit apertures 14 lie in the object plane of the imaging lens 70, while their images 60 lie on the photosensitive medium 50, which coincides with the image plane of lens 70.As far as all light spots 60 are images of substantially identical objects—the exit apertures 14 of the MLPs 12—they too will be substantially identical.",
"Due to the relatively uniform illumination of the exit apertures 14, their images 60 will also feature a relatively uniform distribution of illumination.",
"Thus, substantially identical light spots with uniform energy distribution are achieved on the medium 50.A very important parameter of the imaging head in electro-optical plotters is the depth of focus; higher depth of focus requires lesser mechanical accuracy.",
"The depth of focus is in direct dependence to the numerical aperture NAim of lens 70 image plane, such that the higher NAim, the lower is the depth of focus.",
"The numerical aperture (NA) at the image side of lens 70 is NA im = NA o K , where K<1 (usually) is the magnification of imaging lens 70 and NAo is the object side NA, i.e.",
"the NA of the beam emerging from the MLP 12 exit aperture 14.Assuming that the MLP is a prism, i.e.",
"all its walls are parallel to a central axis 16 (FIGS.",
"1b, 1c), it can be proven out of simple geometrical considerations, that a beam entering the MLP 12 at a certain angle with respect to the axis 16, will exit the MLP at the same angle with respect to the axis 16, but with altered direction due to the MLP scrambling effect.",
"In other words, the numerical aperture at the MLP exit will be equal in all directions and will be NAo=NAi, and hence NA im = NA i K .",
"It is therefore apparent that for achieving bigger depth of focus, the designer's goal will be to work with as low as possible NAi and as low as possible MLP 12 dimension a, allowing for higher K values.",
"The minimum value for a is determined by the beam divergence in the slow axis direction and the distance d between the IALDA 20 and the MIA 10 (FIG.",
"1), and it is obviously achieved when d≅0.In this case, all energy emitted by the emitter will enter the MLP, but also NAi will have its maximum value corresponding to the emitter's beam divergence in the fast axis direction.",
"In other words, working with a low numerical aperture and accommodating all emitted energy are contradictory conditions in the embodiment of FIG.",
"1a-1c.",
"Depending on the specific requirements of the optical system, a compromise between these two parameters should be made.",
"As an example, in the optical system of FIGS.",
"1a-1c the cross sectional dimension a of the MLPs is chosen to be approximately the length l of the LD emitters, while the NAi is chosen to correspond to the FWHM divergence angle of the LD in the emitter's slow axis direction.",
"The latter is done by proper choice of the IALDA-MLPA distance d and the MLPs' cross sectional dimension h: NAi=sin(atan(h/d)).",
"It is important to point out that the minimum value for h depends also on the mutual displacement of the emitters 21 in the fast axis direction (often call “smile” of the array—dashed lines 21a in FIG.",
"1a).",
"This configuration, however, is far from being optimal, because, as mentioned above, the fast axis divergence angle is much larger than the slow axis one, and all the energy emitted in this direction outside NAi is lost The loss of energy described above is avoided, to a great extent, in the optical system presented in FIGS.",
"2a, 2b and 2c.",
"FIG.",
"2a is a schematic isometric view of an IALDA 120 with anamorphic correcting lens 130 and beam-shaping MLPA 110.The difference between the systems of FIG.",
"1a and FIG.",
"2a is in the lens 130, placed between the IALDA 120 and the MLPA 110.For simplicity of the illustration, a cylindrical lens is shown.",
"It will be, however, appreciated by any person skilled in the art, that anamorphic lenses of different types can be used with the same success.",
"The function of the anamorphic lens 130 in the optical system is illustrated in FIGS.",
"2b and 2c, which are illustrations of the beams propagation in an optical imaging head 105, in a plane coinciding with the fast axis direction, and the beams propagation in a plane coinciding with the slow axis direction, respectively.",
"The power of the anamorphic lens 130 in the fast axis direction is chosen such that the beam divergence in the fast axis direction beyond the lens 130 will approximately equal the beam divergence in the slow axis direction (in FIGS.",
"2a and 2b both values are designated by NAi).",
"Thus, the numerical aperture NAi of the beam that enters the MLP 112 entrance aperture 113 contains most of the energy emitted by the diode.",
"In FIGS.",
"2a-2c, the lens 130 is chosen to be common for all the emitters of the array.",
"It will be, however, appreciated by any person skilled in the art, that other solutions may be implemented, for example a micro-lens array.",
"Since the role of the lens 130 is only to decrease the beam divergence in the fast axis direction and to direct as much energy as possible to the MLP for a given NA, no constraint for imaging with minimum optical aberrations are placed here.",
"This makes the design of the system easy and flexible in the choice of the lens 130.The imaging lens 170 is preferably telecentric and images the exit apertures 114 of the MLPs 113.As the apertures 114 are substantially identical objects with very even spatial energy distribution, their resulting images 160 on the photosensitive medium 150 will also be substantially identical with even spatial energy distribution.",
"The system of FIGS.",
"2a-2c still does not provide optimum performance in terms of efficiency.",
"Adding the lens 130 increases the distance d (FIG.",
"2a) between the IALDA 120 and the MLPA 110.Therefore, as can be seen in FIG.",
"2c, it is necessary to increase the cross-sectional dimension a of the MLP array to a value a>l+2d.NAi, in order to accommodate the entire energy emitted in the slow axis direction.",
"This increased dimension is marked as a1 and leads to a higher demagnification ratio (smaller K) of lens 170, hence to a larger image size numerical aperture NAim and a reduced depth of focus.",
"The minimum value for the cross sectional dimension h is determined by the array smile and the magnification of the anamorphic lens 130; the spot 121b produced by lens 130 from the most displaced emitter 121a, should be within the entrance aperture 113a of the corresponding MLP (FIG.",
"2a).",
"The increase in the cross sectional dimensions a of the MLPs, which is necessary in the system of FIGS.",
"2a-2c for collecting the entire emitted energy, can be avoided by employing an optical system that produces an image of the emitters in close vicinity to the entrance aperture of the MLPs.",
"Because of the emitter's different beam divergence in the fast and slow axis directions, such system should have different power in these two directions.",
"In the examples below (FIGS.",
"3a-3c and 4a-4c), the numerical aperture of the beam NAi at the entrance of the MLP is chosen to be approximately identical in all directions and approximately equal to the numerical aperture NAs of the emitter in the slow axis direction: NAi≅NAs.",
"From the preservation of Etendue principle, it follows that the magnification in the slow axis direction will be approximately 1, while the magnification in the fast axis direction will be approximately NAf/NAs>1.The emitters' image length l1 (FIG.",
"3c) will equal approximately the emitters' length l, and from considerations of preserving the brightness it will follow that the MLPs' dimension a in the slow axis direction will be: a≅l 1≅l.",
"It will be appreciated by any person skilled in the art that other efficient configurations are also possible: a≅l 1<l; NAi>NAs or a≅l 1>l; NAi<NAs.",
"The designer, however, should bear in mind that the numerical aperture NAo of the beam exiting the MLP will be identical in all directions and will correspond to the maximum angle of the entrance beam with respect to the MLPs' axis, and in conjunction with the magnification K of the imaging lens 270, will determine the system's depth of focus.",
"FIG.",
"3a is a schematic isometric view of an optical system consisting of an IALDA 220, an anamorphic correcting lens 230, a lenslet array 240 and a understood with the help of FIGS.",
"3b and 3c, which are illustrations of the beams propagation in an optical imaging head 200, in a plane coinciding with the emitter's fast axis direction, and the beams propagation in a plane coinciding with the emitter's slow axis direction, respectively: The anamorphic lens 230 is designed so as to create virtual images 222 of the LD emitters 221.Because of the anamorphic properties of the lens 230, the NA beyond the lens (designated by NAv in FIGS.",
"3b, 3c) is identical in all directions perpendicular to the system axis of symmetry.",
"The virtual image 222 serves as an object for lens 240, which produces a real image 223 in the vicinity of the entrance aperture 213 of the MLP 212.Because of the imaging properties of the lens combination 230-240 and the controlled NA, the dimensions of the image 223, as explained above, can be made to equal the MLP dimension a.",
"Thus, the energy losses are minimized and no increase in the MLP cross sectional dimension is required.",
"The anamorphic lens 230 can be produced by extrusion, a method used by Bluesky Research Inc., of San Jose, Calif.",
"The imaging lens 270 is preferably telecentric and images the exit apertures 214 of the MLPs 212.As the apertures 214 are substantially identical objects with a very even spatial energy distribution, their resulting images 260 on the photosensitive medium 250 will also be substantially identical, with an even spatial energy distribution.",
"The minimum value for the cross sectional dimension h of the MLP's 212 is determined by the array smile and the magnification of lens system 230-240 in the fast axis direction; the image 221b of the most displaced emitter 221a should be within the entrance aperture 213a of the corresponding MLP (FIG.",
"3a).",
"It will be understood that some loss of energy will occur for displaced emitters.",
"This loss can be compensated by choosing the individual emitters' working regimes such as to obtain the same power yield for each channel of the optical head 200.Another system producing an image of the emitters in proximity to the MLP entrance aperture is illustrated in FIGS.",
"4a-4c.",
"Here, the anamorphic lens 330 has its focal plane approximately coinciding with the entrance aperture 313 of the MLP and decreases the numerical aperture in the fast axis direction to a value close to the numerical aperture in the slow axis direction.",
"The array 340 is an array of anamorphic lenses with power only in the slow axis direction.",
"For each emitter 321, there is a member 341 of the array 340 associated with it.",
"The imaging planes of anamorphic lenses 330 and 340 coincide.",
"Thus, a real image of the emitter 321 is produced in close vicinity to the MLP entrance aperture 313, with numerical aperture approximately identical in all directions.",
"It will be also appreciated that the same optical effect can be achieved by designing the lens 340 not as a lenslet array, but as an assembly of bulk optical elements.",
"It will also be appreciated that the anamorphic lens 330 and the focusing lens 340 can be combined in a single lenslet array of anamorphic elements.",
"As in the previously described systems, the imaging lens 370 is preferably telecentric and images the exit apertures 314 of the MLPs 312.As the apertures 314 are substantially identical objects with very even spatial energy distribution, their resulting images 360 on the photosensitive medium 350 will also be substantially identical with even spatial energy distribution.",
"The minimum value for the cross sectional dimension h of the MLPs 312 is determined by the array smile and the magnification of lens system 330-340 in the fist axis direction; the image 321b of the most displaced emitter 321a should be within the entrance aperture 313a of the corresponding MLP (FIG.",
"4a).",
"The loss of energy due to the displacement can be compensated, as in the previously described systems, by choosing the individual emitter's working regimes such as to obtain the same power yield for each channel of the optical head 300.Reference is now made to FIGS.",
"5a and 5b, illustrating the light scrambling of the optical system of FIGS.",
"4a, 4b and 4c.",
"FIG.",
"5a shows the light distribution in the real image 323 of LD emitter 321, in the entrance aperture 313 of the MLP 312.The length of emitter 321 in this example was 80 microns and the diameter of the aperture 313 was also chosen to be 80 microns.",
"The MLP was chosen to be with a hexagonal cross section and with length L=1.5 mm.",
"FIG.",
"5b shows the energy distribution in the same MLP exit aperture 314.It is obvious, that as far as the spot 360 on the photosensitive medium 350 (FIGS.",
"4b and 4c) is an image of the MLP exit aperture 314, the light energy distribution in it will be similar to that in the aperture 314, i.e.",
"relatively uniform.",
"Same results can be obtained with the optical systems of FIGS.",
"1a-1c, 2a-2c, and 3a-3c.",
"In all the previous embodiments, the MLP used is a prism, i.e.",
"it does not alter the beam angle with respect to the optical axis.",
"There is another type of light-pipe, known as tapered, which can be used not only for scrambling the light energy, but also for altering the numerical aperture of the beam.",
"These light-pipes have a shape of a truncated pyramid, tapered in one or more directions.",
"In the direction in which the light-pipe is tapered, the beam angle with respect to the axis will be changed.",
"Reference is now made to FIG.",
"6a, presenting an IALDA 420, working in conjunction with a MLPA 410 of tapered MLPs 412.The dimension h1 of the MLP 412 entrance aperture 413 and the corresponding dimension h2 of the exit aperture 414, are chosen to be different: h1<h2.Thus, because the MLPs 412 are tapered in the fast axis direction, the fist axis numerical aperture NAf1 of the beam at the entrance of the MLPs will be decreased at the exit to a value NA f2 = NA f1 h1 h2 .",
"Thus, by proper choice of h1, the numerical aperture of the beam at the exit aperture 414 can be made identical in all directions.",
"The embodiment of FIG.",
"6a can be integrated in an optical imaging head 400, shown in FIGS.",
"6b and 6c.",
"The possibility of altering the beam's numerical aperture, allows for choosing the MLPs' dimension in the slow axis direction a=l, where l is the emitter length and for minimizing the IALDA−MLPA distance d≅0 without loss of energy due to system geometry and without loss of depth of focus, in contrast to the optical head of FIGS.",
"1a-1c.",
"The optical imaging head of FIGS.",
"6a-6c is an improved variant of the imaging head of FIGS.",
"1a-1c.",
"In this embodiment, however, there still could be significant energy losses.",
"Because of the high entrance NA in the fast axis direction, the beam experiences more reflections in a tapered MLP than in a regular MLP, which leads to increased losses of energy in the reflective coating.",
"If the tapered MLPs are of bulk-type, then the high entrance numerical aperture can lead to unfulfilled conditions for TIR and hence, once again, to energy losses.",
"These drawbacks of the previously described embodiment are avoided in the system presented in FIGS.",
"7a-7c, which is an improved variant of the embodiment of FIGS.",
"2a-2c.",
"In this preferred embodiment, the MLPs 512 of the array 510 are designed as ‘funnels’, consisting of two parts, I and II, with lengths L1 and L2 respectively, as shown in FIG.",
"7a.",
"Part I is a tapered MLP with entrance aperture 513, with dimensions l1×h1 in the fast and slow axis directions respectively, and exit aperture 515 with dimensions l2×h2 in the fast and slow axis directions respectively.",
"Part II is a regular MLP with identical entrance and exit apertures, 515 and 514 respectively.",
"Imaging head 500, with array 510 of funnel type MLPs, illustrates the beam propagation in the fast axis direction.",
"The correcting anamorphic lens 530 reduces the beam NA in the fast axis direction to a value approximately equal to the NA in the slow axis direction: NAf≅NAs and images the emitter 521 in close proximity to the part I of the MLP 512 exit aperture 515.The dimension h1 of the part I entrance aperture in the fast axis direction and the tapering angle α are chosen such that the MLPs in this part do not alter the NA in the fast axis direction, but allow for accommodation of the entire energy emitted in this direction, even when the emitter is displaced to some extent in vertical direction.",
"Part I of the MLPs does not scramble the light in the fast axis direction.",
"The light in this direction is scrambled by part II of the MLPs.",
"The length L1 is chosen so that at the given angle α, the resulting height at aperture 515 is h2.h2 is a parameter determining the spot 560 dimension on the photosensitive medium 550 in the fast axis direction.",
"FIG.",
"7c illustrates the beam propagation in the slow axis direction.",
"The dimension l1 of the part I entrance aperture in this direction is chosen such that the MLPs in this part accommodate the full energy emitted in numerical aperture NAs, accounting for the beam divergence and the distance between the IALDA 520 and the MLPA 510.Thus, the distance between the correcting anamorphic lens 530 and the MLPA 510 can be made approximately zero: d≅0.The light in this direction is scrambled in both parts I and II of the MLPs.",
"The tapering angle β can be chosen to be zero and in this case l1=l2 and the full energy is accommodated with loss of brightness in this direction.",
"If β is not zero, then l1≠l2 and the NA in this direction is altered by factor l1/l2 and there is once again a loss of brightness.",
"As far as l1 depends on the distance d1 between the IALDA 520 and the MLPA 510, it is advisable to use correcting lens 530 with as small as possible cross section dimension, for example Luneburg type lens with diameter 60μ to 100μ produced by DORIC LENSES of Canada Production Method Hollow MLPAs can be produced by using standard photolithography technology on silicon wafers, or deep X-ray lithography on polymers.",
"Both technologies are well mastered in many companies around the world, for example in the Institute for Micromechanics in Mainz, Germany or MicroDevices Inc of Radford, Va.",
"FIG.",
"8 is an exploded isometric view of a MLPA 610.The array consists of two base plates 617 and 611, in which half-hexagonal grooves are etched.",
"The grooved surfaces are coated with a highly reflective coating, for example enhanced aluminum or bare gold, depending on the LD wavelength.",
"The mechanical keys 615 and 616 are formed by the same photolithography process and are used for easy alignment of the two parts 617 and 611.By etching along the Si crystallographic planes, a diamond-like shape can be achieved as illustrated in FIG.",
"9b.",
"Other shapes can be achieved and other materials can also be used.",
"For example, shapes as illustrated in FIGS.",
"9a, 9c and 9d, as well as non-symmetrical shapes can be made by using lithography or micro-molding techniques.",
"An additional method for producing MLPAs is by shaping a coherent bundle of optical fibers.",
"This technology involves the steps of: 1.Arranging the fibers in a coherent bundle; 2.Heating the bundle to a predetermined temperature; and 3.Extrusion under predetermined pressure.",
"This technology is mastered for example in Schott Optical Fibers of Germany (www.schott.com).",
"The resulting optical element is a coherent bundle of optical fibers with hexagonal cross-section.",
"Such optical elements are usually used as image tapers or extenders.",
"However, if a thin plate of the bundle is cut-off and optically polished on both sides, it can be used as an MLPA, as illustrated in FIG.",
"10.The initial dimensions of the fibers' cladding 712a and core 712b and the production process parameters (temperature, pressure, extrusion, etc.)",
"are chosen such as to achieve a certain distance p between each two or three or four, etc.",
"fibers, to match the pitch of emitters 721 of IALDA 720.Thus, a small number (cross-hatched) of elements 712 in the cut-off plate 700 constitute the MLPA 710.Another method of producing MLP is by means of standard glass technology, to form macro rods with desired cross section shape and then by extrusion to reduce the cross sectional dimension to a desired value.",
"Such techniques are widely used for producing anamorphic lenses with small cross sectional dimensions.",
"Optical imaging heads incorporating IALDA and MLPA can be used, as mentioned hereinabove, in electro-optical plotters for offset plates, laser transfer mediums, etc.",
"FIG.",
"11 illustrates the basic design of such electro-optical plotter.",
"The photosensitive medium (offset plate, etc.)",
"801 is wrapped around a rotating drum 800.Optical head 804, incorporating IALDA and MLPA, produces a plurality of spots 803 on the photosensitive medium 801.The drum rotates with substantially constant speed in the direction shown by arrow 805, while the optical head 804 moves parallel to the drum axis (not shown) in the direction marked by arrow 806.The system is driven by a central processor 809, which by means of control unit 807 synchronizes the two movements 806 and 805, and the data transfer between the image data bank 808 and the optical head 804.The digital equivalent of the image to be written on the photosensitive medium is stored in the image data bank 808, from where it is transferred to the optical head 804, which by means of producing a plurality of light spots 803 on the photosensitive medium 801, forms the desired image 802.FIG.",
"12 illustrates an electro-optical plotter of flatbed type, with optical head 903 incorporating IALDA and MLPA.",
"The photosensitive medium 904 is placed on a flat surface of an X-Y scanning engine 900.The digital equivalent of the image to be written on the photosensitive medium is stored in the image data bank 808, from where it is transferred to the optical head 903, which by means of producing a plurality of light spots 901 on the photosensitive medium 904, forms the desired image 902.The scanning movement of the optical head 903 in two perpendicular direction 905 and 906, is controlled by a central processor 809, through control unit 807.The CPU 809 also synchronizes the data flow from the image data bank 808 to the optical head 903 with the scanning movements 905 and 906."
]
] | Patent_10450660 |
[
[
"Focussing of compound libraries using atomic electrotopological values",
"The present invention relates to a method for generating a focussed compound library containing an enriched amount of ligand compounds being capable of binding to a predetermined receptor."
],
[
"1.A method for generating a focussed compound library from a starting compound library wherein said focussed compound library contains an enriched amount of ligand compounds being capable of binding to a predetermined receptor, comprising the steps: (a) providing at least one structure of a ligand, a ligand-receptor complex or a ligand binding site geometry for the predetermined receptor, (b) generating a computer-readable code of said at least one structure, (c) providing a description of said at least one structure in the form of its three-dimensional geometry or/and of its bond distance matrix, (d) providing atomic eletrotopological values for the atoms of said at least one structure; (e) generating atomic types based on said atomic electrotopological values, (f) generating pharmacophores based on said atomic types, (g) sorting a starting database with said pharmacophores, using a similarity index by (g1) providing a description of the structure of the compounds contained in the database in the form of their three-dimensional geometry or/and of their bond distance matrix, (g2) providing atomic electrotopological values for the atoms of said at least one structure, (g3) generating atomic types based on said atomic electrotopological values, (g4) generating pharmacophores based on said atomic types, and (g5) comparing the pharmacophores of said at least one ligand structure with the pharmacophores of the database compounds, (h) determining a ranking of the database compounds according to the detected similarities, and (i) obtaining a focussed compound libary having an enriched amount of ligand compounds.",
"2.A method according to claim 1 for generating a focussed compound library from a starting compound library wherein said focussed compound library contains an enriched amount of ligand compounds being capable of binding to a predetermined receptor, comprising the steps: (a) providing at least one ligand structure for the predetermined receptor, (b) generating a predetermined number of possible ligand conformers of said ligand structure, (c) generating a computer-readable code of the possible ligand conformers, (d) providing a description of the possible ligand conformers in the form of their three-dimensional geometry or/and of their bond distance matrix, (e) providing atomic electrotopological values for the atoms of the possible ligand conformers, (f) generating atomic types based on said atomic electrotopological values, (g) generating pharmacophores based on said atomic types, (h) sorting a starting database with said pharmacophores using a similarity index by (h1) generating a predetermined number of conformers for compounds contained in the database, (h2) providing a description of the structure of said conformers in the form of their three-dimensional geometry or/and their bond distance matrix, (h3) providing atomic electrotopological values for the atoms of said conformers, (h4) generating atomic types based on said atomic electrotopological values, (h5) generating pharmacophores for said conformers of said database compounds based on said atomic types and (h6) comparing the pharmacophores of the ligand structure with the pharmacophores of the database compounds and (i) determining a ranking of the database compounds according to the detected similarities, (j) obtaining a focussed compound library having an enriched amount of ligand compounds.",
"3.The method according to claim 1, wherein the similarity index used for sorting the database is selected from the group consisting of Tanimoto coefficient, Eucledian distance, Manhattan distance and any combination thereof.",
"4.The method according to claim 1, wherein two-point pharmacophores (2PP), three-point pharmacophores (3PP) and/or four-point pharmacophores (4PP) are generated.",
"5.The method according to claim 2, wherein the conformers are generated by force field or rule based methods or combinations thereof.",
"6.The method according to claim 2, wherein the predetermined number of conformers is a number between 5 and 20.7.The method according to claim 1, wherein the starting compound library is selected from a database of known individual compounds, a database of synthesized combinatorial libraries and/or a database of virtual combinatorial libraries.",
"8.The method according to claim 1, wherein specific atomic electrotopological values that are included in an atomic type and/or the total number of atomic types are optimized by screening a test library.",
"9.A method according to claim 1 for generating a focussed compound library from a starting compound library wherein said focussed compound library contains an enriched amount of ligand compounds being capable of binding to a predetermined receptor, comprising the steps: (a) providing a three-dimensional ligand-receptor-complex structure for the predetermined receptor, (b) deriving the ligand structure from the three-dimensional ligand-receptor-complex structure, (c) generating pharmacophores of the ligand structure based on intermolecular interactions between ligand and receptor based on atomic types generated using atomic electrotopological values, (d) sorting a starting database with said pharmacophores, using a similarity index by (d1) generating a predetermined number of conformers for compounds contained in the database, (d2) determining pharmacophores for said conformers of said database compounds based on atomic types generated using atomic electrotopological values, and (d3) comparing the pharmacophores of the ligand structure with the pharmacophores of the database compound and (e) determining a ranking of the database compounds according to the detected similarities and (f) obtaining a focussed compound library having an enriched amount of ligand compounds.",
"10.A method according to claim 1 for generating a focussed compound library from a starting compound library wherein said focussed compound library contains an enriched amount of ligand compounds being capable of binding to a predetermined receptor, comprising the steps: (a) providing a binding site geometry of said predetermined receptor, (b) inverting the binding site geometry of said receptor to create a ligand candidate, (c) generating a predetermined number of possible ligand conformers of said ligand candidate structure, (d) generating pharmacophores of the possible ligand conformers based on atomic types generated using atomic electrotopological values and (e) sorting a starting database with said pharmacophores using a similarity index by (e1) generating a predetermined number of conformers for compounds contained in the database, (e2) determining pharmacophores for said conformers of said database compounds based on atomic types generated using atomic electrotopological values, and (e4) comparing the pharmacophores of the ligand structure with the pharmacophores of the database compound, (f) determining a ranking of the database compounds according to the detected similarities and (g) obtaining a focussed compound library having an enriched amount of ligand compounds."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The search and evaluation of new drugs or drug targets on short time scales requires the use of high-throughput screening (HTS) in pharmaceutical research.",
"Beside the screening of real compounds, it is also possible to determine new drug targets using computational screening methods.",
"The application of computational screening is often called virtual screening.",
"The bottleneck of current drug discovery and drug development is the large screening demand of pharmaceutically relevant targets.",
"To circumvent this difficulty a lot of effort is put on improvement of effective virtual (computer) screening tools.",
"Currently combinatorial chemistry and high-throughput screening is effective in the search of new lead structures.",
"However, these processes are still very expensive and time-consuming.",
"Therefore, computer-based algorithms can be a significant improvement in minimizing costs and time.",
"In the current screening processes a hit rate of about 0.1% can be reached.",
"Thus, an increase of the hit rate is desirable, which will improve the screening process and reduce costs and time.",
"Tools that can screen databases for molecules with biological activity are of tremendous value for the pharmaceutical and biotechnological industry., A common scenario is that one or several molecules showing biological activity for a specific target is known.",
"This/These molecule(s) are used to screen a database of molecules for other molecules with similar activity.",
"Methods that have been developed in this area, for instance, are CATS (Schneider et al., Angewandte Chemie int.ed.",
"1999, 38, 2894-2896), CATALYST (Barnum et al., J.Chem.Inf.Comput.Sci.",
"1996, 36, 563-571) and PHACIR (U.S. application Ser.",
"No.09/634,586).",
"These methods have three essential parts: 1.Description of the molecular geometry.",
"2.Translation of the atomic properties of the molecule into a pharmacophore model.",
"3.Comparisons of the pharmacophores for different molecules.",
"So far many methods are based on a search of individual chemical structures as new drug-lead compounds.",
"The information used is derived either from existing active compounds or from the target structure.",
"These conventional methods consider each structure separately and search for similarities with the known active compound or for complementarities to a protein-target binding site.",
"However, these methods can be applied only to databases having a limited number of members, since the algorithms used are rather time consuming.",
"However, an increasing number of potential drug candidates can be synthesized, e.g.",
"by combinatorial chemistry or can be virtually generated.",
"It is desirable to provide methods, allowing fast screening of large databases or reducing the total number of members of databases while at the same time increasing the percentage of hits within the database.",
"It is therefore an objective of the present invention to provide a method to further improve and accelerate the virtual screening of databases for specific ligand compounds.",
"It is a further objective of the present invention to provide a method which allows for generating a focussed compound library containing an enriched amount of ligand compounds being capable of binding to a predetermined receptor."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is related to a novel ligand detection system.",
"The method according to the invention identifies ligands, which are capable of specific binding to a given receptor or to given protein domains.",
"An algorithm, preferably computer-based, can generate a focussed compound library which enriches the active compounds.",
"Screening of such an enriched library results in a significant increase of the hit rate.",
"The basic concept underlying the applied algorithms is a new way of translating the atomic information of a molecule into a pharmacophore model.",
"The normal way of translating information about the properties of a molecule into pharmacophores is to assign atoms as different atomic types, traditionally atoms have been described as hydrogen bond donors, hydrogen bond acceptors, lipophilic, aromatic, positive or negative.",
"In general, a pharmacophore describes the three-dimensional order of all atoms within a ligand which can interact with a receptor (Böhm, H.-J., Klebe, G., Kubinyi, H.; Wirkstoffdesign, Spektrum Verlag, Heidelberg, 1996).",
"The pharmacophore concept, suitable interaction types and distance ranges are described e.g.",
"by S. D. Pickett et al., J. Chem.",
"Inf.",
"Comput.",
"Sci.",
"36 (1996), 1214-1232; H.-J.",
"Böhm, G. Klebe, H. Kubinyi, Wirkstoffdesign, Spektrum Verlag, Heidelberg, 1996; S. M. Brocklehurst et al., Creating Integrated Computer Systems for Target Discovery and Drug Discovery, Pharmainformatics Elsevier Science Ltd., (1999), pages 12-15 and J. S. Mason, Computational Screening: Large-scale Drug Discovery, Pharmainformatics Elservier Science Ltd., (1999), pages 34-36.The invention particularly includes the enrichment of biologically active compounds in a focussed substance library, which is obtained from given starting databases.",
"First the relevant biological properties are extracted from compounds with known biological activity and described in the form of pharmacophores.",
"Pharmacophores produced this way can then be used for the extraction of similar compounds from a substance library or for focussing a database according to specific similarity criteria.",
"It is very advantageous that the use of pharmacophores enables very rapid screening also of large databases.",
"During the screening process a focussed substance library is generated, which is smaller than the originally used starting database and which contains an enormously increased proportion of active substances compared to the original substance library.",
"An essential feature of the method according to the invention is the generation of pharmacophores based on atomic types obtained using atomic electrotopological (AET) values.",
"According to the method of the invention, which is also named PHATS, pharmacophores are evaluated in a completely different way than in the state of the art.",
"Atomic electrotopological (AET) values are calculated for each atom and used as atomic types.",
"AET values are usually between −2 and 12.Atoms with AET values between two specified boundaries are assigned to belong to the same atomic type.",
"The method of calculating AET values has been developed by Kier & Hall (Molecular Structure Description, The Electrotopological State, Academic Press, London, 2000).",
"However, AET values have never been used in conjunction with the molecular geometry to screen for biologically active molecules.",
"The atomic types are then used to construct a pharmacophore model.",
"The new way of describing the atomic types has several advantages compared to the old one.",
"Atomic types are assigned automatically by the computer, there is no need for a person to predefine what is a hydrogen bond donor, a lipophilic atom and so on.",
"Most important, since AET values are assigned by the computer, the number of atomic types and the boundary values between different atomic types can be optimized for each specific target.",
"The values can be optimized for a small test library and can then be used to screen large databases to find new lead compounds.",
"This means that a specific pharmacophoric model can be developed for each target type instead of using the same for each target as in earlier models.",
"A specifically designed pharmacophore model will screen a database more efficiently for active molecules of that specific target type.",
"Calculations have shown that this new way of creating a pharmacophore model is superior to the traditional one.",
"Further, with the method according to the invention biologically relevant interactions can be considered.",
"The method according to the invention for the generation of a focussed substance library generally comprises the following steps: 1.The molecules (i.e.",
"the known ligand(s) and compound(s) of a starting database) are described with a code in computer-readable form, e.g.",
"the SMILES code (D. Weininger, J.Chem.Inf.Comput.Sci.",
"28 (1988), 31-36).",
"2.The geometry of the molecules is described either with their bond distance matrix or with their three-dimensional coordinates.",
"3.Atomic electrotopological indexes are evaluated for each atom, the values are used to assign the atoms as different atomic types.",
"4.The molecule is described with pharmacophores, e.g.",
"two-, three-, or four-point pharmacophores, using these atomic types.",
"5.The collection of the pharmacophores is compared using a similarity index, e.g.",
"with the distance between two vectors or with the Tanimoto coefficient.",
"For the preparation of the pharmacophores particularly used are: one or more compounds having known biological activity, one or more compounds each having a known active three-dimensional conformation, one or more compounds, whose intermolecular interactions are derived from known interactions of ligand-protein complexes together with the receptor or one or more receptors with known three-dimensional structures for each a given biologically relevant property, e.g.",
"the binding affinity to a receptor.",
"The method according to the invention can be used for the virtual screening of substance libraries and can be applied in particular for the development of drugs or biologically active compounds.",
"It is particularly suitable for applications in human or veterinary medicine and in plant protection.",
"Examples for indications are oncology, cardiovascular diseases, neurology, metabolic diseases, infectious diseases and virology.",
"The is method can also be applied in searches of substances, which shall be used for the modulation or inhibition of receptor-ligand interactions.",
"Thus, the invention particularly can be applied to find new lead structures for pharmaceutical, biotechnological and agrochemical targets.",
"The method according to the invention allows to convert a database of arbitrary size into a focussed substance library of much smaller size, the members of which can be further evaluated, e.g in experimental tests.",
"With the method according to the invention a database is sorted according to other substances capable of binding, wherein it was detected that more than 40% and in particular 60-80% of the actual hits are placed in the focussed substance library.",
"When the substances of a database are sorted using the inventive method, a substance library of arbitrary size can be generated, e.g.",
"by selecting the 10% best hits, the 20% best hits or the 50% best hits.",
"Preferably a reduction of the starting database to less than ⅓ of its original size, more preferably to less than ⅕ of its original size is carried out.",
"Due to a limiting pre-selection, e.g.",
"using the criterium 1% best hits or just the very best hit, it is further possible to synthesize also complicated structures from this focussed substance library, optionally in a multi-step process and subsequently investigate them experimentally.",
"This synthesizing step requires only little effort, compared to conventional approaches taking into account the large amount of molecules contained in the original database and the large amount of molecules having little or no binding affinity existing therein.",
"The described procedures of virtual screening are unique and innovative and result in an efficient sorting of databases, which allows a significant enrichment for biologically active molecules even within very large databases.",
"The method of the invention preferably provides an enhancement of the proportion of molecules having the desired activity or properties.",
"The enhancement can be described as enrichment factor (EF), which is defined as: EF=δ (focussed library)/δ (whole library) with the density δ=number of active compounds/total number of compounds.",
"The enrichment factor EI is preferably greater than 2, more preferably greater than 3.The virtual screening methods can be applied to three point pharmacophores and can easily be extended to four point pharmacophores to be able to consider the chirality of the molecules, which play an additional role for binding specifity.",
"The term “pharmacophore” as used herein refers to the sum of all ligand atoms which have intermolecular interactions to the receptor.",
"The invention can be summarized as follows: The invention, in general, relates to the identification of biologically active molecules with virtual screening, using the following steps: Description of the molecules as a computer-readable code, e.g.",
"a SMILES string.",
"Description of the molecule e.g.",
"with its three-dimensional geometry or with a bond distance matrix.",
"This information can be extracted from the SMILES code.",
"Description of atomic types based on the atomic electrotopological values.",
"The AET values are calculated for each atom and are usually between −2 and 12.The AET value is specific for each atom depending on the environment/surroundings of the atom.",
"Usually 3-5 different atomic typs are defined within the invention.",
"The grouping of AET values into atomic types can be performed, for example, as follows: Atomic type I Δ AET values from −∞ to <0; atomic type II Δ AET values from 0 to <5; atomic type III Δ AET values from 5 to <10 and atomic type IV Δ AET values from 10 to +∞.",
"However, other groupings are also possible and might prove advantageous for specific target types.",
"The pharmacophore is based on these atomic types.",
"Construction and enumeration of pharmacophores, e.g.",
"of all possible two-, three- or 4-point pharmacophores.",
"Optionally, the specific atomic electrotopological values that are included in an atomic type and the total number of atomic types can be optimized by screening a test libary containing known binding (hits) and non-binding structures.",
"The optimized atomic types can be used to construct pharmacophores.",
"The optimized pharmacophore models then are used to screen large databases for active molecules.",
"With the method according to the invention one can sort a molecular database according to the molecules similarity and/or according to biological activity.",
"The method according to the invention can be used to find new lead structures for pharmaceutical, biotechnological and agrochemical targets.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"The present invention relates to a method for generating a focussed compound library containing an enriched amount of ligand compounds being capable of binding to a predetermined receptor.",
"The focussing of the library can be performed according to predetermined biological activities or properties of the compounds.",
"BACKGROUND OF THE INVENTION The search and evaluation of new drugs or drug targets on short time scales requires the use of high-throughput screening (HTS) in pharmaceutical research.",
"Beside the screening of real compounds, it is also possible to determine new drug targets using computational screening methods.",
"The application of computational screening is often called virtual screening.",
"The bottleneck of current drug discovery and drug development is the large screening demand of pharmaceutically relevant targets.",
"To circumvent this difficulty a lot of effort is put on improvement of effective virtual (computer) screening tools.",
"Currently combinatorial chemistry and high-throughput screening is effective in the search of new lead structures.",
"However, these processes are still very expensive and time-consuming.",
"Therefore, computer-based algorithms can be a significant improvement in minimizing costs and time.",
"In the current screening processes a hit rate of about 0.1% can be reached.",
"Thus, an increase of the hit rate is desirable, which will improve the screening process and reduce costs and time.",
"Tools that can screen databases for molecules with biological activity are of tremendous value for the pharmaceutical and biotechnological industry., A common scenario is that one or several molecules showing biological activity for a specific target is known.",
"This/These molecule(s) are used to screen a database of molecules for other molecules with similar activity.",
"Methods that have been developed in this area, for instance, are CATS (Schneider et al., Angewandte Chemie int.ed.",
"1999, 38, 2894-2896), CATALYST (Barnum et al., J.Chem.Inf.Comput.Sci.",
"1996, 36, 563-571) and PHACIR (U.S. application Ser.",
"No.09/634,586).",
"These methods have three essential parts: 1.Description of the molecular geometry.",
"2.Translation of the atomic properties of the molecule into a pharmacophore model.",
"3.Comparisons of the pharmacophores for different molecules.",
"So far many methods are based on a search of individual chemical structures as new drug-lead compounds.",
"The information used is derived either from existing active compounds or from the target structure.",
"These conventional methods consider each structure separately and search for similarities with the known active compound or for complementarities to a protein-target binding site.",
"However, these methods can be applied only to databases having a limited number of members, since the algorithms used are rather time consuming.",
"However, an increasing number of potential drug candidates can be synthesized, e.g.",
"by combinatorial chemistry or can be virtually generated.",
"It is desirable to provide methods, allowing fast screening of large databases or reducing the total number of members of databases while at the same time increasing the percentage of hits within the database.",
"It is therefore an objective of the present invention to provide a method to further improve and accelerate the virtual screening of databases for specific ligand compounds.",
"It is a further objective of the present invention to provide a method which allows for generating a focussed compound library containing an enriched amount of ligand compounds being capable of binding to a predetermined receptor.",
"SUMMARY OF THE INVENTION The present invention is related to a novel ligand detection system.",
"The method according to the invention identifies ligands, which are capable of specific binding to a given receptor or to given protein domains.",
"An algorithm, preferably computer-based, can generate a focussed compound library which enriches the active compounds.",
"Screening of such an enriched library results in a significant increase of the hit rate.",
"The basic concept underlying the applied algorithms is a new way of translating the atomic information of a molecule into a pharmacophore model.",
"The normal way of translating information about the properties of a molecule into pharmacophores is to assign atoms as different atomic types, traditionally atoms have been described as hydrogen bond donors, hydrogen bond acceptors, lipophilic, aromatic, positive or negative.",
"In general, a pharmacophore describes the three-dimensional order of all atoms within a ligand which can interact with a receptor (Böhm, H.-J., Klebe, G., Kubinyi, H.; Wirkstoffdesign, Spektrum Verlag, Heidelberg, 1996).",
"The pharmacophore concept, suitable interaction types and distance ranges are described e.g.",
"by S. D. Pickett et al., J. Chem.",
"Inf.",
"Comput.",
"Sci.",
"36 (1996), 1214-1232; H.-J.",
"Böhm, G. Klebe, H. Kubinyi, Wirkstoffdesign, Spektrum Verlag, Heidelberg, 1996; S. M. Brocklehurst et al., Creating Integrated Computer Systems for Target Discovery and Drug Discovery, Pharmainformatics Elsevier Science Ltd., (1999), pages 12-15 and J. S. Mason, Computational Screening: Large-scale Drug Discovery, Pharmainformatics Elservier Science Ltd., (1999), pages 34-36.The invention particularly includes the enrichment of biologically active compounds in a focussed substance library, which is obtained from given starting databases.",
"First the relevant biological properties are extracted from compounds with known biological activity and described in the form of pharmacophores.",
"Pharmacophores produced this way can then be used for the extraction of similar compounds from a substance library or for focussing a database according to specific similarity criteria.",
"It is very advantageous that the use of pharmacophores enables very rapid screening also of large databases.",
"During the screening process a focussed substance library is generated, which is smaller than the originally used starting database and which contains an enormously increased proportion of active substances compared to the original substance library.",
"An essential feature of the method according to the invention is the generation of pharmacophores based on atomic types obtained using atomic electrotopological (AET) values.",
"According to the method of the invention, which is also named PHATS, pharmacophores are evaluated in a completely different way than in the state of the art.",
"Atomic electrotopological (AET) values are calculated for each atom and used as atomic types.",
"AET values are usually between −2 and 12.Atoms with AET values between two specified boundaries are assigned to belong to the same atomic type.",
"The method of calculating AET values has been developed by Kier & Hall (Molecular Structure Description, The Electrotopological State, Academic Press, London, 2000).",
"However, AET values have never been used in conjunction with the molecular geometry to screen for biologically active molecules.",
"The atomic types are then used to construct a pharmacophore model.",
"The new way of describing the atomic types has several advantages compared to the old one.",
"Atomic types are assigned automatically by the computer, there is no need for a person to predefine what is a hydrogen bond donor, a lipophilic atom and so on.",
"Most important, since AET values are assigned by the computer, the number of atomic types and the boundary values between different atomic types can be optimized for each specific target.",
"The values can be optimized for a small test library and can then be used to screen large databases to find new lead compounds.",
"This means that a specific pharmacophoric model can be developed for each target type instead of using the same for each target as in earlier models.",
"A specifically designed pharmacophore model will screen a database more efficiently for active molecules of that specific target type.",
"Calculations have shown that this new way of creating a pharmacophore model is superior to the traditional one.",
"Further, with the method according to the invention biologically relevant interactions can be considered.",
"The method according to the invention for the generation of a focussed substance library generally comprises the following steps: 1.The molecules (i.e.",
"the known ligand(s) and compound(s) of a starting database) are described with a code in computer-readable form, e.g.",
"the SMILES code (D. Weininger, J.Chem.Inf.Comput.Sci.",
"28 (1988), 31-36).",
"2.The geometry of the molecules is described either with their bond distance matrix or with their three-dimensional coordinates.",
"3.Atomic electrotopological indexes are evaluated for each atom, the values are used to assign the atoms as different atomic types.",
"4.The molecule is described with pharmacophores, e.g.",
"two-, three-, or four-point pharmacophores, using these atomic types.",
"5.The collection of the pharmacophores is compared using a similarity index, e.g.",
"with the distance between two vectors or with the Tanimoto coefficient.",
"For the preparation of the pharmacophores particularly used are: one or more compounds having known biological activity, one or more compounds each having a known active three-dimensional conformation, one or more compounds, whose intermolecular interactions are derived from known interactions of ligand-protein complexes together with the receptor or one or more receptors with known three-dimensional structures for each a given biologically relevant property, e.g.",
"the binding affinity to a receptor.",
"The method according to the invention can be used for the virtual screening of substance libraries and can be applied in particular for the development of drugs or biologically active compounds.",
"It is particularly suitable for applications in human or veterinary medicine and in plant protection.",
"Examples for indications are oncology, cardiovascular diseases, neurology, metabolic diseases, infectious diseases and virology.",
"The is method can also be applied in searches of substances, which shall be used for the modulation or inhibition of receptor-ligand interactions.",
"Thus, the invention particularly can be applied to find new lead structures for pharmaceutical, biotechnological and agrochemical targets.",
"The method according to the invention allows to convert a database of arbitrary size into a focussed substance library of much smaller size, the members of which can be further evaluated, e.g in experimental tests.",
"With the method according to the invention a database is sorted according to other substances capable of binding, wherein it was detected that more than 40% and in particular 60-80% of the actual hits are placed in the focussed substance library.",
"When the substances of a database are sorted using the inventive method, a substance library of arbitrary size can be generated, e.g.",
"by selecting the 10% best hits, the 20% best hits or the 50% best hits.",
"Preferably a reduction of the starting database to less than ⅓ of its original size, more preferably to less than ⅕ of its original size is carried out.",
"Due to a limiting pre-selection, e.g.",
"using the criterium 1% best hits or just the very best hit, it is further possible to synthesize also complicated structures from this focussed substance library, optionally in a multi-step process and subsequently investigate them experimentally.",
"This synthesizing step requires only little effort, compared to conventional approaches taking into account the large amount of molecules contained in the original database and the large amount of molecules having little or no binding affinity existing therein.",
"The described procedures of virtual screening are unique and innovative and result in an efficient sorting of databases, which allows a significant enrichment for biologically active molecules even within very large databases.",
"The method of the invention preferably provides an enhancement of the proportion of molecules having the desired activity or properties.",
"The enhancement can be described as enrichment factor (EF), which is defined as: EF=δ (focussed library)/δ (whole library) with the density δ=number of active compounds/total number of compounds.",
"The enrichment factor EI is preferably greater than 2, more preferably greater than 3.The virtual screening methods can be applied to three point pharmacophores and can easily be extended to four point pharmacophores to be able to consider the chirality of the molecules, which play an additional role for binding specifity.",
"The term “pharmacophore” as used herein refers to the sum of all ligand atoms which have intermolecular interactions to the receptor.",
"The invention can be summarized as follows: The invention, in general, relates to the identification of biologically active molecules with virtual screening, using the following steps: Description of the molecules as a computer-readable code, e.g.",
"a SMILES string.",
"Description of the molecule e.g.",
"with its three-dimensional geometry or with a bond distance matrix.",
"This information can be extracted from the SMILES code.",
"Description of atomic types based on the atomic electrotopological values.",
"The AET values are calculated for each atom and are usually between −2 and 12.The AET value is specific for each atom depending on the environment/surroundings of the atom.",
"Usually 3-5 different atomic typs are defined within the invention.",
"The grouping of AET values into atomic types can be performed, for example, as follows: Atomic type I Δ AET values from −∞ to <0; atomic type II Δ AET values from 0 to <5; atomic type III Δ AET values from 5 to <10 and atomic type IV Δ AET values from 10 to +∞.",
"However, other groupings are also possible and might prove advantageous for specific target types.",
"The pharmacophore is based on these atomic types.",
"Construction and enumeration of pharmacophores, e.g.",
"of all possible two-, three- or 4-point pharmacophores.",
"Optionally, the specific atomic electrotopological values that are included in an atomic type and the total number of atomic types can be optimized by screening a test libary containing known binding (hits) and non-binding structures.",
"The optimized atomic types can be used to construct pharmacophores.",
"The optimized pharmacophore models then are used to screen large databases for active molecules.",
"With the method according to the invention one can sort a molecular database according to the molecules similarity and/or according to biological activity.",
"The method according to the invention can be used to find new lead structures for pharmaceutical, biotechnological and agrochemical targets.",
"DETAILED DESCRIPTION OF THE INVENTION The invention can be applied for generating a focussed library either when only ligands are known, when the three-dimensional structure of a receptor-ligand complex is known or when the sole receptor structure is available.",
"Thus, the invention relates to a method for generating a focussed compound library from a starting compound library wherein said focussed compound library contains an enriched amount of ligand compounds being capable of binding to a predetermined receptor, comprising the steps: (a) providing at least one structure of a ligand, a ligand-receptor complex or a ligand binding site geometry for the predetermined receptor, (b) generating a computer-readable code of said at least one structure, (c) providing a description of said at least one structure in the form of its three-dimensional geometry or/and of its bond distance matrix, (d) providing atomic eletrotopological values for the atoms of said at least one structure; (e) generating atomic types based on said atomic electrotopological values, (f) generating pharmacophores based on said atomic types, (g) sorting a starting database with said pharmacophores, using a similarity index by (g1) providing a description of the structure of the compounds contained in the database in the form of their three-dimensional geometry or/and of their bond distance matrix, (g2) providing atomic electrotopological values for the atoms of said at least one structure, (g3) generating atomic types based on said atomic electrotopological values, (g4) generating pharmacophores based on said atomic types, and (g5) comparing the pharmacophores of said at least one ligand structure with the pharmacophores of the database compounds, (h) determining a ranking of the database compounds according to the detected similarities, and (i) obtaining a focussed compound libary having an enriched amount of ligand compounds.",
"In a first preferred embodiment the invention comprises a method for generating a focussed compound library from a starting compound library wherein said focussed compound library contains an enriched amount of ligand compounds being capable of binding to a predetermined receptor, comprising the steps: (a) providing at least one ligand structure for the predetermined receptor, (b) generating a predetermined number of possible ligand conformers of said ligand structure, (c) generating a computer-readable code of the possible ligand conformers, (d) providing a description of the possible ligand conformers in the form of their three-dimensional geometry or/and of their bond distance matrix, (e) providing atomic eletrotopological values for the atoms of the possible ligand conformers, (f) generating atomic types based on said atomic eletrotopological values, (g) generating pharmacophores based on said atomic types, (h) sorting a starting database with said pharmacophores using a similarity index by (h1) generating a predetermined number of conformers for compounds contained in the database, (h2) providing a description of the structure of said conformers in the form of their three-dimensional geometry or/and their bond distance matrix, (h3) providing atomic eletrotopological values for the atoms of said conformers, (h4) generating atomic types based on said atomic electrotopological values, (h5) generating pharmacophores for said conformers of said database compounds based on said atomic types and (h6) comparing the pharmacophores of the ligand structure with the pharmacophores of the database compounds and (i) determining a ranking of the database compounds according to the detected similarities, (j) obtaining a focussed compound library having an enriched amount of ligand compounds.",
"The starting point in this approach is that one or more ligands for a predetermined receptor, for which possible ligand compounds are to be searched for, is known.",
"A given compound library is screened by the information obtained from the known ligand structure(s).",
"In a first step a ligand structure for the predetermined receptor is provided.",
"Next, optionally a certain predetermined number of possible ligand conformers of said known ligand structure is generated.",
"Preferably, the predetermined number (m) of generated conformers for the known ligand structure is at least 1, more preferably at least 5 and most preferably at least 10.Theoretically, the upper value of the predetermined number of possible ligand conformers is not limited and preferably is less than 1.000, more preferably less than 100 and most preferably less than 20.The conformers can be generated by computational methods, such as force-field, rule-based, semiempiric, or ab initio methods.",
"However, it is also possible to take experimentally determined conformer structures of the known ligand(s).",
"In a next step a pharmacophore for each of the possible ligand conformers is generated.",
"The pharmocophores are decribed by the atomic types generated from the AET values for each atom.",
"Preferably, the pharmacophores are based on all possible ligand atoms that could have intermolecular interactions to the receptor and distances.",
"According to the invention it is preferred to use three-point pharmacophores.",
"However, it is also possible to use two-point or four-point pharmacophores or even higher point pharmacophores.",
"According to the invention said ligand pharmacophores and preferably all found ligand pharmacophores can be combined to a fingerprint (FP).",
"This fingerprint is an abstract binary representation of the pharmacophores or a common pharmacophore, respectively.",
"The fingerprint concept is described e.g.",
"in Vincent J. van Geerestein, Hans Hamersma and Steven P. van Helden: Exploiting Molecular Diversity: Pharmacophore Searching and Compound Clustering, in: Han van de Waterbeemd et al., Computer-Assisted Lead Finding and Optimization, Verlag Helvetica Chimica Acta Basel (1997), pages 159-178.The use of one fingerprint for screening instead of a large number of pharmacophores greatly reduces the effort necessary for screening large amounts of compounds in a database.",
"Within the fingerprint the characteristic features of the known structures are represented in a simple computer-readable binary form facilitating the comparison of the known ligand structure with possible ligand candidates.",
"The pharmacophores generated and/or said fingerprint can then be used for a similarity search.",
"Thereby a database can be sorted with the pharmacophores or/and the active fingerprint as a query.",
"For this, a starting database, such as a database of known individual compounds, a database of synthesized combinatorial libraries and/or a database of virtual combinatorial libraries is first converted into a database represented by pharmacophores or/and fingerprints.",
"For this purpose in a first step a predetermined maximum number (m) of conformers for all compounds of the database is generated.",
"Preferably, the predetermined number is at least 1, more preferably at least 5 and most preferably at least 10.While the maximum for the predetermined number is theoretically not limited, it is preferred to generated not more than 1.000, preferably not more than 100, more preferably not more than 50 and most preferably not more than 20 conformers for each compound of the database.",
"Next, a potential pharmacophore is determined for said conformers of each database compound in the same way as described above with respect of the generation of a pharmacophore for the known ligand structure.",
"If a fingerprint database is desired, fingerprints are generated for the pharmacophores of each conformer of the database compounds and preferably for all pharmacophores of each conformer of the database compounds.",
"The pharmacophores or/and the active fingerprint of the query molecule is then compared with the pharmacophores or/and the fingerprints of the compounds of the library.",
"A similarity index, such as the Tanimoto coefficent, the Eucledian distance or the Manhattan distance is used to sort all library compounds.",
"Suitable similarity indices are described e.g.",
"in Peter Willet, J. M. Barnard, G. M. Downs: Chemical Similarity Searching, J. Chem.",
"Inf.",
"Comput.",
"Sci.",
"38 (1998), pages 983-996.During this step a ranking of the database compounds according to the detected similarities is performed and the order of the compounds can be fixed according to their similarity to the known ligand structure.",
"It is also possible to use vectors for the similarity comparison.",
"The principle of using vectors is explained in the following for an example comprising a model with three atomic types (derived from the AET values) and bond distances between 1 and 10 and a two-point pharmacophore model.",
"From three atomic types six different two-point pharmacophores can be constructed, and starting with distances up to 10 bond lengths a 60 (6*10) dimensional vector is obtained.",
"Each dimension of the vector describes a specific bond distance between two atoms and the atom types of the two atoms.",
"All possible two-atom combinations in a molecule are calculated and one is added to the dimension that corresponds to each two atom combination.",
"All combinations are summed giving a vector that describes the molecule.",
"Since different molecules have different structure and atomic types, the summed vectors for the two molecules will be different and the Euclidian distance can be calculated between those two vectors.",
"If the distance between the vectors is short (in particular below a predetermined limit), the molecules are considered to be similar to each other.",
"This means that several compounds of a library can be ranked with respect to their similarity to one (or several) active molecule(s).",
"A focussed compound library with a reduced number of total members compared to the original database can be obtained by simply selecting a specific number, such as e.g.",
"1, 10, 100, 1.000 or 2.000 or a specific percentage, e.g.",
"10% or 20% of the compounds according to their ranking.",
"It was found that a considerable amount of hits, such as at least 30%, more preferably at least 50% and typically between 60 and 80% are contained in such a focussed compound library giving a significant enrichment with respect to the biologically active molecules.",
"If more than one ligand with the same function is available, the information of all ligand structures can be used.",
"When two or more ligand structures for the predetermined receptor are known, for each of the N ligands (wherein N is the number of known ligand structures) a certain maximum number (m) of conformers can be generated, e.g.",
"by calculation with an appropriate method as described above.",
"Next, for each conformer the pharmacophores are generated as described above.",
"The pharmacophores then can be combined to a common pharmacophore which is used to query a starting database.",
"Alternatively, a fingerprint for each ligand conformer can be created.",
"These fingerprints are combined with logical operations to a common fingerprint, representing a group of ligands for the same binding site.",
"A database can be sorted with this common fingerprint in the same way as described above with respect to sorting a database with a pharmacophore.",
"The method of the invention also can be applied, if the three-dimensional structure of a ligand-receptor complex for the predetermined receptor is known.",
"From the three-dimensional structure of the ligand-receptor complex the ligand structure can be derived and the informations for the intermolecular interactions between ligand and receptor are used to build up the pharmacophore model.",
"The generation of conformers is not necessary in this case, since a correct structure of the ligand is already known and fixed by the arrangement within the ligand-receptor complex.",
"The three-dimensional structure can be obtained from experimental data, such as X-ray diffraction, NMR, or they can be obtained by calculation methods.",
"Preferably, the three-dimensional structure is derived from experimental results.",
"If more than one three-dimensional ligand-receptor-complex structures are known, the ligand structures are derived from the three-dimensional ligand-receptor-complex structures.",
"The pharmacophores of the ligand structures can be generated based on the intermolecular interactions between ligand and receptor, calculating AET values for the atoms involved, while it is not necessary to create possible conformers.",
"The information of the multiple three-dimensional known ligand-receptor-complex structures can be used by combining at least two and preferably all individual pharmacophores or fingerprints to a common pharmacophore or fingerprint, respectively.",
"This common pharmacophore or fingerprint is then used to sort a given database as described above.",
"Alternatively, information of the multiple three-dimensional known ligand-receptor-complex structures can be used by building a common pharmacophore out of at least two and preferably all individual pharmacophores, to combine the information of the different structures.",
"A common fingerprint can then be derived from the common pharmacophore of at least two and preferably of all known three-dimensional structures.",
"In a further embodiment a pharmacophore model can be derived from a known binding site geometry of the predermined receptor.",
"In this approach the binding site geometry of said receptor is inverted to create pharmacophores.",
"With these pharmacophores a given database can be sorted as described above.",
"The invention also comprises systems, apparatuses, disks, computers or other hardware which are adapted or set up to carry out or control one of the methods of the invention."
]
] | Patent_10450665 |
[
[
"Transmitter and transmission control method ,and receiver and reception control method",
"Radio resources are saved, and power consumption is reduced.",
"A base station 101 carries out training for obtaining correlation information that correlates the reception quality of a packet channel transmitted from a terminal 102 and the transmission power of an associated channel, and based on the correlation information, uses the transmission power of the associated channel to obtain a predicted value of the reception quality of the packet channel, and controls the transmission of data through the packet channel based on that predicted value.",
"In addition, the base station 101 transmits through the associated channel transmission control information for controlling the transmission of the reception quality of the packet channel by the terminal 102.",
"The terminal 102 controls the transmission of the reception quality of the packet channel based on the transmission control information included in the data of the associated channel transmitted from the base station 101.",
"The present invention can be applied to, for example, a communications system of a W-CDMA system."
],
[
"1.A transmission apparatus which uses a first channel through which data is transmitted and a second channel on which power control is performed to transmit said data, said transmission apparatus characterized by comprising: transmission power control means for controlling the transmission power of said second channel; training means for carrying out training to obtain correlation information that correlates the reception quality of said first channel transmitted from a reception apparatus for receiving said data and the transmission power of said second channel; predicting means for obtaining a predicted value of the reception quality of said first channel using the transmission power of said second channel based on said correlation information; and transmission controlling means for controlling the transmission of said data through said first channel based on the predicted value of the reception quality of said first channel.",
"2.The transmission apparatus according to claim 1, characterized in that said training means correlates the reception quality of said first channel and the transmission power of said second channel so as to minimize a statistical error between the reception quality of said first channel transmitted from said reception apparatus and the predicted value of the reception quality of said first channel obtained using the transmission power of said second channel.",
"3.The transmission apparatus according to claim 1, characterized in that said training means obtains said correlation information during a training period in which said reception apparatus is made to transmit said first reception quality, and said predicting means calculates the predicted value of the reception quality of said first channel using the transmission power of said second channel based on said correlation information after a lapse of said training period, and by further comprising a transmission control information transmitting means for transmitting transmission control information, which, with respect to said reception apparatus, controls the transmission of the reception quality of said first channel from said reception apparatus after a lapse of said training period.",
"4.The transmission apparatus according to claim 3, characterized in that said transmission control information is information that instructs the starting or the stopping of transmission of the reception quality of said first channel by said reception apparatus.",
"5.The transmission apparatus according to claim 3, characterized in that said transmission control information is information that indicates the transmission frequency of the reception quality of said first channel by said reception apparatus.",
"6.The transmission apparatus according to claim 1, characterized in that said training means, each time the reception quality of said first channel is transmitted from said reception apparatus, updates said correlation information by obtaining said correlation information using the reception quality of that first channel.",
"7.The transmission apparatus according to claim 1, characterized in that said transmission controlling means controls a code rate or a modulation method of said data transmitted through said first channel based on the predicted value of the reception quality of said first channel.",
"8.The transmission apparatus according to claim 1, characterized by further comprising transmission control information transmitting means for transmitting to said reception apparatus transmission control information, which controls the transmission of the reception quality of said first channel by said reception apparatus.",
"9.The transmission apparatus according to claim 8, characterized in that said transmission control information is information that instructs the starting or the stopping of transmission of the reception quality of said first channel by said reception apparatus.",
"10.The transmission apparatus according to claim 8, characterized in that said transmission control information is information that instructs the transmission frequency of the reception quality of said first channel by said reception apparatus.",
"11.The transmission apparatus according to claim 10, characterized by further comprising generating means for setting the transmission frequency of the reception quality of said first channel based on an error in the predicted value of the reception quality of the first channel, and for generating said transmission control information indicating that transmission frequency.",
"12.The transmission apparatus according to claim 10, characterized by further comprising generating means for setting the transmission frequency of the reception quality of said first channel based on the frequency of proper reception of said data at said reception apparatus, and for generating said transmission control information indicating that transmission frequency.",
"13.The transmission apparatus according to claim 8, characterized in that said transmission control information transmitting means transmits said transmission control information through said second channel.",
"14.The transmission apparatus according to claim 1, characterized in that said transmission power controlling means controls the transmission power of said second channel in accordance with power control information for controlling the transmission power of said second channel, which is transmitted from said reception apparatus.",
"15.A transmission control method for a transmission apparatus that uses a first channel through which data is transmitted and a second channel on which power control is performed to transmit said data, said transmission control method characterized by comprising: a transmission power controlling step for controlling the transmission power of said second channel; a training step for carrying out training to obtain correlation information that correlates the reception quality of said first channel transmitted from a reception apparatus for receiving said data and the transmission power of said second channel; a predicting step for calculating a predicted value of the reception quality of said first channel using the transmission power of said second channel based on said correlation information; and a transmission controlling step for controlling the transmission of said data through said first channel based on the predicted value of the reception quality of said first channel.",
"16.A program for making a computer execute a transmission control process for a transmission apparatus that uses a first channel through which data is transmitted and a second channel on which power control is performed, and transmits said data, said program characterized by comprising: a transmission power controlling step for controlling the transmission power of said second channel; a training step for carrying out training to obtain correlation information that correlates the reception quality of said first channel transmitted from a reception apparatus for receiving said data and the transmission power of said second channel; a predicting step for obtaining a predicted value of the reception quality of said first channel using the transmission power of said second channel based on said correlation information; and a transmission controlling step for controlling the transmission of said data through said first channel based on the predicted value of the reception quality of said first channel 17.A reception apparatus that uses a first channel through which data is transmitted and a second channel on which power control is performed, in order to receive said data transmitted from a transmission apparatus for transmitting said data, said reception apparatus characterized by comprising: reception quality obtaining means for obtaining the reception quality of said first channel; reception quality transmitting means for transmitting the reception quality of said first channel to said transmission apparatus; power control information transmitting means for transmitting power control information that controls the transmission power of said second channel to said transmission apparatus; extracting means for extracting transmission control information that controls transmission of the reception quality of said first channel from the data of said second channel transmitted from said transmission apparatus; and transmission controlling means for controlling the transmission of the reception quality of said first channel based on said transmission control information.",
"18.The reception apparatus according to claim 17, characterized in that said transmission controlling means starts or stops the transmission of the reception quality of said first channel based on said transmission control information.",
"19.The reception apparatus according to claim 17, characterized in that said transmission controlling means controls the transmission frequency of the reception quality of said first channel based on said transmission control information.",
"20.A reception control method for a reception apparatus that uses a first channel through which data is transmitted and a second channel on which power control is performed, and receives said data transmitted from a transmission apparatus for transmitting said data, said reception control method characterized by comprising: a reception quality obtaining step for obtaining the reception quality of said first channel; a reception quality transmitting step for transmitting the reception quality of said first channel to said transmission apparatus; a power control information transmitting step for transmitting power control information for controlling the transmission power of said second channel to said transmission apparatus; an extracting step for extracting transmission control information that controls transmission of the reception quality of said first channel from the data of said second channel transmitted from said transmission apparatus; and a transmission controlling step for controlling the transmission of the reception quality of said first channel based on said transmission control information.",
"21.A program for making a computer perform a reception control process for a reception apparatus that uses a first channel through which a data is transmitted and a second channel on which power control is performed, and receives said data transmitted from a transmission apparatus for transmitting said data, said program characterized by comprising: a reception quality obtaining step for obtaining a reception quality of said first channel; a reception quality transmitting step for transmitting the reception quality of said first channel to said transmission apparatus; a power control information transmitting step for transmitting power control information for controlling the transmission power of said second channel to said transmission apparatus; an extracting step for extracting transmission control information for controlling transmission of the reception quality of said first channel from the data of said second channel transmitted from said transmission apparatus; and a transmission controlling step for controlling the transmission of the reception quality of said first channel based on said transmission control information.",
"22.A communications system, comprising: a transmission apparatus that uses a first channel through which data is transmitted and a second channel on which power control is performed, and transmits said data; and a reception apparatus for receiving said data transmitted from said transmission apparatus, said communications system characterized in that said transmission apparatus includes: transmission power controlling means for controlling the transmission power of said second channel in accordance with power control information for controlling the transmission power of said second channel; training means for carrying out training to obtain correlation information that correlates the reception quality of said first channel transmitted from said reception apparatus and the transmission power of said second channel; predicting means for obtaining a predicted value of the reception quality of said first channel using the transmission power of said second channel based on said correlation information; data transmission controlling means for controlling the transmission of said data through said first channel based on the predicted value of the reception quality of said first channel; and transmission control information transmitting means for transmitting to said reception apparatus through said second channel transmission control information that controls the transmission of the reception quality of said first channel by said reception apparatus; and said reception apparatus includes: reception quality obtaining means for obtaining the reception quality of said first channel; reception quality transmitting means for transmitting the reception quality of said first channel to said transmission apparatus; power control information transmitting means for transmitting said power control information to said transmission apparatus; extracting means for extracting said transmission control information from the data of said second channel transmitted from said transmission apparatus; and reception quality transmission controlling means for controlling the transmission of the reception quality of said first channel based on said transmission control information."
],
[
"<SOH> BACKGROUND ART <EOH>In recent radio communications systems, instead of accommodating a single modulation and encoding system, using an adaptive modulation and code rate communications system that controls so as to perform communications by way of an optimum system depending on the conditions for communications is proposed.",
"Communications systems using adaptive modulation and code rate (hereafter, referred to as an adaptive encoding modulation system where appropriate) change the code rate of an error correction code and the degree of multi-valued modulation depending on the quality of a propagation path, and provides high-speed data communications to a user, whose propagation path has a high quality, at the expense of noise durability characteristics, and provide low-speed data communications to a user, whose propagation path has poor quality, putting noise durability characteristics ahead.",
"Such adaptive encoding modulation systems are expected to be additionally employed even in W-CDMA (Wideband-Code Division Multiple Access) which is attracting attention as a third generation mobile communications system.",
"In adaptive encoding modulation systems, adaptive modulation and code rate between a base station and a terminal is realized through the basic procedures below.",
"1.The terminal measures the reception quality of a signal transmitted from the base station.",
"2.The terminal returns to the base station a reception quality message that indicates the measured result of the reception quality.",
"3.The base station determines the optimal modulation system and code rate from the reception quality message transmitted from the terminal, and transmits to the terminal a transmission parameter that indicates the determined modulation system and code rate.",
"4.The base station transmits user data based on the transmission parameter.",
"5.The terminal receives the transmission parameter and carries out a data receiving process based on the transmission parameter.",
"6.1˜5 mentioned above are repeated periodically.",
"An outline illustrating this procedure is shown in FIG.",
"1 .",
"In FIG.",
"1 , the relationship between a downlink control channel for notifying the transmission parameter from the base station to the terminal, a downlink data channel for transmitting the user data from the base station to the terminal and an uplink control channel for transmitting the reception quality message from the terminal is shown.",
"In the present figure, an example in which the steps 1˜5 mentioned above are carried out at predetermined frame periods is shown.",
"That is, in FIG.",
"1 , the terminal measures the present reception quality at the terminal and transmits a reception quality message indicating the reception quality to the base station through the uplink control channel.",
"The base station determines, from the reception quality message transmitted from the terminal, a combination of modulation method and code rate with which, for example, the error rate of the received data at the terminal is at or below a predetermined value, and transmits, as a transmission parameter, information that indicates the modulation method and the code rate to the terminal through the downlink control channel.",
"Moreover, the base station transmits user data to the terminal through the downlink data channel in accordance with the code rate and the modulation method corresponding to the transmission parameter transmitted to the terminal.",
"Then, the terminal receives the transmission parameter transmitted from the base station in advance and thereby recognizes the code rate, the modulation method and the like of the data transmitted from the base station thereafter.",
"Moreover, the terminal receives the user data transmitted from the base station thereafter, and carries out demodulation by a demodulation method corresponding to the modulation method indicated by the transmission parameter received in advance, and carries out decoding based on a decoding method corresponding to the code rate.",
"The words “downlink” and “uplink” in the downlink data channel, the downlink control channel and the uplink control channel in FIG.",
"1 are used for channels for signals transmitted to the terminal from the base station and for channels for signals transmitted to the base station from the terminal, respectively.",
"That is, the word “downlink” is used in the names for channels for signals transmitted to the terminal from the base station.",
"Also, the word “uplink” is used in the names for channels for signals transmitted to the base station from the terminal.",
"Also, the transmission parameter comprises various parameters which are necessary for the transmission of data from the base station to the terminal.",
"FIG.",
"2 shows an example of a configuration of a conventional base station that realizes a communications system using adaptive modulation and code rate (adaptive encoding modulation method).",
"The base station comprises a transmission/reception compatible device 1 , an inverse spreading section 2 , a power control bit extracting section 3 , a control data inserting section 4 , a reception quality message extracting section 5 , a mode judging section 6 , a control section 7 , a control data generating section 8 , an encoding modulation section 9 , a power adjusting section 10 , a spreading section 11 , an adaptive encoding modulation section 13 and an antenna 14 .",
"The base station demodulates the transmission signal from the user, at the transmission/reception compatible device 1 and the inverse spreading section 2 .",
"That is, a spread spectrum transmission signal is transmitted to the base station from, for example, a terminal as a mobile station capable of radio communications comprising a portable telephone, other PDAs (Personal Digital Assistant) or the like.",
"This transmission signal is received by the antenna 14 and is supplied to the transmission/reception compatible device 1 .",
"The transmission/reception compatible device 1 receives the transmission signal from the antenna 14 , performs necessary processing and supplies it to the inverse spreading section 2 .",
"The inverse spreading section 2 performs an inverse spread spectrum on the signal supplied from the transmission/reception compatible device 1 and supplies it to the power control bit extracting section 3 .",
"The power control bit extracting section 3 extracts a power control bit from the signal supplied from the inverse spreading section 2 .",
"In other words, there is included in the transmission signal transmitted to the base station from the terminal a power control bit, which is a one-bit flag that requests an increase or a decrease in the transmission power of the downlink control channel explained in FIG.",
"1 .",
"The power control bit extracting section 3 extracts such a power control bit from the signal supplied from the inverse spreading section 2 and transfers it to the power adjusting section 10 .",
"The power control bit extracting section 3 extracts the power control bit from the signal supplied from the inverse spreading section 2 , while at the same time supplying the signal to the reception quality message extracting section 5 .",
"The reception quality message extracting section 5 obtains a reception quality message from the signal supplied from the power control bit extracting section 3 .",
"That is, there is included in the transmission signal transmitted to the base station from the terminal a reception quality message that indicates the reception quality (SIR (Signal to Interference Ratio)) at the terminal, as explained in FIG.",
"1 .",
"The reception quality message extracting section 5 obtains, through extraction, the reception quality message from the signal sent from the power control bit extracting section 3 , and transfers it to the mode judging section 6 .",
"Here, the signal that is exchanged between the terminal and the base station is composed in frames of a predetermined duration.",
"Moreover, a frame is configured such that, for example, a slot in units of 0.667 msec (milliseconds) is arranged in a plurality of slots.",
"The power control bit mentioned above is such that it is transmitted from the terminal to the base station per slot.",
"Thus, the power control bit extracting section 3 extracts the power control bit for each slot.",
"Also, at the terminal, the reception quality message is such that it is transmitted in units of one frame.",
"Hence, the reception quality message extracting section 5 extracts the reception quality message in units of one frame.",
"The mode judging section 6 determines the optimal modulation method and code rate from the reception quality message and the condition of the resources the base station has, and assigns encoding resources and power resources to the user (the terminal).",
"That is, if the combination of modulation method and code rate is now taken to be a transmission mode, the mode judging section 6 determines the transmission mode from the resources of the base station and the reception quality message supplied from the reception quality message extracting section 5 , and supplies it to the control section 7 .",
"Here, a large number of transmission modes can be provided through combinations of code rates and modulation methods.",
"However, here, in order to simplify the explanation, three transmission modes # 0 to # 2 shown in FIG.",
"3 are explained.",
"In FIG.",
"3 , R=½ and R=¾ are provided for the code rate (the encoding method).",
"The code rate R=½ signifies that one redundant bit is added for each input data of one bit.",
"The code rate R=¾ signifies that one redundant bit is added for each input data of three bits.",
"In the code rate R=½, although the error correction performance is stronger to the extent that there are more redundant bits in relation to the input data, the number of transmittable data becomes smaller.",
"On the other hand, in the code rate R=¾, although the error correction performance is inferior to the code rate R=½ since the number of redundant bits in relation to the input data is smaller, the number of transmittable data can be increased.",
"Also, in FIG.",
"3 , QPSK (Quadrature Phase Shift Keying) and 16 QAM (Quadrature Amplitude Modulation) are provided for the modulation methods.",
"As shown in FIGS.",
"4A and 4B , in QPSK modulation, two bits of encoded data are mapped to one symbol among four symbols ( FIG.",
"4A ), and in 16 QAM, four bits of data are mapped to one symbol among sixteen symbols ( FIG.",
"4B ).",
"If a transmittable symbol rate is assumed to be constant, data that is actually transmittable is greater for 16 QAM than it is for QPSK.",
"However, in 16 QAM because the distance between the symbols becomes shorter as compared to QPSK, it has a feature that noise characteristics become worse.",
"In FIG.",
"3 , the combination of R=½ and QPSK, the combination of R=½ and 16 QAM and the combination of R=¾ and 16 QAM are defined as the transmission modes # 0 , # 1 and # 2 , respectively.",
"Thus, the relationship in terms of data transfer amount would be transmission mode # 0 (R=½, QPSK)<transmission mode # 1 (R=½, 16 QAM)<transmission mode # 2 (R=¾, 16 QAM).",
"On the other hand, the relationship in terms of noise durability characteristics would be transmission mode # 0 (R=½, QPSK)>transmission mode # 1 (R=½, 16 QAM)>transmission mode # 2 (R=¾, 16 QAM).",
"According to the adaptive encoding modulation method, if the noise is small and the propagation path is good (in a case where the reception quality at the terminal is good), by selecting a combination (a transmission mode) of modulation method and code rate whose data transfer amount is large, it is possible to carry out efficient data transmission.",
"Also, if the noise is large and the propagation path is poor (in a case where the reception quality at the terminal is poor), by selecting a combination of modulation method and code rate whose noise durability characteristics are high, it is possible to suppress data transfer amount and enhance error characteristics.",
"The mode judging section 6 selects, for example as shown in FIG.",
"5 , a transmission mode in which the error rate of the user data received by the terminal is at or below a predetermined value.",
"In other words, FIG.",
"5 shows the relation between the reception quality (SIR) and the error rate of the user data (FER; Frame Error Rate), for each of the three transmission modes # 0 (R=½, QPSK), # 1 (R=½, 16 QAM) and # 3 (R=¾, 16 QAM) mentioned above.",
"The mode judging section 6 judges and selects, for example, a transmission mode in which the error rate of the user data (FER) is 10% or below in relation to reception quality.",
"In this case, the transmission modes # 0 (R=½, QPSK), # 1 (R=½, 16 QAM) and # 2 (R=¾, 16 QAM) are selected respectively at the mode judging section 6 when the reception quality is −8 dB or lower, higher than −8 dB and lower than −4 dB, and −4 dB or higher.",
"Returning to FIG.",
"2 , the control section 7 transfers the transmission mode determined by the mode judging section 6 to the control data generating section 8 and the adaptive encoding modulation section 13 .",
"The control data generating section 8 generates control data including the transmission mode supplied from the control section 7 , and supplies it to the control data inserting section 4 .",
"The control data inserting section 4 is such that besides having the control data from the control data generating section 8 supplied thereto, audio data transferred from a different base station, NW (NetWork) control data used to judge and control a hand-off for shifting control of the terminal from one base station to another base station and the like are sent thereto.",
"The control data inserting section 4 inserts the control data supplied from the control data generating section 8 into the audio data and the NW control data supplied thereto, and supplies it to the encoding modulation section 9 .",
"The encoding modulation section 9 performs an encoding modulation process on the signal supplied from the control data inserting section 4 through a predetermined method, and sends the modulation signal obtained as a result to the power adjusting section 10 .",
"At the power adjusting section 10 , the transmission power of the data through the downlink control channel explained in FIG.",
"1 is determined in accordance with the power control bit supplied from the power control bit extracting section 3 .",
"In other words, the power control bit is, for example, as mentioned above, a 1-bit flag, and the power adjusting section 10 processes the modulation signal from the encoding modulation section 9 so as to increase the transmission power in the downlink control channel by 1 dB if the power control bit is 1, and decrease the transmission power in the downlink control channel by 1 dB if the power control bit is 0.Thus, a mechanism for transmitting data in the downlink control channel to the terminal at an optimal power is provided.",
"In addition, the signal in this downlink control channel is transmitted in such a form that it is always associated with the downlink data channel explained in FIG.",
"1 .",
"Here, in the W-CDMA system, the base station carries out such control of the transmission power in the downlink control channel in accordance with the power control bit transmitted from the terminal with each slot.",
"The modulation signal whose transmission power is adjusted at the power adjusting section 10 is supplied to the spreading section 11 .",
"On the other hand, packet data in which the user data transmitted through the downlink data channel explained in FIG.",
"1 is placed is supplied to the adaptive encoding modulation section 13 .",
"Then, the adaptive encoding modulation section 13 encodes the packet data in accordance with the code rate represented by the transmission mode supplied from the control section 7 , and further carries out a modulation process in accordance with the modulation method represented by the transmission mode.",
"The adaptive encoding modulation section 13 supplies to the spreading section 11 the modulation signal thus obtained by encoding and modulating the packet data.",
"Here, FIG.",
"6 shows a configuration example of the adaptive encoding modulation section 13 in a case where, as shown in FIG.",
"3 , the three transmission modes # 0 to # 2 are arranged.",
"The packet data inputted to the adaptive encoding modulation section 13 is supplied to a switch 21 .",
"Then, if the transmission mode supplied from the control section 7 is transmission mode # 0 , the switch 21 selects a terminal 21 a , and a switch 24 selects a terminal 24 a.",
"The terminal 21 a is connected to an encoding section 22 a .",
"Therefore, if the transmission mode is # 0 , the packet data is supplied from the switch 21 to the encoding section 22 a .",
"The encoding section 22 a encodes the packet data supplied thereto at a code rate of R=½, thereby adding an error correcting code, and supplying the encoded data obtained as a result thereof to a QPSK modulation section 23 a .",
"The QPSK modulation section 23 a performs QPSK modulation on the encoded data from the encoding section 22 a , thereby carrying out a modulation symbol mapping, and supplies a modulation signal obtained as a result thereof to the terminal 24 a of the switch 24 .",
"If the transmission mode is # 0 , because the switch 24 has selected the terminal 24 a as mentioned above, the modulation signal outputted by the QPSK modulation section 23 a is supplied to the spreading section 11 ( FIG.",
"2 ) via the switch 24 .",
"In addition, if the transmission mode supplied from the control section 7 is transmission mode # 1 , the switch 21 selects a terminal 21 b , and the switch 24 selects a terminal 24 b .",
"The terminal 21 b is connected to an encoding section 22 b .",
"Therefore, if the transmission mode is # 1 , the packet data is supplied from the switch 21 to the encoding section 22 b .",
"The encoding section 22 b encodes the packet data supplied thereto at a code rate of R=½, and supplies the encoded data obtained as a result thereof to a 16 QAM modulation section 23 b .",
"The 16 QAM modulation section 23 b performs 16 QAM modulation on the encoded data from the encoding section 22 b , and supplies the modulation signal obtained as a result thereof to the terminal 24 b of the switch 24 .",
"If the transmission mode is # 1 , because the switch 24 has selected the terminal 24 b as mentioned above, the modulation signal outputted by the 16 QAM modulation section 23 b is supplied to the spreading section 11 ( FIG.",
"2 ) via the switch 24 .",
"Moreover, if the transmission mode supplied from the control section 7 is transmission mode # 2 , the switch 21 selects a terminal 21 c, and the switch 24 selects a terminal 24 c .",
"The terminal 21 c is connected to an encoding section 22 c .",
"Therefore, if the transmission mode is # 2 , the packet data is supplied from the switch 21 to the encoding section 22 c .",
"The encoding section 22 c encodes the packet data supplied thereto at a code rate of R=¾, and supplies the encoded data obtained as a result thereof to a 16 QAM modulation section 23 c .",
"The 16 QAM modulation section 23 c performs 16 QAM modulation on the encoded data from the encoding section 22 c , and supplies the modulation signal obtained as a result thereof to the terminal 24 c of the switch 24 .",
"If the transmission mode is # 2 , because the switch 24 has selected the terminal 24 c as mentioned above, the modulation signal outputted by the 16 QAM modulation section 23 c is supplied to the spreading section 11 ( FIG.",
"2 ) via the switch 24 .",
"Again, returning to FIG.",
"2 , using separate spreading codes, the spreading section 11 performs a spread spectrum on the modulation signal supplied from the power adjusting section 10 and the modulation signal supplied from the adaptive encoding modulation section 13 , and supplies the thus obtained spread signals to the transmission/reception compatible device 1 .",
"The transmission/reception compatible device 1 performs the necessary process on the spread signals from the spreading section 11 , and transmits them to the terminal as radio waves from the antenna 14 .",
"Of the signals thus transmitted, the modulation signal supplied from the power adjusting section 10 is the signal in the downlink control channel of FIG.",
"1 , and the modulation signal supplied from the adaptive encoding modulation section 13 is the signal in the downlink data channel of FIG.",
"1 .",
"In addition, in the downlink data channel, as mentioned above, the user data is transmitted in the form of packet data.",
"As such, the downlink data channel will hereinafter be referred to as a packet channel as deemed appropriate.",
"Also, the downlink control channel is transmitted in such a manner as to be always associated with the downlink data channel (the packet channel) as mentioned above.",
"As such, the downlink control channel will hereinafter be referred to as an associated channel as deemed appropriate.",
"Here, the packet channel, in which the user data is transmitted and adaptive encoding modulation is performed, is referred to as, for example, HS-DSCH (High Speed Downlink Shared CHannel).",
"Also, the associated channel, in which the audio data, the NW control data and the control data including the transmission mode are transmitted and on which transmission power control through the power control bit is performed, is referred to as, for example, DPCH (Dedicated Physical CHannel).",
"Next, FIG.",
"7 shows a configuration example of a conventional terminal which realizes a communications system using adaptive modulation and coding rate (adaptive encoding modulation method).",
"A terminal (a user terminal) comprises a transmission/reception compatible device 31 , an inverse spreading section 32 , an associated channel reception quality estimating section 33 , a power control bit generating section 34 , a packet channel reception quality estimating section 35 , a reception quality message generating section 36 , an associated channel demodulation decoding section 37 , a control section 38 , a user data demodulation decoding section 39 , an error detecting section 40 , a reception quality message inserting section 43 , a power control bit inserting section 44 , a spreading section 45 , and an antenna 47 .",
"A transmission signal sent out from the base station is received by the antenna 47 and supplied to the inverse spreading section 32 after necessary processing is performed by the transmission/reception compatible device 31 .",
"The inverse spreading section 32 performs an inverse spread spectrum on the signal from the transmission/reception compatible device 31 to thereby separate it into a packet channel signal and an associated channel signal.",
"Then, the inverse spreading section 32 supplies the associated channel signal to the associated channel reception quality estimating section 33 and the associated channel demodulation decoding section 37 .",
"Moreover, the inverse spreading section 32 supplies the packet channel signal to the packet channel reception quality estimating section 35 and the user data demodulation decoding section 39 .",
"The associated channel reception quality estimating section 33 estimates the signal to noise ratio (SNR) from a pilot signal time-multiplexed on the associated channel.",
"In other words, although not explained in FIG.",
"2 , for example, the control data inserting section 4 is such that it time-multiplexes a predetermined pilot signal as the associated channel signal.",
"Thus, that pilot signal is included in the associated channel signal.",
"The associated channel reception quality estimating section 33 estimates the SNR of the associated channel signal supplied from the inverse spreading section 32 using the pilot signal included in the signal, and supplies to the power control bit generating section 34 the estimated SNR as the reception quality of the associated channel.",
"The power control bit generating section 34 outputs to the power control bit inserting section 44 a power control bit of a value of 0 if the estimated SNR of the associated channel (the reception quality of the associated channel) is better than a reference quality of the associated channel which is the desired SNR, or a power control bit of a value of 1 if it is worse.",
"Here, the estimation of the SNR at the associated channel reception quality estimating section 33 and the generation of the power control bit at the power control bit generating section 34 are executed for each slot.",
"In the base station in FIG.",
"2 , the power adjusting section 10 controls the transmission power of the associated channel based on the power control bit such that the associated channel can be received by the terminal always at a constant SNR.",
"The associated channel demodulation decoding section 37 demodulates and decodes the associated channel signal supplied from the inverse spreading section 32 and separates the audio data, the NW control data and the control data.",
"The audio data, the W control data and the control data are supplied to a circuit not shown in the drawings and are also supplied to the control section 38 .",
"The control section 38 detects information on the modulation method and the code rate which are placed in the control data supplied from the associated channel demodulation decoding section 37 and applied to the packet channel, namely, the transmission mode, and carries out mode setting (control) of the user data demodulation decoding section 39 .",
"In other words, if the transmission mode is # 0 , the control section 38 controls the user data demodulation decoding section 39 so as to QPSK demodulate the packet channel signal and further decode it at a code rate of R=½.",
"Also, if the transmission mode is # 1 , the control section 38 controls the user data demodulation decoding section 39 so as to 16 QAM demodulate the packet channel signal and further decode it at a code rate of R=½.",
"Alternatively, if the transmission mode is # 2 , the control section 38 controls the user data demodulation decoding section 39 so as to 16 QAM demodulate the packet channel signal and further decode it at a code rate of R=¾.",
"On the other hand, the packet channel reception quality estimating section 35 estimates the SNR of the packet channel signal supplied from the inverse spreading section 32 .",
"A pilot symbol that is time-multiplexed on the packet channel or a pilot channel symbol transmitted in parallel with the packet channel is used in this SNR estimation.",
"In other words, although not explained in FIG.",
"2 , the spreading section 11 is such that it time-multiplexes the predetermined pilot signal on the modulation signal supplied from the adaptive encoding modulation section 13 and then carries out a spread spectrum.",
"Thus, the packet channel signal includes the pilot signal.",
"Also, the spreading section 11 is such that it performs a spread spectrum on a different pilot signal with a spreading code different from the spreading code used in the spread spectrum of the modulation signal supplied from the power adjusting section 10 or the adaptive encoding modulation section 13 , and transmits it via the transmission/reception compatible device 1 and the antenna 14 in parallel with the packet channel or the associated channel.",
"The packet channel reception quality estimating section 35 estimates the SNR of the packet channel signal supplied from the inverse spreading section 32 using the pilot signal included in that signal or the pilot signal that is transmitted in parallel with the packet channel signal, and supplies to the reception quality message generating section 36 the estimated SNR as the reception quality of the packet channel.",
"The reception quality message generating section 36 generates a reception quality message of a predetermined message format representing the estimated SNR of the packet channel (the reception quality of the packet channel) supplied from the packet channel reception quality estimating section 35 , and supplies it to the reception quality message inserting section 43 .",
"Here, the estimation of the SNR of the packet channel by the packet channel reception quality estimating section 35 and the generation of the reception quality message by the reception quality message generating section 36 are executed for each frame.",
"On the other hand, the user data demodulation decoding section 39 decodes and demodulates the packet channel signal supplied from the inverse spreading section 32 in accordance with the control of the control section 38 , and supplies the packet data obtained as a result thereof to the error detecting section 40 .",
"In addition, the user data demodulation decoding section 39 , upon decoding the packet channel signal, carries out error correction of the packet data using the error correcting code included in that signal as a redundant bit.",
"The error detecting section 40 carries out, for example, parity detection using cyclic redundancy check (CRC) and judges whether or not there is an error in the packet data decoded by the user data demodulation decoding section 39 .",
"Then, the error detecting section 40 outputs ACK (ACKnowledge), which is a message indicating that the packet data was received properly, if there is no error in the packet data, and outputs NACK, which is a message indicating that the packet data could not be received properly, if there is an error in the packet data.",
"In addition, although not shown in FIG.",
"7 (which similarly applies to FIG.",
"20 described later), the ACK/NACK outputted by the error detecting section 40 is supplied to the spreading section 45 and is transmitted to the base station.",
"The reception quality message inserting section 43 frames the reception quality message, which is supplied from the reception quality message generating section 36 , in the uplink control channel signal explained in FIG.",
"1 , and supplies it to the power control bit inserting section 44 .",
"The power control bit inserting section 44 frames the power control bit, which is supplied from the power control bit generating section 34 , in the uplink control channel signal supplied from the reception quality message inserting section 43 , and supplies it to the spreading section 45 .",
"The spreading section 45 performs a spread spectrum on the uplink control channel signal from the power control bit inserting section 44 and supplies the spread signal obtained as a result thereof to the transmission/reception compatible device 31 .",
"The transmission/reception compatible device 31 performs necessary processing on the spread signal from the spreading section 45 and transmits it from the antenna 47 as a radio wave.",
"In addition, at the terminal, the reception quality message is transmitted per frame, and the power control bit is transmitted per slot.",
"According to the adaptive encoding modulation method, the data transmission speed can be changed in accordance with the reception condition (the reception quality) of the terminal, and data can be transmitted to the terminal side more efficiently.",
"By the way, in the adaptive encoding modulation method, for example, the terminal, which is the mobile station, reports the estimated result of the reception quality of the packet channel to the base station, and the base station selects the optimal combination of the modulation method and encoding method based on the reported value (the reception quality indicated by the reception quality message).",
"For this reason, the reception quality accuracy reported to the base station becomes important.",
"However, because there arises a delay in the estimation and reporting of the reception quality of the packet channel and the message reception at the base station, there are instances where there is some difference between the actual reception quality of the packet channel at the terminal at the time when the base station has demodulated the reception quality message and the reception quality represented by the reception quality message.",
"In other words, at the terminal, as mentioned above, the reception quality of the packet channel is estimated in periods of frames and is transmitted to the base station.",
"For this reason, from when the reception quality at the terminal is estimated till when that reception quality is recognized at the base station, there is a time lag corresponding to several frames.",
"The reception quality recognized by the base station is a reception quality that is behind by a period of time represented by the time lag.",
"Therefore, there are cases where the reception quality recognized by the base station differs from the current reception quality at the terminal.",
"In such cases, the base station cannot select the optimal combination of the modulation and the encoding methods, which may lower system efficiency.",
"This phenomenon presents itself most notably when reception propagation path characteristics change rapidly, such as when, in particular, the terminal, which is the mobile station, is moving at high speed.",
"As such, a method may be considered in which the reception quality message representing the reception quality of the packet channel is transmitted more frequently from the terminal to the base station.",
"However, if the transmission frequency of the reception quality message is made higher, the usage of radio resources increases, and further, power consumption at the terminal also increases.",
"Therefore, in order to save radio resources and carry out a more effective system operation, it is effective to slow (extend) the reception quality report period.",
"However, by slowing the period, the delay from the time of estimation of the reception quality to when the reported value (the reception quality message) reaches the base station further increases, which results in a larger difference between the reported value and the actual reception quality."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.",
"1 is a view explaining conventional data transmission; FIG.",
"2 is a view showing a configuration of an example of a conventional base station; FIG.",
"3 is a view explaining transmission modes; FIG.",
"4A is a view showing the mapping of data by a QPSK modulation method; FIG.",
"4B is a view showing the mapping of data by a 16 QAM modulation method; FIG.",
"5 is a view showing the relationship between reception quality and error rate; FIG.",
"6 is a block diagram showing a configuration example of an adaptive encoding modulation section 13 ; FIG.",
"7 is a view showing a configuration of an example of a conventional terminal; FIG.",
"8 is a block diagram showing a configuration example of an embodiment of a communications system to which the present invention is applied; FIG.",
"9 is a block diagram showing a configuration example of a base station 101 ; FIG.",
"10 is a flowchart explaining a process at the base station 101 ; FIG.",
"11 is a flowchart explaining a process of a training mode; FIG.",
"12 is a view explaining power control of an associated channel; FIG.",
"13A is a view showing propagation characteristics; FIG.",
"13B is a view showing the transmission power of the associated channel; FIG.",
"13C is a view showing the reception quality of the associated channel; FIG.",
"14A is a view showing the transmission power of a packet channel; FIG.",
"14B is a view showing propagation characteristics; FIG.",
"14C is a view showing the reception quality of the packet channel; FIG.",
"15 is a view showing the transmission power of the associated channel and the reception quality of the packet channel; FIG.",
"16 is a flowchart explaining a process of a normal mode; FIG.",
"17 is a flowchart explaining another embodiment of a process at the base station 101 ; FIG.",
"18 is a flowchart explaining a process for setting a transmission period of a reception quality message; FIG.",
"19 is a view showing the transmission period of the reception quality message becoming longer; FIG.",
"20 is a block diagram showing a configuration example of a terminal 102 ; FIG.",
"21 is a flowchart explaining a process at the terminal 102 ; FIG.",
"22 is a flowchart explaining a process at the terminal 102 ; FIG.",
"23 is a flowchart explaining a process at the terminal 102 ; and FIG.",
"24 is a block diagram showing a configuration example of an embodiment of a computer to which the present invention is applied.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to a method of estimating reception quality, and more particularly to a method of estimating the reception quality of a packet channel for communicating data from the transmission power of an associated channel for communicating audio and the like in a system for carrying out simultaneous communications in a plurality of channels.",
"That is, the present invention relates to a transmission apparatus, a transmission control method, a reception apparatus and a reception control method.",
"More particularly, the present invention relates to a transmission apparatus, a transmission control method, a reception apparatus and a reception control method which, for example, in a communications system comprising a terminal for reporting the reception quality of a channel to a base station, and the base station for controlling transmission of data through the channel based on the reception quality from the terminal, reduces the power consumption of the terminal and also makes it possible to save radio resources.",
"BACKGROUND ART In recent radio communications systems, instead of accommodating a single modulation and encoding system, using an adaptive modulation and code rate communications system that controls so as to perform communications by way of an optimum system depending on the conditions for communications is proposed.",
"Communications systems using adaptive modulation and code rate (hereafter, referred to as an adaptive encoding modulation system where appropriate) change the code rate of an error correction code and the degree of multi-valued modulation depending on the quality of a propagation path, and provides high-speed data communications to a user, whose propagation path has a high quality, at the expense of noise durability characteristics, and provide low-speed data communications to a user, whose propagation path has poor quality, putting noise durability characteristics ahead.",
"Such adaptive encoding modulation systems are expected to be additionally employed even in W-CDMA (Wideband-Code Division Multiple Access) which is attracting attention as a third generation mobile communications system.",
"In adaptive encoding modulation systems, adaptive modulation and code rate between a base station and a terminal is realized through the basic procedures below.",
"1.The terminal measures the reception quality of a signal transmitted from the base station.",
"2.The terminal returns to the base station a reception quality message that indicates the measured result of the reception quality.",
"3.The base station determines the optimal modulation system and code rate from the reception quality message transmitted from the terminal, and transmits to the terminal a transmission parameter that indicates the determined modulation system and code rate.",
"4.The base station transmits user data based on the transmission parameter.",
"5.The terminal receives the transmission parameter and carries out a data receiving process based on the transmission parameter.",
"6.1˜5 mentioned above are repeated periodically.",
"An outline illustrating this procedure is shown in FIG.",
"1.In FIG.",
"1, the relationship between a downlink control channel for notifying the transmission parameter from the base station to the terminal, a downlink data channel for transmitting the user data from the base station to the terminal and an uplink control channel for transmitting the reception quality message from the terminal is shown.",
"In the present figure, an example in which the steps 1˜5 mentioned above are carried out at predetermined frame periods is shown.",
"That is, in FIG.",
"1, the terminal measures the present reception quality at the terminal and transmits a reception quality message indicating the reception quality to the base station through the uplink control channel.",
"The base station determines, from the reception quality message transmitted from the terminal, a combination of modulation method and code rate with which, for example, the error rate of the received data at the terminal is at or below a predetermined value, and transmits, as a transmission parameter, information that indicates the modulation method and the code rate to the terminal through the downlink control channel.",
"Moreover, the base station transmits user data to the terminal through the downlink data channel in accordance with the code rate and the modulation method corresponding to the transmission parameter transmitted to the terminal.",
"Then, the terminal receives the transmission parameter transmitted from the base station in advance and thereby recognizes the code rate, the modulation method and the like of the data transmitted from the base station thereafter.",
"Moreover, the terminal receives the user data transmitted from the base station thereafter, and carries out demodulation by a demodulation method corresponding to the modulation method indicated by the transmission parameter received in advance, and carries out decoding based on a decoding method corresponding to the code rate.",
"The words “downlink” and “uplink” in the downlink data channel, the downlink control channel and the uplink control channel in FIG.",
"1 are used for channels for signals transmitted to the terminal from the base station and for channels for signals transmitted to the base station from the terminal, respectively.",
"That is, the word “downlink” is used in the names for channels for signals transmitted to the terminal from the base station.",
"Also, the word “uplink” is used in the names for channels for signals transmitted to the base station from the terminal.",
"Also, the transmission parameter comprises various parameters which are necessary for the transmission of data from the base station to the terminal.",
"FIG.",
"2 shows an example of a configuration of a conventional base station that realizes a communications system using adaptive modulation and code rate (adaptive encoding modulation method).",
"The base station comprises a transmission/reception compatible device 1, an inverse spreading section 2, a power control bit extracting section 3, a control data inserting section 4, a reception quality message extracting section 5, a mode judging section 6, a control section 7, a control data generating section 8, an encoding modulation section 9, a power adjusting section 10, a spreading section 11, an adaptive encoding modulation section 13 and an antenna 14.The base station demodulates the transmission signal from the user, at the transmission/reception compatible device 1 and the inverse spreading section 2.That is, a spread spectrum transmission signal is transmitted to the base station from, for example, a terminal as a mobile station capable of radio communications comprising a portable telephone, other PDAs (Personal Digital Assistant) or the like.",
"This transmission signal is received by the antenna 14 and is supplied to the transmission/reception compatible device 1.The transmission/reception compatible device 1 receives the transmission signal from the antenna 14, performs necessary processing and supplies it to the inverse spreading section 2.The inverse spreading section 2 performs an inverse spread spectrum on the signal supplied from the transmission/reception compatible device 1 and supplies it to the power control bit extracting section 3.The power control bit extracting section 3 extracts a power control bit from the signal supplied from the inverse spreading section 2.In other words, there is included in the transmission signal transmitted to the base station from the terminal a power control bit, which is a one-bit flag that requests an increase or a decrease in the transmission power of the downlink control channel explained in FIG.",
"1.The power control bit extracting section 3 extracts such a power control bit from the signal supplied from the inverse spreading section 2 and transfers it to the power adjusting section 10.The power control bit extracting section 3 extracts the power control bit from the signal supplied from the inverse spreading section 2, while at the same time supplying the signal to the reception quality message extracting section 5.The reception quality message extracting section 5 obtains a reception quality message from the signal supplied from the power control bit extracting section 3.That is, there is included in the transmission signal transmitted to the base station from the terminal a reception quality message that indicates the reception quality (SIR (Signal to Interference Ratio)) at the terminal, as explained in FIG.",
"1.The reception quality message extracting section 5 obtains, through extraction, the reception quality message from the signal sent from the power control bit extracting section 3, and transfers it to the mode judging section 6.Here, the signal that is exchanged between the terminal and the base station is composed in frames of a predetermined duration.",
"Moreover, a frame is configured such that, for example, a slot in units of 0.667 msec (milliseconds) is arranged in a plurality of slots.",
"The power control bit mentioned above is such that it is transmitted from the terminal to the base station per slot.",
"Thus, the power control bit extracting section 3 extracts the power control bit for each slot.",
"Also, at the terminal, the reception quality message is such that it is transmitted in units of one frame.",
"Hence, the reception quality message extracting section 5 extracts the reception quality message in units of one frame.",
"The mode judging section 6 determines the optimal modulation method and code rate from the reception quality message and the condition of the resources the base station has, and assigns encoding resources and power resources to the user (the terminal).",
"That is, if the combination of modulation method and code rate is now taken to be a transmission mode, the mode judging section 6 determines the transmission mode from the resources of the base station and the reception quality message supplied from the reception quality message extracting section 5, and supplies it to the control section 7.Here, a large number of transmission modes can be provided through combinations of code rates and modulation methods.",
"However, here, in order to simplify the explanation, three transmission modes #0 to #2 shown in FIG.",
"3 are explained.",
"In FIG.",
"3, R=½ and R=¾ are provided for the code rate (the encoding method).",
"The code rate R=½ signifies that one redundant bit is added for each input data of one bit.",
"The code rate R=¾ signifies that one redundant bit is added for each input data of three bits.",
"In the code rate R=½, although the error correction performance is stronger to the extent that there are more redundant bits in relation to the input data, the number of transmittable data becomes smaller.",
"On the other hand, in the code rate R=¾, although the error correction performance is inferior to the code rate R=½ since the number of redundant bits in relation to the input data is smaller, the number of transmittable data can be increased.",
"Also, in FIG.",
"3, QPSK (Quadrature Phase Shift Keying) and 16 QAM (Quadrature Amplitude Modulation) are provided for the modulation methods.",
"As shown in FIGS.",
"4A and 4B, in QPSK modulation, two bits of encoded data are mapped to one symbol among four symbols (FIG.",
"4A), and in 16 QAM, four bits of data are mapped to one symbol among sixteen symbols (FIG.",
"4B).",
"If a transmittable symbol rate is assumed to be constant, data that is actually transmittable is greater for 16 QAM than it is for QPSK.",
"However, in 16 QAM because the distance between the symbols becomes shorter as compared to QPSK, it has a feature that noise characteristics become worse.",
"In FIG.",
"3, the combination of R=½ and QPSK, the combination of R=½ and 16 QAM and the combination of R=¾ and 16 QAM are defined as the transmission modes #0, #1 and #2, respectively.",
"Thus, the relationship in terms of data transfer amount would be transmission mode #0 (R=½, QPSK)<transmission mode #1 (R=½, 16 QAM)<transmission mode #2 (R=¾, 16 QAM).",
"On the other hand, the relationship in terms of noise durability characteristics would be transmission mode #0 (R=½, QPSK)>transmission mode #1 (R=½, 16 QAM)>transmission mode #2 (R=¾, 16 QAM).",
"According to the adaptive encoding modulation method, if the noise is small and the propagation path is good (in a case where the reception quality at the terminal is good), by selecting a combination (a transmission mode) of modulation method and code rate whose data transfer amount is large, it is possible to carry out efficient data transmission.",
"Also, if the noise is large and the propagation path is poor (in a case where the reception quality at the terminal is poor), by selecting a combination of modulation method and code rate whose noise durability characteristics are high, it is possible to suppress data transfer amount and enhance error characteristics.",
"The mode judging section 6 selects, for example as shown in FIG.",
"5, a transmission mode in which the error rate of the user data received by the terminal is at or below a predetermined value.",
"In other words, FIG.",
"5 shows the relation between the reception quality (SIR) and the error rate of the user data (FER; Frame Error Rate), for each of the three transmission modes #0 (R=½, QPSK), #1 (R=½, 16 QAM) and #3 (R=¾, 16 QAM) mentioned above.",
"The mode judging section 6 judges and selects, for example, a transmission mode in which the error rate of the user data (FER) is 10% or below in relation to reception quality.",
"In this case, the transmission modes #0 (R=½, QPSK), #1 (R=½, 16 QAM) and #2 (R=¾, 16 QAM) are selected respectively at the mode judging section 6 when the reception quality is −8 dB or lower, higher than −8 dB and lower than −4 dB, and −4 dB or higher.",
"Returning to FIG.",
"2, the control section 7 transfers the transmission mode determined by the mode judging section 6 to the control data generating section 8 and the adaptive encoding modulation section 13.The control data generating section 8 generates control data including the transmission mode supplied from the control section 7, and supplies it to the control data inserting section 4.The control data inserting section 4 is such that besides having the control data from the control data generating section 8 supplied thereto, audio data transferred from a different base station, NW (NetWork) control data used to judge and control a hand-off for shifting control of the terminal from one base station to another base station and the like are sent thereto.",
"The control data inserting section 4 inserts the control data supplied from the control data generating section 8 into the audio data and the NW control data supplied thereto, and supplies it to the encoding modulation section 9.The encoding modulation section 9 performs an encoding modulation process on the signal supplied from the control data inserting section 4 through a predetermined method, and sends the modulation signal obtained as a result to the power adjusting section 10.At the power adjusting section 10, the transmission power of the data through the downlink control channel explained in FIG.",
"1 is determined in accordance with the power control bit supplied from the power control bit extracting section 3.In other words, the power control bit is, for example, as mentioned above, a 1-bit flag, and the power adjusting section 10 processes the modulation signal from the encoding modulation section 9 so as to increase the transmission power in the downlink control channel by 1 dB if the power control bit is 1, and decrease the transmission power in the downlink control channel by 1 dB if the power control bit is 0.Thus, a mechanism for transmitting data in the downlink control channel to the terminal at an optimal power is provided.",
"In addition, the signal in this downlink control channel is transmitted in such a form that it is always associated with the downlink data channel explained in FIG.",
"1.Here, in the W-CDMA system, the base station carries out such control of the transmission power in the downlink control channel in accordance with the power control bit transmitted from the terminal with each slot.",
"The modulation signal whose transmission power is adjusted at the power adjusting section 10 is supplied to the spreading section 11.On the other hand, packet data in which the user data transmitted through the downlink data channel explained in FIG.",
"1 is placed is supplied to the adaptive encoding modulation section 13.Then, the adaptive encoding modulation section 13 encodes the packet data in accordance with the code rate represented by the transmission mode supplied from the control section 7, and further carries out a modulation process in accordance with the modulation method represented by the transmission mode.",
"The adaptive encoding modulation section 13 supplies to the spreading section 11 the modulation signal thus obtained by encoding and modulating the packet data.",
"Here, FIG.",
"6 shows a configuration example of the adaptive encoding modulation section 13 in a case where, as shown in FIG.",
"3, the three transmission modes #0 to # 2 are arranged.",
"The packet data inputted to the adaptive encoding modulation section 13 is supplied to a switch 21.Then, if the transmission mode supplied from the control section 7 is transmission mode #0, the switch 21 selects a terminal 21a, and a switch 24 selects a terminal 24a.",
"The terminal 21a is connected to an encoding section 22a.",
"Therefore, if the transmission mode is #0, the packet data is supplied from the switch 21 to the encoding section 22a.",
"The encoding section 22a encodes the packet data supplied thereto at a code rate of R=½, thereby adding an error correcting code, and supplying the encoded data obtained as a result thereof to a QPSK modulation section 23a.",
"The QPSK modulation section 23a performs QPSK modulation on the encoded data from the encoding section 22a, thereby carrying out a modulation symbol mapping, and supplies a modulation signal obtained as a result thereof to the terminal 24a of the switch 24.If the transmission mode is #0, because the switch 24 has selected the terminal 24a as mentioned above, the modulation signal outputted by the QPSK modulation section 23a is supplied to the spreading section 11 (FIG.",
"2) via the switch 24.In addition, if the transmission mode supplied from the control section 7 is transmission mode #1, the switch 21 selects a terminal 21b, and the switch 24 selects a terminal 24b.",
"The terminal 21b is connected to an encoding section 22b.",
"Therefore, if the transmission mode is #1, the packet data is supplied from the switch 21 to the encoding section 22b.",
"The encoding section 22b encodes the packet data supplied thereto at a code rate of R=½, and supplies the encoded data obtained as a result thereof to a 16 QAM modulation section 23b.",
"The 16 QAM modulation section 23b performs 16 QAM modulation on the encoded data from the encoding section 22b, and supplies the modulation signal obtained as a result thereof to the terminal 24b of the switch 24.If the transmission mode is #1, because the switch 24 has selected the terminal 24b as mentioned above, the modulation signal outputted by the 16 QAM modulation section 23b is supplied to the spreading section 11 (FIG.",
"2) via the switch 24.Moreover, if the transmission mode supplied from the control section 7 is transmission mode #2, the switch 21 selects a terminal 21c, and the switch 24 selects a terminal 24c.",
"The terminal 21c is connected to an encoding section 22c.",
"Therefore, if the transmission mode is #2, the packet data is supplied from the switch 21 to the encoding section 22c.",
"The encoding section 22c encodes the packet data supplied thereto at a code rate of R=¾, and supplies the encoded data obtained as a result thereof to a 16 QAM modulation section 23c.",
"The 16 QAM modulation section 23c performs 16 QAM modulation on the encoded data from the encoding section 22c, and supplies the modulation signal obtained as a result thereof to the terminal 24c of the switch 24.If the transmission mode is #2, because the switch 24 has selected the terminal 24c as mentioned above, the modulation signal outputted by the 16 QAM modulation section 23c is supplied to the spreading section 11 (FIG.",
"2) via the switch 24.Again, returning to FIG.",
"2, using separate spreading codes, the spreading section 11 performs a spread spectrum on the modulation signal supplied from the power adjusting section 10 and the modulation signal supplied from the adaptive encoding modulation section 13, and supplies the thus obtained spread signals to the transmission/reception compatible device 1.The transmission/reception compatible device 1 performs the necessary process on the spread signals from the spreading section 11, and transmits them to the terminal as radio waves from the antenna 14.Of the signals thus transmitted, the modulation signal supplied from the power adjusting section 10 is the signal in the downlink control channel of FIG.",
"1, and the modulation signal supplied from the adaptive encoding modulation section 13 is the signal in the downlink data channel of FIG.",
"1.In addition, in the downlink data channel, as mentioned above, the user data is transmitted in the form of packet data.",
"As such, the downlink data channel will hereinafter be referred to as a packet channel as deemed appropriate.",
"Also, the downlink control channel is transmitted in such a manner as to be always associated with the downlink data channel (the packet channel) as mentioned above.",
"As such, the downlink control channel will hereinafter be referred to as an associated channel as deemed appropriate.",
"Here, the packet channel, in which the user data is transmitted and adaptive encoding modulation is performed, is referred to as, for example, HS-DSCH (High Speed Downlink Shared CHannel).",
"Also, the associated channel, in which the audio data, the NW control data and the control data including the transmission mode are transmitted and on which transmission power control through the power control bit is performed, is referred to as, for example, DPCH (Dedicated Physical CHannel).",
"Next, FIG.",
"7 shows a configuration example of a conventional terminal which realizes a communications system using adaptive modulation and coding rate (adaptive encoding modulation method).",
"A terminal (a user terminal) comprises a transmission/reception compatible device 31, an inverse spreading section 32, an associated channel reception quality estimating section 33, a power control bit generating section 34, a packet channel reception quality estimating section 35, a reception quality message generating section 36, an associated channel demodulation decoding section 37, a control section 38, a user data demodulation decoding section 39, an error detecting section 40, a reception quality message inserting section 43, a power control bit inserting section 44, a spreading section 45, and an antenna 47.A transmission signal sent out from the base station is received by the antenna 47 and supplied to the inverse spreading section 32 after necessary processing is performed by the transmission/reception compatible device 31.The inverse spreading section 32 performs an inverse spread spectrum on the signal from the transmission/reception compatible device 31 to thereby separate it into a packet channel signal and an associated channel signal.",
"Then, the inverse spreading section 32 supplies the associated channel signal to the associated channel reception quality estimating section 33 and the associated channel demodulation decoding section 37.Moreover, the inverse spreading section 32 supplies the packet channel signal to the packet channel reception quality estimating section 35 and the user data demodulation decoding section 39.The associated channel reception quality estimating section 33 estimates the signal to noise ratio (SNR) from a pilot signal time-multiplexed on the associated channel.",
"In other words, although not explained in FIG.",
"2, for example, the control data inserting section 4 is such that it time-multiplexes a predetermined pilot signal as the associated channel signal.",
"Thus, that pilot signal is included in the associated channel signal.",
"The associated channel reception quality estimating section 33 estimates the SNR of the associated channel signal supplied from the inverse spreading section 32 using the pilot signal included in the signal, and supplies to the power control bit generating section 34 the estimated SNR as the reception quality of the associated channel.",
"The power control bit generating section 34 outputs to the power control bit inserting section 44 a power control bit of a value of 0 if the estimated SNR of the associated channel (the reception quality of the associated channel) is better than a reference quality of the associated channel which is the desired SNR, or a power control bit of a value of 1 if it is worse.",
"Here, the estimation of the SNR at the associated channel reception quality estimating section 33 and the generation of the power control bit at the power control bit generating section 34 are executed for each slot.",
"In the base station in FIG.",
"2, the power adjusting section 10 controls the transmission power of the associated channel based on the power control bit such that the associated channel can be received by the terminal always at a constant SNR.",
"The associated channel demodulation decoding section 37 demodulates and decodes the associated channel signal supplied from the inverse spreading section 32 and separates the audio data, the NW control data and the control data.",
"The audio data, the W control data and the control data are supplied to a circuit not shown in the drawings and are also supplied to the control section 38.The control section 38 detects information on the modulation method and the code rate which are placed in the control data supplied from the associated channel demodulation decoding section 37 and applied to the packet channel, namely, the transmission mode, and carries out mode setting (control) of the user data demodulation decoding section 39.In other words, if the transmission mode is #0, the control section 38 controls the user data demodulation decoding section 39 so as to QPSK demodulate the packet channel signal and further decode it at a code rate of R=½.",
"Also, if the transmission mode is #1, the control section 38 controls the user data demodulation decoding section 39 so as to 16 QAM demodulate the packet channel signal and further decode it at a code rate of R=½.",
"Alternatively, if the transmission mode is #2, the control section 38 controls the user data demodulation decoding section 39 so as to 16 QAM demodulate the packet channel signal and further decode it at a code rate of R=¾.",
"On the other hand, the packet channel reception quality estimating section 35 estimates the SNR of the packet channel signal supplied from the inverse spreading section 32.A pilot symbol that is time-multiplexed on the packet channel or a pilot channel symbol transmitted in parallel with the packet channel is used in this SNR estimation.",
"In other words, although not explained in FIG.",
"2, the spreading section 11 is such that it time-multiplexes the predetermined pilot signal on the modulation signal supplied from the adaptive encoding modulation section 13 and then carries out a spread spectrum.",
"Thus, the packet channel signal includes the pilot signal.",
"Also, the spreading section 11 is such that it performs a spread spectrum on a different pilot signal with a spreading code different from the spreading code used in the spread spectrum of the modulation signal supplied from the power adjusting section 10 or the adaptive encoding modulation section 13, and transmits it via the transmission/reception compatible device 1 and the antenna 14 in parallel with the packet channel or the associated channel.",
"The packet channel reception quality estimating section 35 estimates the SNR of the packet channel signal supplied from the inverse spreading section 32 using the pilot signal included in that signal or the pilot signal that is transmitted in parallel with the packet channel signal, and supplies to the reception quality message generating section 36 the estimated SNR as the reception quality of the packet channel.",
"The reception quality message generating section 36 generates a reception quality message of a predetermined message format representing the estimated SNR of the packet channel (the reception quality of the packet channel) supplied from the packet channel reception quality estimating section 35, and supplies it to the reception quality message inserting section 43.Here, the estimation of the SNR of the packet channel by the packet channel reception quality estimating section 35 and the generation of the reception quality message by the reception quality message generating section 36 are executed for each frame.",
"On the other hand, the user data demodulation decoding section 39 decodes and demodulates the packet channel signal supplied from the inverse spreading section 32 in accordance with the control of the control section 38, and supplies the packet data obtained as a result thereof to the error detecting section 40.In addition, the user data demodulation decoding section 39, upon decoding the packet channel signal, carries out error correction of the packet data using the error correcting code included in that signal as a redundant bit.",
"The error detecting section 40 carries out, for example, parity detection using cyclic redundancy check (CRC) and judges whether or not there is an error in the packet data decoded by the user data demodulation decoding section 39.Then, the error detecting section 40 outputs ACK (ACKnowledge), which is a message indicating that the packet data was received properly, if there is no error in the packet data, and outputs NACK, which is a message indicating that the packet data could not be received properly, if there is an error in the packet data.",
"In addition, although not shown in FIG.",
"7 (which similarly applies to FIG.",
"20 described later), the ACK/NACK outputted by the error detecting section 40 is supplied to the spreading section 45 and is transmitted to the base station.",
"The reception quality message inserting section 43 frames the reception quality message, which is supplied from the reception quality message generating section 36, in the uplink control channel signal explained in FIG.",
"1, and supplies it to the power control bit inserting section 44.The power control bit inserting section 44 frames the power control bit, which is supplied from the power control bit generating section 34, in the uplink control channel signal supplied from the reception quality message inserting section 43, and supplies it to the spreading section 45.The spreading section 45 performs a spread spectrum on the uplink control channel signal from the power control bit inserting section 44 and supplies the spread signal obtained as a result thereof to the transmission/reception compatible device 31.The transmission/reception compatible device 31 performs necessary processing on the spread signal from the spreading section 45 and transmits it from the antenna 47 as a radio wave.",
"In addition, at the terminal, the reception quality message is transmitted per frame, and the power control bit is transmitted per slot.",
"According to the adaptive encoding modulation method, the data transmission speed can be changed in accordance with the reception condition (the reception quality) of the terminal, and data can be transmitted to the terminal side more efficiently.",
"By the way, in the adaptive encoding modulation method, for example, the terminal, which is the mobile station, reports the estimated result of the reception quality of the packet channel to the base station, and the base station selects the optimal combination of the modulation method and encoding method based on the reported value (the reception quality indicated by the reception quality message).",
"For this reason, the reception quality accuracy reported to the base station becomes important.",
"However, because there arises a delay in the estimation and reporting of the reception quality of the packet channel and the message reception at the base station, there are instances where there is some difference between the actual reception quality of the packet channel at the terminal at the time when the base station has demodulated the reception quality message and the reception quality represented by the reception quality message.",
"In other words, at the terminal, as mentioned above, the reception quality of the packet channel is estimated in periods of frames and is transmitted to the base station.",
"For this reason, from when the reception quality at the terminal is estimated till when that reception quality is recognized at the base station, there is a time lag corresponding to several frames.",
"The reception quality recognized by the base station is a reception quality that is behind by a period of time represented by the time lag.",
"Therefore, there are cases where the reception quality recognized by the base station differs from the current reception quality at the terminal.",
"In such cases, the base station cannot select the optimal combination of the modulation and the encoding methods, which may lower system efficiency.",
"This phenomenon presents itself most notably when reception propagation path characteristics change rapidly, such as when, in particular, the terminal, which is the mobile station, is moving at high speed.",
"As such, a method may be considered in which the reception quality message representing the reception quality of the packet channel is transmitted more frequently from the terminal to the base station.",
"However, if the transmission frequency of the reception quality message is made higher, the usage of radio resources increases, and further, power consumption at the terminal also increases.",
"Therefore, in order to save radio resources and carry out a more effective system operation, it is effective to slow (extend) the reception quality report period.",
"However, by slowing the period, the delay from the time of estimation of the reception quality to when the reported value (the reception quality message) reaches the base station further increases, which results in a larger difference between the reported value and the actual reception quality.",
"DISCLOSURE OF THE INVENTION The present invention is proposed in view of such circumstances, and is one which makes it possible to achieve an improvement in system efficiency, in other words a reduction in power consumption by the terminal, conservation of radio resources and the like, by accurately predicting the reception quality of a packet channel for data communications from, for example, the transmission power of an associated channel for audio and the like.",
"A transmission apparatus of the present invention is characterized in that it comprises training means for carrying out training to obtain correlation information that correlates the reception quality of a first channel transmitted from a reception apparatus for receiving data and a transmission power of a second channel, predicting means for obtaining a predicted value of the reception quality of the first channel based on the correlation information and using the transmission power of the second channel, and transmission control means for controlling the transmission of the data through the first channel based on the predicted value of the reception quality of the first channel.",
"A transmission control method of the present invention is characterized in that it comprises a training step for carrying out training to obtain correlation information that correlates a reception quality of a first channel transmitted from a reception apparatus for receiving data and a transmission power of a second channel, a predicting step for calculating a predicted value of the reception quality of the first channel based on the correlation information and using the transmission power of the second channel, and a transmission controlling step for controlling the transmission of data through the first channel based on the predicted value of the reception quality of the first channel.",
"A first program of the present invention is characterized in that it comprises a training step for carrying out training to obtain correlation information that correlates a reception quality of a first channel transmitted from a reception apparatus for receiving data and a transmission power of a second channel, a predicting step for obtaining a predicted value of the reception quality of the first channel based on the correlation information and using the transmission power of the second channel, and a transmission controlling step for controlling the transmission of data through the first channel based on the predicted value of the reception quality of the first channel.",
"A reception apparatus of the present invention is characterized in that it comprises extracting means for extracting transmission control information for controlling the transmission of the reception quality of a first channel from data of a second channel transmitted from a transmission apparatus, and transmission controlling means for controlling the transmission of the reception quality of the first channel based on the transmission control information.",
"A reception control method of the present invention is characterized in that it comprises an extracting step for extracting transmission control information for controlling the transmission of the reception quality of a first channel from data of a second channel transmitted from a transmission apparatus, and a transmission controlling step for controlling the transmission of the reception quality of the first channel based on the transmission control information.",
"A second program of the present invention is characterized in that it comprises an extracting step for extracting transmission control information for controlling the transmission of the reception quality of a first channel from data of a second channel transmitted from a transmission apparatus, and a transmission controlling step for controlling the transmission of the reception quality of the first channel based on the transmission control information.",
"A communications system of the present invention is characterized in that a transmission apparatus has training means for carrying out training to obtain correlation information that correlates the reception quality of a first channel transmitted from a reception apparatus and the transmission power of a second channel, prediction means for obtaining a predicted value of the reception quality of the first channel based on the correlation information and using the transmission power of the second channel, data transmission controlling means for controlling the transmission of data through the first channel based on the predicted value of the reception quality of the first channel, and transmission control information transmitting means for transmitting to the reception apparatus transmission control information for controlling the transmission of the reception quality of the first channel by the reception apparatus through the second channel, and in that the reception apparatus has extracting means for extracting the transmission control information from the data of the second channel transmitted from the transmission apparatus and reception quality transmission controlling means for controlling the transmission of the reception quality of the first channel based on the transmission control information.",
"In the transmission apparatus, the transmission control method and the first program of the present invention, the training for obtaining the correlation information that correlates the reception quality of the first channel transmitted from the reception apparatus for receiving data and the transmission power of the second channel is performed.",
"Based on the correlation information, the transmission power of the second channel is used to thereby calculate the predicted value of the reception quality of the first channel.",
"Then, the transmission of data through the first channel is controlled based on the predicted value of the reception quality of that first channel.",
"In the reception apparatus, the reception control method and the second program of the present invention, the transmission control information that controls the transmission of the reception quality of the first channel is extracted from the data of the second channel transmitted from the transmission apparatus.",
"The transmission of the reception quality of the first channel is controlled based on the transmission control information.",
"In the communications system of the present invention, the training for obtaining the correlation information that correlates the reception quality of the first channel transmitted from the reception apparatus and the transmission power of the second channel is performed, and based on the correlation information, the predicted value of the reception quality of the first channel is calculated at the transmission apparatus using the transmission power of the second channel.",
"Then, the transmission of data through the first channel is controlled based on the predicted value of the reception quality of the first channel, while on the other hand, the transmission control information that controls the transmission of the reception quality of the first channel by the reception apparatus is transmitted through the second channel to the reception apparatus.",
"In the reception apparatus, the transmission control information is extracted from the data of the second channel transmitted from the transmission apparatus.",
"The transmission of the reception quality of the first channel is controlled based on the transmission control information.",
"BRIEF DESCRIPTION OF DRAWINGS FIG.",
"1 is a view explaining conventional data transmission; FIG.",
"2 is a view showing a configuration of an example of a conventional base station; FIG.",
"3 is a view explaining transmission modes; FIG.",
"4A is a view showing the mapping of data by a QPSK modulation method; FIG.",
"4B is a view showing the mapping of data by a 16 QAM modulation method; FIG.",
"5 is a view showing the relationship between reception quality and error rate; FIG.",
"6 is a block diagram showing a configuration example of an adaptive encoding modulation section 13; FIG.",
"7 is a view showing a configuration of an example of a conventional terminal; FIG.",
"8 is a block diagram showing a configuration example of an embodiment of a communications system to which the present invention is applied; FIG.",
"9 is a block diagram showing a configuration example of a base station 101; FIG.",
"10 is a flowchart explaining a process at the base station 101; FIG.",
"11 is a flowchart explaining a process of a training mode; FIG.",
"12 is a view explaining power control of an associated channel; FIG.",
"13A is a view showing propagation characteristics; FIG.",
"13B is a view showing the transmission power of the associated channel; FIG.",
"13C is a view showing the reception quality of the associated channel; FIG.",
"14A is a view showing the transmission power of a packet channel; FIG.",
"14B is a view showing propagation characteristics; FIG.",
"14C is a view showing the reception quality of the packet channel; FIG.",
"15 is a view showing the transmission power of the associated channel and the reception quality of the packet channel; FIG.",
"16 is a flowchart explaining a process of a normal mode; FIG.",
"17 is a flowchart explaining another embodiment of a process at the base station 101; FIG.",
"18 is a flowchart explaining a process for setting a transmission period of a reception quality message; FIG.",
"19 is a view showing the transmission period of the reception quality message becoming longer; FIG.",
"20 is a block diagram showing a configuration example of a terminal 102; FIG.",
"21 is a flowchart explaining a process at the terminal 102; FIG.",
"22 is a flowchart explaining a process at the terminal 102; FIG.",
"23 is a flowchart explaining a process at the terminal 102; and FIG.",
"24 is a block diagram showing a configuration example of an embodiment of a computer to which the present invention is applied.",
"BEST MODE FOR CARRYING OUT THE INVENTION FIG.",
"8 shows a configuration example of an embodiment of a communications system to which the present invention is applied (a system refers to something in which a plurality of apparatuses are logically aggregated, regardless of whether or not each constituent apparatus exists in a single body).",
"The communications system in FIG.",
"8 comprises a base station 101 and a terminal 102, for example, such as a portable telephone or the like.",
"The communication system is such that data is exchanged between the base station 101 and the terminal 102 through radio communications.",
"In addition, in the embodiment of FIG.",
"8, one each of the base station 101 and the terminal 102 is illustrated.",
"However, the communications system may comprise a plurality of base stations and terminals.",
"FIG.",
"9 shows a configuration example of the base station 101 of FIG.",
"8.In addition, the same symbols are given to the portions corresponding to the case in FIG.",
"2, and hereinafter explanations thereof are omitted as deemed appropriate.",
"In FIG.",
"9, the base station 101 comprises a transmission/reception compatible device 1, an inverse spreading section 2, a power control bit extracting section 3, a reception quality message extracting section 5, a mode judging section 6, a control section 7, an encoding modulation section 9, a power adjusting section 10, a spreading section 11, an adaptive encoding modulation section 13, a transmission power converting section 111, a multiplexer 112, a control data generating section 113 and control data inserting sections 114 and 115.Thus, the base station 101 of FIG.",
"9 is basically designed similar to the base station of FIG.",
"2 except in that the transmission power converting section 111, the multiplexer 112 and the control data generating section 113 are newly provided and in that the control data inserting sections 114 and 115 are provided in place of the control data inserting section 4.To the transmission power converting section 111 are supplied a reception quality message from the reception quality message extracting section as well as transmission power (absolute transmission power) of an associated channel from the power adjusting section 10.The transmission power converting section 111 carries out training to obtain a parameter as correlation information that correlates reception quality y[n] of a packet channel represented by the reception quality message supplied from the reception quality message extracting section 5 and transmission power x[n] of the associated channel supplied from the power adjusting section 10.Here, with the reception quality represented by a reception quality message extracted by the reception quality message extracting section 5 as a reference, the reception quality y[n] of the packet channel signifies the reception quality represented by the n+1h reception quality message extracted from the reception quality message that serves as the reference.",
"Also, the transmission power x[n] of the associated channel signifies the transmission power of the associated channel when the reception quality y[n] of the packet channel is obtained.",
"Once the parameter that correlates the reception quality y[n] and the transmission power x[n] is obtained through the training, based on that parameter, the transmission power converter 111 obtains a predicted value y′[n] of the reception quality of the packet channel using the transmission power of the associated channel supplied from the power adjusting section 10, and supplies it to the multiplexer 112.Moreover, the transmission power converting section 111 controls the multiplexer 112 and the control data generating section 113.In addition to having the predicted value y′[n] of the reception quality supplied from the transmission power converting section 111, the reception quality message is supplied from the reception quality message extracting section 5 to the multiplexer 112.Then, in accordance with the control of the transmission power converting section 111, the multiplexer 112 selects either one of the predicted value y′[n] of the reception quality supplied from the transmission power converting section 111 and the reception quality message supplied from the reception quality message extracting section 5, and supplies it to the mode judging section 6.Thus, in the base station 101 of FIG.",
"9, the mode judging section 6 determines the transmission mode based on either the predicted value y′[n] of the reception quality or the reception quality represented by the reception quality message.",
"The control section 7 controls the code rate and the modulation method of the packet data in the adaptive encoding modulation section 13 based on that transmission mode.",
"In other words, in the embodiment of FIG.",
"9, the code rate and the modulation method of the packet data are controlled on the basis of the reception quality represented by the reception quality message transmitted from the terminal 102 and the predicted value of the reception quality obtained by the transmission power converting section 111.The control data generating section 113, in accordance with the control of the transmission power converting section 111, generates control data as transmission control information for controlling the transmission of the reception quality message by the terminal 102, and supplies it to the control data inserting section 114.Here, the transmission control information is start/stop information that instructs the starting or stopping of transmission of the reception quality message by the terminal 102, or is transmission frequency information which instructs the transmission frequency of the reception quality message by the terminal 102.However, for example, now, if it is assumed that the transmission frequency information indicates a transmission period for the reception quality message, when the transmission period is a finite value, the reception quality message is transmitted, but if the transmission period is infinite, the reception quality message is not transmitted.",
"Thus, it can be said that the transmission frequency information is information which encompasses the start/stop information.",
"Other than the transmission control information as the control data supplied from the control data generating section 113, to the control data inserting section 114 are supplied audio data transferred from other base stations, NW control data used to judge and control a hand-off for transferring the control of the terminal 102 from the base station 101 to another base station or from another base station to the base station 101, and the like.",
"The control data inserting section 114 inserts into the audio data and the NW control data sent thereto the control data supplied from the control data generating section 113, and supplies it to the control data inserting section 115.In addition to output data from the control data inserting section 114, the transmission mode as control data outputted by the control data generating section 8 is also supplied to the control data inserting section 115.Then, the control data inserting section 115 inserts the control data supplied from the control data generating section 8 into the data from the control data inserting section 114, and outputs it.",
"The data that the control data inserting section 115 outputs is supplied to the encoding modulation section 9, and thereafter is transmitted as an associated channel signal as in the case of FIG.",
"2 mentioned above.",
"Thus, the transmission control information as the control data generated by the control data generating section 113 is transmitted to the terminal 102 as the associated channel signal.",
"In the base station 101 thus configured, for example, the following process is carried out.",
"1.Transmission and reception of data at the base station 101 are executed in accordance with a training mode and a normal mode.",
"Here, the training mode is an operation mode for obtaining (learning) a parameter to reduce prediction errors in the predicted value of the reception quality of the packet channel obtained at the base station 101.In the present embodiment, although it is supposed to be such that, for example, the training mode is entered each time it enters a call, it is not limited as such, and the process of the training mode may be performed periodically to obtain the parameter for reducing prediction errors.",
"Also, the process of the training mode may be performed only once if the possibility of entering a situation in which prediction errors increase is slim.",
"Also, the normal mode refers to a normal communication state after the training is finished (the state in which the predicted value of the reception quality of the packet channel is obtained on the basis of the parameter obtained in the training mode, and the transmission mode is determined on the basis of the predicted value).",
"2.The multiplexer 112 is controlled so as to select the output from the reception quality message extracting section 5 during training mode or the output from the transmission power converting section 111 during normal mode.",
"This selection control is performed based on a flag (information) indicating whether the current operation mode inputted to the multiplexer 112 from the transmission power converting section 111 is the training mode or the normal mode.",
"This flag is also inputted to the control data generating section 113 from the transmission power converting section 111.Based on the flag from the transmission power converting section 111, the control data generating section 113 generates the transmission control information as the control data, and transmits it to the terminal 102 via the control data inserting sections 114, 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11, the transmission/reception compatible device 1 and the antenna 14.Thus, the transmission control information together with the audio data and the NW control data which are supplied to the control data inserting section 114, as well as the transmission mode as the control data supplied to the control data inserting section 115 from the control data generating section 8 are transmitted to the terminal 102.3.During training mode, the transmission power converting section 111 receives the absolute transmission power value x[n] of the associated channel from the power adjusting section 10, and the packet channel reception quality value y[n] from the reception quality message extracting section 5.Here, in FIG.",
"15 which will be described later, there are shown examples of the absolute transmission power value x[n] of the associated channel and the packet channel reception quality value y[n].",
"The absolute transmission power x[n] of the associated channel is dependent upon an associated channel reference quality (a value requested by the terminal 102 as the reception quality of the associated channel) set on the terminal 102 side.",
"When the propagation characteristics of a radio propagation path are represented by H[t], x[n] assumes inverse characteristics to H[t], as explained in FIG.",
"13A, FIG.",
"13B and FIG.",
"13C as well as FIG.",
"14A, FIG.",
"14B and FIG.",
"14C which will be described later.",
"In addition, the associated channel reference quality on the terminal 102 side is different for each call.",
"If the associated channel reference quality increases by an amount corresponding to k [dB], the absolute transmission power x[n] of the associated channel in the base station 101 also increases relatively by an amount corresponding to k [dB].",
"4.The transmission power converting section 111 carries out the training to generate, for example, a conversion equation of equation (1) which will be described later that correlates the transmission power of the associated channel and the reception quality of the packet channel from sample values ((x[0J, y[0]), (x[1], y[1]), .",
".",
".",
", (x[N−1], y[N−1])) of a set of the received transmission power x[n] of the associated channel and the reception quality y[n] of the packet channel.",
"These sample values comprise the transmission power value of the associated channel determined by the base station 101 during training mode and the reception quality value of the packet channel reported (fed back) from the terminal 102.The sample value y[n] is given as an impulse response with respect to the wireless propagation path characteristics of the transmission power of the (high speed) packet channel, the appearance of which is as illustrated in FIG.",
"14A, FIG.",
"14B and FIG.",
"14C which will be described later.",
"Here, samples of the transmission power of the associated channel and the reception quality of the packet channel, which are used in the training, are referred to hereafter as training samples where appropriate.",
"5.The transmission power converting section 111 ends the training, for example, after a predetermined N samples of the training samples are collected, or at a point when a square error, which will be described later, becomes lower than a predetermined threshold.",
"Then, it shifts the operation mode from the training mode to the normal mode.",
"6.After the training is finished, the transmission power converting section 111 obtains a predicted value (y′) of the packet channel reception quality from the output of the power adjusting section 110 in accordance with equation (1) (or, as described later, a table that corresponds to equation (1)), and outputs it to the multiplexer 112.At the same time, the multiplexer 112 is controlled so that the output from the transmission power converting section 111 is selected.",
"7.Once the training is finished, the transmission power converting section 111 outputs that fact as a flag to the control data generating section 113.This flag is a flag that indicates the necessity or otherwise of transmission of a reception quality message y or the period of transmission, that is, the length of the transmission period which is considered based on the square error and the like which will be described later.",
"8.Once it receives the fact that the training has finished, the control data generating section 113 generates the transmission control information that is the message for stopping the reception quality message transmission by the terminal 102, and supplies it to the control data inserting section 114.The control data inserting section 114 inserts the transmission control information into the associated channel.",
"Then transmission control information is thereafter transmitted to the terminal 102 as mentioned above.",
"9.If the training is again needed, for example, in a case where, judging from the reception quality message transmitted in predetermined periods, the prediction error in the predicted value of the reception quality of the packet channel is judged to be higher than the predetermined threshold, or in a case where NACK is frequently transmitted from the terminal 102, the transmission control information that is a message to start the reception quality message transmission by the terminal 102 is generated at the control data generating section 113 is inserted into the associated channel by the control data inserting section 114 and transmitted.",
"10.In addition, although only the ON/OFF states of the reception quality message transmission is defined in the process above, it is also possible to change the reception quality message transmission frequency of the terminal 102 during the training mode or the normal mode.",
"The transmission power converting section 111 derives the predicted value of the reception quality of the packet channel from equation (1) (or the table), and it enters the training mode and updates equation (1) (or the table) in accordance with the reception quality value of the packet channel represented by the reception quality message that is occasionally transmitted.",
"In addition, in this case, the control data generating section 113 generates the transmission control information that is a message which specifies the transmission period of the reception quality message (FIG.",
"19).",
"In FIG.",
"19, which will be described later, there is shown how the periods in which the reception quality message is transmitted from the terminal 102 to the base station 101 are controlled so as to be lengthened gradually as the square error becomes stable (approaches 0).",
"After the end of the training period, it becomes the normal mode, and basically, the reception quality value of the packet channel is predicted based on the transmission power of the associated channel.",
"Next, the process at the base station 101 in FIG.",
"9 will be further described with reference to the flowchart of FIG.",
"10.In the base station 101, when a call with the terminal 102 is established, at first, in step Si, the transmission power converting section 111 sets the operation mode to the training mode, and supplies a flag representing that fact to the multiplexer 112, and proceeds to step S2.Here, immediately after the call with the base station 101 is established, the terminal 102, for example, transmits the reception quality message, in frame periods as in the conventional terminal shown in FIG.",
"7.Thus, at the base station 102, which has a call with the terminal 102, established and is in the training mode, the reception quality message transmitted from the terminal 102 at frame periods can be received.",
"In step S2, transmission of the uplink control channel signal from the terminal is awaited, and that uplink control channel signal is received.",
"In other words, the uplink control channel signal transmitted from the terminal is received by the antenna 14, and is supplied to the power control bit extracting section 3 via the transmission/reception compatible device 1 and the inverse spreading section 2.Then, it proceeds to step S3, and the power control bit extracting section 3 judges if the current timing is the timing of the slot period.",
"In step S3, if the current timing is judged to be the timing of the slot period, it proceeds to step S4.The power control bit extracting section 3 extracts a power control bit transmitted at the timing of the slot period from the uplink control channel signal supplied from the spreading section 2, and supplies it to the power adjusting section 10, and also supplies the uplink control channel signal to the reception quality message extracting section 5.Then, it proceeds to step S5.In step S5, the power adjusting section 10 changes the transmission power of the associated channel so as to become higher or lower by 1 dB than the current transmission power in accordance with the power control bit supplied from the power control bit extracting section 3, and supplies the altered transmission power x[n] to the transmission power converting section 111.Then, it proceeds to step S6.On the other hand, in step S3, if the current timing is judged not to be the timing of the slot period, steps S4 and S5 are skipped to proceed to step S6, and the reception quality message extracting section 5 judges if the current timing is the timing of the frame period.",
"In step S6, if the current timing is judged not to be the timing of the frame period, it returns to step S2, and thereafter, similar processes are repeated.",
"In addition, in step S6, if the current timing is judged to be the timing of the frame period, that is, if the current timing is the timing of the frame period at which the reception quality message is supposed to be transmitted, it proceeds to step S7.The transmission power converting section 111 judges which of the training mode or the normal mode the current operation mode is.",
"In step S7, if the current operation mode is judged to be the training mode, it proceeds to step S8.A training mode process which will be described later is carried out.",
"Then, it proceeds to step S9.In step S9, the transmission power converting section 111 judges whether or not a training mode terminating event, which is an event that is to end the training mode and make the transition to the normal mode, has occurred.",
"Here, for example, it is possible to adopt as the training mode terminating event the fact that the predetermined N samples of the training samples are gathered as mentioned above, or the fact that the square error, which will be described later, becomes lower than the predetermined threshold.",
"In step S9, if it is judged that the training terminating event has not occurred, it returns to step S2 with the operation mode remaining in the training mode.",
"Thereafter, similar processes are repeated.",
"In addition, in step S9, if it is judged that the training terminating event has occurred, it proceeds to step S10.By controlling the control data generating section 113, the transmission power converting section 111 generates transmission control information that instructs the transmission of the reception quality message from the terminal 102 to stop (for example, transmission control information instructing to make the transmission period infinite).",
"Then, it proceeds to step S11.In step S11, the transmission control information generated in step S10 is transmitted by the associated channel from the control data generating section 113 to the terminal 102 via the control data inserting sections 114 and 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11, the transmission/reception compatible device 1 and the antenna 14.Then, it proceeds to step S12.In step S12, the transmission power converting section 111 sets (changes) the operation mode from the training mode to the normal mode.",
"It then returns to step S2, and thereafter, similar processes are repeated.",
"As mentioned above, in step S11, because the transmission control information that instructs the transmission of the reception quality message to stop is transmitted, thereafter, until the base station 101 transmits, in step S16, which will be described later, to the terminal 102 the transmission control information that instructs the transmission of the reception quality message to start, the terminal 102 stops the transmission of the reception quality message.",
"Thus, it is possible to reduce power consumption at the terminal 102 and also to save radio resources for transmitting the reception quality message.",
"On the other hand, in step S7, if the current operation mode is judged to be the normal mode, it proceeds to step S13.A normal mode process which will be described later is carried out.",
"Then, it proceeds to step S14.In step S14, the transmission power converting section 111 judges if a training mode transition event, which is an event that is to end the normal mode and make the transition to the training mode, has occurred.",
"Here, it is possible do adopt as the training mode transition event, for example, as mentioned above, the fact that the square error of the predicted value of the reception quality is judged to be higher than the predetermined threshold, or the fact that NACK is frequently sent from the terminal 102.In step S14, if it is judged that the training transition event has not occurred, it returns back to step S2 with the operation mode remaining in the normal mode, and thereafter, similar processes are repeated.",
"Also, in step S14, if it is judged that the training transition event has occurred, it proceeds to step S15.By controlling the control data generating section 113, the transmission power converting section 111 generates the transmission control information that instructs the transmission of the reception quality message from the terminal 102 to start (for example, transmission control information specifying that the transmission period is to be made per frame).",
"Then, it proceeds to step S16.In step S16, the transmission control information generated in step S15 is transmitted by the associated channel from the control data generating section 113 to the terminal 102 via the control data inserting sections 114 and 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11, the transmission/reception compatible device 1 and the antenna 14.Then, it proceeds to step S17.In other words, if the operation mode is set to the normal mode from the training mode in step S12 mentioned above, in the immediately preceding step S11, the transmission control information that instructs the transmission of the reception quality message to stop is transmitted to the terminal 102.Consequently, during the normal mode, the terminal 102 stops the transmission of the reception quality message.",
"On the other hand, in step S17 to follow, if the operation mode is set to the training mode from the normal mode, because the reception quality message becomes necessary in the training mode as will be described later, in step S16, the transmission control information that instructs the transmission of the reception quality message to start is transmitted to the terminal 102.Consequently, during the training mode, the terminal 102, for example, carries out transmission of the reception quality message in frame periods as in the conventional case.",
"In step S17, the transmission power converting section 111 sets (changes) the operation mode from the normal mode to training.",
"It returns to step S2.Then, similar processes are repeated.",
"In addition, the process of the flowchart shown in FIG.",
"10 (as well as FIG.",
"17 which will be described later) is ended, for example, when the call between the base station 101 and the terminal 102 is disconnected.",
"Next, the process of the training mode in step S8 of FIG.",
"10 will be described with reference to the flowchart of FIG.",
"11.In the training mode process, at first, in step S21, the reception quality message extracting section 5 extracts the reception quality message representing the reception quality of the packet channel from the uplink control channel signal supplied from the power control bit extracting section 3, and supplies it to the transmission power converting section 111 and the multiplexer 112.In other words, as explained in steps S14 to S17 of FIG.",
"10, when the operation mode is set to the training mode, the transmission control information that instructs the transmission of the reception quality message to start is transmitted.",
"Therefore, during the training mode, the reception quality message is transmitted from the terminal 102 to the base station 102, but the reception quality message extracting section 5 thus extracts the reception quality message transmitted from the terminal 102, and supplies it to the transmission power converting section 111 and the multiplexer 112.When the transmission power converting section 111 receives the reception quality message from the reception quality message extracting section 5, it proceeds to step S22.The transmission power converting section 111 uses the reception quality y[n] of the packet channel represented by the reception quality message and the transmission power x[n] of the associated channel outputted by the power adjusting section 10 to carry out the training (learning) of the predictive equation for calculating the predicted value y′[n] of the reception quality of the packet channel.",
"Here, from the base station 101 to the terminal 102, as shown in FIG.",
"12, at least signals of the packet channel (HS-DSCH) and the associated channel (DPCH) are transmitted.",
"However, as for the associated channel of these two channels, the terminal 102 generates the power control bit which requests the increase or decrease in the transmission power thereof, and transmits it to the base station 101.Then, the base station 101 adjusts the transmission power of the associated channel in accordance with this power control bit so that the reception quality of the associated channel in the terminal 102 becomes constant as a result.",
"In other words, if it is assumed that t represents time and the propagation path characteristics H[t] of the radio waves are, for example, as shown in FIG.",
"13A, the base station 101 controls (adjusts) the transmission power of the associated channel to have inverse characteristics to the propagation path characteristics H[t].",
"Consequently, the reception quality of the associated channel at the terminal 102 is maintained constant, as shown in FIG.",
"13C.",
"On the other hand, with respect to the packet channel, the base station 101 does not perform transmission power control like that for the associated channel.",
"It carries out transmission at a constant transmission power, for example, as shown in FIG.",
"14A.",
"Thus, if the propagation characteristics H[t] of the radio waves are assumed to be something like those shown FIG.",
"14B which is identical to FIG.",
"13A, the reception quality of the packet channel at the terminal 102, as shown in FIG.",
"14C, becomes one that is directly influenced by the propagation characteristics H[t].",
"Thus, because the transmission power of the associated channel becomes the inverse characteristics to the propagation characteristics H[t] as shown in FIG.",
"13B, and the reception quality of the packet channel at the terminal 102 is one that is directly influenced by the propagation characteristics H[t] as shown in FIG.",
"14C, the transmission power of the associated channel and the reception quality of the packet channel are correlated such that they have what could be described as a complementary relation.",
"Thus, the transmission power of the associated channel can be mapped to the reception quality of the packet channel correlated with that transmission power.",
"Through this mapping, it is possible to predict the reception quality of the packet channel from the transmission power of the associated channel.",
"As such, now, the predicted value y′[n] of the reception quality of the packet channel is calculated using the transmission power x[n] of the associated channel with, for example, a linear expression defined by parameters a and b below as a predictive equation.",
"y′[n]=ax[n]+b (1) If the prediction error of the predicted value y′[n] of the reception quality calculated with the predictive equation of equation (1) is represented as e[n], this prediction error e[n] can be expressed by equation (2).",
"e[n]=y[n]−y′[n] (2) Since it is difficult to determine the parameters a and b that make the prediction error e[n] of equation (2) to always be 0, here, parameters a and b that minimize, for example, the square error as statistical errors (error metrics) calculated from the prediction error e[n] of equation (2) are determined.",
"Now, if the square error of the prediction error e [n] in equation (2) is represented by E, the square error E can be expressed through equation (3).",
"E = ∑ n = 0 N - 1 { e [ n ] } 2 ( 3 ) In addition, in equation (3), sets of N samples of the transmission power x[n] of the associated channel and the reception quality y[n] of the packet channel ((x[0], y[0]), (x[1], y[1]), .",
".",
".",
", (x[N−1], y[N−1])) are used as training samples.",
"When equation (2) is substituted into equation (3), equation (4) can be obtained.",
"E = ∑ n = 0 N - 1 ( y [ n ] - y ′ [ n ] ) 2 ( 4 ) Moreover, by substituting equation (1) into equation (4), equation (5) can be obtained.",
"E = ∑ n = 0 N - 1 ( y [ n ] - ( ax [ n ] + b ) ) 2 ( 5 ) When equation (5) is partially differentiated with respect to each of the parameters a and b, equations (6) and (7) can be obtained.",
"∂ E ∂ a = - 2 ∑ n = 0 N - 1 x [ n ] ( y [ n ] - ( ax [ n ] + b ) ) ( 6 ) ∂ E ∂ b = - 2 ∑ n = 0 N - 1 ( y [ n ] - ( ax [ n ] + b ) ) ( 7 ) The minimum value (the smallest value) of the square error E of equation (3) are given by the parameters a and b which make the right sides of equations (6) and (7) 0.As such, if the right sides of the equations (6) and (7) are set to 0, equation (8) and equation (9) can be obtained from equation (6) and equation (7), respectively.",
"N ∑ n = 0 N - 1 x [ n ] y [ n ] = Nb ∑ n = 0 N - 1 x [ n ] + Na ∑ n = 0 N - 1 { x [ n ] } 2 ( 8 ) Nb = ∑ n = 0 N - 1 y [ n ] - a ∑ n = 0 N - 1 x [ n ] ( 9 ) By substituting equation (9) into equation (8), the parameter a, which minimizes the square error E, can be determined by equation (10).",
"a = N ∑ n = 0 N - 1 x [ n ] y [ n ] - ∑ n = 0 N - 1 x [ n ] ∑ n = 0 N - 1 y [ n ] N ∑ n = 0 N - 1 { x [ n ] } 2 - { ∑ n = 0 N - 1 x [ n ] } 2 ( 10 ) In addition, from equations (9) and (10), the parameter b that minimizes the square error E can be calculated with equation (11).",
"b = ∑ n = 0 N - 1 y [ n ] - a ∑ n = 0 N - 1 x [ n ] N ( 11 ) In step S22 of FIG.",
"11, by computing equations (10) and (11) using the reception quality y[n] of the packet channel and the transmission power x[n] of the associated channel, the transmission power converting section 111 carries out training to determine the parameters a and b that define the predictive equation of equation (1).",
"Then, it proceeds to step S23.Here, x[n] of equation (1) is the absolute transmission power of the associated channel, and is dependent upon the data transmitted through the associated channel, namely, the QoS (Quality of Service) of a provided service, the process gain of a physical channel and the characteristics of a receiver (the terminal 102).",
"However, according to equation (1), it is possible to, taking those above into consideration, correlate (map) the absolute transmission power of the associated channel with the reception quality of the packet channel.",
"In addition, here, although the predicted value y′[n] of the reception quality of the packet channel is calculated with the linear expression of equation (1), this predicted value y′[n] can also be calculated with an equation of an order of or above a quadratic expression.",
"Moreover, here, although the absolute transmission power x[n] of the associated channel is used to calculate the predicted value y′[n] of the reception quality of the packet channel, this predicted value y′[n] can also be calculated, in addition, for example, using changes in the transmission power of the associated channel and the like.",
"In other words, if the reception quality represented by the previous reception quality message transmitted from the terminal 102 is represented by SIRprv and the change in the transmission power of the associated channel caused by the power control bits received from when that reception quality message was received up to the present is represented by Δx[n], then the predicted value y′[n] of the reception quality of the packet channel can be predicted, for example, in accordance with the following equation.",
"y′[n]=aΔx[n]+b +SIRprv (12) In step S23, the transmission power converting section 111 controls the multiplexer 112 so that the output of the reception quality message extracting section 5 is selected and supplied to the mode judging section 6.Consequently, the reception quality message outputted by the reception quality message extracting section 5 is supplied to the mode judging section 6 via the multiplexer 112.Then, it proceeds to step S24.The mode judging section 6 determines the code rate and the modulation method (the transmission mode) in the adaptive encoding modulation section 13 based on the reception quality represented by the reception quality message supplied from the multiplexer 112, and supplies it to the control section 7.Then, it proceeds to step S25.In step S25, the control section 7 supplies the transmission mode supplied from the mode judging section 6 to the control data generating section 8, and the control data generating section 8 generates control data including that transmission mode.",
"This control data is supplied from the control data generating section 8 to the antenna 14 via the control data inserting section 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11 and the transmission/reception compatible device 1, and is transmitted as an associated channel signal from the antenna 14.After that, it proceeds to step S26.The packet data in which the user data addressed to the terminal 102 is arranged is transmitted in the transmission mode determined by the mode judging section 6 and it is returned.",
"In other words, in step S26, the user data addressed to the terminal 102 is arranged in a packet and is sent as packet data to the adaptive encoding modulation section 13.Then, the control section 7 controls the adaptive encoding modulation section 13 so as to encode and modulate the packet data addressed to the terminal 102 in the transmission mode determined by the mode judging section 6.The adaptive encoding modulation section 13, in accordance with the control of the control section 7, encodes and modulates the packet data addressed to the terminal 102 that is supplied thereto, and supplies it to the spreading section 11.Thereafter, processes similar to the case explained in FIG.",
"2 are performed in the spreading section 11 and the transmission/reception compatible device 1.Consequently, the packet data is transmitted from the antenna 14 to the terminal 102 as a packet channel signal.",
"In addition, in the training of the parameters a and b in step S22 of FIG.",
"11, by calculating equation (10) and (11) using only the transmission power x[n] of the associated channel and the reception quality y[n] represented by the reception quality message obtained during the most recent training mode period, new parameters a and b are obtained.",
"With the new parameters a and b, the parameters a and b obtained in the training performed previously are updated.",
"However, the updating of the parameters a and b can be performed using the transmission power x[n] of the associated channel and the reception quality y[n] represented by the reception quality message obtained in past training mode periods as well.",
"In other words, in the training of the parameters a and b, by calculating equations (10) and (11) using, for example, all of the transmission powers x[n] of the associated channel and the reception qualities y[n] represented by the reception quality messages obtained in the training periods up to that point, new parameters a and b can be obtained.",
"With the new parameters a and b, the parameters a and b obtained in the previously performed training can be updated.",
"Next, the normal mode process in step S13 of FIG.",
"10 will be described below with reference to the flowchart of FIG.",
"16.In the normal mode process, at first, in step S31, the transmission power converting section 111 uses the transmission power x[n] of the associated channel outputted by the power adjusting section 10 and calculates the predicted value y′[n] of the reception quality of the packet channel in the terminal 102.In other words, during the normal mode, as explained in FIG.",
"10, the terminal 102 stops transmission of the reception quality message.",
"Thus, the base station 101 cannot recognize the reception quality at the terminal 102 from the reception quality message.",
"As such, in step S31, the transmission power converting section 111 calculates the predicted value y′[n] of the reception quality of the packet channel using the transmission power x[n] of the associated channel in accordance with equation (1) defined by the most recent parameters a and b obtained through the training in step S22 of FIG.",
"11 performed most recently.",
"Then, the transmission power converting section 111 supplies the predicted value y′[n] of the reception quality to the multiplexer 112.It proceeds from step S31 to step S32.Here, equation (1) defined by the parameters a and b minimizes the square error E of the prediction error of the predicted value y′[n] of the reception quality of the packet channel obtained through equation (1), as mentioned above.",
"Thus, the reception quality of the packet channel can be predicted accurately.",
"In addition, in step S31, instead of calculating equation (1), it is possible to obtain the predicted value of the reception quality using a table.",
"In other words, a plurality of representative values of the transmission power of the associated channel are substituted into equation (1), and representative values of the predicted values of the reception quality of the packet channel are calculated.",
"Moreover, a table that correlates the representative values of the transmission power of the associated channel with the representative values of the predicted values of the reception quality of the packet channel is prepared.",
"In step S31, the predicted value y′[n] of the reception quality correlated with the transmission power x[n] of the associated channel may be obtained by referring to such a table.",
"In step S32, the transmission power converting section 111 controls the multiplexer 112 so that the output of the transmission power converting section 111 is selected and supplied to the mode judging section 6.Consequently, the predicted value of the reception quality calculated at the transmission power converting section 111 is supplied to the mode judging section 6 via the multiplexer 112.Then, it proceeds to step S33.The mode judging section 6 determines the code rate and the modulation method (the transmission mode) in the adaptive encoding modulation section 13 based on the predicted value of the reception quality supplied from the multiplexer 112, and supplies it to the control section 7.Then, it proceeds to step S34.In step S34, the control section 7 supplies to the control data generating section 8 the transmission mode supplied from the mode judging section 6, and the control data generating section 8 generates the control data including that transmission mode.",
"This control data is supplied from the control data generating section 8 to the antenna 14 via the control data inserting section 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11 and the transmission/reception compatible device 1, and is transmitted from the antenna 14 as an associated channel signal.",
"Thereafter, it proceeds to step S35.The packet data, in which user data addressed to the terminal 102 is arranged, is transmitted in the transmission mode determined by the mode judging section 6 and it returns.",
"In other words, in step S35, the user data addressed to the terminal 102 is arranged in the packet and is supplied to the adaptive encoding modulation section 13 as packet data.",
"Then, the control section 7 controls the adaptive encoding modulation section 13 so as to encode and modulate the packet data addressed to the terminal 102 in the transmission mode determined by the mode judging section 6.In accordance with the control of the control section 7, the adaptive encoding modulation section 13 encodes and modulates the packet data supplied thereto that is addressed to the terminal 102, and supplies it to the spreading section 11.Thereafter, at the spreading section 11 and the transmission/reception compatible device 1, processes similar to the case explained in FIG.",
"2 are performed.",
"Consequently, the packet data is transmitted from the antenna 14 to the terminal 102 as a packet channel signal.",
"Next, in the processes at the base station 101 explained in FIG.",
"10 to FIG.",
"16, the training mode and the normal mode are provided as operation modes and the parameters a and b of equation (1) are updated each time the operation mode becomes the training mode.",
"However, since the training of the parameters a and b can be done if the reception quality message and the transmission power of the associated channel can be recognized, it is possible to update the parameters a and b with each reception of the reception quality message transmitted from the terminal 102 without providing the operation modes such as the training mode and the normal mode.",
"In addition, in the processes at the base station 101 explained in FIG.",
"10 to FIG.",
"16, the transmission control information that instructs the transmission of the reception quality message to start is transmitted to the terminal 102 during the training mode, thereby making the transmission of the reception quality message be performed in frame periods as is done conventionally, and also the transmission control information that instructs the transmission of the reception quality message to stop is transmitted to the terminal 102 during the normal mode, thereby stopping the transmission of the reception quality message.",
"However, as for the transmission of the reception quality message from the terminal 102, it is possible to control the transmission frequency thereof, and not just whether the transmission thereof is performed or not performed.",
"As such, another embodiment of the process at the base station 101 is described with reference to the flowchart of FIG.",
"17.At the base station 101, when a call between the terminal 102 is established, firstly in step S41, transmission of the uplink control channel signal from the terminal is awaited, and the uplink control channel signal is received.",
"In other words, the uplink control channel signal transmitted from the terminal is received by the antenna 14 and is supplied to the power control bit extracting section 3 via the transmission/reception compatible device 1 and the inverse spreading section 2.Then, it proceeds to step S42.The power control bit extracting section 3 judges whether or not the current timing is the timing of the slot period.",
"In step S42, if the current timing is judged to be the timing of the slot period, it proceeds to step S43.The power control bit extracting section 3 extracts from the uplink control channel signal supplied from the spreading section 2 the power control bit transmitted at the timing of the slot period, and supplies it to the power adjusting section 10, and also supplies the uplink control channel signal to the reception quality message extracting section 5.Then, it proceeds to step S44.In step S44, the power adjusting section 10 changes the transmission power of the associated channel so as to be higher or lower than the current transmission power by 1 dB in accordance with the power control bit supplied from the power control bit extracting section 3, and supplies the altered transmission power x[n] to the transmission power converting section 111.Then, it proceeds to step S45.On the other hand, in step S42, if the current timing is judged not to be the timing of the slot period, steps S43 and S44 are skipped and it proceeds to step S45.The reception quality message extracting section 5 judges whether or not the current timing is the timing of the frame period.",
"In step S45, if the current timing is judged not to be the timing of the frame period, it returns to step S41.Thereafter, similar processes are repeated.",
"In addition, in step S45, if the current timing is judged to be the timing of the frame period, that is, if the current timing is the timing of the frame period at which the reception quality message is supposed to be transmitted, it proceeds to step S46.The transmission power converting section 111 judges if the current timing is a message reception timing for receiving the reception quality message transmitted from the terminal 102.Here, in the embodiment of FIG.",
"17, in step S56, which will be described later, transmission control information, for example, which indicates the transmission period as the transmission frequency of the reception quality message, is generated and transmitted to the terminal 102.The terminal 102 transmits the reception quality message at the transmission period indicated by the transmission control information.",
"Thus, at the base station 101, the timing at which the reception quality message is transmitted from the terminal 102 can be recognized through the transmission control information transmitted to the terminal 102.Thus, in step S46, it is judged if the current timing is the message reception timing.",
"In addition, immediately after the process at the base station 101 of FIG.",
"17 is started, the transmission period indicated by the transmission control information is initially set to the frame period that is, for example, the period in which conventional terminals transmit the reception quality message.",
"In step S45, if the current timing is judged to be the message reception timing, it proceeds to step S47.The reception quality message extracting section 5 extracts the reception quality message from the uplink control channel signal supplied from the power control bit extracting section 3, and supplies it to the transmission power converting section 111 and the multiplexer 112.Then, it proceeds to step S48.In step S48, the transmission power converting section 111 uses the reception quality y[n] of the packet channel represented by the reception quality message from the reception quality message extracting section 5 and the transmission power x[n] of the associated channel outputted by the power adjusting section 10 to perform the training (learning) of the parameters a and b that define the predictive equation of equation (1) to calculate the predicted value y′[n] of the reception quality of the packet channel.",
"With the new parameters a and b obtained by the training, the parameters obtained by the training in step S48 of the previous round are updated (overwritten).",
"Thus, in step S48, for example, the reception qualities y[n] indicated by the reception quality messages transmitted from the terminal 102 from when the call is established between the base station 101 and the terminal 102 up to the present, and the transmission powers x[n] of the associated channel outputted by the power adjusting section 10 are used to carry out the training of the parameters a and b.",
"However, in the training of step S48, in addition, it is also possible to obtain the parameters a and b by computing equations (10) and (11) using, for example, the transmission powers x[n] and the reception qualities y[n] obtained within a timing going back by a predetermined time, or the latest predetermined number of the transmission powers x[n] and the reception qualities y[n] among the transmission powers x[n] and the reception qualities y[n] obtained up until the present.",
"Moreover, in step S48, in computing equations (10) and (11) using the reception quality y[n] and the transmission power x[n], it is possible to, for example, applying a larger weighting factor the more recent the reception quality y[n] and the transmission power x[n] are.",
"After the process of step S48, it proceeds to step S49.The transmission power converting section 111 controls the multiplexer 112 so that the output of the reception quality message extracting section 5 is selected and supplied to the mode judging section 6.Consequently, the reception quality message outputted by the reception quality message extracting section 5 is supplied to the mode judging section 6 via the multiplexer 112.Then, it proceeds to step S52.The mode judging section 6 determines the code rate and the modulation method (the transmission mode) in the adaptive encoding modulation section 13 based on the reception quality represented by the reception quality message supplied from the multiplexer 112, and supplies it to the control section 7.Then, it proceeds to step S53.In step S53, the control section 7 supplies to the control data generating section 8 the transmission mode supplied from the mode judging section 6, and the control data generating section 8 generates control data including the transmission mode.",
"This control data is supplied from the control data generating section 8 to the antenna 14 via the control data inserting section 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11 and the transmission/reception compatible device 1, and is transmitted from the antenna 14 as an associated channel signal.",
"Thereafter, it proceeds to step S54.Packet data in which user data addressed to the terminal 102 is arranged is transmitted in the transmission mode determined by the mode judging section 6.In other words, in step S54, the user data addressed to the terminal 102 is arranged in a packet and is supplied to the adaptive encoding modulation section 13 as packet data.",
"Then, the control section 7 controls the adaptive encoding modulation section 13 so as to encode and modulate the packet data addressed to the terminal 102 in the transmission mode determined by the mode judging section 6.Thus, if the reception quality message from the terminal 102 is received, the adaptive encoding modulation section 13, in accordance with the transmission mode determined based on the reception quality represented by the reception quality message from the terminal 102, encodes and modulates the packet data supplied thereto that is addressed to the terminal 102, and supplies it to the spreading section 11.Thereafter, in the spreading section 11 and the transmission/reception compatible device 1, processes similar to the case explained in FIG.",
"2 are performed.",
"Consequently, the packet data is transmitted from the antenna 14 to the terminal 102 as the packet channel signal.",
"On the other hand, in step S46, if the current timing is judged not to be the message reception timing at which the reception quality message is received, it proceeds to step S50.The transmission power converting section 111 uses the transmission power x[n] of the associated channel outputted by the power adjusting section 10 and calculates the predicted value y′[n] of the reception quality of the packet channel at the terminal 102.In other words, since the current timing is not the message reception timing, the reception quality message is not transmitted from the terminal 102.Thus, at the base station 101, the reception quality at the terminal 102 cannot be recognized from the reception quality message.",
"As such, in step S50, the transmission power converting section 111 calculates the predicted value y′[n] of the reception quality of the packet channel using the transmission power x[n] of the associated channel in accordance with equation (1) defined by the most recent parameters a and b determined through the training in step S48 performed most recently.",
"Then, the transmission power converting section 111 supplies the predicted value y′[n] of the reception quality to the multiplexer 112.It proceeds from step S50 to step S51.In step S51, the transmission power converting section 111 controls the multiplexer 112 so that the output of the transmission power converting section 111 is selected and supplied to the mode judging section 6.Thus, the predicted value of the reception quality calculated by the transmission power converting section 111 is supplied to the mode judging section 6 via the multiplexer 112.Then, it proceeds to step S52.The mode judging section 6 determines the code rate and the modulation method (the transmission mode) in the adaptive encoding modulation section 13 based on the predicted value of the reception quality supplied from the multiplexer 112, and supplies it to the control section 7.Then, it proceeds to step S53.In step S53, the control section 7 supplies to the control data generating section 8 the transmission mode supplied from the mode judging section 6, and the control data generating section 8 generates control data including the transmission mode.",
"This control data is supplied from the control data generating section 8 to the antenna 14 via the control data inserting section 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11 and the transmission/reception compatible device 1, and is transmitted from the antenna 14 as an associated channel signal.",
"Thereafter, it proceeds to step S54.Packet data in which user data addressed to the terminal 102 is arranged is transmitted in the transmission mode determined by the mode judging section 6.In other words, in step S54, the user data addressed to the terminal 102 is arranged in a packet and supplied to the adaptive encoding modulation section 13 as packet data.",
"Then, the control section 7 controls the adaptive encoding modulation section 13 so as to encode and modulate the packet data addressed to the terminal 102 in the transmission mode determined by the mode judging section 6.Thus, if the reception quality message is not sent from the terminal 102, the adaptive encoding modulation section 13 encodes and modulates the packet data addressed to the terminal 102 that is supplied thereto in accordance with the transmission mode determined based on the predicted value of the reception quality, and supplies it to the spreading section 11.Thereafter, at the spreading section 11 and the transmission/reception compatible device 1, processes similar to the case explained in FIG.",
"2 are performed.",
"Thus, the packet data is transmitted from the antenna 14 to the terminal 102 as a packet channel signal.",
"After the process of step S54, it proceeds to step S55.The transmission power converting section 111 performs a process for setting the transmission period (the transmission frequency) of the reception quality message by the terminal 102, and supplies the transmission period obtained as a result to the control data generating section 113.Then, it proceeds to step S56.In step S56, the control data generating section 113 generates transmission control information that indicates the transmission period of the reception quality message supplied from the transmission power converting section 111.Then, it proceeds to step S57.In step S57, the transmission control information generated in step S56 is transmitted from the control data generating section 113 to the terminal 102 by the associated channel via the control data inserting sections 114 and 115, the encoding modulation section 9, the power adjusting section 10, the spreading section 11, the transmission/reception compatible device 1 and the antenna 14.It returns to step S41, and thereafter, similar processes are repeated.",
"In addition, in the embodiment of FIG.",
"17, although in steps S56 and S57, the transmission control information is always generated and transmitted to the terminal 102, alternatively, it is possible to generate and transmit the transmission control information, for example, only when a transmission period different from the transmission period indicated by the transmission control information transmitted in the previous step S55 is set.",
"The setting process for the transmission period (the transmission frequency) of the reception quality message performed in step S55 of FIG.",
"17 will be described next with reference to the flowchart of FIG.",
"18.In the setting process for the transmission period (transmission frequency) of the reception quality message, first, in step S61, the transmission power converting section 111 judges if the reception frequency of NACK transmitted from the terminal 102 is updated.",
"In other words, as in the conventional terminal of FIG.",
"7, the terminal 102, if the packet data could be received properly, transmits ACK which indicates so, and if the packet data could not be received properly, transmits NACK which indicates so to the base station 101, for example, through the uplink control channel.",
"The transmission power converting section 111, when it receives a new ACK/NACK, uses the most recent K ACK/NACKs received up to that point to update the reception frequency of NACK.",
"In other words, the transmission power converting section 111, when it receives a new ACK/NACK, if it is assumed that of the K most recent ACK/NACKs received up to that point there are K′ and K−K′ ACKs and NACKs, respectively, obtains a new NACK reception frequency, for example, by calculating the equation (K−K′)/K×100%, and according to the new reception frequency of NACK, updates the reception frequency of NACK obtained when the previous ACK/NACK was received.",
"In step S61, after the process of the previous step S61 is performed, it is judged if such an update of the reception frequency of NACK as mentioned above has been carried out.",
"If, in step S61, it is judged that the reception frequency of NACK has not been updated, steps S62 and S63 are skipped and it proceeds to step S64.In addition, in step S61, if it is judged that the reception frequency of NACK has been updated, it proceeds to step S62.The transmission power converting section 111 judges if the reception frequency of NACK is less than (or at or below) a predetermined threshold TH1.In step S62, if the reception frequency of NACK is judged not to be less than the predetermined threshold TH1, that is, if the reception frequency of NACK is high, and therefore packet data often cannot be received properly at the terminal 102, it proceeds to step S63.The transmission power converting section 111 sets the transmission period of the reception quality message to be shorter than the current period.",
"Then, it proceeds to step S64.Thus, in this case, due to the fact that the transmission period of the reception quality message is shortened, the frequency at which the reception quality message is transmitted from the terminal 102 becomes high.",
"On the other hand, in step S62, if the reception frequency of NACK is judged to be less than the predetermined threshold TH1, that is, if the reception frequency of NACK is low, and therefore packet data can often be received properly at the terminal 102, step S63 is skipped, and it proceeds to step S64.In step S64, the transmission power converting section 111 judges if the predictive equation of equation (1) (the parameters a and b defining it) is updated.",
"In other words, by performing the process of step S48 of FIG.",
"17, the predictive equation of equation (1) is updated.",
"However, in step S64, it is judged if such an update of the predictive equation has been performed after the process in step S64 of the previous round was performed.",
"In step S64, if it is judged that the updating of the predictive equation of equation (1) has not been performed, steps S65 through S68 are skipped, and it returns.",
"In this case, the transmission period of the reception quality message remains as it is.",
"In addition, in step S64, if it is judged that the predictive equation of equation (1) has been performed, it proceeds to step S65.The transmission power converting section 111 judges if the square error E of equation (4) obtained with the predictive equation is less than (or at or below) a predetermined low threshold TH2.In addition, the square error E of equation (4) is calculated using the transmission power x[n] and the reception quality y[n] which are the training samples used in updating (training) the predictive equation of equation (1).",
"In step S65, if it is judged that the square error E is not less than the predetermined threshold TH2, that is, if the square error E is relatively large and, therefore, the accuracy of the predicted value of the reception quality obtained with the predictive equation of equation (1) is not bad but is not highly accurate either, it proceeds to step S66.The transmission power converting section 111 sets the transmission period of the reception quality message to be shorter than the current period, and it returns.",
"Thus, in this case, due to the fact that the transmission period of the reception quality message is shortened, the frequency at which the reception quality message is transmitted from the terminal 102 becomes high.",
"In addition, in step S66 (as in step S68 which will be described later), if the transmission period is already altered, for example, the transmission period is set (altered) anew with the altered transmission period as the current period.",
"On the other hand, in step S65, if the square error E is judged to be less than the predetermined threshold TH2, that is, if the square error E is relatively small and, therefore, the accuracy of the predicted value of the reception quality calculated with the predictive equation of equation (1) is relatively high in accuracy, it proceeds to step S67.The transmission power converting section 111 judges if the square error E is less than (or at or below) a threshold TH3 still smaller than the threshold TH2.In step S67, if it is judged that the square error E is not less than the predetermined threshold TH3, that is, if the square error E is smaller than the threshold TH2 but is not smaller than the threshold TH3, and therefore, the accuracy of the predicted value of the reception quality calculated with the predictive equation of equation (1) is relatively high in accuracy, step S68 is skipped and it returns.",
"In this case, the transmission period of the reception quality message is kept as it is.",
"In addition, in step S67, if the square error E is judged to be less than the predetermined threshold TH3, that is, if the square error E is smaller than the threshold TH3 that is smaller than the threshold TH2 and, therefore, the accuracy of the predicted value of the reception quality calculated with the predictive equation of equation (1) is extremely high in accuracy, it proceeds to step S68.The transmission power converting section 111 sets the transmission period of the reception quality message to be longer than the current period, and it returns.",
"Thus, in this case, due to the fact that the transmission period of the reception quality message is lengthened, the frequency at which the reception quality message is transmitted from the terminal 102 becomes low.",
"In addition, in step S62, instead of the reception frequency of NACK, the magnitude of the reception frequency of ACK may be judged.",
"Moreover, in steps S65, S67, instead of the square error E, for example, the size of the prediction error e[n], expressed by equation (2), between the reception quality y[n] represented by the most recently received reception quality message and the predicted value y′[n] of the reception quality calculated with equation (1) may be judged.",
"Thus, by setting the transmission period of the reception quality message in accordance with the NACK reception frequency and the square error E, at the terminal 102, for example, as shown in FIG.",
"19, although the reception quality message is transmitted in frame periods at first, the transmission period gradually becomes longer, and ultimately (ideally), it is no longer transmitted.",
"In other words, if the predicted value y′[n] of the reception quality calculated from equation (1) is of a high accuracy, the square error E is reduced.",
"Moreover, at the base station 101, because the transmission mode determined based on that predicted value y′[n] corresponds to the actual reception quality at the terminal 102, at the terminal 102, the likelihood of being able to receive packet data properly becomes high, and the NACK reception frequency becomes small.",
"Hence, the transmission period of the reception quality message gradually becomes long.",
"Ultimately, it is no longer transmitted (the transmission period becomes infinite).",
"In addition, if the propagation path characteristics between the base station 101 and the terminal 102 change rapidly, at the terminal 102, there may be cases where packet data cannot be received properly.",
"Moreover, the accuracy of the predicted value y′[n] of the reception quality calculated from equation (1) may deteriorate.",
"In this case, according to the process in accordance with the flowchart of FIG.",
"18, the transmission period of the reception quality message becomes short.",
"Consequently, at the base station 101, it becomes more frequent that the transmission mode is determined in accordance with the reception quality indicated by the reception quality message actually transmitted from the terminal 102.Moreover, in this case, according to the process in accordance with the flowchart of FIG.",
"17, in step S48, the chances of the training of the predictive equation of equation (1) being performed increases.",
"Consequently, a predictive equation (the parameters a and b defining it) that is capable of calculating a highly accurate predicted value y′[n] in accordance with the rapidly changed propagation path characteristics is quickly obtained.",
"Then, after the predictive equation that is capable of obtaining the highly accurate predicted value y′[n] in accordance with the rapidly changed propagation path characteristics, as shown in FIG.",
"19, the transmission period of the reception quality message is gradually made longer again.",
"Thus, in this case, it is possible to reduce the power consumption at the terminal 102, and also to save radio resources for transmitting the reception quality message, and further it is possible to adaptively change the transmission frequency of the reception quality message depending on whether or not the terminal 102 is receiving packet data properly, or in accordance with the accuracy of the predicted value y′[n].",
"Next, FIG.",
"20 shows a configuration example of the terminal 102 of FIG.",
"8.In addition, in the figure, the same symbols are assigned to portions corresponding to the case in FIG.",
"7, and hereinafter, explanations thereof are omitted as deemed appropriate.",
"In FIG.",
"20, the terminal (the user terminal) comprises a transmission/reception compatible device 31, an inverse spreading section 32, an associated channel reception quality estimating section 33, a power control bit generating section 34, a packet channel reception quality estimating section 35, a reception quality message generating section 36, an associated channel demodulation decoding section 37, a control section 38, a user data demodulation decoding section 39, an error detecting section 40, a reception quality transmission control section 41, a reception quality message inserting section 43, a power control bit inserting section 44, a spreading section 45, and an antenna 47.Therefore, the terminal 102 of FIG.",
"20 is configured basically similar to the terminal of FIG.",
"7 except in that the reception quality transmission control section 41 is additionally provided.",
"The control data, which is demodulated and decoded by the associated channel demodulation decoding section 37, is supplied to the reception quality transmission control section 41.Based on the transmission control information included in the control data, the reception quality transmission control section 41 controls the transmission of the reception quality message representing the reception quality of the packet channel by controlling the packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception quality message inserting section 43.The outline of the process at the terminal 102 thus configured is as follows.",
"1.The associated channel data is inputted to the associated channel demodulation decoding section 37, and the control data, the audio data and the NW control data are demodulated and decoded.",
"The control section 38 detects the encoding method and the modulation method information (the transmission mode) applied to the packet channel, and carries out the mode setting (control) of the user data demodulation decoding section 39 based on the detection result.",
"2.The packet channel separated by the inverse spreading section 32 has a demodulation decoding process performed thereon at the user data demodulation section 39 and is outputted as reception packet data.",
"The transmission control information inserted into the associated channel is extracted by the reception quality transmission control section 41.3.The reception quality transmission control section 41, in accordance with the transmission control information, reports to the packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception message inserting section 43 the presence or absence (or the period) of a transmission of the reception quality message of the packet channel.",
"The presence/absence of a transmission or the period of transmission of the reception quality message of the packet channel mentioned above is reported from the side of the base station 101.More specifically, the associated channel demodulation decoding section 37 reports to the reception quality transmission control section 41 the information regarding the presence/absence or the period of a transmission obtained by demodulating and decoding the control data, which was inserted into the associated channel and transmitted, in other words, the transmission control information.",
"4.The packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception message inserting section 43 execute respective processes in specified periods.",
"In other words, the packet channel reception quality estimating section 35 estimates the signal to noise ratio as the reception quality (SIR) of the packet channel.",
"In the method of estimating the signal to noise ratio is used a pilot symbol time-multiplexed on the packet channel or a pilot channel symbol transmitted in parallel with the packet channel.",
"The estimated reception quality (SIR) is converted into a predetermined message format (for example, the SIR is converted into binary digits) by the reception quality message generating section 36.5.The associated channel reception quality estimating section 33 estimates the signal to noise ratio as the reception quality (SIR) of the associated channel from the pilot signal time multiplexed on the associated channel.",
"The estimated reception quality is inputted to the power control generating section 34.If the estimated reception quality is better than the desired SNR, −1 is outputted, and if worse, +1 is outputted.",
"This process is executed for each slot and is used for the purpose of controlling the transmission power of the channel such that the associated channel can always be received at a constant SNR.",
"6.The reception quality message of the packet channel and the power control bit of the associated channel are inserted into the uplink control channel data by the reception quality inserting section 43 and the power control bit inserting section 44, respectively, and are transmitted to the base station 101 via the spreading section 45, the transmission/reception compatible device 31 and the antenna 47.Thus, in the communications system of FIG.",
"8, the reception quality of a first channel (the packet channel) is estimated (predicted) from the transmission power of a second channel (the associated channel) on which the power control is performed.",
"In other words, the transmission power of the second channel is mapped to the reception quality of the first channel transmitted from the receiving side, and the reception quality estimate value (predicted value) of the first channel obtained through this mapping is obtained.",
"Then, this reception quality estimate value is used to determine the modulating and encoding method, and adaptive control of the modulating and encoding methods is carried out.",
"In other words, the base station 101 or the base station control station serving as the transmitting side controls the presence or absence of the transmission of the reception quality message.",
"Immediately after a packet data call is generated, the mobile station (the terminal 102) transmits the reception quality message, and the base station 101 or the base station control station derives the packet channel reception quality from the reception quality message.",
"In addition, the base station 101 maps (trains) the transmission power of the associated channel to the reception quality value indicated by the reception quality message, and instructs the stopping and the like of the reception quality message to the mobile station after training is finished.",
"Then, it makes a transition to a mode that derives the packet channel reception quality directly from the associated channel transmission power.",
"Thus, by providing a mechanism for correlating the associated channel transmission power and the packet channel reception quality, it becomes possible to eliminate the transmission of the reception quality message without sacrificing transmission efficiency and to thereby contribute to a reduction in the power consumption of the mobile station and the effective utilization of radio resources.",
"Next, the process at the terminal 102 of FIG.",
"20 will further be described with reference to the flowcharts of FIG.",
"21 through FIG.",
"23.First, the process performed by the terminal 102, which is related to the packet channel and the associated channel that process the signals of the packet channel and the associated channel, is explained with reference to the flowchart of FIG.",
"21.First, in step S81, the signals of the packet channel and the associated channel transmitted from the base station 101 are received.",
"In other words, in step S81, the signals of the packet channel and the associated channel from the base station 101 are received by the antenna 47, and supplied to the inverse spreading section 32 after necessary processing is performed at the transmission/reception compatible device 31.Moreover, in step S81, the inverse spreading section 32 performs a spread spectrum on the signal from the transmission/reception compatible device 31 and thereby separates it into the signal of the packet channel of FIG.",
"1 and the signal of the associated channel.",
"Then, the inverse spreading section 32 supplies the signal of the associated channel to the associated channel reception quality estimating section 33 and the associated channel demodulation decoding section 37.Moreover, the inverse spreading section 32 supplies the signal of the packet channel to the packet channel reception quality estimating section 35 and the user data demodulation decoding section 39.After that, it proceeds to step S82.The associated channel demodulation decoding section 37 demodulates and decodes the signal of the associated channel supplied from the inverse spreading section 32 and outputs the control data, the audio data and the NW control data obtained as a result.",
"It proceeds to step S83.In step S83, the reception quality transmission control section 41 extracts the transmission control information included in the control data outputted by the associated channel demodulation decoding section 37, and sets a transmission period P of the reception quality message indicated by the transmission control information in the packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception quality message inserting section 43.Then, it proceeds to step S84.In step S84, the control section 38 recognizes the transmission mode included in the control data supplied from the associated channel demodulation decoding section 37.Then, it proceeds to step S85.In step S85, the control section 38 judges if the transmission mode is #0.In step S85, if the transmission mode is judged to be #0, it proceeds to step S86.The control section 38 controls the user data demodulation decoding section 39 such that the packet channel signal supplied from the inverse spreading section 32 is QPSK demodulated and further decoded at a code rate of R=½.",
"Moreover, in step S86, the user data demodulation decoding section 39 supplies to the error detecting section 40 the user data arranged in the packet data obtained by demodulating and decoding the signal of the packet channel.",
"Then, it proceeds to step S91.In addition, in step S85, if it is judged that the transmission mode is not #0, it proceeds to step S87.The control section 38 judges if the transmission mode is #1.In step S87, if the transmission mode is judged to be #1, it proceeds to step S88.The control section 38 controls the user data demodulation decoding section 39 such that the packet channel signal supplied from the inverse spreading section 32 is 16 QAM demodulated and further decoded at a code rate of R=½.",
"Moreover, in step S88, the user data demodulation decoding section 39 supplies to the error detecting section 40 the user data arranged in the packet data obtained by demodulating and decoding the signal of the packet channel.",
"Then, it proceeds to step S91.In addition, in step S87, if it is judged that the transmission mode is not #1, it proceeds to step S89.The control section 38 judges if the transmission mode supplied from the associated channel demodulation decoding section 37 is #2.In step S89, if it is judged that the transmission mode is not #2, steps S91 to S94 are skipped, and it returns to step S81.In addition, in step S89, if the transmission mode is judged to be #2, it proceeds to step S90.The control section 38 controls the user data demodulation decoding section 39 such that the packet channel signal supplied from the inverse spreading section 32 is 16 QAM demodulated and further decoded at a code rate of R=¾.",
"Moreover, in step S90, the user data demodulation decoding section 39 supplies to the error detecting section 40 the user data arranged in the packet data obtained by demodulating and decoding the signal of the packet channel.",
"Then, it proceeds to step S91.In step S91, the error detecting section 40 carries out error detection with regard to the user data supplied from the user data demodulation decoding section 39, and it proceeds to step S92.In step S92, the error detecting section 40 judges if an error is detected from the user data through the process in step S91.In step S92, if it is judged that an error is not detected from the user data, it proceeds to step S93.The error detecting section 40 outputs ACK indicating that the user data is received properly.",
"Then, it returns to step S81.Thereafter, similar processes are repeated.",
"In addition, in step S92, if it is judged that an error is detected from the user data, it proceeds to step S94.The error detecting section 40 outputs NACK indicating that the user data is not received properly.",
"Then, it returns to step S81.Thereafter, similar processes are repeated.",
"In addition, ACK/NACK outputted by the error detecting section 40 is transmitted to the base station 101 as explained in FIG.",
"7.Next the reception quality message transmission process carried out by the terminal 102 and for transmitting the reception quality message will be described below with reference to the flowchart of FIG.",
"22.First, in step S101, the packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception quality message inserting section 43 set, for example, 0 as an initial value in a variable p that counts frames, and it proceeds to step S102.In step S102, the packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception quality message inserting section 43 increment the variable p by just 1, and it proceeds to step S103.In step S103, the packet channel reception quality estimating section 35, the reception quality message generating section 36 and the reception quality message inserting section 43 judge if the variable p is equal to or greater than the transmission period P that was set in step S83 of FIG.",
"21.In step S103, if it is judged that the variable p is not equal to nor greater than the transmission period P, a lapse of time corresponding to one frame of the uplink control channel is awaited, and it returns to step S102.Thereafter, similar processes are repeated.",
"In addition, in step S103, if the variable p is judged to be equal to or greater than the transmission period P, that is, if a period of time equal to or greater than P has lapsed since the last transmission of the reception quality message, it proceeds to step S104.The packet channel reception quality estimating section 35 estimates the SNR, in other words the reception quality, of the signal of the packet channel supplied from the inverse spreading section 32, and supplies it to the reception quality message generating section 36.Then, it proceeds to step S105.In step S105, the reception quality message generating section 36 generates a reception quality message indicating the reception quality of the packet channel at the terminal supplied from the packet channel reception quality estimating section 35, and supplies it to the reception quality message inserting section 43.Then, it proceeds to step S106.In step S106, the reception quality message inserting section 43 inserts (frames) the reception quality message supplied from the reception quality message generating section 36 into the data of the uplink control channel.",
"Thus, the reception quality message is transmitted through the uplink control channel.",
"It returns to step S101.In other words, in this case, the reception quality message is transmitted to the base station 101 from the reception quality message inserting section 43 by the uplink control channel via the power control bit inserting section 44, the spreading section 45, the transmission/reception compatible device 31 and the antenna 47.Thus, in the reception quality message transmitting process of FIG.",
"22, because it is transmitted per P frames represented by the transmission period P set in step S83 of FIG.",
"21, in other words, at the terminal 102, because the reception quality message is transmitted in transmission periods indicated by the transmission control information transmitted from the base station 101, power consumption can be reduced to thereby save radio resources.",
"In addition, in cases where the reception quality message is to be transmitted in periods of a frame or is not to be transmitted at all, the transmission period P is set to 1 or to infinity.",
"In addition, the transmission period P can be set to a value smaller than 1.In this case, the reception quality message is updated more frequently than the conventional terminal of FIG.",
"7.Next, the power control bit transmitting process carried out by the terminal 102 and for transmitting the power control bit will be described below with reference to the flowchart of FIG.",
"23.First, in step S111, the associated channel reception quality estimating section 33 estimates the reception quality that is the SNR of the signal of the associated channel, and supplies the estimated reception quality to the power control bit generating section 34.Then, it proceeds to step S112.In step S112, the power control bit generating section 34 judges if the estimated reception quality of the associated channel is better than a desired reception quality that is a desired SNR.",
"In step S112, if it is judged that the estimated reception quality is not better than the desired reception quality, it proceeds to step S113.The power control bit generating section 34 generates a power control bit whose value is 1.This power control bit is transmitted from the power control bit generating section 34 to the base station 101 by the uplink control channel via the power control bit inserting section 44, the spreading section 45, the transmission/reception compatible device 31 and the antenna 47.After that, it returns to step S111.Thereafter, similar processes are repeated.",
"In addition, in step S112, if the estimated reception quality is judged to be better than the desired reception quality, it proceeds to step S114.The power control bit generating section 34 generates a power control bit whose value is 0.This power control bit is transmitted from the power control bit generating section 34 to the base station 101 by the uplink control channel via the power control bit inserting section 44, the spreading section 45, the transmission/reception compatible device 31 and the antenna 47.After that, it returns to step S111.Thereafter, similar processes are repeated.",
"Next, the series of processes at the transmission power converting section 111 of the base station 101, the reception quality transmission control section 41 of the terminal 102 and the like may be carried out with hardware and they can also be carried out through software.",
"If the series of processes are carried out through software, a program constituting the software is installed in a general purpose computer and the like.",
"As such, FIG.",
"24 shows a configuration example of an embodiment of a computer in which a program for executing the series of processes mentioned above is installed.",
"The program can be recorded in advance on a hard disk 205 or in a ROM 203 as recording media built into the computer.",
"Alternatively, the program can be temporarily or permanently stored (recorded) on a removable recording medium 211, such as a flexible disk, a CD-ROM (Compact Disc Read Only Memory), an MO (Magneto Optical) disc, a DVD (Digital Versatile Disc), a magnetic disk, a semiconductor memory and the like.",
"Such a removable recording medium 211 can be provided as so-called packaged software.",
"In addition, the program, aside from being installed in the computer from the removable recording medium 211 as mentioned above, can be transferred wirelessly from a download site to the computer through an artificial satellite for digital satellite broadcasting, or be transferred to the computer by wire via a network such as a LAN (Local Area Network) or the Internet.",
"Then, at the computer, the program thus transferred can be received by a communication section 208 and installed in a built-in hard disk 205.A CPU (Central Processing Unit) 202 is built into the computer.",
"An input output interface 210 is connected to the CPU 202 via a bus 201.When command is inputted via the input output interface 210 by having an input section 207 comprising a keyboard, a mouse, a microphone or the like operated on by a user, the CPU 202, in accordance therewith, executes the program stored in the ROM (Read Only Memory) 203.Alternatively, the CPU 202 loads into a RAM (Random Access Memory) and executes the program stored in the hard disk 205, the program that is transferred from a satellite or a network and received by the communication section 208 and then installed in the hard disk 205, or the program that is read out from the removable recording medium 211 inserted into a drive 209 and then installed in the hard disk 205.Thus, the CPU 202 carries out the process in accordance with the above-mentioned flowchart, or the process that is performed with the configuration of the above-mentioned block diagram.",
"Then, the CPU 202 outputs the process result, as required, for example, from an output section 206 comprising an LCD (Liquid Crystal Display), a speaker or the like via the input output interface 210, or transmits it from the communication section 208, and further records it on the hard disk 205.Here, in the present specification, the process steps that describe the program for making the computer execute various processes do not necessarily have to be processed chronologically in the order listed in the flowchart and also include processes to be executed in parallel or individually (for example, parallel processing or processing by object).",
"In addition, the program may be processed by one computer or the process may be distributed among a plurality of computers.",
"Moreover, the program may be transferred to a remote computer and be executed.",
"In addition, the present invention can be applied to any communications system in which data is exchanged between the base station 101 and the terminal 102 using a plurality of channels, and the reception quality of one channel among the plurality of channels is reported from the terminal 102 to the base station 101, and the base station 101 controls the transmission power of one other channel.",
"Here, for example, because in the W-CDMA system, at the terminal, the reception quality message indicating the reception quality of the packet channel is transmitted to the base station, it is a matter of course that the present invention can be applied to a communications system, such as the W-CDMA system, in which the terminal 102 transmits a reception quality message to the base station 101 but it is also applicable to, for example, a communications system, such as an HDR system.",
"In other words, in an HDR system and the like, at the terminal 102, the transmission mode is determined based on the reception quality of the packet channel, and a mode request message indicating that the transmission mode is transmitted to the base station 101.However, because the mode request message is determined based on the reception quality of packets, the reception quality of the packet channel can be estimated from the mode request message.",
"Hence, the mode request message can be considered equivalent to the reception quality message.",
"The present invention can also be applied to a communications system of an HDR system in which the mode request message that is equivalent to such a reception quality message is transmitted from the terminal 102 to the base station 101.Also, it is possible to make the base station control station, not shown in drawing, for controlling the base station 101 perform the process at the base station 101.Moreover, the present invention can be applied to both wireless and wired communications systems.",
"In addition, whether the terminal 102 is of a portable type or a stationary type does not impede the application of the present invention.",
"Industrial Applicability Thus, according to the present invention, wireless resources can be saved and power consumption can also be reduced."
]
] | Patent_10450689 |
[
[
"Oxidation dyeing composition for keratinous fibres on amphiphilic polymers of at least an ethylenically unsaturated monomer with sulphonic group and comprising a hydrophobic part",
"The invention concerns an oxidation dyeing composition for keratinous fibres, in particular for human keratinous fibres and more particularly hair, comprising, in a medium suited for dyeing, at least an oxidation dyeing agent, and also at least an amphiphilic polymer including at least an ethylenically unsaturated monomer with a sulphonic group, in free form or partly or completely neutralised and further at least a hydrophobic part.",
"The invention also concerns dyeing methods and devices using said composition."
],
[
"1.An oxidation dye composition for keratin fibers, comprising: at least one oxidation dye in a medium that is suitable for dyeing and at least one amphiphilic polymer comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form, and also at least one hydrophobic portion.",
"2.The composition as claimed in claim 1, wherein the hydrophobic portion of the amphiphilic polymer comprises from 6 to 50 carbon atoms.",
"3.The composition as claimed in claim 2, wherein the hydrophobic portion of the amphiphilic polymer comprises from 6 to 22 carbon atoms.",
"4.The composition as claimed in claim 3, wherein the hydrophobic portion of the amphiphilic polymer comprises from 6 to 18 carbon atoms.",
"5.The composition as claimed in claim 4, characterized in that the hydrophobic portion of the amphiphilic polymer comprises from 12 to 18 carbon atoms.",
"6.The composition as claimed in claim 1, wherein the amphiphilic polymers are partially or totally neutralized with a mineral or organic base.",
"7.The composition as claimed in 6 claim 1, wherein the amphiphilic polymers have a number-average molecular weight ranging from 1,000 to 20,000,000 g/mol.",
"8.The composition as claimed in claim 7, wherein the number-average molecular weight ranges from 20,000 to 5,000,000 g/mol.",
"9.The composition as claimed in claim 8, wherein the number-average molecular weight ranges from 100,000 to 1,500,000 g/mol.",
"10.The composition as claimed in claim 1, wherein an aqueous solution at 1% by weight of said polymers has, at a temperature of 25° C., a viscosity, measured using a Brookfield viscometer with a No.",
"7 needle, ranging from 20,000 mPa·s to 100,000 mPa·s.",
"11.The composition as claimed in claim 1, wherein the amphiphilic polymers are prepared by free-radical precipitation polymerization in tert-butanol.",
"12.The composition as claimed in claim 1, wherein the amphiphilic polymers are crosslinked or noncrosslinked.",
"13.The composition as claimed in claim 12, wherein the amphiphilic polymers are crosslinked.",
"14.The composition as claimed in claim 13, wherein one or more crosslinking agents is a polyolefinically unsaturated compound.",
"15.The composition as claimed in claim 14, wherein one or more crosslinking agent is selected from the group consisting of methylenebisacrylamide, allyl methacrylate, and trimethylolpropane triacrylate (TMPTA).",
"16.The composition as claimed in claim 13, wherein the degree of crosslinking ranges from 0.01 mol % to 10 mol % relative to the polymer.",
"17.The composition as claimed in claim 1, wherein the ethylenically unsaturated monomer comprising a sulfonic group is selected from the group consisting of a vinylsulfonic acid, a styrenesulfonic acid, a (meth)acrylamido(C1-C22)alkylsulfonic acid, and a N-(C1-C22)alkyl(meth)acrylamido(C1-C22)alkylsulfonic acid, and also partially or totally neutralized forms thereof.",
"18.The composition as claimed in claim 17, wherein the ethylenically unsaturated comprising a sulfonic group is selected from the group consisting of acrylamidomethanesulfonic acid, acrylamidoethanesulfonic acid, acrylamidopropane sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, methacrylamido-2-methylpropanesulfonic acid, 2-acrylamido-n-butanesulfonic acid, 2-acrylamido-2,4,4-trimethylpentanesulfonic acid, 2-methacrylamidododecylsulfonic acids and 2-acrylamido-2,6-dimethyl-3-heptanesulfonic acid, and also partially or totally neutralized forms thereof.",
"19.The composition as claimed in claim 17, wherein the ethylenically unsaturated monomer comprising a sulfonic group is 2-acrylamido-2-methylpropanesulfonic acid (AMPS), and also partially or totally neutralized forms thereof.",
"20.The composition as claimed in claim 19, wherein the amphiphilic polymers comprise random AMPS polymers modified by reaction with an n-mono(C6-C22)alkylamine or a di-n-(C6-C22)alkylamine.",
"21.The composition as claimed in claim 19, wherein the amphiphilic AMPS polymers further comprise at least one ethylenically unsaturated monomer not comprising a fatty chain.",
"22.The composition as claimed in claim 20, wherein the ethylenically unsaturated monomer not comprising a fatty chain is or selected from the group consisting of a (meth)acrylic acid a (meth)acrylamide, a vinylpyrrolidone, maleic anhydride, itaconic acid, maleic acid, and mixtures of these compounds: wherein said (meth)acrylic acid is an alkyl derivative thereof or an ester thereof as obtained with monoalcohols or mono-or polyalkylene glycols.",
"23.The composition as claimed in claim 1, wherein the amphiphilic AMPS polymers are amphiphilic copolymers of AMPS or at least one ethylenically unsaturated hydrophobic monomer comprising at least one hydrophobic comprises from 6 to 50 carbon atoms.",
"24.The composition as claimed in claim 23, wherein the hydrophobic portion comprises from 6 to 22 carbon atoms.",
"25.The composition as claimed in claim 24, wherein the hydrophobic portion comprises from 6 to 18 carbon atoms.",
"26.The composition as claimed in claim 25, wherein the hydrophobic portion comprises from 12 to 18 carbon atoms.",
"27.The composition as claimed in any one of claims 23 to 26 claim 23, wherein the ethylenically unsaturated hydrophobic monomer is an acrylate or an acrylamide of formula (I) below: in which R1 and R3, which may be identical or different, denote a hydrogen atom or a linear or branched C1-C6 alkyl radical; Y denotes O or NH; R2 denotes a hydrophobic hydrocarbon-based radical comprising at least from 6 to 50 carbon atoms; x denotes a number of moles of alkylene oxide and ranges from 0 to 100.28.The composition as claimed in claim 27, wherein the hydrophobic radical R2 is selected from the group consisting of a linear C6-C18 alkyl radical, a branched C6-C18 alkyl radical ea cyclic C6-C18 alkyl radical: a C6-C18 alkylperfluoro radical, a cholesteryl radical, an ester of a cholesterol radical; and an aromatic polycyclic group.",
"29.The composition as claimed in claim 27, wherein the monomer of formula (I) has a value of x that ranges from 1 to 100.30.The composition as claimed in claim 27, wherein the monomer of formula (I) further comprises at least one polyoxyalkylenated chain.",
"31.The composition as claimed in claim 30, wherein the polyoxyalkylenated chain consists of ethylene oxide units and/or of propylene oxide units.",
"32.The composition as claimed in claim 31, wherein the polyoxyalkylenated chain consists of ethylene oxide units.",
"33.The composition as claimed in claim 27, wherein the number of oxyalkylene units ranges from 3 to 100.34.The composition as claimed in claim 33, wherein the number of oxyalkylene units ranges from 3 to 50.35.The composition as claimed in claim 34, wherein the number of oxyalkylene units ranges from 7 to 25.36.The composition as claimed in claim 23, wherein the amphiphilic AMPS polymer is selected from the group consisting of A and B. wherein A is defined by crosslinked or noncrosslinked, neutralized or nonneutralized copolymers comprising from 15% to 60% by weight of AMPS units and from 40% to 85% by weight of (C8-C16)alkyl(meth)acrylamide units or of (C8-C16)alkyl (meth)acrylate units relative to the polymer; and B is defined by terpolymers comprising from 10 mol % to 90 mol % of acrylamide units, from 0.1 mol % to 10 mol % of AMPS units and from 5 mol % to 80 mol % of n-(C6-C18)alkylacrylamide units relative to the polymer.",
"37.The composition as claimed in claim 23, wherein the amphiphilic AMPS polymer is selected from the group consisting of A and B; wherein A is defined by noncrosslinked copolymers of partially or totally neutralized AMPS and of n-dodecyl methacrylate; and B is defined by crosslinked or noncrosslinked copolymers of partially or totally neutralized AMPS and of n-dodecylmethacrylamide.",
"38.The composition as claimed in claim 23, wherein the amphiphilic AMPS polymer is a copolymer consisting of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) units of formula (II) below: in which X+ is a proton, an alkali metal cation, an alkaline-earth metal cation or the an ammonium ion, and of units of formula (III) below: in which x denotes an integer ranging from 3 to 100; R1 has the same meaning as that given above in formula (I) and R4 denotes a linear or branched C6-C22.39.The composition as claimed in claim 38, wherein x=25, R1 is methyl and R4 is n-dodecyl.",
"40.The composition as claimed in claim 27 wherein the molar percentage proportion of units of formula (I) or of units of formula (III) in the polymers ranges from 50.1% to 99.9%.",
"41.The composition as claimed in claim 27 wherein the molar percentage proportion of units of formula (I) or of units of formula (III) in the polymers ranges from 0.1% to 50%.",
"42.The composition as claimed in claim 1, wherein the amphiphilic polymers are present in concentrations ranging from 0.01% to 30% by weight, relative to the total weight of the composition.",
"43.The composition as claimed in claim 1, wherein the oxidation dye is an oxidation base and/or a coupler.",
"44.The composition as claimed in claim 43, wherein the composition comprises at least one oxidation base.",
"45.The composition as claimed in claim 43, wherein the oxidation base is selected from the group consisting of an ortho-phenylenediamine, a para-phenylenediamine, a double base, an ortho-aminophenol, a para-aminophenol, and a heterocyclic base, and also addition salts of these compounds with an acid.",
"46.The composition as claimed in claim 45, wherein the para-phenylenediamine is defined by structure (I) below: in which: R1 represents a hydrogen atom, a C1-C4 alkyl radical, a C1-C4 monohydroxyalkyl radical, a C2-C4 polyhydroxyalkyl radical, a (C1-C4)alkoxy(C1-C4)alkyl radical or a C1-C4 alkyl radical substituted with a nitrogenous, phenyl or 4′-aminophenyl group; R2 represents a hydrogen atom, a C1-C4 alkyl radical, a C1-C4 monohydroxyalkyl radical, a C2-C4 polyhydroxyalkyl radical, a (C1-C4)alkoxy(C1-C4)alkyl radical or a C1-C4 alkyl radical substituted with a nitrogenous group; R1 and R2 may also form, with the nitrogen atom that bears them, a 5- or 6-membered nitrogenous heterocycle optionally substituted with one or more alkyl, hydroxyl or ureido groups; R3 represents a hydrogen atom, a halogen atom such as a chlorine atom, a C1-C4 alkyl radical, a sulfo radical, a carboxy radical, a C1-C4 monohydroxyalkyl radical, a C1-C4 hydroxyalkoxy radical, an acetylamino(C1-C4)alkoxy radical, a mesylamino(C1-C4)-alkoxy radical or a carbamoylamino(C1-C4)alkoxy radical, R4 represents a hydrogen or halogen atom or a C1-C4 alkyl radical.",
"47.The composition as claimed in claim 45, wherein the double base is defined by structure (II) below: in which: Z1 and Z2, which may be identical or different, represent a hydroxyl or —NH2 radical which may be substituted with a C1-C4 alkyl radical or with a linker arm Y; the linker arm Y represents a linear or branched alkylene chain containing from 1 to 14 carbon atoms, which may be interrupted by or terminated with one or more nitrogenous groups and/or one or more hetero atoms such as oxygen, sulfur or nitrogen atoms, and optionally substituted with one or more hydroxyl or C1-C6 alkoxy radicals; R5 and R6 represent a hydrogen or halogen atom, a C1-C4 alkyl radical, a C1-C4 monohydroxyalkyl radical, a C2-C4 polyhydroxyalkyl radical, a C1-C4 aminoalkyl radical or a linker arm Y; R7, R8, R9, R10, R11 and R12, which may be identical or different, represent a hydrogen atom, a linker arm Y or a C1-C4 alkyl radical; it being understood that the compounds of formula (II) contain only one linker arm Y per molecule.",
"48.The composition as claimed in claim 45, wherein the para-aminophenol is defined by structure (III) below: in which: R13 represents a hydrogen atom, a halogen atom, such as fluorine, or a C1-C4 alkyl, C1-C4 monohydroxyalkyl, (C1-C4)alkoxy(C1-C4)alkyl, C1-C4 aminoalkyl or hydroxy(C1-C4)-alkylamino(C1-C4)alkyl radical, R14 represents a hydrogen atom, a halogen atom, such as fluorine, or a C1-C4 alkyl, C1-C4 monohydroxyalkyl, C2-C4 polyhydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl or (C1-C4)alkoxy(C1-C4)alkyl radical.",
"49.The composition as claimed in claim 45, wherein the heterocyclic base is selected from the group consisting of a pyridine derivative, a pyrimidine derivative a pyrazolopyrimidine, and a pyrazole derivative.",
"50.The composition as claimed in claim 45, wherein the oxidation base is present in concentrations ranging from 0.0005% to 12% by weight relative to the total weight of the composition.",
"51.The composition as claimed in claim 43, wherein the coupler is selected from the group consisting of a meta-phenyldiamine, a meta-aminophenol, a meta-diphenol, and a heterocyclic coupler, and the addition salts of these compounds with an acid.",
"52.The composition as claimed in claim 43, wherein the coupler is present in concentrations ranging from 0.0001% to 10% by weight relative to the total weight of the composition.",
"53.The composition as claimed in claim 43, wherein an acid addition salt of the oxidation dye comprises an anion which is selected from the group consisting of a chloride, a bromide, a sulfate, a hydrogensulfate, a tartrate, a lactate, and an acetate.",
"54.The composition as claimed in claim 1, wherein the composition further comprises direct dyes.",
"55.The composition as claimed in claim 1, wherein the composition further comprises at least one amphoteric polymer or one cationic polymer.",
"56.The composition as claimed in claim 55, wherein the cationic polymer is a polyquaternary ammonium consisting of repeating units corresponding to formula (W) below: 57.The composition as claimed in claim 55, wherein the cationic polymer is a polyquaternary ammonium consisting of repeating units corresponding to formula (U) below: 58.The composition as claimed in claim 55, wherein the amphoteric polymer is a copolymer comprising at least acrylic acid and a dimethyldiallylammonium salt as monomers.",
"59.The composition as claimed in claim 55, wherein the cationic or amphoteric polymer is present from 0.01% to 10% by weight relative to the total weight of the composition.",
"60.The composition as claimed in claim 1, wherein it comprises at least one surfactant chosen from anionic, cationic, nonionic and amphoteric surfactants.",
"61.The composition as claimed in claim 60, wherein the surfactants represent 0.01% to 40% by weight relative to the total weight of the composition.",
"62.The composition as claimed in claim 1, wherein the composition further comprises at least one reducing agent in amounts ranging from 0.05% to 3% by weight relative to the total weight of the composition.",
"63.The composition as claimed in claim 1, wherein the composition further comprises an oxidizing agent.",
"64.The composition as claimed in claim 63, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide, urea peroxide, an alkali metal bromate, an alkali metal ferricyanide, a persalt, and a redox enzyme, wherein said redox enzyme optionally comprises a respective donor or cofactor thereof.",
"65.The composition as claimed in claim 64, wherein the oxidizing agent is hydrogen peroxide.",
"66.The composition as claimed in claim 65, wherein the oxidizing agent is an aqueous hydrogen peroxide solution with a titer ranging from 1 to 40 volumes.",
"67.The composition as claimed in claim 63, wherein it the composition has a pH ranging from 4 to 11.68.A process for dyeing keratin fibers, which comprises: applying to the keratin fibers at least one dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, the color being developed at alkaline, neutral or acidic pH using an oxidizing composition comprising at least one oxidizing agent, which is mixed with the dye composition just at the time of use or which is applied sequentially without intermediate rinsing, at least one amphiphilic polymer as defined in claim 1 being present in the dye composition or in the oxidizing composition or in each of the two compositions.",
"69.The process as claimed in claim 68, which comprises applying to the wet or dry keratin fibers the composition prepared extemporaneously at the time of use from the dye composition and oxidizing compositions leaving the composition to act for an action time ranging from 1 to 60 minutes approximately; rinsing the fibers and then optionally washing them with shampoo, and then rinsing them again and drying them.",
"70.A process for dyeing keratin fibers, which comprises: applying to the wet or dry keratin fibers the ready to use a composition prepared extemporaneously at the time of use from a dye composition comprising at least one oxidation dye, another composition comprising at least one amphiphilic polymer as defined according to claim 1 and an oxidizing composition, leaving the composition to act for an action time ranging from 1 to 60 minutes approximately, rinsing the fibers and then optionally washing them with shampoo, and then rinsing them again and drying them.",
"71.The process as claimed in claim 70, wherein one or the other of said compositions also comprises at least one cationic or amphoteric polymer and at least one surfactant.",
"72.A two-compartment device or kit for dyeing keratin fibers, wherein one compartment contains comprises a dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, and another compartment comprises an oxidizing composition comprising an oxidizing agent in a medium that is suitable for dyeing, at least one amphiphilic polymer as defined in claim 1 being present in the dye composition or in the oxidizing composition or in each of the two compositions.",
"73.A three-compartment device for dyeing keratin fibers, wherein a first compartment comprises a dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, a second compartment comprises an oxidizing composition comprising at least one oxidizing agent in a medium that is suitable for dyeing, and a third compartment comprises a composition comprising at least one amphiphilic polymer as defined in claim 1, in a medium that is suitable for dyeing, the dye composition and/or the oxidizing composition also optionally comprises the amphiphilic polymer.",
"74.The composition as claimed in claim 13, wherein the degree of crosslinking ranges from 0.2 mol % to 2 mol % relative to the polymer.",
"75.The composition as claimed in claim 23, wherein the ethylenically unsaturated hydrophobic monomer is an acrylate or an acrylamide of formula (I) below: in which R1 and R3, which may be identical or different, denote a hydrogen atom or a methyl radical; Y denotes O or NH; R2 denotes a hydrophobic hydrocarbon-based radical comprising at least from 6 to 50 carbon atoms; x denotes a number of moles of alkylene oxide and ranges from 0 to 100.76.The composition as claimed in claim 23, wherein the ethylenically unsaturated hydrophobic monomer is an acrylate or an acrylamide of formula (I) below: in which R1 and R3, which may be identical or different, denote a hydrogen atom or a linear or branched C1-C6 alkyl radical; Y denotes O or NH; R2 denotes a hydrophobic hydrocarbon-based radical comprising comprising at least from 6 to 22 carbon atoms; x denotes a number of moles of alkylene oxide and ranges from 0 to 100.77.The composition as claimed in claim 23, wherein the ethylenically unsaturated hydrophobic monomer is an acrylate or an acrylamide of formula (I) below: in which R1 and R3, which may be identical or different, denote a hydrogen atom or a linear or branched C1-C6 alkyl radical; Y denotes O or NH; R2 denotes a hydrophobic hydrocarbon-based radical comprising comprising at least from 6 to 18 carbon atoms; x denotes a number of moles of alkylene oxide and ranges from 0 to 100.78.The composition as claimed in claim 23, wherein the ethylenically unsaturated hydrophobic monomer is an acrylate or an acrylamide of formula (I) below: in which R1 and R3, which may be identical or different, denote a hydrogen atom or a linear or branched C1-C6 alkyl radical; Y denotes O or NH; R2 denotes a hydrophobic hydrocarbon-based radical comprising comprising at least from 12 to 18 carbon atoms; x denotes a number of moles of alkylene oxide and ranges from 0 to 100.79.The composition as claimed in claim 23, wherein the amphiphilic AMPS polymer is copolymer consisting of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) units of formula (II) below: in which X+ is a proton, an alkali metal cation, an alkaline-earth metal cation or an ammonium ion, and of units of formula (III) below: in which x denotes an integer ranging from 5 to 80; R1 has the same meaning as that given above in formula (I) and R4 denotes a linear or branched C6-C22.80.The composition as claimed in claim 23, wherein the amphiphilic AMPS polymer is a copolymer consisting of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) units of formula (II) below: in which X+ is a proton, an alkali metal cation, an alkaline-earth metal cation or an ammonium ion, and of units of formula (III) below: in which x denotes an integer ranging from 7 to 25; R1 has the same meaning as that given above in formula (I) and R4 denotes a linear or branched C6-C22.81.The composition as claimed in claim 23, wherein the amphiphilic AMPS polymer is a copolymer consisting of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) units of formula (II) below: in which X+ is a proton, an alkali metal cation, an alkaline-earth metal cation or an ammonium ion, and of units of formula (III) below: in which x denotes an integer ranging from 3 to 100; R1 has the same meaning as that given above in formula (I) and R4 denotes a linear or branched C10-C22.82.The composition as claimed in claim 1, wherein the amphiphilic polymers are present in concentrations ranging from 0.1% to 10% by weight relative to the total weight of the composition.",
"83.The composition as claimed in claim 1, wherein the amphiphilic polymers are present in concentrations ranging from 0.1% to 5% by weight relative to the total weight of the composition.",
"84.The composition as claimed in claim 1, wherein the amphiphilic polymers are present in concentrations ranging from 0.5% to 2% by weight relative to the total weight of the composition.",
"85.The composition as claimed in claim 55, wherein the cationic or amphoteric polymer is present from 0.05% to 5% by weight relative to the total weight of the composition.",
"86.The composition as claimed in claim 55, wherein the cationic or amphoteric polymer is present from 0.1% to 3% by weight relative to the total weight of the composition.",
"87.The composition as claimed in claim 60, wherein the surfactants represent 0.1% to 30% by weight relative to the total weight of the composition.",
"88.A process for dyeing keratin fibers, which comprises: applying to the wet or dry keratin fibers a composition prepared extemporaneously at the time of use from a dye composition comprising at least one oxidation dye, another composition comprising at least one amphiphilic polymer as defined according to claim 1, and an oxidizing composition, leaving the composition to act for an action time ranging from 10 to 45 minutes approximately rinsing the fibers and then optionally washing them with shampoo, and then rinsing them again and drying them.",
"89.A process for dyeing human keratin fibers, which comprises: applying to the wet or dry keratin fibers a composition prepared extemporaneously at the time of use from a dye composition comprising at least one oxidation dye, another composition comprising at least one amphiphilic polymer as defined according to claim 1, and an oxidizing composition, leaving the composition to act for an action time ranging from 1 to 60 minutes approximately rinsing the fibers and then optionally washing them with shampoo, and then rinsing them again and drying them.",
"90.A process for dyeing human keratin fibers, which comprises: applying to the wet or dry keratin fibers a composition prepared extemporaneously at the time of use from a dye composition comprising at least one oxidation dye, another composition comprising at least one amphiphilic polymer as defined according to claim 1, and an oxidizing composition, leaving the composition to act for an action time ranging from 10 to 45 minutes approximately rinsing the fibers and then optionally washing them with shampoo, and then rinsing them again and drying them.",
"91.A two-compartment device or kit for dyeing human keratin fibers, wherein one compartment comprises a dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, and another compartment comprises an oxidizing composition comprising an oxidizing agent in a medium that is suitable for dyeing, at least one amphiphilic polymer as defined in claim 1 being present in the dye composition or in the oxidizing composition or in each of the two compositions.",
"92.A three-compartment device for dyeing human keratin fibers, wherein a first compartment comprises a dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, a second compartment comprises an oxidizing composition comprising at least one oxidizing agent in a medium that is suitable for dyeing, and a third compartment comprises a composition comprising at least one amphiphilic polymer as defined in claim 1, in a medium that is suitable for dyeing, the dye composition and/or the oxidizing composition also optionally comprises the amphilic polymer."
],
[
"The present invention relates to a composition for the oxidation dyeing of keratin fibers, in particular human keratin fibers and more particularly the hair, comprising at least one oxidation dye and at least one amphiphilic polymer comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form, and also at least one hydrophobic portion.",
"It is known practice to dye keratin fibers, and in particular human hair, with dye compositions containing oxidation dye precursors, generally known as “oxidation bases”, in particular ortho- or para-phenylenediamines, ortho- or para-aminophenols, and heterocyclic bases.",
"Oxidation dye precursors are compounds that are initially uncolored or only weakly colored, which develop their dyeing power on the hair in the presence of oxidizing agents, leading to the formation of colored compounds.",
"The formation of these colored compounds results either from an oxidative condensation of the “oxidation bases” with themselves or from an oxidative condensation of the “oxidation bases” with coloration modifiers, or “couplers”, which are generally present in the dye compositions used in oxidation dyeing and are represented more particularly by meta-phenylenediamines, meta-aminophenols and meta-diphenols, and certain heterocyclic compounds.",
"The variety of molecules used, which consist on the one hand of the “oxidation bases” and on the other hand of the “couplers”, allows a very wide range of colors to be obtained.",
"To localize the oxidation dye product on application to the hair so that it does not run down the face or beyond the areas that it is proposed to dye, use has been made hitherto of conventional thickeners such as crosslinked polyacrylic acid, hydroxyethylcelluloses, certain polyurethanes, waxes or mixtures of nonionic surfactants with an HLB (Hydrophilic-Lipophilic Balance) value, which, when suitably chosen, give rise to a gelling effect when they are diluted with water and/or surfactants.",
"However, the Applicant has found that the thickening systems mentioned above do not make it possible to obtain intense and chromatic shades of low selectivity and good staying power, while at the same time leaving the treated hair in a good cosmetic condition.",
"Moreover, the Applicant has also found that ready-to-use dye compositions containing the oxidation dye(s) and the thickening systems of the prior art do not allow a sufficiently precise application without running or reductions in viscosity over time.",
"After considerable research conducted in this matter, the Applicant has now discovered that it is possible to obtain ready-to-use oxidation dye compositions that do not run and thus remain satisfactorily localized at the point of application, and that also make it possible to obtain powerful and chromatic (luminous) shades with low selectivity and good staying power with respect to chemical agents (shampoos, permanent-waving agents, etc.)",
"or natural agents (light, perspiration, etc.)",
"while at the same time giving the hair good cosmetic properties, if an effective amount of an amphiphilic polymer comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form, and also at least one hydrophobic portion, is introduced either (i) into the composition containing the oxidation dye(s) and optionally the coupler(s) [or composition A], or (ii) into the oxidizing composition [or composition B], or (iii) into both compositions at the same time.",
"The Applicant has also found that the mixing of the dye compositions with the oxidizing agents is facilitated by the presence of the amphiphilic polymers according to the invention.",
"These discoveries form the basis of the present invention.",
"One subject of the present invention is thus an oxidation dye composition for keratin fibers, in particular for human keratin fibers and particularly the hair, comprising at least one oxidation dye in a medium that is suitable for dyeing, which is characterized in that it also comprises at least one amphiphilic polymer comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form, and also at least one hydrophobic portion.",
"Another subject of the invention relates to a ready-to-use composition for dyeing keratin fibers, which comprises at least one oxidation dye, at least one oxidizing agent and at least one amphiphilic polymer comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form, and also at least one hydrophobic portion.",
"For the purposes of the invention, the expression “ready-to-use composition” means the composition intended to be applied in unmodified form to the keratin fibers, i.e.",
"it may be stored in unmodified form before use, or may result from the extemporaneous mixing of two or more compositions.",
"The invention is also directed toward a process for dyeing keratin fibers, in particular human keratin fibers such as the hair, which consists in applying to the fibers at least one dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, the color being developed at alkaline, neutral or acidic pH using an oxidizing composition comprising at least one oxidizing agent, which is mixed with the dye composition just at the time of use or which is applied sequentially without intermediate rinsing, at least one amphiphilic polymer comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form, and also at least one hydrophobic portion, being present in the dye composition or in the oxidizing composition or in each of the two compositions.",
"A subject of the invention is also dyeing devices or multicompartment “kits”.",
"A two-compartment device according to the invention comprises one compartment containing a dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, and another compartment containing an oxidizing composition comprising an oxidizing agent in a medium that is suitable for dyeing, the amphiphilic polymer according to the invention being present in the dye composition or in the oxidizing composition or in each of the two compositions.",
"Another device, containing three compartments, according to the invention comprises a first compartment containing a dye composition comprising at least one oxidation dye in a medium that is suitable for dyeing, a second compartment containing an oxidizing composition comprising at least one oxidizing agent in a medium that is suitable for dyeing, and a third compartment containing a composition comprising at least one amphiphilic polymer according to the invention described above, in a medium that is suitable for dyeing, the dye composition and/or the oxidizing composition also possibly comprising an amphiphilic polymer according to the invention.",
"However, other characteristics, aspects, subjects and advantages of the invention will emerge even more clearly on reading the description and the examples that follow.",
"Amphiphilic Polymers According to the Invention The polymers in accordance with the invention are amphiphilic polymers comprising at least one ethylenically unsaturated monomer containing a sulfonic group, in free form or partially or totally neutralized form and comprising at least one hydrophobic portion.",
"The expression “amphiphilic polymer” means any polymer comprising both a hydrophilic portion and a hydrophobic portion and especially a fatty chain.",
"The hydrophobic portion present in the polymers of the invention preferably contains from 6 to 50 carbon atoms, more preferably from 6 to 22 carbon atoms, even more preferably from 6 to 18 carbon atoms and more particularly from 12 to 18 carbon atoms.",
"Preferably, the polymers in accordance with the invention are partially or totally neutralized with a mineral base (sodium hydroxide, potassium hydroxide or aqueous ammonia) or an organic base such as mono-, di- or triethanolamine, an aminomethylpropanediol, N-methylglucamine, basic amino acids, for instance arginine and lysine, and mixtures of these compounds.",
"The amphiphilic polymers in accordance with the invention generally have a number-average molecular weight ranging from 1000 to 20 000 000 g/mol, preferably ranging from 20 000 to 5 000 000 and even more preferably from 100 000 to 1 500 000 g/mol.",
"The amphiphilic polymers according to the invention may or may not be crosslinked.",
"Crosslinked amphiphilic polymers are preferably chosen.",
"When they are crosslinked, the crosslinking agents may be chosen from polyolefinically unsaturated compounds commonly used for the crosslinking of polymers obtained by free-radical polymerization.",
"Mention may be made, for example, of divinylbenzene, diallyl ether, dipropylene glycol diallyl ether, polyglycol diallyl ethers, triethylene glycol divinyl ether, hydroquinone diallyl ether, ethylene glycol di(meth)acrylate or tetraethylene glycol di(meth)acrylate, trimethylolpropane triacrylate, methylenebisacrylamide, methylenebismethacrylamide, triallylamine, triallyl cyanurate, diallyl maleate, tetraallylethylenediamine, tetraallyloxyethane, trimethylolpropane diallyl ether, allyl (meth)acrylate, allylic ethers of alcohols of the sugar series, or other allyl or vinyl ethers of polyfunctional alcohols, and also allylic esters of phosphoric and/or vinylphosphonic acid derivatives, or mixtures of these compounds.",
"Methylenebisacrylamide, allyl methacrylate or trimethylolpropane triacrylate (TMPTA) will be used more particularly.",
"The degree of crosslinking will generally range from 0.01 mol % to 10 mol % and more particularly from 0.2 mol % to 2 mol % relative to the polymer.",
"The ethylenically unsaturated monomers containing a sulfonic group are chosen especially from vinylsulfonic acid, styrenesulfonic acid, (meth)acrylamido(C1-C22)alkylsulfonic acids, and N-(C1-C22)alkyl(meth)acrylamido(C1-C22)alkylsulfonic acids, for instance undecylacrylamidomethanesulfonic acid, and also partially or totally neutralized forms thereof.",
"(Meth)acrylamido(C1-C22)alkylsulfonic acids such as, for example, acrylamidomethanesulfonic acid, acrylamidoethanesulfonic acid, acrylamidopropane-sulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, methacrylamido-2-methylpropanesulfonic acid, 2-acrylamido-n-butanesulfonic acid, 2-acrylamido-2,4,4-trimethylpentanesulfonic acid, 2-methacrylamidododecylsulfonic acid or 2-acrylamido-2,6-dimethyl-3-heptanesulfonic acid, and also partially or totally neutralized forms thereof, will more preferably be used.",
"2-Acrylamido-2-methylpropanesulfonic acid (AMPS), and also partially or totally neutralized forms thereof, will more particularly be used.",
"The amphiphilic polymers in accordance with the invention may be chosen especially from random amphiphilic AMPS polymers modified by reaction with a C6-C22 n-monoalkylamine or di-n-alkylamine, and such as those described in patent application WO-A-00/31154 (forming an integral part of the content of the description).",
"These polymers may also contain other ethylenically unsaturated hydrophilic monomers chosen, for example, from (meth)acrylic acids, β-substituted alkyl derivatives thereof or esters thereof obtained with monoalcohols or mono- or polyalkylene glycols, (meth)acrylamides, vinylpyrrolidone, maleic anhydride, itaconic acid or maleic acid, or mixtures of these compounds.",
"The preferred polymers of the invention are chosen from amphiphilic copolymers of AMPS and of at least one ethylenically unsaturated hydrophobic monomer comprising at least one hydrophobic portion containing from 6 to 50 carbon atoms, more preferably from 6 to 22 carbon atoms, even more preferably from 6 to 18 carbon atoms and more particularly 12 to 18 carbon atoms.",
"These same copolymers may also contain one or more ethylenically unsaturated monomers not comprising a fatty chain, such as (meth)acrylic acids, β-substituted alkyl derivatives thereof or esters thereof obtained with monoalcohols or mono- or polyalkylene glycols, (meth)acrylamides, vinylpyrrolidone, maleic anhydride, itaconic acid or maleic acid, or mixtures of these compounds.",
"These copolymers are described especially in patent application EP-A-750 899, patent U.S. Pat.",
"No.",
"5,089,578 and in the following publications from Yotaro Morishima: “Self-assembling amphiphilic polyelectrolytes and their nanostructures—Chinese Journal of Polymer Science Vol.18, No.",
"40, (2000), 323-336”; “Micelle formation of random copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and a nonionic surfactant macromonomer in water as studied by fluorescence and dynamic light scattering—Macromolecules 2000, Vol.",
"33, No.",
"10-3694-3704”; “Solution properties of micelle networks formed by nonionic moieties covalently bound to a polyelectrolyte: salt effects on rheological behavior—Langmuir, 2000, Vol.16, No.",
"12, 5324-5332”; “Stimuli responsive amphiphilic copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and associative macromonomers—Polym.",
"Preprint, Div.",
"Polym.",
"Chem.",
"1999, 40(2), 220-221”.",
"The ethylenically unsaturated hydrophobic monomers of these particular copolymers are preferably chosen from the acrylates or acrylamides of formula (I) below: in which R1 and R3, which may be identical or different, denote a hydrogen atom or a linear or branched C1-C6 alkyl radical (preferably methyl); Y denotes O or NH; R2 denotes a hydrophobic hydrocarbon-based radical containing at least from 6 to 50 carbon atoms, more preferably from 6 to 22 carbon atoms, even more preferably from 6 to 18 carbon atoms and more particularly from 12 to 18 carbon atoms; x denotes a number of moles of alkylene oxide and ranges from 0 to 100.The radical R2 is preferably chosen from linear C6-C18 alkyl radicals (for example n-hexyl, n-octyl, n-decyl, n-hexadecyl and n-dodecyl) and branched or cyclic C6-C18 alkyl radicals (for example cyclododecane (C12) or adamantane (C10)); C6-C18 alkylperfluoro radicals (for example the group of formula —(CH2)2—(CF2)9—CF3); the cholesteryl radical (C27) or a cholesterol ester residue, for instance the cholesteryl oxyhexanoate group; aromatic polycyclic groups, for instance naphthalene or pyrene.",
"Among these radicals, the ones that are more particularly preferred are linear alkyl radicals and more particularly the n-dodecyl radical.",
"According to one particularly preferred form of the invention, the monomer of formula (I) comprises at least one alkylene oxide unit (x≧1) and preferably a polyoxyalkylenated chain.",
"The polyoxyalkylenated chain preferably consists of ethylene oxide units and/or of propylene oxide units and even more particularly consists of ethylene oxide units.",
"The number of oxyalkylene units generally ranges from 3 to 100, more preferably from 3 to 50 and even more preferably from 7 to 25.Among these polymers, mention may be made of: crosslinked or noncrosslinked, neutralized or normeutralized copolymers comprising from 15% to 60% by weight of AMPS units and from 40% to 85% by weight of (C8-C16)alkyl(meth)acrylamide units or of (C8-C16)alkyl (meth)acrylate units relative to the polymer, such as those described in patent application EP-A-750 899; terpolymers comprising from 10 mol % to 90 mol % of acrylamide units, from 0.1 mol % to 10 mol % of AMPS units and from 5 mol % to 80 mol % of n-(C6-C18)alkylacrylamide units, such as those described in patent U.S. Pat.",
"No.",
"5,089,578.Mention may also be made of copolymers of totally neutralized AMPS and of dodecyl methacrylate, and also crosslinked and noncrosslinked copolymers of AMPS and of n-dodecylmethacrylamide, such as those described in the Morishima articles mentioned above.",
"Mention will be made more particularly of the copolymers consisting of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) units of formula (II) below: in which X+ is a proton, an alkali metal cation, an alkaline-earth metal cation or the ammonium ion, and of units of formula (III) below: in which x denotes an integer ranging from 3 to 100, preferably from 5 to 80 and more preferably from 7 to 25; R1 has the same meaning as that given above in formula (I) and R4 denotes a linear or branched C6-C22 and more preferably C10-C22 alkyl.",
"The polymers that are particularly preferred are those for which x=25, R1 denotes methyl and R4 represents n-dodecyl; they are described in the Morishima articles mentioned above.",
"The polymers for which X+ denotes sodium or ammonium are more particularly preferred.",
"The preferred amphiphilic polymers in accordance with the invention may be obtained according to the standard free-radical polymerization processes in the presence of one or more initiators such as, for example, azobisisobutyronitrile (AIBN), azobisdimethylvaleronitrile, ABAH (2,2-azobis[2-amidinopropane]hydrochloride), organic peroxides such as dilauryl peroxide, benzoyl peroxide, tert-butyl hydroperoxide, etc., mineral peroxide compounds such as potassium persulfate or ammonium persulfate, or H2O2 optionally in the presence of reducing agents.",
"The amphiphilic polymers are obtained especially by free-radical polymerization in tert-butanol medium in which they precipitate.",
"Using precipitation polymerization in tert-butanol, it is possible to obtain a size distribution of the polymer particles that is particularly favorable for its uses.",
"The size distribution of the polymer particles may be determined, for example, by laser diffraction or image analysis.",
"An advantageous distribution for this type of polymer, determined by image analysis, is as follows: 60.2% less than 423 microns, 52.0% less than 212 microns, 26.6% less than 106 microns, 2.6% less than 45 microns and 26.6% greater than 850 microns.",
"The reaction may be performed at a temperature of between 0 and 150° C., preferably between 10 and 100° C., either at atmospheric pressure or under reduced pressure.",
"It may also be performed under inert atmosphere, and preferably under nitrogen.",
"According to this process 2-acrylamido-2-methylpropanesulfonic acid (AMPS) or a sodium or ammonium salt thereof was especially polymerized with a (meth)acrylic acid ester and a C10-C18 alcohol oxyethylenated with 8 mol of ethylene oxide (Genapole C-080 from the company Hoechst/Clariant), a C11 oxo alcohol oxyethylenated with 8 mol of ethylene oxide (Genapole UD-080 from the company Hoechst/Clariant), a C11 oxo alcohol oxyethylenated with 7 mol of ethylene oxide (Genapol® UD-070 from the company Hoechst/Clariant), a C12-C14 alcohol oxyethylenated with 7 mol of ethylene oxide (Genapol® LA-070 from the company Hoechst/Clariant), a C12-C14 alcohol oxyethylenated with 9 mol of ethylene oxide (Genapol® LA-090 from the company Hoechst/Clariant), a C12-C14 alcohol oxyethylenated with 11 mol of ethylene oxide (Genapol® LA-110 from the company Hoechst/Clariant), a C16-C18 alcohol oxyethylenated with 8 mol of ethylene oxide (Genapol® T-080 from the company Hoechst/Clariant), a C16-C18 alcohol oxyethylenated with 15 mol of ethylene oxide (Genapol® T-150 from the company Hoechst/Clariant), a C16-C18 alcohol oxyethylenated with 11 mol of ethylene oxide (Genapol® T-110 from the company Hoechst/Clariant), a C16-C18 alcohol oxyethylenated with 20 mol of ethylene oxide (Genapol® T-200 from the company Hoechst/Clariant), a C16-C18 alcohol oxyethylenated with 25 mol of ethylene oxide (Genapol® T-250 from the company Hoechst/Clariant), a C18-C22 alcohol oxyethylenated with 25 mol of ethylene oxide and/or a C16-C18 iso alcohol oxyethylenated with 25 mol of ethylene oxide.",
"The molar % concentration of the units of formula (II) and of the units of formula (III) in the polymers according to the invention will vary as a function of the desired cosmetic use and of the desired rheological properties of the formulation.",
"It may range between 0.1 mol % and 99.9 mol %.",
"Preferably, for the most hydrophobic polymers, the molar proportion of units of formula (I) or (III) ranges from 50.1% to 99.9%, more particularly from 70% to 95% and even more particularly from 80% to 90%.",
"Preferably, for the sparingly hydrophobic polymers, the molar proportion of units of formula (I) or (III) ranges from 0.1% to 50%, more particularly from 5% to 25% and even more particularly from 10% to 20%.",
"The monomer distribution in the polymers of the invention may be, for example, alternating, block (including multiblock) or random.",
"According to the invention, it is preferable for the polymers to contain heat-sensitive pendant chains and for the aqueous solution thereof to have a viscosity that, beyond a certain threshold temperature, increases or remains virtually constant as the temperature increases.",
"Even more particularly, the preferred polymers are those whose aqueous solution has a viscosity that is low below a first threshold temperature and that, above this first threshold temperature, increases to a maximum as the temperature increases, and that, above a second threshold temperature, decreases again as the temperature increases.",
"From this perspective, it is preferable for the viscosity of the polymer solutions below the first threshold temperature to be from 5% to 50%, in particular from 10% to 30% of the maximum viscosity at the second threshold temperature.",
"These polymers preferably lead in water to a phenomenon of demixing by heating, reflected by curves showing, as a function of the temperature and the concentration, a minimum known as the LCST (Lower Critical Solution Temperature).",
"The viscosities (measured at 25° C. using a Brookfield viscometer, needle No.",
"7) of the aqueous 1% solutions preferably range from 20 000 mPa.s to 100 000 mPa.s and more particularly from 60 000 mPa.s to 70 000 mPa.s.",
"The amphiphilic polymers in accordance with the invention are present in the compositions in concentrations ranging from 0.01% to 30% by weight of active material, more preferably from 0.1% to 10% of active material, even more preferably from 0.1% to 5% by weight of active material and even more particularly from 0.5% to 2% by weight.",
"Oxidation Dyes The oxidation dyes that may be used according to the invention are chosen from oxidation bases and/or couplers.",
"Preferably, the compositions according to the invention contain at least one oxidation base.",
"The oxidation bases that may be used in the context of the present invention are chosen from those conventionally known in oxidation dyeing, and among which mention may be made in particular of the ortho- and para-phenylenediamines, double bases, ortho- and para-aminophenols and heterocyclic bases below, and also the addition salts thereof with an acid.",
"Mention may be made in particular of: (I) the para-phenylenediamines of formula (I) below, and the addition salts thereof with an acid: in which: R1 represents a hydrogen atom, a C1-C4 alkyl radical, a C1-C4 monohydroxyalkyl radical, a C2-C4 polyhydroxyalkyl radical, a (C1-C4)alkoxy(C1-C4)alkyl radical or a C1-C4 alkyl radical substituted with a nitrogenous, phenyl or 4′-aminophenyl group; R2 represents a hydrogen atom, a C1-C4 alkyl radical, a C1-C4 monohydroxyalkyl radical, a C2-C4 polyhydroxyalkyl radical, a (C1-C4)alkoxy(C1-C4)alkyl radical or a C1-C4 alkyl radical substituted with a nitrogenous group; R1 and R2 may also form, with the nitrogen atom that bears them, a 5- or 6-membered nitrogenous heterocycle optionally substituted with one or more alkyl, hydroxyl or ureido groups; R3 represents a hydrogen atom, a halogen atom such as a chlorine atom, a C1-C4 alkyl radical, a sulfo radical, a carboxy radical, a C1-C4 monohydroxyalkyl radical, a C1-C4 hydroxyalkoxy radical, an acetylamino(C1-C4)alkoxy radical, a mesylamino(C1-C4)-alkoxy radical or a carbamoylamino(C1-C4)alkoxy radical, R4 represents a hydrogen or halogen atom or a C1-C4 alkyl radical.",
"Among the nitrogenous groups of formula (I) above, mention may be made in particular of amino, mono(C1-C4)alkylamino, di(C1-C4)alkylamino, tri(C1-C4)alkylamino, monohydroxy(C1-C4)alkylamino, imidazolinium and ammonium radicals.",
"Among the para-phenylenediamines of formula (I) above, mention may be made more particularly of para-phenylenediamine, para-tolylenediamine, 2-chloro-para-phenylenediamine, 2,3-dimethyl-para-phenylenediamine, 2,6-dimethyl-para-phenylenediamine, 2,6-diethyl-para-phenylenediamine, 2,5-dimethyl-para-phenylenediamine, N,N-dimethyl-para-phenylenediamine, N,N-diethyl-para-phenylenediamine, N,N-dipropyl-para-phenylenediamine, 4-amino-N,N-diethyl-3-methylaniline, N,N-bis(β-hydroxyethyl)-para-phenylenediamine, 4-N,N-bis(β-hydroxyethyl)amino-2-methylaniline, 4-N,N-bis(β-hydroxyethyl)amino-2-chloroaniline, 2-β-hydroxyethyl-para-phenylenediamine, 2-fluoro-para-phenylenediamine, 2-isopropyl-para-phenylenediamine, N-(β-hydroxypropyl)-para-phenylenediamine, 2-hydroxymethyl-para-phenylenediamine, N,N-dimethyl-3-methyl-para-phenylenediamine, N,N-(ethyl-β-hydroxyethyl)-para-phenylenediamine, N-(β,γ-dihydroxypropyl)-para-phenylenediamine, N-(4′-aminophenyl)-para-phenylenediamine, N-phenyl-para-phenylenediamine, 2-β-hydroxyethyloxy-para-phenylenediamine, 2-β-acetylaminoethyloxy-para-phenylene diamine, N-(β-methoxyethyl)-para-phenylenediamine and 2-methyl-1-N-β-hydroxyethyl-para-phenylenediamine, and the addition salts thereof with an acid.",
"Among the para-phenylenediamines of formula (I) above, para-phenylenediamine, para-tolylenediamine, 2-isopropyl-para-phenylenediamine, 2-β-hydroxyethyl-para-phenylenediamine, 2-β-hydroxyethyloxy-para-phenylenediamine, 2,6-dimethyl-para-phenylenediamine, 2,6-diethyl-para-phenylenediamine, 2,3-dimethyl-para-phenylenediamine, N,N-bis(β-hydroxyethyl)-para-phenylenediamine and 2-chloro-para-phenylenediamine, and the addition salts thereof with an acid are most particularly preferred.",
"(II) According to the invention, the term double bases is understood to refer to compounds containing at least two aromatic nuclei bearing amino and/or hydroxyl groups.",
"Among the double bases which can be used as oxidation bases in the dye compositions in accordance with the invention, mention may be made in particular of the compounds corresponding to formula (II) below, and the addition salts thereof with an acid: in which: Z, and Z2, which may be identical or different, represent a hydroxyl or —NH2 radical which may be substituted with a C1-C4 alkyl radical or with a linker arm Y; the linker arm Y represents a linear or branched alkylene chain containing from 1 to 14 carbon atoms, which may be interrupted by or terminated with one or more nitrogenous groups and/or one or more hetero atoms such as oxygen, sulfur or nitrogen atoms, and optionally substituted with one or more hydroxyl or C1-C6 alkoxy radicals; R5 and R6 represent a hydrogen or halogen atom, a C1-C4 alkyl radical, a C1-C4 monohydroxyalkyl radical, a C2-C4 polyhydroxyalkyl radical, a C1-C4 aminoalkyl radical or a linker arm Y; R7, R8, R9, R10, R11, and R12, which may be identical or different, represent a hydrogen atom, a linker arm Y or a C1-C4 alkyl radical; it being understood that the compounds of formula (II) contain only one linker arm Y per molecule.",
"Among the nitrogenous groups of formula (II) above, mention may be made in particular of amino, mono(C1-C4)alkylamino, di(C1-C4)alkylamino, tri(C1-C4)alkylamino, monohydroxy(C1-C4)alkylamino, imidazolinium and ammonium radicals.",
"Among the double bases of formula (II) above, mention may be made more particularly of N,N′-bis(β-hydroxyethyl)-N,N′-bis(4′-aminophenyl)-1,3-diaminopropanol, N,N′-bis(β-hydroxyethyl)-N,N′-bis(4′-aminophenyl)ethylenediamine, N,N′-bis(4-amino phenyl)tetramethylenediamine, N,N′-bis(β-hydroxyethyl)-N,N′-bis(4-aminophenyl) tetramethylenediamine, N,N′-bis(4-methylaminophenyl)tetramethylenediamine, N,N′-bis-(ethyl)-N,N′-bis(4′-amino-3′-methylphenyl)ethylenediamine and 1,8-bis(2,5-diamino phenoxy)-3,5-dioxaoctane, and the addition salts thereof with an acid.",
"Among these double bases of formula (II), N,N′-bis(β-hydroxyethyl)-N,N′-bis(4′-aminophenyl)-1,3-diaminopropanol and 1,8-bis(2,5-diaminophenoxy)-3,5-dioxaoctane, or one of the addition salts thereof with an acid, are particularly preferred.",
"(III) The para-aminophenols corresponding to formula (III) below, and the addition salts thereof with an acid: in which: R13 represents a hydrogen atom, a halogen atom, such as fluorine, or a C1-C4 alkyl, C1-C4 monohydroxyalkyl, (C1-C4)alkoxy(C1-C4)alkyl, C1-C4 aminoalkyl or hydroxy(C1-C4)-alkylamino(C1-C4)alkyl radical, R14 represents a hydrogen atom, a halogen atom, such as fluorine, or a C1-C4 alkyl, C1-C4 monohydroxyalkyl, C2-C4 polyhydroxyalkyl, C1-C4 aminoalkyl, C1-C4 cyanoalkyl or (C1-C4)alkoxy(C1-C4)alkyl radical.",
"Among the para-aminophenols of formula (III) above, mention may be made more particularly of para-aminophenol, 4-amino-3-methylphenol, 4-amino-3-fluorophenol, 4-amino-3-hydroxymethylphenol, 4-amino-2-methylphenol, 4-amino-2-hydroxy methyl phenol, 4-amino-2-methoxymethylphenol, 4-amino-2-aminomethylphenol and 4-amino-2-(β-hydroxyethylaminomethyl)phenol, and the addition salts thereof with an acid.",
"(IV) The ortho-aminophenols that can be used as oxidation bases in the context of the present invention are chosen in particular from 2-aminophenol, 2-amino-1-hydroxy-5-methylbenzene, 2-amino-1-hydroxy-6-methylbenzene and 5-acetamido-2-aminophenol, and the addition salts thereof with an acid.",
"(V) Among the heterocyclic bases which can be used as oxidation bases in the dye compositions in accordance with the invention, mention may be made more particularly of pyridine derivatives, pyrimidine derivatives and pyrazole derivatives, and the addition salts thereof with an acid.",
"Among the pyridine derivatives, mention may be made more particularly of the compounds described, for example, in patents GB 1 026 978 and GB 1 153 196, such as 2,5-diaminopyridine, 2-(4-methoxyphenyl)amino-3-aminopyridine, 2,3-diamino-6-methoxypyridine, 2-(β-methoxyethyl)amino-3-amino-6-methoxypyridine and 3,4-diamino pyridine, and the addition salts thereof with an acid.",
"Among the pyrimidine derivatives, mention may be made more particularly of the compounds described, for example, in German patent DE 2 359 399 or Japanese patents JP 88-169 571 and JP 91-10659 or patent application WO 96/15765, such as 2,4,5,6-tetraminopyrimidine, 4-hydroxy-2,5,6-triaminopyrimidine, 2-hydroxy-4,5,6-triamino pyrimidine, 2,4-dihydroxy-5,6-diaminopyrimidine and 2,5,6-triaminopyrimidine, and pyrazolopyrimidine derivatives such as those mentioned in patent application FR-A-2 750 048 and among which mention may be made of pyrazolo[1,5-a]pyrimidine-3,7-diamine; 2,5-dimethylpyrazolo[1,5-a]pyrimidine-3,7-diamine; pyrazolo[1,5-a]pyrimidine-3,5-diamine; 2,7-dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine; 3-aminopyrazolo[1,5-a]pyrimidin-7-ol; 3-aminopyrazolo[1,5-a]pyrimidin-5-ol; 2-(3-aminopyrazolo[1,5-a]pyrimidin-7-ylamino)ethanol; 2-(7-aminopyrazolo[1,5-a]pyrimidin-3-ylamino)ethanol; 2-[(3-aminopyrazolo[1,5-a]pyrimidin-7-yl)(2-hydroxyethyl)amino]ethanol; 2-[(7-amino pyrazolo[1,5-a]pyrimidin-3-yl)(2-hydroxyethyl)amino]ethanol; 5,6-dimethylpyrazolo[1,5-a]pyrimidine-3,7-diamine; 2,6-dimethylpyrazolo[1,5-a]pyrimidine-3,7-diamine; 2,5,N7,N7-tetramethylpyrazolo[1,5-a]pyrimidine-3,7-diamine and 3-amino-5-methyl-7-imidazolyl propylaminopyrazolo[1,5-a]pyrimidine, and the addition salts thereof and the tautomeric forms thereof, when a tautomeric equilibrium exists, and the addition salts thereof with an acid.",
"Among the pyrazole derivatives, mention may be made more particularly of the compounds described in patents DE 3 843 892, DE 4 133 957 and patent applications WO 94/08969, WO 94/08970, FR-A-2 733 749 and DE 195 43 988, such as 4,5-diamino-1-methylpyrazole, 3,4-diaminopyrazole, 4,5-diamino-1-(4′-chlorobenzyl)pyrazole, 4,5-diamino-1,3-dimethylpyrazole, 4,5-diamino-3-methyl-1-phenylpyrazole, 4,5-diamino-1-methyl-3-phenylpyrazole, 4-amino-1,3-dimethyl-5-hydrazinopyrazole, 1-benzyl-4,5-diamino-3-methylpyrazole, 4,5-diamino-3-tert-butyl-1-methylpyrazole, 4,5-diamino-1-tert-butyl-3-methylpyrazole, 4,5-diamino-1-(β-hydroxyethyl)-3-methylpyrazole, 4,5-diamino-1-(β-hydroxyethyl)pyrazole, 4,5-diamino-1-ethyl-3-methylpyrazole, 4,5-diamino-1-ethyl-3-(4′-methoxyphenyl)pyrazole, 4,5-diamino-1-ethyl-3-hydroxymethylpyrazole, 4,5-diamino-3-hydroxymethyl-1-methylpyrazole, 4,5-diamino-3-hydroxymethyl-1-isopropylpyrazole, 4,5-diamino-3-methyl-1-isopropylpyrazole, 4-amino-5-(2′-aminoethyl)amino-1,3-dimethyl-pyrazole, 3,4,5-triaminopyrazole, 1-methyl-3,4,5-triaminopyrazole, 3,5-diamino-1-methyl-4-methylaminopyrazole and 3,5-diamino-4-(β-hydroxyethyl)amino-1-methylpyrazole, and the addition salts thereof with an acid.",
"According to the present invention, the oxidation bases preferably represent from 0.0005% to 12% by weight approximately relative to the total weight of the composition and even more preferably from 0.005% to 8% by weight approximately relative to this weight.",
"The couplers which may be used in the dye composition according to the invention are those conventionally used in oxidation dye compositions, that is to say meta-aminophenols, meta-phenylenediamines, meta-diphenols, naphthols and heterocyclic couplers such as, for example, indole derivatives, indoline derivatives, sesamol and its derivatives, pyridine derivatives, pyrazolotriazole derivatives, pyrazolones, indazoles, benzimidazoles, benzothiazoles, benzoxazoles, 1,3-benzodioxoles and quinolines, and the addition salts thereof with an acid.",
"These couplers are chosen more particularly from 2,4-diamino-1-(β-hydroxyethyloxy)benzene, 2-methyl-5-aminophenol, 5-N-(β-hydroxyethyl)amino-2-methylphenol, 3-aminophenol, 2-methyl-3-amino-6-methylphenol, 3,5-diamino-2,6-dimethoxypyridine, 6-hydroxybenzomorpholine, 1-β-hydroxyethylamino-3,4-methylene dioxybenzene, 1-methyl-2,6-bis(β-hydroxyethylamino)benzene, 1,3-dihydroxybenzene, 1,3-dihydroxy-2-methylbenzene, 4-chloro-1,3-dihydroxybenzene, 2-amino-4-(β-hydroxyethylamino)-1-methoxybenzene, 1,3-diaminobenzene, 1,3-bis(2,4-diamino phenoxy)propane, sesamol, 1-amino-2-methoxy-4,5-methylenedioxybenzene, α-naphthol, 6-hydroxyindole, 4-hydroxyindole, 4-hydroxy-N-methylindole, 6-hydroxyindoline, 2,6-dihydroxy-4-methylpyridine, 1H-3-methylpyrazol-5-one, 1-phenyl-3-methylpyrazol-5-one, 2-amino-3-hydroxypyridine, 3,6-dimethylpyrazolo[3,2-c]-1,2,4-triazole and 2,6-dimethyl pyrazolo[1,5-b]-1,2,4-triazole, and the addition salts thereof with an acid.",
"When they are present, these couplers preferably represent from 0.0001% to 10% by weight approximately relative to the total weight of the composition, and even more preferably from 0.005% to 5% by weight approximately.",
"In general, the addition salts with an acid of the oxidation bases and couplers are chosen in particular from the hydrochlorides, hydrobromides, sulfates, tartrates, lactates and acetates.",
"In addition to the oxidation dyes defined above, the composition according to the invention may also contain direct dyes to enrich the shades with glints.",
"In this case, these direct dyes may be chosen in particular from neutral, cationic or anionic nitro, azo or anthraquinone dyes, in a weight proportion from about 0.001% to 20% and preferably from 0.01% to 10% relative to the total weight of the composition.",
"The dye composition and/or the oxidizing composition may also more particularly comprise at least one amphoteric or cationic polymer.",
"Cationic Polymers For the purposes of the present invention, the expression “cationic polymer” denotes any polymer containing cationic groups and/or groups which may be ionized into cationic groups.",
"The cationic polymers which may be used in accordance with the present invention may be chosen from any of those already known per se as improving the cosmetic properties of the hair, namely, in particular, those described in patent application EP-A-337 354 and in French patents FR-2 270 846, 2 383 660, 2 598 611, 2 470 596 and 2 519 863.The cationic polymers that are preferred are chosen from those containing units comprising primary, secondary, tertiary and/or quaternary amine groups, which may either form part of the main polymer chain or may be borne by a side substituent directly attached thereto.",
"The cationic polymers used generally have a number-average molecular mass of between 500 and 5×106 approximately and preferably between 103 and 3×106 approximately.",
"Among the cationic polymers which may be mentioned more particularly are polymers of the polyamine, polyamino amide and polyquaternary ammonium type.",
"These are known products.",
"They are described in particular in French patents 2 505 348 and 2 542 997.Among said polymers, mention may be made of: (1) homopolymers or copolymers derived from acrylic or methacrylic esters or amides and comprising at least one of the units of formula (I), (II), (III) or (IV) below: in which: R3, which may be identical or different, denote a hydrogen atom or a CH3 radical; A, which may be identical or different, represent a linear or branched alkyl group of 1 to 6 carbon atoms, preferably 2 or 3 carbon atoms, or a hydroxyalkyl group of 1 to 4 carbon atoms; R4, R5 and R6, which may be identical or different, represent an alkyl group containing from 1 to 18 carbon atoms or a benzyl radical and preferably an alkyl group containing from 1 to 6 carbon atoms; R1 and R2, which may be identical or different, represent hydrogen or an alkyl group containing from 1 to 6 carbon atoms, and preferably methyl or ethyl; X denotes an anion derived from an inorganic or organic acid, such as a methosulfate anion or a halide such as chloride or bromide.",
"The polymers of family (1) can also contain one or more units derived from comonomers which may be chosen from the family of acrylamides, methacrylamides, diacetoneacrylamides, acrylamides and methacrylamides substituted on the nitrogen with lower (C1-C4)alkyls, acrylic or methacrylic acids or esters thereof, vinyllactams such as vinylpyrrolidone or vinylcaprolactam, and vinyl esters.",
"Thus, among these polymers of family (1), mention may be made of: copolymers of acrylamide and of dimethylaminoethyl methacrylate quaternized with dimethyl sulfate or with a dimethyl halide, such as the product sold under the name Hercofloc by the company Hercules, the copolymers of acrylamide and of methacryloyloxyethyltrimethylammonium chloride described, for example, in patent application EP-A-080 976 and sold under the name Bina Quat P 100 by the company Ciba Geigy, the copolymer of acrylamide and of methacryloyloxyethyltrimethylammonium methosulfate sold under the name Reten by the company Hercules, quaternized or nonquaternized vinylpyrrolidone/dialkylaminoalkyl acrylate or methacrylate copolymers, such as the products sold under the name “Gafquat” by the company ISP, such as, for example, “Gafquat 734” or “Gafquat 755”, or alternatively the products known as “Copolymer 845, 958 and 937”.",
"These polymers are described in detail in French patents 2 077 143 and 2 393 573, dimethylaminoethyl methacrylate/vinylcaprolactam/vinylpyrrolidone terpolymers, such as the product sold under the name Gaffix VC 713 by the company ISP, vinylpyrrolidone/methacrylamidopropyldimethylamine copolymers sold in particular under the name Styleze CC 10 by ISP, and quaternized vinylpyrrolidone/dimethylaminopropylmethacrylamide copolymers such as the product sold under the name “Gafquat HS 100” by the company ISP.",
"(2) The cellulose ether derivatives containing quaternary ammonium groups, described in French patent 1 492 597, and in particular the polymers sold under the names “JR” (JR 400, JR 125 and JR 30M) or “LR” (LR 400, or LR 30M) by the company Union Carbide Corporation.",
"These polymers are also defined in the CTFA dictionary as quaternary ammoniums of hydroxyethylcellulose that has reacted with an epoxide substituted with a trimethylammonium group.",
"(3) Cationic cellulose derivatives such as cellulose copolymers or cellulose derivatives grafted with a water-soluble monomer of quaternary ammonium, and described in particular in U.S. Pat.",
"No.",
"4,131,576, such as hydroxyalkylcelluloses, for instance hydroxymethyl-, hydroxyethyl- or hydroxypropylcelluloses grafted, in particular, with a methacryloyloxyethyltrimethylammonium, methacrylamidopropyltrimethylammonium or dimethyldiallylammonium salt.",
"The commercial products corresponding to this definition are more particularly the products sold under the names “Celquat L 200” and “Celquat H 100” by the company National Starch.",
"(4) The cationic polysaccharides described more particularly in U.S. Pat.",
"Nos.",
"3,589,578 and 4,031,307, such as guar gums containing cationic trialkylammonium groups.",
"Guar gums modified with a salt (e.g.",
"chloride) of 2,3-epoxypropyltrimethylammonium are used, for example.",
"Such products are sold in particular under the trade names Jaguar C13 S, Jaguar C 15, Jaguar C 17 or Jaguar C162 by the company Meyhall.",
"(5) Polymers consisting of piperazinyl units and of divalent alkylene or hydroxyalkylene radicals containing straight or branched chains, optionally interrupted by oxygen, sulfur or nitrogen atoms or by aromatic or heterocyclic rings, and also the oxidation and/or quaternization products of these polymers.",
"Such polymers are described, in particular, in French patents 2 162 025 and 2 280 361.",
"(6) Water-soluble polyamino amides prepared in particular by polycondensation of an acidic compound with a polyamine; these polyamino amides can be crosslinked with an epihalohydrin, a diepoxide, a dianhydride, an unsaturated dianhydride, a bis-unsaturated derivative, a bis-halohydrin, a bis-azetidinium, a bis-haloacyldiamine, a bis-alkyl halide or alternatively with an oligomer resulting from the reaction of a difunctional compound which is reactive with a bis-halohydrin, a bis-azetidinium, a bis-haloacyldiamine, a bis-alkyl halide, an epihalohydrin, a diepoxide or a bis-unsaturated derivative; the crosslinking agent being used in proportions ranging from 0.025 to 0.35 mol per amine group of the polyamino amide; these polyamino amides can be alkylated or, if they contain one or more tertiary amine functions, they can be quaternized.",
"Such polymers are described, in particular, in French patents 2 252 840 and 2 368 508.",
"(7) The polyamino amide derivatives resulting from the condensation of polyalkylene polyamines with polycarboxylic acids followed by alkylation with difunctional agents.",
"Mention may be made, for example, of adipic acid/dialkylaminohydroxyalkyldialkylene-triamine polymers in which the alkyl radical contains from 1 to 4 carbon atoms and preferably denotes methyl, ethyl or propyl.",
"Such polymers are described in particular in French patent 1 583 363.Among these derivatives, mention may be made more particularly of the adipic acid/dimethylaminohydroxypropyl/diethylenetriamine polymers sold under the name “Cartaretine F, F4 or F8” by the company Sandoz.",
"(8) The polymers obtained by reaction of a polyalkylene polyamine containing two primary amine groups and at least one secondary amine group with a dicarboxylic acid chosen from diglycolic acid and saturated aliphatic dicarboxylic acids having from 3 to 8 carbon atoms.",
"The molar ratio between the polyalkylene polyamine and the dicarboxylic acid is between 0.8:1 and 1.4:1; the polyamino amide resulting therefrom is reacted with epichlorohydrin in a molar ratio of epichlorohydrin relative to the secondary amine group of the polyamino amide of between 0.5:1 and 1.8:1.Such polymers are described in particular in U.S. Pat.",
"Nos.",
"3,227,615 and 2,961,347.Polymers of this type are sold in particular under the name “Hercosett 57” by the company Hercules Inc. or alternatively under the name “PD 170” or “Delsette 101” by the company Hercules in the case of the adipic acid/epoxypropyl/diethylenetriamine copolymer.",
"(9) Cyclopolymers of alkyldiallylamine or of dialkyldiallylammonium, such as the homopolymers or copolymers containing, as main constituent of the chain, units corresponding to formula (V) or (VI): in which formulae k and t are equal to 0 or 1, the sum k+t being equal to 1; R9 denotes a hydrogen atom or a methyl radical; R7 and R8, independently of each other, denote an alkyl group having from 1 to 8 carbon atoms, a hydroxyalkyl group in which the alkyl group preferably has 1 to 5 carbon atoms, a lower C1-C4 amidoalkyl group, or R7 and R8 can denote, together with the nitrogen atom to which they are attached, heterocyclic groups such as piperidyl or morpholinyl; R7 and R8, independently of each other, preferably denote an alkyl group having from 1 to 4 carbon atoms; Y− is an anion such as bromide, chloride, acetate, borate, citrate, tartrate, bisulfate, bisulfite, sulfate or phosphate.",
"These polymers are described in particular in French patent 2 080 759 and in its Certificate of Addition 2 190 406.Among the polymers defined above, mention may be made more particularly of the dimethyldiallylammonium chloride homopolymer sold under the name “Merquat 100” by the company Calgon (and its homologues of low weight-average molecular mass) and copolymers of diallyldimethylammonium chloride and of acrylamide, sold under the name “Merquat 550”.",
"(10) The quaternary diammonium polymer containing repeating units corresponding to the formula: in which formula (VII): R10, R11, R12 and R13, which may be identical or different, represent aliphatic, alicyclic or arylaliphatic radicals containing from 1 to 20 carbon atoms or lower hydroxyalkylaliphatic radicals, or alternatively R10, R11, R12 and R13, together or separately, constitute, with the nitrogen atoms to which they are attached, heterocycles optionally containing a second hetero atom other than nitrogen, or alternatively R10, R11, R12 and R13 represent a linear or branched C1-C6 alkyl radical substituted with a nitrile, ester, acyl or amide group or a group —CO—O—R14-D or —CO—NH—R14-D where R14 is an alkylene and D is a quaternary ammonium group; A1 and B1 represent polymethylene groups containing from 2 to 20 carbon atoms which may be linear or branched, saturated or unsaturated, and which may contain, linked to or intercalated in the main chain, one or more aromatic rings or one or more oxygen or sulfur atoms or sulfoxide, sulfone, disulfide, amino, alkylamino, hydroxyl, quaternary ammonium, ureido, amide or ester groups, and X−,denotes an anion derived from a mineral or organic acid; A1, R10 and R12 can form, with the two nitrogen atoms to which they are attached, a piperazine ring; in addition, if A1 denotes a linear or branched, saturated or unsaturated alkylene or hydroxyalkylene radical, B1 can also denote a group —(CH2), —CO-D-OC—(CH2)n—in which D denotes: a) a glycol residue of formula: —O-Z-O—, where Z denotes a linear or branched hydrocarbon-based radical or a group corresponding to one of the following formulae: —(CH2—CH2—O)x—CH2—CH2— —[CH2—CH(CH3)—O]y—CH2—CH(CH3)— where x and y denote an integer from 1 to 4, representing a defined and unique degree of polymerization or any number from 1 to 4 representing an average degree of polymerization; b) a bis-secondary diamine residue such as a piperazine derivative; c) a bis-primary diamine residue of formula: —NH—Y—NH—, where Y denotes a linear or branched hydrocarbon-based radical, or alternatively the divalent radical —CH2—CH2—S—S—CH2—CH2—; d) a ureylene group of formula: —NH—CO—NH—.",
"Preferably, X− is an anion such as chloride or bromide.",
"These polymers generally have a number-average molecular mass of between 1000 and 100 000.Polymers of this type are described in particular in French patents 2 320 330, 2 270 846, 2 316 271, 2 336 434 and 2 413 907 and U.S. Pat.",
"Nos.",
"2,273,780, 2,375,853, 2,388,614, 2,454,547, 3,206,462, 2,261,002, 2,271,378, 3,874,870, 4,001,432, 3,929,990, 3,966,904, 4,005,193, 4,025,617, 4,025,627, 4,025,653, 4,026,945 and 4,027,020.It is more particularly possible to use polymers that consist of repeating units corresponding to formula (VIII) below: in which R10, R11, R12 and R13, which may be identical or different, denote an alkyl or hydroxyalkyl radical containing from 1 to 4 carbon atoms approximately, n and p are integers ranging from 2 to 20 approximately, and X− is an anion derived from a mineral or organic acid.",
"(11) Polyquaternary ammonium polymers consisting of repeating units of formula (IX): in which p denotes an integer ranging from 1 to 6 approximately, D may be nothing or may represent a group —(CH2), —CO— in which r denotes a number equal to 4 or to 7, X is an anion.",
"Such polymers may be prepared according to the processes described in U.S. Pat.",
"Nos.",
"4,157,388, 4,702,906 and 4,719,282.They are described in particular in patent application EP-A-122 324.Among these products, mention may be made, for example, of “Mirapol A 15”, “Mirapol AD1”, “Mirapol AZ1” and “Mirapol 175” sold by the company Miranol.",
"(12) Quaternary polymers of vinylpyrrolidone and of vinylimidazole, such as, for example, the products sold under the names Luviquat FC 905, FC 550 and FC 370 by the company BASF.",
"(13) Polyamines such as Polyquart H sold by Henkel, which are given under the reference name “Polyethylene glycol (15) tallow polyamine” in the CTFA dictionary.",
"(14) Crosslinked methacryloyloxy(C1-C4)alkyltri(C1-C4)alkylammonium salt polymers such as the polymers obtained by homopolymerization of dimethylaminoethyl methacrylate quaternized with methyl chloride, or by copolymerization of acrylamide with dimethylaminoethyl methacrylate quaternized with methyl chloride, the homo- or copolymerization being followed by crosslinking with an olefinically unsaturated compound, in particular methylenebisacrylamide.",
"A crosslinked acrylamide/methacryloyl-oxyethyltrimethylammonium chloride copolymer (20/80 by weight) in the form of a dispersion containing 50% by weight of said copolymer in mineral oil can be used more particularly.",
"This dispersion is sold under the name “Salcare® SC 92” by the company Allied Colloids.",
"A crosslinked methacryloyloxyethyltrimethylammonium chloride homopolymer containing about 50% by weight of the homopolymer in mineral oil or in a liquid ester can also be used.",
"These dispersions are sold under the names “Salcare® SC 95” and “Salcare® SC 96” by the company Allied Colloids.",
"Other cationic polymers which can be used in the context of the invention are polyalkyleneimines, in particular polyethyleneimines, polymers containing vinylpyridine or vinylpyridinium units, condensates of polyamines and of epichlorohydrin, quaternary polyureylenes and chitin derivatives.",
"Among all the cationic polymers which may be used in the context of the present invention, it is preferred to use the polymers of families (1), (9), (10), (11) and (14) and even more preferably the polymers consisting of repeating units of formulae (W) and (U) below: and in particular those whose weight-average molar mass, determined by gel permeation chromatography, is between 9500 and 9900; and in particular those whose weight-average molar mass, determined by gel permeation chromatography, is about 1200.The concentration of cationic polymer in the composition according to the present invention may range from 0.01% to 10% by weight relative to the total weight of the composition, preferably from 0.05% to 5% and even more preferably from 0.1% to 3%.",
"Amphoteric Polymers The amphoteric polymers which may be used in accordance with the present invention may be chosen from polymers comprising units K and M randomly distributed in the polymer chain, in which K denotes a unit derived from a monomer comprising at least one basic nitrogen atom and M denotes a unit derived from an acidic monomer comprising one or more carboxylic or sulfonic groups, or alternatively K and M may denote groups derived from zwitterionic carboxybetaine or sulfobetaine monomers; K and M may also denote a cationic polymer chain comprising primary, secondary, tertiary or quaternary amine groups, in which at least one of the amine groups bears a carboxylic or sulfonic group linked via a hydrocarbon-based radical, or alternatively K and M form part of a chain of a polymer containing an α,β-dicarboxylic ethylene unit in which one of the carboxylic groups has been made to react with a polyamine comprising one or more primary or secondary amine groups.",
"The amphoteric polymers corresponding to the above definition that are more particularly preferred are chosen from the following polymers: (1) polymers resulting from the copolymerization of a monomer derived from a vinyl compound bearing a carboxylic group such as, more particularly, acrylic acid, methacrylic acid, maleic acid, α-chloroacrylic acid, and a basic monomer derived from a substituted vinyl compound containing at least one basic atom, such as, more particularly, dialkylaminoalkyl methacrylate and acrylate, dialkylaminoalkylmethacrylamide and -acrylamide.",
"Such compounds are described in U.S. Pat.",
"No.",
"3,836,537.Mention may also be made of the sodium acrylate/acrylamidopropyl trimethylammonium chloride copolymer sold under the name Polyquart KE 3033 by the company Henkel.",
"The vinyl compound may also be a dialkyldiallylammonium salt such as dimethyldiallylammonium chloride.",
"The copolymers of acrylic acid and of the latter monomer are sold under the names Merquat 280, Merquat 295 and Merquat Plus 3330 by the company Calgon.",
"(2) polymers containing units derived from: a) at least one monomer chosen from acrylamides and methacrylamides substituted on the nitrogen with an alkyl radical, b) at least one acidic comonomer containing one or more reactive carboxylic groups, and c) at least one basic comonomer such as esters containing primary, secondary, tertiary and quaternary amine substituents of acrylic and methacrylic acids and the product of quaternization of dimethylaminoethyl methacrylate with dimethyl or diethyl sulfate.",
"The N-substituted acrylamides or methacrylamides which are more particularly preferred according to the invention are groups in which the alkyl radicals contain from 2 to 12 carbon atoms and more particularly N-ethylacrylamide, N-tert-butylacrylamide, N-tert-octylacrylamide, N-octylacrylamide, N-decylacrylamide, N-dodecylacrylamide and the corresponding methacrylamides.",
"The acidic comonomers are chosen more particularly from acrylic acid, methacrylic acid, crotonic acid, itaconic acid, maleic acid and fumaric acid and alkyl monoesters, having 1 to 4 carbon atoms, of maleic or fumaric acids or anhydrides.",
"The preferred basic comonomers are aminoethyl, butylaminoethyl, N,N′-dimethylaminoethyl and N-tert-butylaminoethyl methacrylates.",
"The copolymers whose CTFA (4th edition, 1991) name is octylacrylamide/acrylates/butylaminoethyl methacrylate copolymer such as the products sold under the name Amphomer or Lovocryl 47 by the company National Starch are particularly used.",
"(3) crosslinked and alkylated polyamino amides partially or totally derived from polyamino amides of general formula: —[—CO—R19—CO-Z]— (X) in which R19 represents a divalent radical derived from a saturated dicarboxylic acid, a mono- or dicarboxylic aliphatic acid containing an ethylenic double bond, an ester of a lower alkanol, having 1 to 6 carbon atoms, of these acids or a radical derived from the addition of any one of said acids to a bis(primary) or bis(secondary) amine, and Z denotes a bis(primary), mono- or bis(secondary) polyalkylene-polyamine radical and preferably represents: a) in proportions of from 60 to 100 mol %, the radical where x=2 and p=2 or 3, or alternatively x=3 and p=2 this radical being derived from diethylenetriamine, from triethylenetetraamine or from dipropylenetriamine; b) in proportions of from 0 to 40 mol %, the radical (XI) above in which x=2 and p=1 and which is derived from ethylenediamine, or the radical derived from piperazine: c) in proportions of from 0 to 20 mol %, the —NH—(CH2)6—NH— radical derived from hexamethylenediamine, these polyamino amines being crosslinked by addition of a difunctional crosslinking agent chosen from epihalohydrins, diepoxides, dianhydrides and bis-unsaturated derivatives, using from 0.025 to 0.35 mol of crosslinking agent per amine group of the polyamino amide and alkylated by the action of acrylic acid, chloroacetic acid or an alkane sultone, or salts thereof.",
"The saturated carboxylic acids are preferably chosen from acids having 6 to 10 carbon atoms, such as adipic acid, 2,2,4-trimethyladipic acid and 2,4,4-trimethyladipic acid, terephthalic acid and acids containing an ethylenic double bond such as, for example, acrylic acid, methacrylic acid and itaconic acid.",
"The alkane sultones used in the alkylation are preferably propane sultone or butane sultone, and the salts of the alkylating agents are preferably the sodium or potassium salts.",
"(4) polymers containing zwitterionic units of formula: in which R20 denotes a polymerizable unsaturated group such as an acrylate, methacrylate, acrylamide or methacrylamide group, y and z represent an integer from 1 to 3, R2, and R22 represent a hydrogen atom, methyl, ethyl or propyl, R23 and R24 represent a hydrogen atom or an alkyl radical such that the sum of the carbon atoms in R23 and R24 does not exceed 10.The polymers comprising such units can also contain units derived from nonzwitterionic monomers such as dimethyl or diethylaminoethyl acrylate or methacrylate or alkyl acrylates or methacrylates, acrylamides or methacrylamides or vinyl acetate.",
"By way of example, mention may be made of the copolymer of butyl methacrylate/dimethylcarboxymethylammonioethyl methacrylate such as the product sold under the name Diaformer Z301 by the company Sandoz.",
"(5) polymers derived from chitosan, described in particular in French patent No.",
"2 137 684 or U.S. Pat.",
"No.",
"3,879,376, containing monomer units corresponding to formulae (XIII), (XIV) and (XV) below: the unit (XIII) being present in proportions of between 0 and 30%, the unit (XIV) in proportions of between 5% and 50% and the unit (XV) in proportions of between 30% and 90%, it being understood that, in this unit (XV), R25 represents a radical of formula: in which q denotes zero or 1; if q=0, R26, R27 and R28, which may be identical or different, each represent a hydrogen atom, a methyl, hydroxyl, acetoxy or amino residue, a monoalkylamine residue or a dialkylamine residue which are optionally interrupted by one or more nitrogen atoms and/or optionally substituted with one or more amine, hydroxyl, carboxyl, alkylthio or sulfonic groups, an alkylthio residue in which the alkyl group bears an amino residue, at least one of the radicals R26, R27 and R28 being, in this case, a hydrogen atom; or, if q=1, R26, R27 and R28 each represent a hydrogen atom, and also the salts formed by these compounds with bases or acids.",
"Polymers of this type that are more particularly preferred comprise from 0 to 20% by weight of units (XIII), from 40% to 50% by weight of units (XIV) and from 40% to 50% by weight of units (XV) in which R25 denotes the radical —CH2—CH2—.",
"(6) polymers derived from the N-carboxyalkylation of chitosan, such as N-carboxymethylchitosan or N-carboxybutylchitosan sold under the name “Evalsan” by the company Jan Dekker.",
"(7) polymers corresponding to the general formula (XI) as described, for example, in French patent 1 400 366: in which R29 represents a hydrogen atom, a CH3O, CH3CH2O or phenyl radical, R30 denotes hydrogen or a lower alkyl radical such as methyl or ethyl, R31 denotes hydrogen or a lower alkyl radical such as methyl or ethyl, R32 denotes a lower alkyl radical such as methyl or ethyl or a radical corresponding to the formula: —R33—N(R31)2, R33 representing a —CH2—CH2—, —CH2—CH2—CH2— or —CH2—CH(CH3)— group, R3, having the meanings mentioned above, and also the higher homologues of these radicals and containing up to 6 carbon atoms, r is such that the molecular weight is between 500 and 6 000 000 and preferably between 1000 and 1 000 000.",
"(8) amphoteric polymers of the type -D-X-D-X- chosen from: a) polymers obtained by the action of chloroacetic acid or sodium chloroacetate on compounds containing at least one unit of formula: -D-X-D-X-D- (XVII) where D denotes a radical and X denotes the symbol E or E′, E or E′, which may be identical or different, denote a divalent radical which is an alkylene radical with a straight or branched chain containing up to 7 carbon atoms in the main chain, which is unsubstituted or substituted with hydroxyl groups and which can contain, in addition to the oxygen, nitrogen and sulfur atoms, 1 to 3 aromatic and/or heterocyclic rings; the oxygen, nitrogen and sulfur atoms being present in the form of ether, thioether, sulfoxide, sulfone, sulfonium, alkylamine or alkenylamine groups, hydroxyl, benzylamine, amine oxide, quaternary ammonium, amide, imide, alcohol, ester and/or urethane groups; b) polymers of formula: -D-X-D-X- (XVIII) where D denotes a radical and X denotes the symbol E or E′ and at least once E′; E having the meaning given above and E′ being a divalent radical which is an alkylene radical with a straight or branched chain having up to 7 carbon atoms in the main chain, which is unsubstituted or substituted with one or more hydroxyl radicals and containing one or more nitrogen atoms, the nitrogen atom being substituted with an alkyl chain which is optionally interrupted by an oxygen atom and necessarily containing one or more carboxyl functions or one or more hydroxyl functions and betainized by reaction with chloroacetic acid or sodium chloroacetate.",
"(9) (C1-C5)alkyl vinyl ether/maleic anhydride copolymers partially modified by semiamidation with an N,N-dialkylaminoalkylamine such as N,N-dimethylamino-propylamine or by semiesterification with an N,N-dialkanolamine.",
"These copolymers can also contain other vinyl comonomers such as vinylcaprolactam.",
"The amphoteric polymers that are particularly preferred according to the invention are those of family (1).",
"According to the invention, the amphoteric polymer(s) may represent from 0.01% to 10% by weight, preferably from 0.05% to 5% by weight and even more preferably from 0.1% to 3% by weight relative to the total weight of the composition.",
"The compositions of the invention preferably comprise one or more surfactants.",
"The surfactants may be chosen, without discrimination, alone or as mixtures, from anionic, amphoteric, nonionic, zwitterionic and cationic surfactants.",
"The surfactants that are suitable for carrying out the present invention are especially the following: (i) Anionic Surfactant(s): By way of example of anionic surfactants which can be used, alone or as mixtures, in the context of the present invention, mention may be made in particular (nonlimiting list) of salts (in particular alkali metal salts, especially sodium salts, ammonium salts, amine salts, amino alcohol salts or magnesium salts) of the following compounds: alkyl sulfates, alkyl ether sulfates, alkylamido ether sulfates, alkylarylpolyether sulfates, monoglyceride sulfates; alkyl sulfonates, alkyl phosphates, alkylamide sulfonates, alkylaryl sulfonates, α-olefin sulfonates, paraffin sulfonates; (C6-C24)alkyl sulfosuccinates, (C6-C24)alkyl ether sulfosuccinates, (C6-C24)alkylamide sulfosuccinates; (C6-C24)alkyl sulfoacetates; (C6-C24)acyl sarcosinates; and (C6-C24)acyl glutamates.",
"It is also possible to use (C6-C24)alkylpolyglycoside carboxylic esters such as alkylglucoside citrates, alkylpoly-glycoside tartrates and alkylpolyglycoside sulfosuccinates, alkylsulfosuccinamates; acyl isethionates and N-acyl taurates, the alkyl or acyl radical of all of these different compounds preferably containing from 12 to 20 carbon atoms and the aryl radical preferably denoting a phenyl or benzyl group.",
"Among the anionic surfactants which can also be used, mention may also be made of fatty acid salts such as oleic, ricinoleic, palmitic and stearic acid salts, coconut oil acid or hydrogenated coconut oil acid; acyl lactylates in which the acyl radical contains 8 to 20 carbon atoms.",
"It is also possible to use alkyl D-galactoside uronic acids and their salts, polyoxyalkylenated (C6-C24)alkyl ether carboxylic acids, polyoxyalkylenated (C6-C24)alkylaryl ether carboxylic acids, polyoxyalkylenated (C6-C24)alkylamido ether carboxylic acids and their salts, in particular those containing from 2 to 50 alkylene oxide groups, in particular ethylene oxide groups, and mixtures thereof.",
"(ii) Nonionic Surfactant(s): The nonionic surfactants are, themselves also, compounds that are well known per se (see in particular in this respect “Handbook of Surfactants” by M. R. Porter, published by Blackie & Son (Glasgow and London), 1991, pp.",
"116-178) and their nature is not a critical factor in the context of the present invention.",
"Thus, they can be chosen in particular from (nonlimiting list) polyethoxylated or polypropoxylated, alkylphenols, alpha-diols or alcohols, having a fatty chain containing, for example, 8 to 18 carbon atoms, it being possible for the number of ethylene oxide or propylene oxide groups to range in particular from 2 to 50.Mention may also be made of copolymers of ethylene oxide and of propylene oxide, condensates of ethylene oxide and of propylene oxide with fatty alcohols; polyethoxylated fatty amides preferably having from 2 to 30 mol of ethylene oxide, polyglycerolated fatty amides containing on average 1 to 5, and in particular 1.5 to 4, glycerol groups; polyethoxylated fatty amines preferably having 2 to 30 mol of ethylene oxide; oxyethylenated fatty acid esters of sorbitan having from 2 to 30 mol of ethylene oxide; fatty acid esters of sucrose, fatty acid esters of polyethylene glycol, alkylpolyglycosides, N-alkylglucamine derivatives, and amine oxides such as (C10-C14)alkylamine oxides or N-acylaminopropylmorpholine oxides.",
"It will be noted that alkylpolyglycosides are nonionic surfactants that are particularly suitable within the context of the present invention.",
"(iii) Amphoteric or Zwitterionic Surfactant(s): The amphoteric or zwitterionic surfactants, the nature of which is not a critical factor in the context of the present invention, can be, in particular (nonlimiting list), aliphatic secondary or tertiary amine derivatives in which the aliphatic radical is a linear or branched chain containing 8 to 18 carbon atoms and containing at least one water-solubilizing anionic group (for example carboxylate, sulfonate, sulfate, phosphate or phosphonate); mention may also be made of (C8-C20)alkylbetaines, sulfobetaines, (C8-C20)alkylamido(C1-C6)alkylbetaines or (C8-C20)alkylamido(C1-C6)alkylsulfobetaines.",
"Among the amine derivatives, mention may be made of the products sold under the name Miranol, as described in U.S. Pat.",
"Nos.",
"2,528,378 and 2,781,354 and classified in the CTFA dictionary, 3rd edition, 1982, under the names Amphocarboxyglycinates and Amphocarboxypropionates, with the respective structures: R2—CONHCH2CH2—N(R3)(R4)(CH2COO−) in which: R2 denotes an alkyl radical of an acid R2—COOH present in hydrolyzed coconut oil, a heptyl, nonyl or undecyl radical, R3 denotes a beta-hydroxyethyl group and R4 denotes a carboxymethyl group; and R2—CONHCH2CH2—N(B)(C) in which: B represents —CH2CH2OX′, C represents —(CH2), —Y′, with z=1 or 2, X′ denotes the —CH2CH2—COOH group or a hydrogen atom, Y′ denotes —COOH or the —CH2—CHOH—SO3H radical, R2′ denotes an alkyl radical of an acid RG-COOH present in coconut oil or in hydrolyzed linseed oil, an alkyl radical, in particular a C7, C9, C11 or C13 alkyl radical, a C1-7 alkyl radical and its iso form, or an unsaturated C17 radical.",
"These compounds are classified in the CTFA dictionary, 5th edition, 1993, under the names Disodium Cocoamphodiacetate, Disodium Lauroamphodiacetate, Disodium Caprylamphodiacetate, Disodium Capryloamphodiacetate, Disodium Cocoampho-dipropionate, Disodium Lauroamphopropionate, Disodium Caprylamphodipropionate, Disodium Caprylamphodipropionate, Lauroamphodipropionic acid and Cocoampho-dipropionic acid.",
"By way of example, mention may be made of the cocoamphodiacetate sold under the trade name Miranol® C2M concentrate by the company Rhodia Chimie.",
"(iv) Cationic Surfactants: Among the cationic surfactants, mention may be made in particular (nonlimiting list) of: salts of optionally polyoxyalkylenated primary, secondary or tertiary fatty amines; quaternary ammonium salts such as tetraalkylammonium, alkylamidoalkyltrialkyl-ammonium, trialkylbenzylammonium, trialkylhydroxyalkylammonium or alkylpyridinium chlorides or bromides; imidazoline derivatives; or amine oxides of cationic nature.",
"The amounts of surfactants present in the composition according to the invention can range from 0.01% to 40% and preferably from 0.1% to 30% relative to the total weight of the composition.",
"The medium for the composition that is suitable for dyeing is preferably an aqueous medium consisting of water and can advantageously contain cosmetically acceptable organic solvents including, more particularly, alcohols such as ethyl alcohol, isopropyl alcohol, benzyl alcohol and phenylethyl alcohol, or polyols or polyol ethers such as, for example, ethylene glycol monomethyl, monoethyl or monobutyl ether, propylene glycol or its ethers such as, for example, propylene glycol monomethyl ether, butylene glycol or dipropylene glycol, and also diethylene glycol alkyl ethers such as, for example, diethylene glycol monoethyl ether or monobutyl ether.",
"The solvents may then be present in concentrations ranging from about 0.5% to about 20% and preferably between about 2% and 10% by weight relative to the total weight of the composition.",
"The dye composition according to the invention may also comprise an effective amount of other agents, known previously elsewhere in oxidation dyeing, such as various common adjuvants, for instance sequestering agents such as EDTA and etidronic acid, UV screening agents, waxes, volatile or nonvolatile, cyclic or linear or branched silicones, which are optionally organomodified (in particular with amine groups), preserving agents, ceramides, pseudoceramides, plant, mineral or synthetic oils, vitamins or provitamins, for instance panthenol, opacifiers, etc.",
"Said composition may also comprise reducing agents or antioxidants.",
"These agents may be chosen in particular from sodium sulfite, thioglycolic acid, thiolactic acid, sodium bisulfite, dehydroascorbic acid, hydroquinone, 2-methylhydroquinone, tert-butylhydroquinone and homogentisic acid, and, in this case, they are generally present in amounts ranging from about 0.05% to 1.5% by weight relative to the total weight of the composition.",
"Needless to say, a person skilled in the art will take care to select the optional additional compound(s) mentioned above such that the advantageous properties intrinsically associated with the dye composition according to the invention are not, or are not substantially, adversely affected by the envisaged addition(s).",
"In the ready-to-use composition or in the oxidizing composition, the oxidizing agent is preferably chosen from [lacuna] urea peroxide, alkali metal bromates or ferricyanides, and persalts such as perborates and persulfates.",
"It is particularly preferred to use hydrogen peroxide.",
"This oxidizing agent advantageously consists of an aqueous hydrogen peroxide solution whose titer may range, more particularly, from about 1 to 40 volumes and even more preferably from about 5 to 40 volumes.",
"Oxidizing agents that may also be used are one or more redox enzymes such as laccases, peroxidases and 2-electron oxidoreductases (such as uricase), where appropriate in the presence of respective donor or cofactor thereof.",
"The pH of the ready-to-use composition applied to the keratin fibers [composition resulting from mixing together the dye composition and the oxidizing composition] is generally from 4 to 11.It is preferably between 6 and 10 and may be adjusted to the desired value using acidifying or basifying agents that are well known in the prior art in the dyeing of keratin fibers.",
"Among the basifying agents which may be mentioned, for example, are aqueous ammonia, alkali metal carbonates, alkanolamines such as monoethanolamine, diethanolamine and triethanolamine and derivatives thereof, oxyethylenated and/or oxypropylenated hydroxyalkylamines and ethylenediamines, sodium hydroxide, potassium hydroxide and the compounds of formula (XIX) below: in which R is a propylene residue optionally substituted with a hydroxyl group or a C1-C4 alkyl radical; R38, R39, R40 and R41, which may be identical or different, represent a hydrogen atom, a C1-C4 alkyl radical or a C1-C4 hydroxyalkyl radical.",
"The acidifying agents are conventionally, for example, mineral or organic acids, for instance hydrochloric acid, orthophosphoric acid, carboxylic acids, for instance tartaric acid, citric acid or lactic acid, or sulfonic acids.",
"The dyeing process according to the invention preferably consists in applying the ready-to-use composition, prepared extemporaneously at the time of use from the dye composition and the oxidizing composition described above, to wet or dry keratin fibers, and leaving the composition to act for an action time preferably ranging from 1 to 60 minutes approximately and more preferably from 10 to 45 minutes approximately, rinsing the fibers and then optionally washing them with shampoo, and then rinsing them again and drying them.",
"One variant of this process consists in taking a composition comprising at least one oxidation dye that is free of amphiphilic polymer according to the invention, and another composition comprising at least one amphiphilic polymer, and mixing these two compositions with the oxidizing composition at the time of use, then applying the mixture obtained and leaving it to act as above.",
"According to said processes, each of these three compositions or the three compositions may also comprise at least one cationic or amphoteric polymer and at least one surfactant.",
"Concrete examples illustrating the invention are given below, without, however, being limiting in nature.",
"PREPARATION EXAMPLES: Preparation of Ethoxylated (Meth)Acrylic Esters: These may especially be obtained by the action of glycidyl (meth)acrylate or (meth)acrylic acid or an alkyl (meth)acrylate or a (meth)acryloyl halide on an ethoxylated fatty alcohol.",
"Nonlimiting examples that may be mentioned include the following preparations: a) starting with glycidyl methacrylate and Genapol T-250; b) starting with (meth)acrylic acid and Genapol UD-070; c) starting with methyl (meth)acrylate and Genapol LA-090; d) starting with (meth)acryloyl chloride and Genapol UD-070.a) 500 g of Genapol T-250 and 75 g of glycidyl methacrylate are placed in a one-liter three-necked reactor equipped with a stirrer, a thermometer and a reflux condenser.",
"The reaction mixture is heated at a temperature of 100° C. for 2 hours and the excess glycidyl methacrylate is removed by distillation under reduced pressure.",
"The monomer obtained may be used for the polymerization without further purification.",
"b) 500 g of Genapol UD-070, 100 g of (meth)acrylic acid and p-toluenesulfonic acid as catalyst are placed in a one-liter three-necked reactor equipped with a stirrer, a thermometer and a reflux condenser.",
"The reaction mixture is refluxed for 2 hours and the excess acid and the water formed during the reaction are separated out by distillation under reduced pressure.",
"The monomer obtained may be used for the polymerization without further purification.",
"c) 500 g of Genapol LA-090, 100 g of methyl (meth)acrylate and 20 g of titanium tetraisopropoxide are placed in a one-liter three-necked reactor equipped with a stirrer, a thermometer and a reflux condenser.",
"The reaction mixture is refluxed for 2 hours and, after separation by distilling off the alcohol formed, the remaining ester is distilled under reduced pressure.",
"The monomer obtained may be used for the polymerization without further purification.",
"d) 500 g of Genapol UD-070, 110 g of (meth)acryloyl chloride and 50 g of sodium carbonate are placed in a one-liter three-necked reactor equipped with a stirrer, a thermometer and a reflux condenser.",
"The reaction mixture is refluxed for 2 hours and the excess acid chloride is separated out by distillation under reduced pressure.",
"The monomer obtained may be used for the polymerization without further purification.",
"Polymerization According to the Precipitation Method in Tert-Butanol 500 ml of tert-butanol and the calculated amount of AMPS are placed in a 2-liter reactor equipped with a reflux condenser, a gas inlet, a thermometer and a stirrer.",
"The mixture is neutralized by introducing NH3, and the monomer prepared above is added to the reaction mixture.",
"The reaction mixture is made inert by passing nitrogen or argon through, and, when the internal temperature has reached 60° C., the initiator (AIBN) is introduced to initiate the polymerization.",
"After a few minutes, the polymer thus prepared precipitates.",
"The mixture is maintained at reflux for 2 hours, and the polymer is separated from the solvent by vacuum filtration and then dried under reduced pressure.",
"The following polymers were prepared in the manner described above: (starting with the following reagents in amounts expressed in grams' Genapol T-250 methacrylate 10 20 30 97 AMPS neutralized with NH3 90 80 90 3 Methylenebisacrylamide (crosslinking agent) 1.5 Allyl methacrylate (crosslinking agent) 1.7 TMPTA (crosslinking agent) 1.8 1.8 Azobisisobutyronitrile (initiator) 1 Dilauryl peroxide (initiator) 1 1 1 tert-Butanol 300 300 300 300 EXAMPLES OF DYE COMPOSITIONS The composition below was prepared: (amounts expressed in grams) Oxidizing composition: Fatty alcohol 2.3 Oxyethylenated fatty alcohol 0.6 Fatty amide 0.9 Glycerol 0.5 Hydrogen peroxide 7.5 Fragrance.",
"qs Demineralized water qs 100 Dye composition: Acrylamido-2-methyl-2-propanesulfonic acid/n- 1 dodecylacrylamide copolymer (3.5%/96.5%) 100% neutralized with sodium hydroxide Isostearyl alcohol (Tego Alkanol 66 sold by the 12 company Goldschmidt) Benzyl alcohol 4 Polyethylene glycol containing 8 mol of ethylene oxide 3 Ethanol 10 para-Phenylenediamine 0.54 2-Methyl-5-aminophenol 0.615 Sodium metabisulfite 0.2 Sequestering agent qs Aqueous ammonia containing 20% NH3 10 Demineralized water qs 100 The dye composition was mixed, at the time of use, in a plastic bowl and for two minutes, with the oxidizing composition given above, in a proportion of one part of dye composition per one part of oxidizing composition.",
"The mixture obtained was applied to locks of natural hair containing 90% white hairs, and was left to act for 30 minutes.",
"The locks were then rinsed with water, washed with a standard shampoo, rinsed again with water and then dried.",
"The hair was dyed in a uniform purple shade.",
"Similar results were obtained by replacing the acrylamido-2-methyl-2-propanesulfonic acid/n-dodecylacrylamide copolymer neutralized with sodium hydroxide, of the above example, with a copolymer crosslinked with methylenebisacrylamide consisting of 75% by weight of AMPS units neutralized with NH3 and 25% by weight of units of formula (III) in which R1=H, R4═C16-C18 and x=25.Similar results were obtained by replacing the acrylamido-2-methyl-2-propanesulfonic acid/n-dodecylacrylamide copolymer neutralized with sodium hydroxide, of the above example, with a copolymer crosslinked with allyl methacrylate consisting of 90% by weight of AMPS units neutralized with NH3 and 10% by weight of Genapol T-250 methacrylate units [units of formula (III) in which R1=CH3, R4=C16-C18 and x=25], or with a copolymer crosslinked with allyl methacrylate consisting of 80% by weight of AMPS units neutralized with NH3 and 20% by weight of Genapol T-250 methacrylate units [units of formula (III) in which R1=CH3, R4=C16-C18 and x=25]."
]
] | Patent_10450703 |
[
[
"Traversing firewalls and nats",
"An incoming UDP packet is allowed to traverse a network address translation (NAT) device or a firewall, wherein first, a TCP connection is opened and a Raw-IP interface is utilized to build the UDP-like packet using the parameters of the TCP connection (e.g., session number, port, etc.)",
"Furthermore, when one of two communicating machines is behind a firewall, a connection is established between each of the machines and a proxy server located in a public network.",
"The proxy then communicates the port and address information while using the proxy server's port and address information as the source port and address, or provides both with an address of an appropriate (potentially based on network proximity) packet forwarder."
],
[
"1.A method for transmitting data between a first and second device by traversing firewalls, said method comprising the steps of: a. said first and second device establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. said proxy server inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. said proxy server notifying said first device regarding said identified external mapped address CP and said proxy server notifying said second device regarding said identified external mapped address BP, and d. said first or second device spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.",
"2.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said step of spoofing TCP packets is done via Raw-IP datagrams.",
"3.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said communication link is established via TCP.",
"4.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said communication link is established via TCP/HTTP.",
"5.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said firewall is equipped with a network address translation device (NAT).",
"6.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.",
"7.A method for transmitting data between a first and second device by traversing firewalls, as per claim 1, wherein said data is streaming multimedia data.",
"8.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, said method comprising the steps of: a. said first and second device establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. said proxy server inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. said proxy server notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and d. said first device forwarding TCP packets via transmitting data with said packet forwarder as said destination address and said packet forwarder forwarding said data with CP as the destination address, or said second device forwarding TCP packets via transmitting data with said packet forwarder as said destination address and said packet forwarder forwarding said data with BP as the destination address.",
"9.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said step of forwarding TCP packets is done via Raw-IP datagrams.",
"10.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per.",
"Claim 8, wherein said communication link is established via TCP.",
"11.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said communication link is established via TCP/HTTP.",
"12.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said firewall is equipped with a network address translation device (NAT).",
"13.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.",
"14.A method for forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, as per claim 8, wherein said data is streaming multimedia data.",
"15.A system for transmitting data between a first and second device by traversing firewalls, said system comprising: a. a first host and an associated first firewall; b. a second host and an associated second firewall; c. a proxy server that establishes a communication link with said first and second host, identifies from said first and second firewalls external mapped addresses BP and CP respectively, and forwards said CP to said first device and forwards said BP to said second device, whereupon said first and second host utilize said forwarded external mapped addresses to spoof TCP packets and forward data directly between each other.",
"16.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said proxy server forwards TCP packets via Raw-IP datagrams.",
"17.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said communication link is established via TCP.",
"18.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said communication link is established via TCP/HTTP.",
"19.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said firewall is equipped with a network address translation device (NAT).",
"20.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.",
"21.A system for transmitting data between a first and second device by traversing firewalls, as per claim 15, wherein said data is streaming multimedia data.",
"22.A system for transmitting data between a first and second device by traversing firewalls, said system comprising: a. a first host and an associated first firewall; b. a second host and an associated second firewall; c. a proxy server that establishes a communication link with said first and second host, identifies from said first and second firewalls external mapped addresses BP and CP respectively; d. a packet forwarder receiving BP and CP from said proxy server and using BP and CP to forward incoming communications from said first device to second device with CP as destination address, or forwarding incoming communications from said second device to first device with BP as the destination address.",
"23.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said packet forwarder forwards TCP packets via Raw-IP datagrams.",
"24.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said communication link is established via TCP.",
"25.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said communication link is established via TCP/HTTP.",
"26.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said firewall is equipped with a network address translation device (NAT).",
"27.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said network is any of: local area network (LAN), wide area network (WAN), wireless network, or the Internet.",
"28.A system for transmitting data between a first and second device by traversing firewalls, as per claim 22, wherein said data is streaming multimedia data.",
"29.An article of manufacture comprising a computer usable medium having computer readable program code embodied therein for assisting in the transmission of data between a first and second device by traversing firewalls, said article further comprising: a. computer readable program code aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. computer readable program code inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. computer readable program code notifying said first device regarding said identified external mapped address CP and computer readable program code notifying said second device regarding said identified external mapped address BP, and d. computer readable program code aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.",
"30.An article of manufacture comprising a computer usable medium having computer readable program code embodied therein for aiding in forwarding data between a first and second device by traversing firewalls, said data forwarded via a packet forwarder, said medium further comprising: a. computer readable program code aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; b. computer readable program code inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; c. computer readable program code notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and d. computer readable program code forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or computer readable program code forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of Invention The present invention relates generally to the field of network communications.",
"More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).",
"2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.",
"Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).",
"The inability to send and receive information when opening TCP communications into the private network.",
"Each of the below described references teach the method of firewalls in general.",
"However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).",
"U.S. Pat.",
"No.",
"5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.",
"Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.",
"A time limit is set and so long as the set time limit does not run out, the communication is allowed.",
"Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.",
"A connection type session does not appear to be initiated for the UDP transport.",
"U.S. Pat.",
"No.",
"5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.",
"The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.",
"The firewall then binds a port and notes it in a link table.",
"The packet is passed to a stack, on the private side, which forwards the packet to the server.",
"Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.",
"U.S. Pat.",
"No.",
"5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.",
"A client on the public network connects to the external machine.",
"A private channel is opened between the external machine and a machine internal to the private network.",
"The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.",
"U.S. Pat.",
"No.",
"5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).",
"When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.",
"The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.",
"It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.",
"Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.",
"Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).",
"Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.",
"Connections are established between each machine and a proxy server sitting on a public network.",
"The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address."
],
[
"RELATED APPLICATIONS The present application claims the benefit of provisional patent application “Traversing Firewalls and NATs”, Ser.",
"No.",
"60/255,422, filed Dec. 14, 2000.In addition, this application incorporates by reference, co-pending U.S. patent application Ser.",
"No.",
"09/867,371, filed May 29, 2001.BACKGROUND OF THE INVENTION 1.Field of Invention The present invention relates generally to the field of network communications.",
"More specifically, the present invention is related to a system and method for traversing firewalls and network address translators (NATs).",
"2.Discussion of Prior Art NATs and firewalls present a challenge to a network software programming, while their functions and operations are different: firewalls filter information into and out of the private network, while NATs hide or encapsulate a private network behind a single (or few) “real” Internet Protocol addresses.",
"Their effect on many network applications is the same: The inability to send and receive information when receiving information using UDP (e.g., UDP data-grams coming into the private network).",
"The inability to send and receive information when opening TCP communications into the private network.",
"Each of the below described references teach the method of firewalls in general.",
"However, none of the references provide or suggest the present invention method of ATM over IP traversing firewalls and network address translators (NATs).",
"U.S. Pat.",
"No.",
"5,898,830, assigned to Network Engineering Software describes a system, which allows connectionless traffic across a firewall.",
"Rule checking is performed on the first packet entering, and if it is determined that the packet needs to be sent, a virtual host sends it to the destination computer.",
"A time limit is set and so long as the set time limit does not run out, the communication is allowed.",
"Addressing is accomplished utilizing name based addressing for end-to-end communication, with virtual hosts/DNS servers providing the intermediate address routing information.",
"A connection type session does not appear to be initiated for the UDP transport.",
"U.S. Pat.",
"No.",
"5,915,087 discloses a firewall system, which allows communication, using a connectionless protocol.",
"The firewall holds a list of servers located on the private side, and intercepts any communications addressed to the servers.",
"The firewall then binds a port and notes it in a link table.",
"The packet is passed to a stack, on the private side, which forwards the packet to the server.",
"Any communications from the server to the originating client is sent to the firewall, where the originating clients address is determined using the link table.",
"U.S. Pat.",
"No.",
"5,778,174 describes a system, which utilizes an external machine, located on a public network to bypass a router firewall.",
"A client on the public network connects to the external machine.",
"A private channel is opened between the external machine and a machine internal to the private network.",
"The internal machine connects to the destination server, and communication between the client and server is conducted through the external and internal machines.",
"U.S. Pat.",
"No.",
"5,941,988 provides for a proxy system that “glues” together two separate TCP connections terminating at a common host (proxy).",
"When communications from one connection are received at the proxy, the headers are altered to address the socket at the end of the second connection, and the sequence numbers of the first connection are mapped to the sequence space of the second connection.",
"The non-patent literature entitled, “A Weakness in the 4.2 BSD Unix TCP/IP Software” describes the spoofing of a trusted host to communicate with a system, having a list of the trusted hosts, from a host that is not on the trusted list.",
"It should however be noted that the prior art described above fails to provide many features, for example an explicit recitation of opening a connection-oriented session in order to allow connectionless data-grams to pass through a NAT/firewall is not provided.",
"Additionally, none of the prior art described above uses a proxy server to exchange respective address information between two hosts and the hosts communicating directly via the address information and spoofing the proxy, in order to traverse at least one firewall.",
"Whatever the precise merits, features and advantages of the above cited references, none of them achieve or fulfills the purposes of the present invention.",
"SUMMARY OF THE INVENTION The present invention provides for a method and a system for allowing an incoming UDP packet to traverse a NAT/firewall comprising, opening a TCP connection and utilizing a Raw-IP interface which builds the UDP packet utilizing the parameters of the TCP connection (e.g., session number, port, etc.).",
"Furthermore, the present system provides for a method and system for allowing communication between two machines, at least one of which is behind a firewall.",
"Connections are established between each machine and a proxy server sitting on a public network.",
"The proxy then communicates the port and address information of each machine to the other machine, after which, each machine sends directly to each other using the supplied port and address information, while using the proxy servers port and address information as the source port and address.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 illustrates the Intranet to Internet data transfer scenario.",
"FIG.",
"2 illustrates the Internet to Intranet data transfer scenario.",
"FIG.",
"3 illustrates the Intranet to Intranet data transfer scenario.",
"FIG.",
"4 illustrates a bi-directional connection, using TCP and HTTP, communicating indirectly with the proxy.",
"FIG.",
"5 illustrates TCP spoofing of the present invention.",
"FIG.",
"6 illustrates TCP spoofing of the present invention in the presence of a packet forwarder.",
"FIG.",
"7 illustrates the methodology associated with the present invention.",
"DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is illustrated and described in a preferred embodiment, the invention may be produced in many different configurations, forms and materials.",
"There is depicted in the drawings, and will herein be described in detail, a preferred embodiment of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and the associated functional specifications for its construction and is not intended to limit the invention to the embodiment illustrated.",
"Those skilled in the art will envision many other possible variations within the scope of the present invention.",
"When a communicating device, such as the Internet phone or a Voice-over-IP Gateway or an IETF MGCP Gateway or an ITU-T H.248 Gateway or a PacketCable Residential Gateway or a CPE Gateway (Customer premises equipment Gateway), opens a signaling connection from a private network to a public network, the TCP channel is bi-directional, and therefore the signaling protocol can execute in both directions.",
"This also allows HTTP to work behind network address translators (NATs) and Firewalls.",
"Connections are always opened from the private network to the public network; taking advantage of the fact that TCP data communications are bi-directional.",
"NATs present an additional translation step when communicating.",
"NATs map the source addresses (in the private network) of the originating computer into a public address and a port number on the public interface of the NAT.",
"As long as TCP is used, the translation can be done in reverse, as the TCP channel is bi-directional.",
"Multimedia signaling and media streaming is usually UDP-based for better efficiency, which introduces the problem—the ingress system sends UDP packets to the public interface on the NAT, and the NAT has no automatic method to map this UDP data-gram to the actual computer that is supposed to receive that data-gram.",
"The solution provided by the present invention is to stream audio and video (and other time-sensitive data) over TCP, but TCP streaming and windowing mechanizing hurts the real-time performance.",
"The present invention opens a TCP connection as usual (using TCP), and then switches to a Raw-IP interface that sends Raw-IP data-grams that are legal TCP messages using just opened TCP channel parameters (e.g., session number, port, etc.)",
"To an intermediate system, these messages will look like standard TCP messages, but as they are sent using Raw-IP, the usual timing issues that TCP introduces to real-time media streaming are not in place.",
"Thus, the present invention uses the protocol software to “spoof” the TCP channel to enable real-time TCP communications.",
"It is impossible with NATs and Firewalls to open ingress connections (e.g., it is impossible to open a TCP connection to a computer behind a Firewall or the NAT.",
"Thus: 1.It is impossible to originate communications from the public network into the private network.",
"2.It is impossible to originate communications from a private network (to a public network) to another private network.",
"Thus, the present invention uses a server proxy that both communication parties open their TCP channels to (using the previous procedure).",
"Then, the proxy communicates to each party the other party's source address/port (of the TCP channel).",
"Finally, each communication element sends information to the other party using the server proxy source address/port.",
"It should be noted that packets are sent directly between the communicating entities, as the proxy is only used to hold the TCP state to “spoof” the NATs and Firewalls.",
"In the preferred embodiment of the present invention, full communication is possible between all types of private and public networks, as long as outgoing TCP channel establishment is allowed.",
"In another embodiment, the server proxy and originating clients use TCP/HTTP, which is universally supported, and in this instance, all information is tunneled over the simulated TCP/HTTP channel.",
"In the Internet as it exists today, using the small address space provided by IPv4, many networks deploy NAT (network address translation) devices to enlarge the internal address space.",
"In addition, many networks deploy firewall devices to block intrusions and hacking.",
"Many firewalls also support integrated NAT capabilities.",
"The end-result of both types of devices is that ingress traffic (one originating outside the network and destined into the network) is usually blocked, as incoming connections are usually blocked for firewalls and are impossible to complete on NAT devices, and as the originating (outside the NAT) IP host is unaware of the destination internal IP address.",
"Thus, users cannot place audio/video calls from NAT protected networks (as the audio and video will not penetrate back into the network from the remote called host), and in many cases users behind corporate firewalls are blocked from using such services.",
"A communications protocol such as the TrulyGlobal™ Protocol (TGP), (as described in the related application, “Communication Protocol”) can be used in conjunction with the present invention to operate over standard HTTP and remote TGP servers to use the HTTP back-channel to send information to the client; and ensuring that all actions carried by TGP traverse both NATs and firewalls.",
"Intranet, as defined in this application, is a network that is protected by a NAT or a firewall device, and blocks all incoming traffic into the protected network (e.g., TCP connections cannot be initiated into the network, and UDP traffic will be blocked at the entry-point into the network).",
"Similarly, Internet is defined as a public addressed, unprotected network, where fill IP communication is possible.",
"In the Intranet to Internet scenario, as illustrated in FIG.",
"1, a user inside an Intranet is attempting to call a user that is outside the Intranet, and the remote user is in the public Intranet.",
"The problem encountered is that while the call will be successfully set-up (as the originating host is allowed to open connection to the outside network), audio and/or video data will not be able to get back into the Intranet, hence the caller will not be able to hear and/or see the called device.",
"Similarly, in an Internet to Intranet scenario, as illustrated in FIG.",
"2, the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet are not allowed.",
"Lastly, in the Intranet to Intranet scenario, as illustrated in FIG.",
"3, the same end-effect as the previous scenario happens, e.g., the caller cannot open a signaling channel at all to the called device, as ingress connections into the Intranet from the Internet are not allowed.",
"The solution provided for by the present invention uses TCP and potentially HTTP, and a service is provided outside the Intranet (in the public Internet) to help both end-points to complete calls.",
"The first assumption made is that the clients inside the Intranet can initiate TCP or at least TCP/HTTP specifically to the public Internet, so some form of communications is possible.",
"HTTP can be used to insure safe traversal via HTTP proxies.",
"Once a TCP/HTTP connection is available, bi-directional communications are possible.",
"Outwards messages use standard HTTP commands to request resources (using URLs), and incoming information flow returns using the HTTP reply channel (as TCP/HTTP is full-duplex).",
"Furthermore, at any time, as illustrated by FIG.",
"4, the caller can initiate a TCP/HTTP connection (or a plain TCP connection) to a service that resides in the public Internet, and that service is responsible to “proxy” the request (using the reply leg of the remote HTTP session) to the called-device.",
"When both devices have bi-directional connections to the proxy, they can communicate indirectly via the proxy.",
"While the present invention shows how proxy-based communication can work, the problem when the communication between the two hosts has to flow via the proxy, which adds delay and has limited scalability.",
"The solution as per the present invention is to spoof the TCP session to allow direct TCP communications between the two machines.",
"This scenario is illustrated in FIG.",
"5.When a machine behind a proxy, NAT or firewall establishes a session with the outside world, the session is mapped on the outside of Intranet 1 and 2 on the public interface address(s) to an internal connection between Host 1 and 2 and their gateways to the Internet.",
"Sending correctly formed TCP packets to that interface will result in the gateway forwarding these packets to the correct host inside the private network.",
"The sequence is as follows: 1.A session is established from Host 1 in Intranet 1 to the Proxy (AB session).",
"2.A session is established from Host 2 in Intranet 2 to the Proxy (DC session).",
"3.The Address BP (public side of session A) is found by inspecting the source address/port of B.",
"4.The Address of CP (public side of session D) is found by inspecting the source address/port of C. 5.The external mapped addresses are provided to the other hosts, i.e., Host 1 is provided with address/pair CP and Host 2 is provided with address/port pair BP.",
"TCP session B parameters are provided to host 2.TCP session C parameters are provided to host 1.6.Hosts 1 and 2 will spoof TCP packets for sessions B and C, sent to target address/port pairs BP and CP.",
"This traffic will go directly between the two networks and not via the Proxy.",
"In effect, a virtual TCP session C1/B1 is created by combining the two existing TCP sessions C and B.",
"Once the spoofed TCP session is handed over to Hosts 1 and 2, the Proxy should not send any information on that session, as session parameters may be out-of-date.",
"The session is kept open for the duration Hosts 1 and 2 requires it, and will be closed by either Host when required.",
"The proxy is only used for establishing session, and does not use the session for anything else once it is “handed over”.",
"In some cases the internal network will filter spoofed packets (for security, e.g., hack prevention) and therefore will not let the packets with the spoofed source address leave the internal network.",
"In such cases, as illustrated by FIG.",
"6, the TCP connections will be handed over to a packet forwarder (that resides in the same server or a separate server) that handles the packet interchange.",
"One of the side effects of spoofing the TCP session between Hosts 1 and 2 is that the TCP session parameters can be changed or completely ignored, as long as packets are synthetically correct (as per TCP), they can be sent without consideration to window-sizes, exponential back-off algorithms or slow-start mechanisms.",
"When such a spoofed TCP session is in place, it can be used to transmit both audio and video with the same time of performance that is expected from UDP.",
"The session-establishment procedures described above allow any session to be established between any two computers.",
"This is done as a result of Host 1 calling Host 2 (or the reverse).",
"The calling host will send a Call-Establishment message to the Proxy, which will (pending, any policy decision) forward the request to the called Host.",
"The called host will receive the Call Answer transaction over the back-channel of the session it already has with the Proxy, requesting it to answer the call.",
"If the called host responses positively, one or more media channel(s) will be established between Host 1 and 2, with the help of the proxy as required by the session's parameters (audio only, audio and video, etc).",
"It should be noted that both IETF SIP and ITU-T H.323 signaling can be used, but are not required.",
"In one embodiment, the Proxy contains all the required functionality (e.g., signaling a RTP:Address:Port destination instead of a H323:Address:Port destination).",
"Furthermore, one skilled in the art can recognize that IETF SIP (by manipulating IETF Session Description Protocol (SDP) parameters) and ITU H.323 (by Manipulating ITU-T H.245 OpenLogicalChannel or FastStart parameters) can be used, with minor changes, to accomplish the required signaling.",
"The present invention is implemented using a raw-IP interfaces that spoofs the TCP sessions.",
"A limited TCP stack is implemented that creates synthetically correct TCP packets, to insure the packets are interpreted and forwarded correctly by the NATs, proxies and firewalls in the way.",
"Such a spoofed-TCP stack does not need to support any reliable transmission, as it is only used for real-time sensitive transmission purposes.",
"FIG.",
"7 summarizes the methodology 700 associated with the present invention.",
"In step 702, both hosts establish a connection (e.g., TCP connection or TCP/HTTP connection) with a TCP proxy server.",
"Next, in step 704, external mapped addresses BP and CP associated with the firewalls of both hosts are identified.",
"Subsequently, in step 706, the identified external mapped addresses are exchanged between the two hosts.",
"Lastly, the TCP packets are spoofed to transmit the data (e.g., streaming multimedia data) between the hosts.",
"Furthermore, the present invention includes a computer program code based product, which is a storage medium having program code stored therein, which can be used to instruct a computer to perform any of the methods associated with the present invention.",
"The computer storage medium includes any of, but not limited to, the following: CD-ROM, DVD, magnetic tape, optical disc, hard drive, floppy disk, ferroelectric memory, flash memory, ferromagnetic memory, optical storage, charge coupled devices, magnetic or optical cards, smart cards, EEPROM, EPROM, RAM, ROM, DRAM, SRAM, SDRAM, or any other appropriate static or dynamic memory, or data storage devices.",
"Implemented in computer program code based products are software modules for: aiding in establishing a communication link with a proxy server over a network, wherein a first and second device can access the network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said first device regarding said identified external mapped address CP and notifying said second device regarding said identified external mapped address BP; and aiding said first or second device in spoofing TCP packets via transmitting data with said notified external mapped address as the destination address.",
"Also implemented in computer program based products are software modules for: aiding in establishing a communication link with a proxy server over a network, each of said first and second devices accessing said network over a firewall; inspecting said firewalls and identifying an external mapped addresses BP associated with said first device and identifying an external mapped address CP associated with said second device; notifying said packet forwarder regarding said identified external mapped addresses CP and BP, and forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with CP as the destination address, or forwarding TCP packets via transmitting data with said packet forwarder as said destination address and computer readable program code aiding said packet forwarder in forwarding said data with BP as the destination address.",
"CONCLUSION A system and method has been shown in the above embodiments for the effective implementation of a method and a system for traversing firewalls and network address translations (NATs).",
"While various preferred embodiments have been shown and described, it will be understood that there is no intent to limit the invention by such disclosure, but rather, it is intended to cover all modifications and alternate constructions falling within the spirit and scope of the invention, as defined in the appended claims.",
"For example, the present invention should not be limited by type of firewall, type of network address translation device, location of packet forwarder, software/program, computing environment, or specific computing hardware.",
"The above enhancements are implemented in various computing environments.",
"For example, the present invention may be implemented on a multi-nodal system (e.g., LAN) or networking system (e.g., Internet, WWW, wireless web).",
"All programming, and data related thereto are stored in computer memory, static or dynamic, and may be retrieved by the user in any of: conventional computer storage, display (i.e., CRT) and/or hardcopy (i.e., printed) formats.",
"The programming of the present invention may be implemented by one of skill in the art of network communications."
]
] | Patent_10450751 |
[
[
"Novel nucleic acids and polypeptides",
"The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof."
],
[
"1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, and complementary sequences thereof.",
"2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.",
"3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.",
"5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.",
"6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.",
"10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-30368.11.A composition comprising the polypeptide of claim 10 and a carrier.",
"12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.",
"14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.",
"15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.",
"16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.",
"17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.",
"18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.",
"19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-30368, a mature protein coding portion of SEQ ID NO: 1-30368, an active domain of SEQ ID NO: 1-30368, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-30368, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).",
"20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30369-60736, the mature protein portion thereof, or the active domain thereof.",
"21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.",
"22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-30368.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.",
"24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.",
"25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.",
"26.The collection of claim 22, wherein the collection is provided in a computer-readable format.",
"27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.",
"28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier."
],
[
"<SOH> 2.BACKGROUND <EOH>Technology aimed at the discovery of protein factors (including e.g.",
"cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.",
"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).",
"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.",
"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences."
],
[
"<SOH> 3.SUMMARY OF THE INVENTION <EOH>The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.",
"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.",
"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.",
"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.",
"These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.",
"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.",
"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.",
"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.",
"The identifying sequence can be 100 base pairs in length.",
"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.",
"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.",
"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.",
"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.",
"The collection can also be provided in a computer-readable format.",
"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.",
"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.",
"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.",
"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.",
"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.",
"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.",
"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.",
"host cells) of the invention.",
"The invention also provides compositions comprising a polypeptide of the invention.",
"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.",
"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.",
"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.",
"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.",
"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.",
"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.",
"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.",
"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.",
"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.",
"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.",
"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.",
"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.",
"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.",
"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.",
"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.",
"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.",
"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.",
"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.",
"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.",
"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.",
"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.",
"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.",
"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.",
"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.",
"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.",
"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).",
"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these-polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.",
"2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g.",
"cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade.",
"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).",
"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.",
"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.",
"3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.",
"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.",
"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.",
"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.",
"These nucleic acid sequences are designated as SEQ ID NO: 1-30368.The polypeptides sequences are designated SEQ ID NO: 30369-60736.The nucleic acids and polypeptides are provided in the Sequence Listing.",
"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.",
"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.",
"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-30368 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-30368.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-30368 or a degenerate variant or fragment thereof.",
"The identifying sequence can be 100 base pairs in length.",
"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-30368.A collection as used in this application can be a collection of only one polynucleotide.",
"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.",
"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.",
"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.",
"The collection can also be provided in a computer-readable format.",
"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.",
"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the arL In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-30368 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-30368; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-30368.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 30369-60736); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.",
"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.",
"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.",
"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-30368; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.",
"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.",
"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.",
"host cells) of the invention.",
"The invention also provides compositions comprising a polypeptide of the invention.",
"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.",
"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.",
"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.",
"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.",
"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.",
"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.",
"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.",
"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.",
"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.",
"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.",
"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.",
"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.",
"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.",
"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.",
"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.",
"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.",
"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.",
"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.",
"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.",
"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.",
"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.",
"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.",
"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.",
"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.",
"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.",
"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).",
"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.",
"4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.",
"The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.",
"According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.",
"Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.",
"The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.",
"The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.",
"For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.",
"Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.",
"The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.",
"The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.",
"The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.",
"The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.",
"PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.",
"Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.",
"The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.",
"As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.",
"EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).",
"One class of EMEs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.",
"The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.",
"These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.",
"In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).",
"It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).",
"Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.",
"The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.",
"The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.",
"Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.",
"Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.",
"A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.",
"Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-30368.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.",
"(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).",
"They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.",
"Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.",
"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-30368.The sequence information can be a segment of any one of SEQ ID NO: 1-30368 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-30368.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.",
"Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.",
"Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.",
"The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.",
"Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.",
"The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).",
"The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.",
"The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.",
"The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.",
"The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.",
"For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.",
"While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.",
"repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.",
"The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.",
"A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.",
"The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.",
"A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.",
"The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.",
"Preferably the peptide is from about 5 to about 200 amino acids.",
"To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.",
"The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.",
"The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.",
"The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.",
"The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.",
"The peptide may have been produced by processing in the cell which removes any leader/signal sequence.",
"The mature protein portion may or may not include an initial methionine residue.",
"The methionine residue may be removed from the protein during processing in the cell.",
"The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.",
"The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.",
"The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.",
"Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.",
"Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.",
"Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.",
"Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.",
"Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.",
"“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.",
"For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.",
"“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.",
"The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.",
"Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.",
"Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.",
"For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.",
"Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.",
"For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.",
"The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.",
"In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).",
"The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.",
"In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.",
"The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.",
"The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.",
"“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.",
"As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.",
"Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.",
"The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.",
"An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.",
"Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.",
"Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.",
"This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.",
"The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.",
"Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.",
"This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.",
"Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.",
"The cells can be prokaryotic or eukaryotic.",
"The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.",
"“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.",
"“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.",
"“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.",
"Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.",
"Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.",
"(1998) Annu.",
"Rev.",
"Immunol.",
"16:27-55).",
"Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.",
"Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.",
"The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.",
"Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).",
"Other exemplary hybridization conditions are described herein in the examples.",
"In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).",
"As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.",
"Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).",
"Such a sequence is said to have 65% sequence identity to the listed sequence.",
"In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).",
"Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.",
"Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.",
"Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.",
"For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.",
"For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.",
"Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.",
"(1990) Methods Enzymol.",
"183:626-645).",
"Identity between sequences can also be determined by other methods known in the art, e.g.",
"by varying hybridization conditions.",
"The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.",
"The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.",
"The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.",
"The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.",
"As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.",
"UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.",
"The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.",
"The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.",
"As described above, a UMF will increase the frequency of uptake of a linked marker sequence.",
"Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.",
"4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.",
"The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-30368; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 30369-60736; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 30369-60736.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-30368; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 30369-60736.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.",
"The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.",
"The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.",
"The present invention also provides genes corresponding to the cDNA sequences disclosed herein.",
"The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.",
"Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.",
"Further 5′ and 3′ sequence can be obtained using methods known in the art.",
"For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-30368 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-30368 or a portion there of as a probe.",
"Alternatively, the polynucleotides of SEQ ID NO: 1-30368 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.",
"The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.",
"The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.",
"The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.",
"Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.",
"Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-30368, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.",
"Fragments of, e.g.",
"15, 17, or 20 nucleotides or more that are selective for (i.e.",
"specifically hybridize to any one of the polynucleotides of the invention) are contemplated.",
"Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.",
"The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.",
"Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-30368, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-30368 with a sequence from another isolate of the same species.",
"Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.",
"In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.",
"The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-30368 can be obtained by searching a database using an algorithm or a program.",
"Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.",
"Evol.",
"36 290-300 (1993) and Altschul S. F. et al.",
"J. Mol.",
"Biol.",
"21:403-410(1990)).",
"Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.",
"Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.",
"Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.",
"The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.",
"The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.",
"These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.",
"There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.",
"Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.",
"These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).",
"Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.",
"Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.",
"Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.",
"Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.",
"Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.",
"In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.",
"This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.",
"In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).",
"A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.",
"10:6487-6500 (1982).",
"PCR may also be used to create amino acid sequence variants of the novel nucleic acids.",
"When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.",
"PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.",
"The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.",
"A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.",
"Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.",
"Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.",
"Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.",
"The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.",
"The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.",
"Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.",
"In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-30368, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.",
"Also included are the cDNA inserts of any of the clones identified herein.",
"A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.",
"(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).",
"Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.",
"Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.",
"In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.",
"Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.",
"A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.",
"The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof or any other polynucleotides of the invention.",
"In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-30368 or a fragment thereof is inserted, in a forward or reverse orientation.",
"In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.",
"Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.",
"The following vectors are provided by way of example.",
"Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).",
"Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).",
"The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.",
"19, 44854490 (1991), in order to produce the protein recombinantly.",
"Many suitable expression control sequences are known in the art.",
"General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).",
"As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.",
"Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.",
"Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.",
"Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.",
"Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.",
"Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TP1gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.",
"Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.",
"The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.",
"Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.",
"Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.",
"The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.",
"Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.",
"As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).",
"Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).",
"These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.",
"Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.",
"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.",
"Polynucleotides of the invention can also be used to induce immune responses.",
"For example, as described in Fan et al, Nat.",
"Biotech.",
"17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.",
"The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.",
"4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-30368, or fragments, analogs or derivatives thereof.",
"An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.",
"In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.",
"Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 30369-60736 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-30368 are additionally provided.",
"In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.",
"The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.",
"In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.",
"The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).",
"Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-30368), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.",
"The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.",
"For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.",
"An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.",
"An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.",
"For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.",
"Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.",
"Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).",
"The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.",
"The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.",
"An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.",
"Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.",
"For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.",
"The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.",
"To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.",
"In yet another embodiment, the antisense nucleic acid molecule of the invention is an-anomeric nucleic acid molecule.",
"An-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual-units, the strands run parallel to each other (Gaultier et al.",
"(1987) Nucleic Acids Res 15: 6625-6641).",
"The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.",
"(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.",
"(1987) FEBS Lett 215: 327-330).",
"4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.",
"Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.",
"Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.",
"A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-30368).",
"For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-30368 (see, e.g., Cech et al.",
"U.S. Pat.",
"No.",
"4,987,071; and Cech et al.",
"U.S. Pat.",
"No.",
"5,116,742).",
"Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.",
"See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.",
"See generally, Helene.",
"(1991) Anticancer Drug Des.",
"6: 569-84; Helene.",
"et al.",
"(1992) Ann.",
"N.Y. Acad.",
"Sci.",
"660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.",
"For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.",
"(1996) Bioorg Med Chem 4: 5-23).",
"As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.",
"The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.",
"The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.",
"(1996) above; Perry-O'Keefe et al.",
"(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.",
"For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.",
"PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymnes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.",
"(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.",
"(1996), above; Perry-OKeefe (1996), above).",
"In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.",
"For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.",
"Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.",
"PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).",
"The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.",
"(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.",
"(1989) Nucl Acid Res 17: 5973-88).",
"PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.",
"(1996) above).",
"Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.",
"See, Petersen et al.",
"(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.",
"Natl.",
"Acad.",
"Sci.",
"U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.",
"Natl.",
"Acad.",
"Sci.",
"84:648-652; PCT Publication No.",
"WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No.",
"WO89/10134).",
"In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.",
"Res.",
"5:539-549).",
"To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.",
"4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.",
"For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.",
"The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.",
"Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.",
"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.",
"The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.",
"See, for example, PCT International Publication No.",
"WO94/12650, PCT International Publication No.",
"WO92/20808, and PCT International Publication No.",
"WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.",
"If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.",
"The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.",
"Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).",
"The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.",
"Any host/vector system can be used to express one or more of the ORFs of the present invention.",
"These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.",
"The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.",
"Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.",
"Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.",
"Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.",
"Various mammalian cell culture systems can also be employed to express recombinant protein.",
"Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).",
"Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.",
"Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.",
"DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.",
"Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.",
"Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.",
"Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.",
"Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.",
"Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.",
"Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.",
"Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.",
"If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.",
"Such covalent attachments may be accomplished using known chemical or enzymatic methods.",
"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.",
"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.",
"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.",
"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.",
"These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.",
"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.",
"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.",
"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.",
"Here, the naturally occurring sequences are deleted and new sequences are added.",
"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.",
"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative, selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.",
"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.",
"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.",
"No.",
"5,272,071 to Chappel; U.S. Pat.",
"No.",
"5,578,461 to Sherwin et al.",
"; International Application No.",
"PCT/US92/09627 (WO93/09222) by Selden et al.",
"; and International Application No.",
"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.",
"4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 30369-60736 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-30368 or the corresponding full length or mature protein.",
"Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-30368 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.",
"The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 30369-60736 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.",
"Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 30369-60736.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.",
"Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.",
"Chem.",
"Soc.",
"114, 9245-9253 (1992), both of which are incorporated herein by reference.",
"Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.",
"The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.",
"The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.",
"The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.",
"The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.",
"Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.",
"In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.",
"Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.",
"The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.",
"By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.",
"Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.",
"A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.",
"At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.",
"The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.",
"This technique is particularly useful in producing small peptides and fragments of larger polypeptides.",
"Fragments are useful, for example, in generating antibodies against the native polypeptide.",
"Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.",
"The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.",
"As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.",
"One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.",
"The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.",
"For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.",
"The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.",
"Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.",
"In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.",
"One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.",
"These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.",
"See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.",
"Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.",
"The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.",
"These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.",
"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.",
"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.",
"In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.",
"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 30369-60736.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.",
"The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.",
"For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.",
"Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.",
"For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.",
"Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.",
"No.",
"4,518,584).",
"Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.",
"Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.",
"This type of analysis determines the importance of the substituted amino acid(s) in biological activity.",
"Regions of the protein that are important for protein function may be determined by the eMATRIX program.",
"Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.",
"Such modifications are encompassed by the present invention.",
"The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.",
"Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.",
"1555 (1987), incorporated herein by reference.",
"As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.",
"The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.",
"The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.",
"Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.",
"For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX), or as a His-tag.",
"Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.",
"), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.",
"The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.",
"One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).",
"Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.",
"Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.",
"The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).",
"This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.",
"Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.",
"Such analogs may exhibit improved properties such as activity and/or stability.",
"Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.",
"Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.",
"Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.",
"4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.",
"Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.",
"Biol.",
"215:403410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.",
"vol.",
"25, pp.",
"3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.",
"Biol., Vol.",
"6, pp.",
"219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.",
"4, pp.",
"202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.",
"26(1), pp.",
"320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.",
"Biol, 157, pp.",
"105-31 (1982), incorporated herein by reference).",
"The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.",
"NCB NLM NIH Bethesda, Md.",
"20894; Altschul, S., et al., J. Mol.",
"Biol.",
"215:403-410 (1990).",
"4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.",
"As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.",
"Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.",
"In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.",
"In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.",
"Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.",
"The polypeptide can be fused to the N-terminus or C-terminus.",
"For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.",
"In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.",
"In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.",
"The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.",
"The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.",
"Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g.",
"promoting or inhibiting) cell survival.",
"Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.",
"A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.",
"For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.",
"In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.",
"Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.",
"(eds.)",
"CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).",
"Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).",
"A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.",
"4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.",
"The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.",
"Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).",
"See, for example, Anderson, Nature, supplement to vol.",
"392, no.",
"6679, pp.",
"25-20 (1998).",
"For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).",
"Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).",
"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.",
"Treated cells can then be introduced in vivo for therapeutic purposes.",
"Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.",
"It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.",
"Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.",
"Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.",
"The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.",
"These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.",
"Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.",
"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.",
"The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.",
"See, for example, PCT International Publication No.",
"WO 94/12650, PCT International Publication No.",
"WO 92/20808, and PCT International Publication No.",
"WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.",
"If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplificationof the desired protein coding sequences in the cells.",
"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.",
"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.",
"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.",
"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.",
"These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.",
"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.",
"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.",
"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.",
"Here, the naturally occurring sequences are deleted and new sequences are added.",
"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.",
"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.",
"Markers useful for this purpose include the Herpes Sinplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphonbosyl-transferase (gpt) gene.",
"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.",
"No.",
"5,272,071 to Chappel; U.S. Pat.",
"No.",
"5,578,461 to Sherwin et al.",
"; International Application No.",
"PCT/US92/09627 (WO93/09222) by Selden et al.",
"; and International Application No.",
"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.",
"4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].",
"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.",
"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.",
"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.",
"No.",
"5,557,032, incorporated herein by reference.",
"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.",
"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.",
"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.",
"No.",
"5,489,743 and PCT Publication No.",
"WO94/28122, incorporated herein by reference.",
"Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.",
"Inactivation can be carried out using homologous recombination methods described above.",
"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.",
"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.",
"The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.",
"Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.",
"In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)).",
"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.",
"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.",
"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.",
"No.",
"5,557,032, incorporated herein by reference.",
"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.",
"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.",
"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.",
"No.",
"5,489,743 and PCT Publication No.",
"WO94/28122, incorporated herein by reference.",
"Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.",
"Inactivation can be carried out using homologous recombination methods described above.",
"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.",
"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.",
"4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.",
"Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).",
"The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.",
"Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.",
"Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.",
"The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.",
"4.10.1 Research uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.",
"The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.",
"Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.",
"The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.",
"Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.",
"Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.",
"Methods for performing the uses listed above are well known to those skilled in the art.",
"References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.",
"Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.",
"In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.",
"In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.",
"4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.",
"Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.",
"The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.",
"Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.",
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"149:3778-3783, 1992; Bowman et al., I. Immunol.",
"152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.",
"J. E. e.a.",
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"J. E. e.a Coligan eds.",
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"173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.",
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"Vol 1 pp.",
"6.13.1, John Wiley and Sons, Toronto.",
"1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 77:6091-6095, 1980; Weinberger et al., Eur.",
"J. Immun.",
"11:405-411, 1981; Takai et al., J. Immunol.",
"137:3494-3500, 1986; Takai et al., J. Immunol.",
"140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.",
"Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.",
"The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.",
"It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).",
"Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.",
"Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.",
"This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.",
"Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.",
"Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.",
"No.",
"5,690,926).",
"Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.",
"This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.",
"These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.",
"These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.",
"Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.",
"For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.",
"The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.",
"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.",
"In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.",
"Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.",
"A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.",
"The selectable marker allows only cells of the desired type to survive.",
"For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.",
"Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.",
"Lanza et al., Academic Press (1997)).",
"Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.",
"In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.",
"Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.",
"Proc.",
"Natl.",
"Acad.",
"Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.",
"The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.",
"as described by Bernstein et al., Blood, 77: 2316-2321 (1991).",
"4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.",
"Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.",
"in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (ie., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.",
"Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.",
"Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.",
"Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.",
"In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.",
"In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.",
"A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.",
"Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.",
"De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.",
"A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.",
"Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)",
"mediated by inflammatory processes may also be possible using the composition of the invention.",
"Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.",
"Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.",
"Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.",
"De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.",
"The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.",
"The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.",
"The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.",
"The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.",
"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.",
"More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.",
"Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.",
"Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.",
"Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.",
"Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.",
"Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.",
"A polypeptide of the present invention may also exhibit angiogenic activity.",
"A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.",
"A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.",
"Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.",
"WO95/16035 (bone, cartilage, tendon); International Patent Publication No.",
"WO95/05846 (nerve, neuronal); International Patent Publication No.",
"WO91/07491 (skin, endothelium).",
"Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.",
"71-112 (Maibach, H. I. and Rovee, D. T., eds.",
"), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.",
"Invest.",
"Dermatol 71:382-84 (1978).",
"4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such activities.",
"A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.",
"These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.",
"More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.",
"and various fungal infections such as candidiasis.",
"Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.",
"Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.",
"Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.",
"Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.",
"The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig slin sensitization test (Vohr et al., Arch.",
"Toxocol.",
"73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.",
"Environ.",
"Health 53: 563-79).",
"Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.",
"Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.",
"The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.",
"Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.",
"Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.",
"Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.",
"Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).",
"For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.",
"Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.",
"The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.",
"Moreover, a lack of costinulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.",
"Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.",
"To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.",
"The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.",
"Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4I g fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.",
"Natl.",
"Acad.",
"Sci USA, 89:11102-11105 (1992).",
"In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.",
"846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.",
"Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.",
"Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.",
"Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.",
"Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.",
"Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.",
"The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.",
"Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.",
"840-856).",
"Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.",
"Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.",
"For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.",
"Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.",
"Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.",
"The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.",
"A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.",
"In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class 1 alpha chain protein and P2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.",
"Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.",
"Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.",
"Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.",
"The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 78:2488-2492, 1981; Herrmnann et al., J. Immunol.",
"128:1968-1974, 1982; Handa et al., J. Immunol.",
"135:1564-1572, 1985; Takai et al., I. Immunol.",
"137:3494-3500, 1986; Takai et al., J. Immunol.",
"140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.",
"153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.",
"144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.",
"J. E. e.a.",
"Coligan eds.",
"Vol 1 pp.",
"3.8.1-3.8.16, John Wiley and Sons, Toronto.",
"1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.",
"137:3494-3500, 1986; Takai et al., J. Immunol.",
"140:508-512, 1988; Bertagnolli et al., J. Immunol.",
"149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.",
"134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260,1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:40374045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.",
"A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.",
"Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).",
"Thus, a polypeptide of the present invention, alone or in heterodirners with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.",
"Administration of sufficient amounts of other inhibins can induce infertility in these mammals.",
"Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.",
"See, for example, U.S. Pat.",
"No.",
"4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.",
"The activity of a polypeptide of the invention may, among other means, be measured by the following methods.",
"Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.",
"Chemotactic and chemolinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.",
"Chemotactic or chemokinetic compositions (e.g.",
"proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.",
"For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.",
"A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.",
"Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.",
"Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.",
"Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.",
"Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.",
"J. Clin.",
"Invest.",
"95:1370-1376, 1995; Lind et al.",
"APMIS 103:140-146, 1995; Muller et al Eur.",
"J. Immunol.",
"25:1744-1748; Gruber et al.",
"J. of Immunol.",
"152:5860-5867, 1994; Johnston et al.",
"J. of Immunol.",
"153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.",
"Compositions may be usefull in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.",
"A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).",
"Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.",
"Pharmacol.",
"26:131-140, 1986; Burdick et al., Thrombosis Res.",
"45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79(1991); Schaub, Prostaglandins 35:467-474,1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.",
"Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.",
"For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.",
"Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.",
"Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.",
"Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.",
"Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.",
"Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.",
"Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.",
"reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.",
"The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.",
"An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.",
"The use of anti-cancer cocktails as a cancer treatment is routine.",
"Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.",
"In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.",
"There are hereditary conditions and/or environmental situations (e.g.",
"exposure to carcinogens) known in the art that predispose an individual to developing cancers.",
"Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.",
"In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.",
"These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.",
"Can.",
"Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.",
"J. Dev.",
"Biol., 40: 1189-97 (1999) and Li et al., Clin.",
"Exp.",
"Metastasis, 17:423-9 (1999), respectively.",
"Suitable tumor cells lines are available, e.g.",
"from American Type Tissue Culture Collection catalogs.",
"4.10.12 RECEPTOR/LIGAND ACTIVITY A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.",
"Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.",
"Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.",
"A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.",
"The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 84:6864-6868, 1987; Bierer et al., J. Exp.",
"Med.",
"168:1145-1156, 1988; Rosenstein et al., J. Exp.",
"Med.",
"169:149-160 1989; Stoltenborg et al., J. Immunol.",
"Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).",
"Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.",
"Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.",
"The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.",
"(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.",
"182 (1990) Academic Press, Inc. San Diego).",
"Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.",
"Examples of toxins include, but are not limited, to ricin.",
"4.10.13 DRUG SCREENING This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.",
"The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.",
"One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.",
"Drugs are screened against such transformed cells in competitive binding assays.",
"Such cells, either in viable or fixed form, can be used for standard binding assays.",
"One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.",
"Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.",
"Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.",
"The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.",
"Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.",
"For a review, see Science 282:63-68 (1998).",
"Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.",
"Of particular interest are peptide and oligonucleotide combinatorial libraries.",
"Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.",
"For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.",
"Opin.",
"Biotechnol.",
"8:701-707 (1997).",
"For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.",
"Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).",
"Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.",
"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.",
"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.",
"The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.",
"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.",
"Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.",
"4.10.14 ASSAY FOR RECEPTOR ACTIVITY The invention also provides methods to detect specific binding of a polypeptide e.g.",
"a ligand or a receptor.",
"The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.",
"For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.",
"As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.",
"There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.",
"Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.",
"The responses of the two cell populations to the addition of ligands(s) are then compared.",
"Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).",
"As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.",
"The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.",
"For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.",
"The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.",
"Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.",
"phosphorylation.",
"Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.",
"4.10.15 Anti-Inflammtory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.",
"The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.",
"Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.",
"Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.",
"4.10.16 LEUKEMIAS Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.",
"Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.",
"B. Lippincott Co., Philadelphia).",
"4.10.17 NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.",
"Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vi) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (vii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.",
"Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.",
"For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.",
"Such effects may be measured by any method known in the art.",
"In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.",
"(1990, J. Neurosci.",
"10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.",
"(1980, Exp.",
"Neurol.",
"70:65-82) or Brown et al.",
"(1981, Ann.",
"Rev.",
"Neurosci.",
"4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.",
"In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).",
"4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fingi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.",
"4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.",
"Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.",
"For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.",
"Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.",
"For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.",
"Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).",
"In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.",
"Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.",
"The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.",
"In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.",
"Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.",
"4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.",
"The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.",
"Arch.",
"Allergy Appl.",
"Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).",
"The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.",
"The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.",
"The control consists of administering PBS only.",
"The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.",
"An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.",
"4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.",
"Examples of therapeutic applications include, but are not limited to, those exemplified herein.",
"4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.",
"While the mode of administration is not particularly important, parenteral administration is preferred.",
"An exemplary mode of administration is to deliver an intravenous bolus.",
"The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.",
"It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.",
"Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.",
"For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.",
"Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.",
"The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.",
"The preparation of such solutions is within the skill of the art.",
"4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.",
"Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.",
"The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).",
"The characteristics of the carrier will depend on the route of administration.",
"The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, ThF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.",
"In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.",
"These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.",
"The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.",
"Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.",
"Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).",
"A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.",
"As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.",
"As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).",
"Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.",
"A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.",
"When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.",
"When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.",
"In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.",
"Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.",
"When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.",
"If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.",
"4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.",
"Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.",
"Intravenous administration to the patient is preferred.",
"Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.",
"In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.",
"Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.",
"The liposomes will be targeted to and taken up selectively by the afflicted tissue.",
"The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.",
"The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.",
"Preferably for wound treatment, one administers the therapeutic compound directly to the site.",
"Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.",
"Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.",
"4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.",
"These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.",
"Proper formulation is dependent upon the route of administration chosen.",
"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.",
"When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.",
"The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.",
"When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.",
"The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.",
"When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.",
"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.",
"The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.",
"A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.",
"The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.",
"For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.",
"Such penetrants are generally known in the art.",
"For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.",
"Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.",
"Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.",
"Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).",
"If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.",
"Dragee cores are provided with suitable coatings.",
"For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.",
"Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.",
"Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.",
"The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.",
"In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.",
"In addition, stabilizers may be added.",
"All formulations for oral administration should be in dosages suitable for such administration.",
"For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.",
"For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.",
"In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.",
"Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.",
"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.",
"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.",
"The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.",
"Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.",
"Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.",
"Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.",
"Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.",
"Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.",
"Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.",
"The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.",
"In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.",
"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.",
"Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.",
"A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.",
"The co-solvent system may be the VPD co-solvent system.",
"VPD is a solution of 3%/o w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.",
"The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.",
"This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.",
"Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.",
"Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.",
"polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.",
"Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.",
"Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.",
"Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.",
"Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.",
"Various types of sustained-release materials have been established and are well known by those skilled in the art.",
"Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.",
"Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.",
"The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.",
"Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.",
"Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.",
"Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.",
"The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.",
"The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.",
"B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.",
"T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.",
"MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.",
"The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.",
"Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.",
"The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.",
"Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.",
"Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.",
"Nos.",
"4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.",
"The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.",
"Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.",
"Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.",
"Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.",
"It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.",
"For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.",
"When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.",
"Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.",
"Topical administration may be suitable for wound healing and tissue repair.",
"Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.",
"Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.",
"Such matrices may be formed of materials presently in use for other implanted medical applications.",
"The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.",
"The particular application of the compositions will define the appropriate formulation.",
"Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.",
"Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.",
"Further matrices are comprised of pure proteins or extracellular matrix components.",
"Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.",
"Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.",
"The biocermics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.",
"Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.",
"In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.",
"A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).",
"Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).",
"The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.",
"In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.",
"These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).",
"The therapeutic compositions are also presently valuable for veterinary applications.",
"Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.",
"The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.",
"The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.",
"For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.",
"Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.",
"Polynucleotides of the present invention can also be used for gene therapy.",
"Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.",
"Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).",
"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.",
"Treated cells can then be introduced in vivo for therapeutic purposes.",
"4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.",
"More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.",
"Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.",
"For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.",
"For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.",
"For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity).",
"Such information can be used to more accurately determine useful doses in humans.",
"A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.",
"Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).",
"The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.",
"The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.",
"The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity.",
"The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.",
"The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.",
"See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.",
"1 p.1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).",
"The MEC will vary for each compound but can be estimated from in vitro data.",
"Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.",
"However, HPLC assays or bioassays can be used to determine plasma concentrations.",
"Dosage intervals can also be determined using MEC value.",
"Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.",
"In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.",
"An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.",
"Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.",
"The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.",
"4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.",
"The pack may, for example, comprise metal or plastic foil, such as a blister pack.",
"The pack or dispenser device may be accompanied by instructions for administration.",
"Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.",
"4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.",
"The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.",
"Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab′)2 fragments, and an Fab expression library.",
"In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.",
"Certain classes have subclasses as well, such as IgG1, IgG2, and others.",
"Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.",
"Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.",
"An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.",
"The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.",
"An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, (for example the amino acid sequence shown in SEQ ID NO: 30369), and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.",
"Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.",
"Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.",
"In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention that is located on the surface of the protein, e.g., a hydrophilic region.",
"A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.",
"As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.",
"See, e.g., Hopp and Woods, 1981, Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.",
"Biol.",
"157: 105-142, each of which is incorporated herein by reference in its entirety.",
"Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.",
"A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.",
"Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).",
"Some of these antibodies are discussed below.",
"5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.",
"An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.",
"Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.",
"Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.",
"The preparation can further include an adjuvant.",
"Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.",
"), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.",
"Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).",
"The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.",
"Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.",
"Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.",
"14, No.",
"8 (Apr.",
"17, 2000), pp.",
"25-28).",
"5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.",
"In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.",
"MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.",
"Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).",
"In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.",
"Alternatively, the lymphocytes can be immunized in vitro.",
"The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.",
"Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.",
"The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.",
"59-103).",
"Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.",
"Usually, rat or mouse myeloma cell lines are employed.",
"The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.",
"For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.",
"Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.",
"More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.",
"Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.",
"51-63).",
"The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.",
"Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).",
"Such techniques and assays are known in the art.",
"The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.",
"Biochem.",
"107:220 (1980).",
"Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.",
"After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.",
"Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.",
"Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.",
"The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.",
"The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.",
"No.",
"4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).",
"The hybridoma cells of the invention serve as a preferred source of such DNA.",
"Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.",
"The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.",
"No.",
"4,816,567; Morrison, Nature 368 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.",
"Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.",
"5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.",
"These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.",
"Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.",
"Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.",
"(See also U.S. Pat.",
"No.",
"5,225,539.)",
"In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.",
"Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.",
"In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.",
"The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.",
"Op.",
"Struct.",
"Biol., 2:593-596 (1992)).",
"5.13.3 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.",
"Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.",
"Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.",
"77-96).",
"Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.",
"77-96).",
"In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.",
"Biol., 227:381 (1991); Marks et al., J. Mol.",
"Biol., 222:581 (1991)).",
"Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.",
"Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.",
"This approach is described, for example, in U.S. Pat.",
"Nos.",
"5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.",
"(Bio/Technology 10, 779-783 (1992)); Lonberg et al.",
"(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.",
"Rev.",
"Immunol.",
"13 65-93 (1995)).",
"Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.",
"(See PCT publication WO94/02602).",
"The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.",
"The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.",
"An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.",
"The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.",
"The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.",
"Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.",
"An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.",
"No.",
"5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.",
"A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.",
"No.",
"5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.",
"The hybrid cell expresses an antibody containing the heavy chain and the light chain.",
"In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.",
"No.",
"4,946,778).",
"In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.",
"Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) FV fragments.",
"5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.",
"In the present case, one of the binding specificities is for an antigenic protein of the invention.",
"The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.",
"Methods for making bispecific antibodies are known in the art.",
"Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).",
"Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.",
"The purification of the correct molecule is usually accomplished by affinity chromatography steps.",
"Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.",
"The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.",
"It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.",
"DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.",
"For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).",
"According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.",
"The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.",
"In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.",
"tyrosine or tryptophan).",
"Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.",
"alanine or threonine).",
"This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.",
"Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.",
"F(ab′)2 bispecific antibodies).",
"Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.",
"For example, bispecific antibodies can be prepared using chemical linkage.",
"Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.",
"These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.",
"The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.",
"One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.",
"The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.",
"Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.",
"Shalaby et al., J. Exp.",
"Med.",
"175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.",
"Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.",
"The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.",
"Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.",
"For example, bispecific antibodies have been produced using leucine zippers.",
"Kostelny et al., J. Immunol.",
"148(5):1547-1553 (1992).",
"The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.",
"The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.",
"This method can also be utilized for the production of antibody homodimers.",
"The “diabody” technology described by Hollinger et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.",
"The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.",
"Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.",
"Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported.",
"See, Gruber et al., J. Immunol.",
"152:5368 (1994).",
"Antibodies with more than two valencies are contemplated.",
"For example, trispecific antibodies can be prepared.",
"Tutt et al., J. Immunol.",
"147:60 (1991).",
"Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.",
"Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.",
"CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.",
"Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.",
"These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.",
"Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (° F.).",
"5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.",
"Heteroconjugate antibodies are composed of two covalently joined antibodies.",
"Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.",
"No.",
"4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).",
"It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.",
"For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.",
"Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.",
"No.",
"4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.",
"For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.",
"The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).",
"See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.",
"Immunol., 148: 2918-2922 (1992).",
"Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.",
"Cancer Research, 53: 2560-2565 (1993).",
"Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.",
"See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).",
"5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).",
"Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.",
"Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.",
"A variety of radionuclides are available for the production of radioconjugated antibodies.",
"Examples include 212Bi, 131I, 90Y, 90Y, and 186Re.",
"Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).",
"For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).",
"Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.",
"See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.",
"4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.",
"As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.",
"Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.",
"As used herein, “recorded” refers to a process for storing information on computer readable medium.",
"A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.",
"A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.",
"The choice of the data storage structure will generally be based on the means chosen to access the stored information.",
"In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.",
"The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.",
"A skilled artisan can readily adapt any number of data processor structuring formats (e.g.",
"text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.",
"By providing any of the nucleotide sequences SEQ ID NO: 1-30368 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-30368 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.",
"Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.",
"The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.",
"Biol.",
"215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.",
"Chem.",
"17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.",
"Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.",
"As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.",
"The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.",
"A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.",
"As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.",
"As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.",
"As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.",
"Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.",
"A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.",
"Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASIN and BLASTA (NPOLYPEPTIDEIA).",
"A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.",
"As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.",
"A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.",
"The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.",
"However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.",
"As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.",
"There are a variety of target motifs known in the art.",
"Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.",
"Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).",
"4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.",
"Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.",
"Acids Res.",
"6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olnno, J. Neurochem.",
"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).",
"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.",
"Both techniques have been demonstrated to be effective in model systems.",
"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.",
"4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.",
"In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.",
"Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.",
"In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.",
"In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.",
"Conditions for incubating a nucleic acid probe or antibody with a test sample vary.",
"Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.",
"One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.",
"Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.",
"1 (1982), Vol.",
"2 (1983), Vol.",
"3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).",
"The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.",
"The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.",
"Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.",
"In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.",
"Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.",
"In detail, a compartment kit includes any kit in which reagents are contained in separate containers.",
"Such containers include small glass containers, plastic containers or strips of plastic or paper.",
"Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.",
"Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.",
"), and containers which contain the reagents used to detect the bound antibody or probe.",
"Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.",
"One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.",
"4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).",
"See, e.g., Kunkel et al., U.S. Pat.",
"No.",
"5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.",
"4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-30368, or bind to a specific domain of the polypeptide encoded by the nucleic acid.",
"In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.",
"In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.",
"Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.",
"Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.",
"Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).",
"Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).",
"Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.",
"The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.",
"The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.",
"For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.",
"Alternatively, agents may be rationally selected or designed.",
"As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.",
"For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.",
"Freeman, N.Y. (1992), pp.",
"289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.",
"In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.",
"As described above, such agents can be randomly screened or rationally designed/selected.",
"Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.",
"One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.",
"Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.",
"Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.",
"Acids Res.",
"6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.",
"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).",
"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.",
"Both techniques have been demonstrated to be effective in model systems.",
"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.",
"Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.",
"Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.",
"4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.",
"The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-30368.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-30368 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.",
"Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.",
"PCR as described in U.S. Pat.",
"Nos.",
"4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.",
"Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.",
"The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.",
"Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.",
"Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.",
"The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.",
"The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.",
"These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.",
"The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.",
"Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f).",
"Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.",
"The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.",
"4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.",
"Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.",
"One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.",
"Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.",
"Microbiol.",
"28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey& Collins, (1989) Mol.",
"Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.",
"Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.",
"For example, Broude et al.",
"(1994) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.",
"Streptavidin-coated beads may be purchased from Dynal, Oslo.",
"Of course, this same linking chemistry is applicableto coating any surface with streptavidin Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).",
"Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.",
"Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.",
"CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.",
"CovaLink Modules may be purchased from Nunc Laboratories.",
"DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.",
"Biochem.",
"198(1) 138-42).",
"The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).",
"In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.",
"1 (8) 6513-29).",
"This is beneficial as immobilization using only a single covalent bond is preferred.",
"The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.",
"To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.",
"It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.",
"More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.",
"at 95° C. and cooling on ice for 10 min.",
"Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.",
"Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well.",
"The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).",
"It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.",
"This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.",
"The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.",
"Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.",
"An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.",
"For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al.",
"(1991) Science 251(4995) 767-73, incorporated herein by reference.",
"Probes may also be immobilized on nylon supports as described by Van Ness et al.",
"(1991) Nucleic Acids Res.",
"19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.",
"Biochem.",
"169(1) 104-8; all references being specifically incorporated herein.",
"To link an oligonucleotide to a nylon support, as described by Van Ness et at (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.",
"One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference).",
"These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).",
"These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.",
"A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.",
"4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.",
"For example, Sambrook et al.",
"(1989) describes three protocols for the isolation ofhigh molecular weight DNA from mammalian cells (p. 9.14-9.23).",
"DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.",
"Samples may be prepared or dispensed in multiwell plates.",
"About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.",
"The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.",
"(1989), shearing by ultrasound and NaOH treatment.",
"Low pressure shearing is also appropriate, as described by Schriefer et at (1.990) Nucleic Acids Res.",
"18(24) 7455-6, incorporated herein by reference).",
"In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.",
"A lever device allows controlled application of low to intermediate pressures to the cell.",
"The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.",
"One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.",
"(1992) Nucleic Acids Res.",
"20(14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.",
"The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.",
"A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).",
"Fitzgerald et al.",
"(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest ofpUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.",
"Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.",
"As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.",
"Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.",
"This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.",
"Phosphate groups must also be removed from genomic DNA by methods known in the art.",
"4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.",
"Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.",
"By offset printing, a density of dots higher than the density of the wells is achieved.",
"One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.",
"By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.",
"Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.",
"Each of the subarrays may represent replica spotting of the same samples.",
"In one example, a selected gene segment may be amplified from 64 patients.",
"For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).",
"A plate for each of the 64 patients is prepared.",
"By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.",
"Subarrays may contain 64 samples, one from each patient.",
"Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.",
"Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.",
"a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.",
"A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.",
"The present invention is illustrated in the following examples.",
"Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.",
"Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.",
"The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.",
"Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.",
"Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.",
"All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.",
"5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.",
"The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.",
"Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.",
"The clones were clustered into groups of similar or identical sequences.",
"Representative clones were selected for sequencing.",
"In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.",
"PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.",
"Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.",
"In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.",
"5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.",
"The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-30368 and are provided in the attached Sequence Listing.",
"The contigs were assembled using an EST sequence as a seed.",
"Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.",
"The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.",
"Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.",
"The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-30368) of the present invention are incorporated in the attached Sequence Listing.",
"A subset the predicted polypeptide sequences contain an unknown amino acid, a stop codon, a possible nucleotide deletion or a possible nucleotide insertion.",
"These sequences have been shown in their entirety with the special characters in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-30368.Table 2 also indicates the method by which the polypeptide was predicted.",
"Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fastabioch.virginia.edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).",
"Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.",
"Biol., 268:78-94(1997), incorporated herein by reference).",
"Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.",
"The nearest neighbor results for SEQ ID NO: 1-30368 were obtained by a BLASTP version 2.0al 19 MP-WashU search against Genpept release 121 and Geneseq release 200103 (Derwent), using BLAST algorithm.",
"The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-30368.The nearest neighbor results for SEQ ID NO: 1-30368 are incorporated in the attached Sequence Listing.",
"Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.",
"Biol., Vol.",
"6 pp.",
"219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions.",
"The attached Sequence Listing provodes the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.",
"Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.",
"26(1) pp.",
"320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.",
"The attached Sequence Listing provides the results obtained by PFAM analysis for each peptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.",
"Tables 1 and 2 follow.",
"Table I shows the various tissue sources of SEQ ID NO: 1-30368.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.",
"Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.",
"No.",
"09/540,217"
]
] | Patent_10450763 |
[
[
"Novel means for the diagnosis and therapy of ctcl",
"A means for the diagnosis and therapy of T lymphomas, in particular Cutaneous T Cell Lymphomas (“CTCL”) are provided.",
"Tumor markers which are universal for CTCL are described.",
"More particularly, a molecule, termed SC5 by the inventors, an allelic form of p140, and biological applications of SC5 and p140 molecules, notably in the diagnosis and therapy of CTCL are described."
],
[
"1 A monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"2 A hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"3 An isolated protein obtainable by: (i) collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating them with PHA at 1 microgram/ml, (ii) lysing the cells by incubation in a lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the compound onto which the mAb of claim 1 binds under conditions enabling this mAb to perform reactions of the antigen-antibody type.",
"4 An isolated protein obtainable by recovering the isolated protein compound of claim 3 under non reducing conditions.",
"5 An isolated cDNA obtainable by: collecting cells selected from the group consisting of total blood cells and PBL5 incubating the collected cell population with a CD3 activator at 1 microgram per ml, extracting and purifying the whole mRNA population from said cells, synthesizing every complementary cDNA, operably cloning each cDNA so that expression of this cDNA in this clone is under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody of claim 1 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or of which lysate would give an immuno-precipitation reaction with this mAb, optionally, amplifying those clones that have been thus selected, and recovering the inserted cDNA from the selected clones.",
"6 An isolated mRNA obtainable by selection among the mRNA population of cells selected from the group consisting of total blood cells and PBL, of a mRNA which is complementary to a cDNA of claim 5.7 An isolated genomic DNA encoding an isolated mRNA of claim 6.8 An engineered cell in which a cDNA according to claim 5, has been transfected.",
"9 An isolated protein encoded by an isolated cDNA according to claim 5.10 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 3, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.",
"11 An isolated polypeptide compound obtainable by: collecting PBL cells or total blood cells, incubating the collected cells with a CD3 activator at 1 microgram par ml, labeling the cells with a polypeptide-specific label, lysing the cells in a lysis buffer comprising Trition X-100 at 1%, and submitting the lysate to an immuno-precipitation reaction with the mAb of claim 1, and recovering from the digested immuno-precipitate any compound onto which bear a label.",
"12 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 3, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells maizy at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with We a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"for binding to a protein according to claim 3, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.",
"13 A fragment of a monoclonal antibody according to claim 1, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.14 An isolated compound, comprising a fragment according to claim 13.15 The isolated compound according to claim 14, wherein it is a humanized antibody.",
"16 The isolated compound according to claim 14, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.",
"17 An isolated protein of SEQ ID No.",
"2.18 The isolated DNA encoding an isolated protein according to claim 17.19 An isolated DNA of SEQ No.",
"1.20 A polypeptidic vector comprising a monoclonal antibody according to claim 1, or a fragment thereof selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2 and CDR3.21 A polypeptidic vector for use in the treatment of CTCL, wherein said vector comprises a p140 binding compound.",
"22 The polypeptidic vector according to claim 20, further comprising an element selected from the group consisting of tumoral toxins and radioelements.",
"23 The polypeptidic vector according to claim 20, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form.",
"24 A medical kit comprising a polypeptidic vector according to claim 23 and an antimitotic pro-drug.",
"25 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.26 A method for manufacturing an anti-CTCL medicament comprising including a p140 binding compound in said medicament.",
"27 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 3 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.",
"28 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 3, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.",
"29 A method for CTCL diagnosis, comprising measuring the percentage of CD4+ cells expressing an element selected from the group consisting of the proteins according to claim 3, in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.",
"30 A method for CTCL diagnosis, comprising determining the presence of CD4+ cells expressing p140 in a biological sample which comprises potential CTCL cells, a CTCL-positive diagnosis being made when such a presence is significantly detected.",
"31 A method according to claim 27, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1 and the DNA encoding SEQ ID No.",
"3 or SEQ ID No.",
"4.32 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 1, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody according to claim 1.33 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 3, or to a p140 molecule, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.",
"34 The isolated protein of claim 4, wherein said non reducing conditions are of chromatography by affinity with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"35 The isolated cDNA of claim 5, wherein said CD3 activator is PHA at 1 microgram per ml.",
"36 An engineered cell in which a mRNA according to claim 6 has been transfected.",
"37 An engineered cell in which a DNA according to claim 7 has been transfected.",
"38 An isolated protein encoded by an isolated mRNA according to claim 6.39 An isolated protein encoded by an isolated cDNA according to claim 7.40 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 4, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.",
"41 A bank of polypeptide compounds, obtainable by cleavage of a protein according to claim 9, with an enzyme selected from the group consisting of V8 protease and alpha chymotrypsine.",
"42 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 4, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"for binding to a protein according to claim 4, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.",
"43 A monoclonal antibody obtainable by: (i) immunizing an animal against a protein according to claim 9, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"for binding to a protein according to claim 9, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.",
"44 A monoclonal antibody obtainable by: (i) immunizing an animal against a polypeptide compound according to claim 11, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the polypeptide compound that has been used as an immunogen in step (i), and which has at least one property selected from the group consisting of: binding resting non-tumoral T cells in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of CTCL cells, modulating the proliferation of CTCL cells in a non-human animal, and competing with a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"for binding to a polypeptide according to claim 11, (iv) selecting and cloning hybridomas producing the desired antibody, and (v) recovering the antibody from the supernatant above said clones.",
"45 A fragment of a monoclonal antibody according to claim 12, selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3.46 The isolated compound according to claim 15, wherein it further comprises at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.",
"47 The polypeptidic vector according to claim 20, wherein said fragment is the Fab or F(ab′)2 fragment.",
"48 A polypeptidic vector comprising an isolated compound according to claim 15, optionally including at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, and CDR3 fragments of anti-CD4 antibodies.",
"49 The polypeptidic vector according to claim 21, further comprising an element selected from the group consisting of tumoral toxins and radioelements.",
"50 The polypeptidic vector according to claim 21, further comprising an enzyme capable of transforming an anti-mitotic pro-drug into an active drug form 51 The polypeptidic vector according to claim 23, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.",
"52 The polypeptidic vector according to claim 50, wherein said enzyme capable of transforming an anti-mitotic pro-drug into an active drug form is a carboxypeptidase.",
"53 The medical kit according to claim 24, wherein said antimitotic pro-drug is phenol mustard pro-drug.",
"54 A medicament comprising, as an active principle, an element selected from the group consisting of a monoclonal antibody according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.",
"55 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.",
"56 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.",
"57 A method for evaluating the percentage of malignant CD4+ CTCL cells preset within a body compartment of a patient, comprising: measuring the percentage of CD4+ cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, wherein the percentage of malignant CD4+ CTCL cells present in said body compartment falls within a ±10% range around said measured percentage.",
"58 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 4, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.",
"59 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a protein according to claim 9, in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.",
"60 A method for CTCL diagnosis, comprising measuring the percentage of T cells expressing a member selected from the group consisting of the polypeptide compounds of claim 11 and the p140 molecules in a biological sample collected from said patient, and comparing said percentage measured to the average percentage observed in non-CTCL humans, a CTCL-positive diagnosis being made when the measured percentage is significantly higher than the average percentage.",
"61 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 4 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.",
"62 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing a protein according to claim 9 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.",
"63 A method for CTCL diagnosis comprising measuring the percentage of CD4+ cells expressing the polypeptide compounds of claim 11 in a biological sample collected from a patient, wherein a CD4+ CTCL positive diagnosis is made when the measured percentage is higher than the average percentage usually observed in non-CTCL humans.",
"64 A method according to claim 28, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.",
"65 A method according to claim 29, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.",
"66 A method according to claim 30, wherein said percentage measurement makes use of an element selected from the group consisting of a monoclonal antibody (mAb) produced by a hybridoma deposited as deposit number I-2575 at the C.N.C.M., a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody, and the DNA encoding SEQ ID No.",
"3 or SEQ ID No.",
"4.67 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting of the monoclonal antibodies according to claim 12, a fragment thereof selected from the group consisting of Fab and F(ab′)2 fragments of said antibody, and a humanized antibody comprising a Fab or F(ab′)2 fragment of said monoclonal antibody.",
"68 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises at least one polypeptide compound selected from the bank of polypeptide compounds according to claim 10.69 A kit for assessing the development level of a CTCL, or for CTCL diagnosis, which comprises an element selected from the group consisting the cDNA according to claim 5, an mRNA complementary to said cDNA, a p140 binding compound, a p140 DNA, an isolated DNA encoding an isolated protein of SEQ ID NO: 2, an isolated DNA of SEQ ID NO: 1, the DNA encoding SEQ ID No.",
"3 and the DNA encoding SEQ ID No.",
"4.70 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 4, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.",
"71 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a protein according to claim 9, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.",
"72 A method for the identification of a compound which is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells, said method comprising detecting a compound that is capable of binding to a polypeptide compound according to claim 10, wherein said compound which binds is useful in the palliation, prevention, relief and/or therapy of a proliferation of malignant CTCL cells.",
"73 The method according to claim 33, wherein said p140 molecule is of SEQ ID No.",
"2 or SEQ ID No.",
"4."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>CTCL is a group of T lymphomas which primarily involve the skin.",
"The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.",
"Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.",
"LP and CD30+ lymphomas also develop in the skin.",
"More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.",
"SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.",
"Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.",
"The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.",
"In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.",
"Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).",
"Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.",
"Such a diagnosis method does also not enable to stage the disease.",
"Technically speaking, it is also time-consuming.",
"Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.",
"There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).",
"The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.",
"It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.",
"The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.",
"(C.N.C.M.",
"Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.",
"deposit number: I-2575).",
"When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.",
"The second marker of the invention, i.e.",
"p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.",
"The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.",
"They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.",
"Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.",
"lupus, lichen), or toxic epidermal necrolysis.",
"At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).",
"Allelic form KIR3D clone 24 (SEQ ID No.",
"1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.",
"1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.",
"3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.",
"2) when compared to p140 clone 1.1.mature protein (SEQ ID No.",
"4).",
"The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.",
"sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.",
"To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.",
"The invention thus provides with the first CTCL universal markers.",
"No prior art product was known to be such a CTCL universal marker.",
"The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.",
"CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.",
"CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.",
"In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.",
"cytokine profile) of non-tumoral T cells.",
"The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.",
"SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.",
"rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).",
"SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.",
"The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).",
"It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.",
"In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).",
"Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).",
"More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.",
"The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.",
"humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity)."
],
[
"FIELD OF THE INVENTION The present application relates to novel means for the diagnosis and therapy of T lymphomas, and more particularly of Cutaneous T Cell Lymphomas (abbreviated into CTCL).",
"The invention indeed provides novel tumor markers which are universal for CTCL, and describes biotechnological and medical uses thereof.",
"BACKGROUND OF THE INVENTION CTCL is a group of T lymphomas which primarily involve the skin.",
"The CTCL group namely comprises transformed Mycosis Fungoides (abbreviated into transformed MF), Sézary Syndrome (abbreviated into SS), Lymphomatoide Papulosis (abbreviated into LP), and CD30+ lymphomas.",
"Transformed MF is characterized by skin invasion of clonally-derived malignant T lymphocytes that phenotypically resemble mature T helper cells.",
"LP and CD30+ lymphomas also develop in the skin.",
"More aggressive forms of CTCL develop when the malignant cells become non-epidermotropic, and are associated with extra-cutaneous involvement.",
"SS is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.",
"Most CTCL are CD4+ CTCL, but some rare CD8+ CTCL cases exist.",
"The biology of CTCL disease remains poorly understood, as it is difficult to identify the malignant cells, due to the lack of specific cell surface markers.",
"In cutaneous lesions, it is therefore difficult to distinguish CTCL cells from reactive infiltrating (non-tumoral) T lymphocytes.",
"Diagnosis of T lymphomas such as CTCL is at present time mainly based on cytological and histological observations of the presence or absence of tumoral cells in a sample collected from a suspected body area (observation of histopathological aspect on skin biopsis and/or presence of SS cells in the blood, via detection of cells showing a cerebriform nucleus).",
"Such a diagnosis method is not fully reliable, notably at the early stages of the transformation of skin lymphocytes into malignant lymphocytes.",
"Such a diagnosis method does also not enable to stage the disease.",
"Technically speaking, it is also time-consuming.",
"Today therapy of CTCL is tentatively achieved by induction of tumoral cell apoptosis via non specific chemotherapy.",
"There is therefore still a need for more accurate and appropriate solutions to the problem of the diagnosis and therapy of diseases involving the proliferation of malignant T cells, such as CTCL.",
"SUMMARY OF THE INVENTION In view of this prior art situation, the inventors now provide with two molecules which give solutions to the problem of CTCL diagnostic and therapy: one has been termed SC5 by the inventors, and the other one is the p140 molecule (p140 is also referred to as KIR3DL2).",
"The SC5 molecule of the invention appears as a biochemically and functionally new protein, of which apparent molecular weight is of 96 kD under reducing conditions.",
"It has been isolated as the antigen of a monoclonal antibody that has been produced by the inventors.",
"The hybridoma producing this monoclonal antibody has been deposited on Oct. 30, 2000 at the C.N.C.M.",
"(C.N.C.M.",
"Institut Pasteur; 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty (C.N.C.M.",
"deposit number: I-2575).",
"When expressed at the cell surface, and made to aggregate, SC5 molecules act as inhibitory receptors for cell activity and proliferation.",
"The second marker of the invention, i.e.",
"p140, was already known to be an inhibitory receptor, but has been previously described only on sub-groups of NK cells and of CD3+ CD8+ cells from healthy humans.",
"The inventors now demonstrate that p140 is expressed at the surface of tumoral T cells such as CTCL cells.",
"They further demonstrate that p140 is expressed at the surface of malignant CD4+ T cells, such as CD4+ CTCL cells, whereas those other receptors that are usually observed at the surface of NK cells (such as p58.1, p58.2, p70 KIRs, CD94/NKG2A) are not found at the surface of malignant CD4+ T cells.",
"Of further note is that p140 has not been observed by the inventors at the surface of CD4+ T cells collected from patients suffering from non-tumoral dermatological diseases such as inflammatory skin diseases (e.g.",
"lupus, lichen), or toxic epidermal necrolysis.",
"At the surface of tumoral CD4+ T cells, two allelic forms of p140 have been identified by the inventors: allelic form KIR3D clone 24 (SEQ ID No.1), and allelic form p140 clone 1.1 (SEQ ID No.3).",
"Allelic form KIR3D clone 24 (SEQ ID No.",
"1) is a new polynucleotide encoding a new protein (SEQ ID No.2): KIR3D clone 24 (SEQ ID No.",
"1) displays five differences when compared to the previously described p140 clone 1.1 DNA sequence (SEQ ID No.",
"3), resulting in four amino acid substitutions in the mature protein (SEQ ID No.",
"2) when compared to p140 clone 1.1.mature protein (SEQ ID No.",
"4).",
"The inventors demonstrate that the SC5 and p140 both share the same technical following features: SC5 and p140 both are membranar differentiation antigens which are characteristic of malignant T cells, and notably of malignant CD4+ T cells, whichever form of CD4+ CTCL is concerned, there are malignant CD4+ T cells which express SC5 or p140 at their surface (usually both SC5 and p140 are expressed): SC5 as well as p140 indeed cover the whole range of CD4+ CTCL, and notably the Sézary Syndrome (abbreviated into “SS”), transformed Mycosis Fungoides (abbreviated into “transformed MF”), Lymphomatoide Papulosis (abbreviated onto “LP”), and CD30+ lymphomas, there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that a CD4+ CTCL diagnosis based on the analysis of the presence of SC5 or p140 at the surface of CD4+ cells collected from the suspected body area (e.g.",
"sample of skin erythroderma when transformed MF is suspected, or sample of peripheral blood when a more aggressive CTCL form, such as SS, is suspected) has a reliability of more than 90%, preferably of more than of 95%, most preferably of 100% (among those patients who were tested up to now, the reliability is of 100%): indeed, according to the invention, it can be concluded that a CD4+ T cell is tumoral as soon as there are p140 molecules detected at the surface of these CD4+ T cells, or as soon as a percentage of SC5+ CD4+ T cells higher than the average standard level is measured (the average standard level is in the 1-15% range, generally in the 5-10% range), and there exists such a link between the presence of SC5 or p140 at the surface of CD4+ T cells and the existence of a CD4+ CTCL, that the percentage of CD4+ SC5+ T cells, as well as of CD4+ p140+ T cells, that is measured in a sample of peripheral blood collected from a patient for whom a Sézary Syndrome (SS) is suspected, substantially corresponds to the percentage of malignant SS cells that are actually present in the peripheral blood of this patient (within a ±10% range for SC5+ CD4+ cells, within a ±5% range for p140+ CD4+ cells): the SC5 and p140 markers of the invention therefore share the common particular technical feature of enabling to assess the staging of a SS.",
"To the best of the inventors' knowledge, SC5 and p140 are the first molecules which are described as having these common technical features.",
"The invention thus provides with the first CTCL universal markers.",
"No prior art product was known to be such a CTCL universal marker.",
"The closest prior art product in this respect would be CD30 of which presence at the surface of malignant CD4+ T cells directs to the conclusion that the patient has a particular form of CD4+ CTCL which is referred to in the art as CD30+ lymphoma.",
"CD30 is therefore a CTCL marker, but its reliability is limited to a particular form of CTCL (CD30+ lymphomas), and CD30 does not cover every form of CD4+ CTCL: for CD4+ CTCL such as SS, transformed MF, or LP, there does not necessarily exist a malignant CD4+ T cell which would express CD30 at its surface.",
"CD30 uses in CTCL diagnosis and therapy is thus restricted to a particular form of CTCL, whereas the markers of the invention advantageously cover the whole range of CD4+ CTCL.",
"In addition to the technical features shared with p140, SC5 has the further characteristic of being a positive indicator of proliferation and/or functional activity (e.g.",
"cytokine profile) of non-tumoral T cells.",
"The presence of SC5 at the surface of T cells is indeed positively linked with the activation status of non-tumoral T cells, such as normal (CD4+ and CD8+) T cells or virus-infected CD4+ T cells.",
"SC5 is thus also a useful novel means for the diagnostic and therapy of T cell viral infections such as HIV-infection, and of inflammatory diseases such as those of the auto-immune type (e.g.",
"rhumatoid arthritis such as spondyloarthropathis, or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).",
"SC5 is also a useful target for modulating graft-host reactions: the activation of SC5 transduces an inhibitory signal that can be used to inhibit the reactions that effector recipient cells may develop against a graft.",
"The invention also provides with products capable of binding to the SC5 new molecule, and in particular with monoclonal antibodies directed against SC5 (anti-SC5 mAbs).",
"It is also provided with anti-SC5 monoclonal antibodies which are capable of modulating the proliferation and/or the functional activity of T cells, and notably of tumoral T cells such as malignant CTCL cells.",
"In particular, the monoclonal antibody produced by hybridoma I-2575 is capable of inhibiting the proliferation and/or functional activity of T cells, and according to a particularly useful feature, is capable of inhibiting the proliferation and/or activity of malignant T cells such as malignant CTCL cells (see examples below).",
"Compounds binding to SC5, but not aggregating them will prevent SC5 inhibitory signal transduction, or will prevent compounds to reach SC5+ cells (target masking).",
"More generally, compounds binding to SC5 or p140 may, according to the invention, be used as complement recruiting agents, as ADCC stimulators, or as vectors for therapeutic agents so as to prevent, palliate, treat T lymphomas, such as CTCL.",
"The invention therefore also relates to the diagnostic and therapeutic uses of SC5- or p140-binding compounds such as anti-SC5 and/or anti-p140 monoclonal antibodies, and of products directly-derived therefrom (e.g.",
"humanized monoclonal antibodies, or monoclonal antibodies with a double CD4-p140/SC5 specificity).",
"DETAILED DESCRIPTION OF THE INVENTION The invention thus provides with an hybridoma deposited on Oct. 30, 2000 as deposit number I-2575 at the C.N.C.M.",
"in accordance with the Budapest Treaty (C.N.C.M.",
"Collection Nationale de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France), and with a method for preparing an anti-SC5 monoclonal antibody which comprises cultivating hybridoma I-2575 under conditions suitable for the metabolism of the hybridoma cells (e.g.",
"culture for three days at 37° C., with 5% CO2 in a humid atmosphere at 2.106 cells par ml of appropriate culture medium such as RPMI 1640 medium Gibco BRL Cat.",
"No21875-034, with 10% Fetal Calf Serum, penicillin 100 U/ml and streptomycin 100 microgram/ml).",
"The invention therefore encompasses the monoclonal antibody (mAb) which is produced by hybridoma I-2575, or which is obtainable from the culture supernatant of this deposited hybridoma.",
"This mAb is a pentameric anti-SC5 IgM.",
"It is a useful tool for the diagnosis and therapy of an inappropriate or undesired T cell proliferation and/or functional activity (cytokine profile), of an inappropriate or undesired IL-2 production.",
"Its inhibits the proliferation and/or functional activity of normal T cells, of virus-infected T cells (e.g.",
"HIV-infected CD4+ T cells), and of malignant T cells, such as notably malignant CTCL cells.",
"The monoclonal antibody produced by hybridoma I-2575 enables the isolation of a is protein, termed SC5 by the inventors.",
"SC5 protein appears as a biochemically and functionally new molecule, with an apparent molecular weight of 96 kD under reducing conditions.",
"It has been located in and on numerous PBL cells: normal T cells (both from CD4+ and CD8+ subsets), and in particular CD3+ T cells, CD45RO+ T cells, but also normal NK cells, B cells, granulocytes and macrophages.",
"In addition, SC5 has been located in the cytoplasm of cells from other lineages and other species such as NIH/3T3 (ATCC CRL-1658) and CHO-K1 (ATCC CRL-9618).",
"In resting normal PBL cells, SC5 is mainly intracellularly located, but CD3 stimulation induces the transfer of SC5 to the cell membrane.",
"Remarkably, SC5 has also been observed by the inventors on malignant T cells, such as malignant CTCL cells, and in particular malignant CD4+ CTCL cells, where they are mainly located at the cell surface (without any ex vivo CD3 stimulation).",
"As a further remarkable feature, SC5 surface expression has been observed for all the CTCL patients who were tested until now (see the examples below).",
"Another further remarkable feature of the invention is that the percentage of CD4+ T cells which co-express SC5 is statistically higher in patients developing tumoral CD4+ T cells than in healthy patients.",
"The percentage of SC5+ CD4+ cells in CD4+ CTCL patients is significantly higher than the percentage observed in healthy patients (average standard percentage of SC5+ CD4+ cells is in the 1-15% range, generally in the 5-10% range).",
"In the particular CD4+ CTCL case of Sézary Syndrome (SS), the percentage of CD4+ cells which co-express SC5 is furthermore statistically closely correlated to the percentage of malignant CTCL cells in the PBL of the CTCL patient: the percentage of SC5+ CD4+ T cells is substantially equal to the percentage of malignant CTCL cells (within ±10% of this percentage).",
"SC5 protein is therefore a new molecule whose transmembrane expression at the cell surface is linked to an activation and/or proliferation status for non-tumoral T cells (such as virus-infected cells, e.g.",
"HIV-infected cells, and such as normal CD4+ or CD8+ T cells), and to a malignant status for non-activated T cells.",
"According to a remarkable feature, it enables a CD4+ CTCL-positive diagnosis with a confidence of more than 90% (actually of 100% of those humans who have been tested up-to-date).",
"A further outstanding feature of the invention is that the SC5 new molecule is capable.",
"of acting as an inhibitory receptor for cell proliferation and/or functional activity when expressed at the cell surface: SC5 aggregation induces a decrease in the proliferation and activity of the cell expressing it.",
"For example, the anti-SC5 monoclonal antibody produced by hybridoma I-2575 (which is a pentameric Ig) inhibits the anti-CD3 induced proliferation of SC5+ cells such as normal CD3+ T cells collected from a human or an animal, or normal CD3+ T cell clones, and also inhibits the anti-CD3 induced in vitro proliferation of CTCL cells collected from a CTCL patient, and the in vivo natural proliferation of human CTCL cells in an animal model.",
"This mAb also binds HIV-infected CD4+ T cells, and is therefore useful as an active principle in an anti-HIV drug intended for killing infected cells, and therefore reducing HIV propagation.",
"Several procedures enabling the isolation of the SC5 protein are available to the skilled person to whom a monoclonal antibody of the invention has been made available.",
"Standard procedures comprise recovering the SC5 molecules from a cell lysate by affinity chromatography, or by electrophoresis under reducing conditions.",
"All human or animal cells which the monoclonal antibody produced by hybridoma I-2575 recognizes as an antigen, or of which lysate gives an immuno-precipitation reaction with this monoclonal antibody, are appropriate cell sources for isolating the SC5 molecule.",
"Such cells notably include normal cells (collected from a human or an animal, or cell clones) such as T cells, CD3+ T cells, CD45RO+ T cells, B cells, NK cells, macrophages, granulocytes.",
"Appropriate cells also include tumoral cells such as malignant T cells, e.g.",
"CD4+ CTCL cells collected from a CTCL patient, or tumoral clones such as the CTCL clone HUT78 (ATCC TIB-161).",
"Due to the wide range of appropriate cell sources, it will be understood that rough total blood cell or PBL may be directly used as a SC5 source without any further purification.",
"Normal cells enables the isolation of both the intracellular form and the transmembrane receptor form of SC5.When it is desired to isolate the SC5 receptor form, or a portion thereof, it may thus be preferable to use a source of SC5+ cells (i.e.",
"cells expressing SC5 at their surface).",
"Such a source include normal cells as above-mentioned but that have been CD3-stimulated so that SC5 expression increases at the cell surface (see examples below for CD3 stimulation procedures).",
"When total blood cells or PBL are used as a source of SC5+ cells, CD3 activation can be induced by incubation with a polyclonal activator such as PHA (obtainable from Wellcome) at a concentration of about 1 microgram per ml, because total blood cells or PBL also contain T cells and mononuclear cells.",
"When a more homogeneous normal cell population is used as a source of SC5+ cells (e.g.",
"only SC5+ NK cells), then CD3 activation is preferably performed by incubation with immobilized anti-CD3 antibodies, or alternatively with both soluble anti-CD3 antibodies and polyclonal activator such as PHA.",
"Optionally, a culture step may be performed before and/or after CD3 activation so as to increase cell amount (e.g.",
"culture for 3 days in RPMI 10% fetal calf serum at 37° C., 95% humidity, and 5% CO2).",
"When it is desired to isolate SC5 in its intracellular form, then CD3 stimulation may be omitted.",
"Tumoral cells such as CTCL cells, and notably malignant transformed MF or SS cells, enable the isolation of SC5 molecules in their receptor form, without any required ex vivo CD3 activation: CTCL cells already express SC5 in a transmembranar location.",
"The invention therefore encompasses any isolated protein obtainable by: (i) collecting cells selected from the group consisting of T cells, CD3+ T cells, CD45RO+ T cells, B cells, NIK cells, macrophages, granulocytes, and stimulating the collected cells with immobilized anti-CD3 antibodies, or with both soluble anti-CD3 antibodies and PHA at 1 micrograimn/ml, or collecting malignant CTCL cells such as malignant CD4+ CTCL cells, or collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), and stimulating the collected cells with PHA at about 1 microgram per ml, (ii) lysing the cells under conditions enabling the dissociation of multimeric polypeptidic complexes, for example by incubation in a drastic lysis buffer containing Triton X-100 at 1%, (iii) recovering from the lysate the protein compound onto which the monoclonal antibody produced by hybridoma I-2575 binds under conditions enabling this monoclonal antibody to perform reactions of the antigen-antibody type (i.e.",
"the compound which is recognized as an antigen by this mAb).",
"The aminoacid sequence of the recovered protein compound can be obtained by any standard technique, such as mass spectrophotometry.",
"As above-indicated, the cells mentioned in step i. are activated cells, and therefore enables the isolation of SC5 in the form it has when it is a transmembrane receptor.",
"If the SC5 source used in step i. consists of resting normal T cells (without ex vivo stimulation), the the protein compound thus isolated will be SC5 in its cytoplamsic form.",
"When the cells selected in step i. are activated or malignant cells, lysis in step ii.",
"therefore may be performed either on whole cells, or on the membranar fraction of these cells.",
"The lysis step itself can be achieved by any technique enabling the dissociation of multimeric polypeptidic complexes from each others.",
"For example, cells (or their membrane fraction) may be incubated in a lysis buffer containing a drastic detergent such as Triton X-100 at about 1% for 1 hour at 4° C.; this enables the dissociation of the SC5 protein from the other cell components.",
"If a mild detergent such as digitonin 1% or Bridg58 1% is used, then SC5 won't dissociate from those compounds which are naturally associated with it; the use of a mild detergent in the above step ii.",
"therefore enables the isolation of SC5, and the further isolation of the SC5 transducers and effectors.",
"The invention therefore also encompasses any isolated compound obtainable by such processes.",
"The lysate obtained from step ii.",
"is then incubated with the monoclonal antibody produced by hybridoma I-2575 under conditions appropriate for this mAb to perform reactions of the antigen-antibody type, such as coating the antibodies into microwells containing the cell lysate (e.g.",
"2 microgrammes of the monoclonal antibody produced by hybridoma I-2575 for a lysate of 107 cells) and incubating the monoclonal antibodies with the cell lysate for two hours at 4° C. The step of recovering the compound onto which the monoclonal antibody produced by hybridoma I-2575 binds (step iii.)",
"can be achieved by any technique available the skilled person.",
"Recovering under non reducing conditions enables the isolation of SC5 in its native conformation, and will be preferred when it is desired to retain the native biological properties of SC5.Appropriate techniques for recovering SC5 protein under non reducing conditions namely include chromatography by affinity, and, for example: percolation of the lysate through a column in which the monoclonal antibody (mAb) produced by hybridoma I-2575 has been immobilized (e.g.",
"by coupling this mAb onto agar beads and placing these beads inside said column); preferably, the lysate is mixed with a buffer which has a.physiological pH, e.g.",
"at a pH comprised between 6 and 8, such as 7, before percolation so as to make the percolation easier without substantially altering the conformation of the compounds contained in the lysate, rinsing the column with a buffer at the same physiological pH, and recovering the compound which has bound to the immobilized mAb, e.g.",
"by elution with a buffer which disrupts the binding of the compound onto the mAb without substantially modifying the native conformation of the compound.",
"Such an elution buffer usually has a slightly acidic pH (usually comprised between 2 and 5, e.g.",
"a pH of 3).",
"For the preparation of a matrix which has an optimal orientation of antibody molecules, and which thereby enables antigen binding at a high efficiency, and for a one-step purification procedure for the isolation of membrane proteins, see e.g.",
"Schneider et al.",
"1982 (J. Biol.",
"Chem.",
"257: 10766-10769) of which content is herein incorporated by reference.",
"The protein compound thus recovered is the isolated SC5 protein in its native form.",
"Alternatively, step iii.",
"under non reducing conditions can be achieved by placing the lysate obtained from step ii.",
"for immuno-precipitation with the monoclonal produced by hybridoma I-2575, and the immuno-precipitate thus obtained can be percolated through a column comprising antibodies capable of binding mice IgM.",
"The protein compound which has bound onto the monoclonal antibody produced by hybridoma I-2575 is then recovered by elution similarly to what has been above described.",
"The protein compound thus recovered is also the isolated SC5 protein in its native form.",
"Another standard way for recovering the SC5 protein (step iii.)",
"is to proceed through electrophoresis under reducing conditions.",
"This notably includes incubating the monoclonal antibody produced by hybridoma I-2575 with the lysate obtained from step ii.",
"under conditions enabling this mAb to perform reactions of the antigen-antibody type, collecting the immuno-precipitate thus formed, separating the compounds contained in this immuno-precipitate under reducing conditions, and recovering the protein compound which has an apparent molecular weight of 96 kD.",
"An alternative way to localize on the electrophoresis gel or membrane the desired SC5 protein (and to subsequently isolate it from the gel or membrane) is to proceed with a labeling step prior to lysis so that surface polypeptidic components of the cells are labeled (e.g.",
"125 iode or fluorescent labeling with e.g.",
"sulfo-NHS-LC-biotin), and to recover from the immuno-precipitate those compounds which bear the label.",
"It has to be noted that if the labeling step is performed after lysis, then both the intra-cellular form and the trans-membranar receptor form of SC5 will be isolated (in this case, as above-indicated, non-activated T cells may be used as a SC5 source).",
"Because denaturing conditions are used, the protein compound thus recovered is in a non native form: it still has the native amino acid sequence, but its native biological properties have been substantially lost.",
"It is however the SC5 protein in its non-native conformation that is observed (and has to be searched for) when the biological sample to be tested has been treated under denaturing conditions (such as e.g.",
"paraffin sections of skin sample).",
"The SC5 protein in its non-native form may therefore be used for the production of monoclonal antibodies directed against it, such monoclonal antibodies being then useful for SC5 detection on denaturated samples.",
"If the SC5 protein in its native conformation is desired, portions of at least 10 amino acids of the denaturated protein may then be sequenced (e.g.",
"by mass spectrophotometry), oligonucleotides deduced from the sequenced portions may be synthetized, and then used as probes to screen a cDNA library obtained from SC5+ cells.",
"The cDNA thus selected may the be produced and cloned into a cell under conditions enabling the production, and preferably the excretion, of the cDNA-encoded product by culture of the clone (e.g.",
"via insertion of the SC5-encoding cDNA in a baculo virus vector and transformation of insect cells—e.g.",
"Sf9 cells—, or via the vaccina virus and EBV cells, see Rindis Bacher 1995, J. Biol.",
"Chem.",
"270(23): 14220-14228).",
"The invention therefore encompasses any isolated protein of which sequence is obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, labeling the stimulated cells with a polypeptide-specific label such as biotin, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which bears the label, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, recovering the protein produced by the transfected cells, e.g.",
"with the monoclonal antibody produced by hybridoma I-2575, or obtainable by: collecting cells selected from the group consisting of total blood cells and peripheral blood lymphocytes (PBL), stimulating the collected cells with PHA at 1 microgram/ml, lysing the labeled cells by incubation in a lysis buffer containing Triton X-100 at 1%, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, recovering from the immuno-precipitate the protein compound which has an apparent molecular weight of 96 kD under reducing conditions, synthesing the cDNA which encodes the protein sequence thus obtained, operably transfecting insect cells with this cDNA, the protein compound produced by the transfected insect cells, e.g.",
"with the monoclonal antibody produced by hybridoma I-2575.The invention thus encompasses any isolated protein compound obtainable by any of the above-mentioned processes, as well as any amino acid sequence of such an isolated protein compound, and any isolated protein of which sequence comprises such a sequence.",
"Falls within the scope of the invention any solid support onto which a protein compound or protein of the invention has been placed.",
"Such solid supports notably include sepharose beads.",
"The invention also encompasses any isolated DNA (or cDNA) of which sequence codes for an isolated protein of the invention.",
"It also relates to engineered cells transfected by such a DNA, to engineered cells which excrete an isolated protein of the invention, and to isolated protein obtainable by isolation from the culture medium of such engineered cells.",
"The invention notably encompasses any cDNA obtainable by: collecting a cell population which consists of, or comprises cells onto which the monoclonal antibody produced by hybridoma I-2575 binds (i.e.",
"onto which this mAb recognizes an antigen), or cells of which lysate would give an immuno-precipitation reaction with this monoclonal antibody, e.g.",
"collecting cells selected from.",
"the group consisting of total blood cells and PBL, if the sole transmembranar form of SC5 is desired: stimulating the CD3 pathway if said cell population mainly comprises or consists of non-activated cells (e.g.",
"resting non-tumoral cells) so as to increase SC5 expression at the cell surface, e.g.",
"by incubating the collected cell population with a CD3 activator (for example a polyclonal activator such as PHA at 1 microgram par ml when the collected cell population is PBL or total blood, or immobilized anti-CD3 antibodies, or both a polyclonal activator such as PHA and soluble anti-CD3 antibodies when the collected cell population is more homogeneous such as a cell population mainly comprising, or consisting of CD3+ T cells, or of CD45RO+ cells, or of NK cells, or of macrophages, or of granulocytes); if the collected cell population consists of, or mainly comprises activated cells (such as CD3-activated non-tumoral cells, or such as tumoral cells such as CD4+ CTCL cells), then CD3 stimulation is not necessary because SC5 location is already mainly transmembranar in these activated cells; if it is desired to isolate both the intracellular form and the trans-membranar form of SC5, then non-activated cells are preferred and CD3 activation is omitted, extracting and purifying the whole mRNA population from said cell population (commercial kits are available for mRNA extraction and purification, e.g.",
"a polydT columns), synthetising every complementary cDNA (with a reverse transcriptase, e.g.",
"such as described in Seed B. and Arrufo A.",
"1987, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 84: 3365-3369, of which content is herewith incorporated by reference), operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions, and cultivating every clone accordingly, selecting those clones which express a compound onto which the monoclonal antibody produced by hybridoma I-2575 binds when placed under conditions suitable for this monoclonal antibody to perform reactions of the antigen-antibody type, and/or those clones of which lysate would give an immuno-precipitation reaction with the monoclonal antibody produced by hybridoma I-2575 will be selected.",
"optionally amplifying those clones that have been thus selected, recovering the inserted cDNA from the selected clones.",
"The cDNA thus recovered is an SC5 cDNA of the invention, it can be sequenced using standard DNA sequencing techniques.",
"Operably cloning each cDNA so that expression of this cDNA in this clone is possible under appropriate clone culture conditions may be achieved by any conventional cloning procedure available to the skilled person.",
"An example of cloning procedure comprises inserting each cDNA in a transfection vector under the control of a promoter suitable for the expression of this cDNA in the host cell, transfecting the host cells with this transfection vector, and cultivating the transfected host cells under conditions appropriate to their metabolism.",
"Appropriate transfection vectors and host cells comprise virus such as Baculo virus and host cells such as insect cells (Sf9 cells), or vaccinia virus as a vector and EBV cells as host cells (see Rindis Bacher reference supra).",
"Such a cDNA can be used for cell transfection, and for SC5 production from the clones thus obtained.",
"It may also be used as probe for identifying or isolating the native SC5 mRNA.",
"The invention therefore also encompasses any isolated mRNA obtainable by selection among the above-mentioned mRNA population, of a mRNA which is complementary to a cDNA of the invention (e.g.",
"via selection of a mRNA which hybridizes to the above-mentioned cDNA under stringent conditions such as described in Freeman G. J. et al.",
"1992, J. Immunol.",
"149: 3745).",
"Are particularly encompassed any isolated mRNA obtainable by using an isolated cDNA of the invention as a probe so as to recover from the whole mRNA population of said cell population, the mRNA which is complementary to this extracted cDNA.",
"Alternatively, the one of ordinary skill in the art may directly sequence the cDNA that is recovered from the selected clones, and deduce from this sequence the sequence of the mRNA.",
"The invention also encompasses any DNA encoding an isolated mRNA according to the invention.",
"It particularly encompasses any genomic DNA obtainable by searching a genomic bank (such as www.ucsc.edu) for a genomic DNA matching with the above-mentioned SC5 cDNA.",
"The invention also encompasses any engineered cell in which a cDNA, a mRNA or a DNA of the invention has been transfected.",
"Are also encompassed SC5-specific probes and primers.",
"Such probes and primers can be obtained by any technique available to the skilled person (e.g.",
"selecting, as candidate sequences, portions of SC5 sequence that may be unique to SC5, comparing these candidate sequences on DNA/RNA banks with the sequences of cell receptors other than SC5, and selecting those candidate sequences which target a SC5 sequence that appears as unique after said comparison, and/or a SC5 sequence of which length appears as unique after said comparison).",
"Any solid support onto which is placed a cDNA, a mRNA or a genomic DNA of the invention falls within the scope of the present invention.",
"Such solid supports notably include DNA chips, or DNA microspheres.",
"The invention also encompasses any isolated protein encoded by an isolated cDNA, mRNA, or DNA according to the invention.",
"The invention further encompasses any isolated portion of a protein of the invention, provided that this portion is still characteristic of CTCL cells by comparison with normal resting cells.",
"It particularly encompasses any portion that is recognized by the monoclonal antibody produced by the hybridoma I-2575 at the C.N.C.M.",
"It also encompasses any isolated portion of a protein compound of the invention, provided that this portion is capable of inducing a modulation (inhibition or stimulation) of the CD3-induced proliferation of a normal T cell, and/or is capable of inducing a modulation of the in vivo proliferation of a malignant T cell such as a CTCL cell, and/or is capable of inducing a modulation of the IL-2 production of a normal T cell or of a malignant T cell, when said portion is expressed by said cell and contacted with the mAb produced by hybridoma I-2575.It particularly encompasses any isolated portion of a protein compound of the invention, provided that this portion is a part of a molecule that is expressed in a trans-membrane location by malignant CTCL cells and that is expressed in the cytoplasm compartment by non-tumoral resting T cells.",
"The invention notably provides with a bank of polypeptidic fragments which is obtainable by enzymatic cleavage of the SC5 protein of the invention.",
"Preferred enzymes comprises proteolytic enzymes such as those serine endopeptidases which cleave at the level of Tyr, Phe and Trp (e.g.",
"alpha-chymotrypsin), and also those enzymes which cleaves at the C-terminus of glutamic acid and aspartic acid (e.g.",
"V8 protease); see e.g.",
"Shesberadaran and Payne 1988, Proc.",
"Natl.",
"Acad.",
"USA 85:1-5, of which content is herein incorporated by reference.",
"Such a bank of SC5 polypeptidic fragments is of particular use in analysis such as mass spectrophotometry analysis: such analysis use the characteristic separation of such a polypeptidic bank, and give a (polypeptide) profile that is characteristic of SC5, and that can be used for SC5 detection.",
"Such a use and such a profile is encompassed by the present application.",
"The invention particularly encompasses the extra-cellular, trans-membranar and intra-cytoplasmic portions of any SC5 protein compound of the invention.",
"Determining the extra-cellular, trans-membranar and intra-cytoplasmic portions of a receptor molecule may be achieved by the skilled person according to any appropriate technique.",
"They may be identified on the whole molecule by identification of the region which contains glycosylation sites (extra-cellular portion), of the region that is hydrophobic (trans-membrane portion), and of the region that is hydrophylic (intra-cytoplasmic region), and they may then be isolated either by cleavage or synthesis.",
"An alternative way to isolate the extra-cellular portion of a protein compound of the invention is to proceed as above-described for the isolation of the whole protein compound, but with an additional step of enzymatic digestion so as to cleave from the whole molecule its extra-cellular portion.",
"Appropriate enzymes for such a cleavage includes enzymes such as V8 protease or alpha-chymotrypsin as above-mentioned.",
"The invention thus encompasses any isolated polypeptide compound obtainable by: collecting a SC5 cell source as above-described, such as e.g.",
"PBL or total blood cells, if it is desired to isolate a polypeptide compound in the form it has when it is naturally expressed at the cell surface, and if the collected SC5 cell source is, or mainly comprises normal resting cells, then stimulating the CD3 pathway, e.g.",
"via incubation with an appropriate CD3 activator (e.g.",
"incubation with PHA at 1 microgramme per ml when said SC5 source is total blood cells or PBL), labeling the cells with a polypeptide-specific label, such as biotin (surface labeling on whole cells), lysing the SC5 cell source (such as PBL or total blood cells) so as to dissociate multimeric polypeptidic complexes (and thereby recovering SC5 dissociated from the transducers and effectors naturally associated therewith) e.g.",
"by incubating the cells in a lysis buffer comprising a drastic detergent such as Triton X-100 at 1%, and submitting the lysate to an enzymatic digestion enabling the cleavage of SC5 into portions, e.g.",
"by incubating the lysate with a proteolytic enzyme such as an enzyme selected from the group consisting of V8 protease or alpha-chemotrypsin, submitting the lysate to immuno-precipitation with the monoclonal antibody produced by hybridoma I-2575, and recovering from the immuno-precipitate a polypeptide compound which bears a label.",
"Such recovered compounds are SC5 portions which either are the complete SC5 extra-cellular portion, or are part of such a complete SC5 extra-cellular portion, or comprise such a SC5 extra-cellular portion.",
"The set of polypeptide compounds that are thus obtainable constitute a bank of SC5 “extra-cellular” portions.",
"Such a bank, and its medical uses, such as e.g.",
"its use for the diagnosis of a T-related disease (e.g.",
"via mass spectrophotometry analysis), falls within the scope of the present invention.",
"The person of ordinary skill in the art will note that lysis and enzymatic digestion may be performed in a single step.",
"Labeling on whole cells is performed prior to cell lysis so as to identify SC5 portions which are extra-cellular portions, or which are part of such extra-cellular portions, or which comprise such portions.",
"The invention also encompasses any isolated compound which comprises a portion of a protein of the invention.",
"The mAb of the invention also enables the identification of the SC5 epitope onto which this mAb binds.",
"Conventional techniques such as directed mutagenesis are appropriate (see e.g.",
"Chang H. C. et al.",
"1989, J. Exp.",
"Med.",
"169: 2073-2083, of which content is herein incorporated by reference).",
"The proteins of the invention and their portions according to the invention, and notably their extra-cellular portions, enable the production of monoclonal antibodies directed against them.",
"An example of such monoclonal antibodies is produced by hybridoma I-2575.The monoclonal antibodies of the invention are particularly useful for the detection of SC5, for the diagnosis of T lymphomas such as CTCL, and are also useful as modulators of an undesired or inappropriate proliferation and/or activity of T cells, such as normal CD4+ and CD8+ T cells, virus-infected T cells such as HIV-infected T cells, T lymphomas such as CTCL.",
"The invention therefore encompasses any monoclonal antibody obtainable by: (i) immunizing an animal against a protein of the invention or against a portion thereof as above-defined, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the protein or protein portion that as been used as an immunogen in step i., and which has at least one property selected from the group consisting of the property of: binding resting non-tumoral T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, modulating the CD3 activation pathway of T cells, modulating IL-2 production from T cells, modulating the CD3-induced proliferation of T cells, modulating the CD3-induced in vitro proliferation of malignant T cells such as CD4+ CTCL cells, modulating the proliferation of malignant T cells such as CD4+ CTCL cells in an animal, and preferably in a human, competing with a monoclonal antibody of the invention, such as the monoclonal antibody produced by hybridoma I-2575, for binding to a protein of the invention, or to a portion thereof as above-defined, and in particular with the extra-cellular portion of such a protein, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.",
"Production of hybridomas may be achieved using any conventional technique.",
"This includes removing the spleen from said animal, making a suspension of spleen cells, fusing the spleen cells with myeloma cells in the presence of a fusion promoter, diluting and cultivating the fused cells in separate wells in a medium which will not support unfused cells.",
"The invention also encompasses any ascite isolated from said immunized animal.",
"For step iii., T and malignant T cells can be collected from a human using standard procedures.",
"Alternatively, clones can be used such as the CD4+ CTCL HUT 28 cell line (ATCC TIB-161).",
"In the case of CD4+ tumors, collection is made from the body part which will display the CD4+ tumoral cells (PBL for SS, cutaneous erythroderma for transformed MF, etc.).",
"The above-mentioned properties can be easily assessed by the skilled person using common knowledge in the art.",
"Examples of appropriate experimental conditions for assessing the properties mentioned in step iii.",
"can be found in the below examples.",
"The person of ordinary skill in the art will adjust them to the particular cells, or concentrations used.",
"Modulation of cell proliferation herein refers to stimulation as well as inhibition of this proliferation.",
"When CTCL cells are used, inhibition of proliferation is preferred in perspective of therapeutic application.",
"The mAbs of the invention are all anti-SC5 mAbs which have either diagnostic or therapeutic applications, or both.",
"For certain applications, it may be desired to immobilize one or several of these mAb onto a support, e.g.",
"for protein purification purposes, or for exerting the modulation property of which they are capable.",
"The present application also encompasses any support onto which a mAb of the invention has been immobilized.",
"Such supports notably include those appropriate for columns of chromatography by affinity (e.g.",
"agar beads).",
"The present application is also directed to any fragment of a mAb of the invention (anti-SC5 fragments) selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3.It is directed to any compound, and namely to any antibody, comprising such a fragment.",
"It notably relates to any humanized antibody comprising at least one of such fragments.",
"Techniques for the production of humanized antibodies have been widely described in the prior art (see e.g.",
"Farah et al.",
"1998, Crit.",
"Rev.",
"Eucaryote Gene Exp.",
"Vol.",
"8 pp 321-345, of which content is incorporated herein by reference).",
"In particular, it relates to any compound, and notably to any antibody and humanized antibody, comprising at least one anti-SC5 fragment as herein defined and also at least one anti-CD4 fragment selected from the group consisting of heavy chains, light chains, VH, VL, Fab, F(ab′)2, CD1, CDR2, CDR3 (see e.g.",
"WO 94/13804 “Multivalent and multispecific binding proteins, their manufacture and use”, Inventors: Holliger et al.",
"Applicants: Cambridge Antibody Technology Ltd and Medical Research Council; see also Merchant et al.",
"“An efficient route to human bi-specific IgG” Nat.",
"Biotechnol.",
"1998 vol.",
"16 pp 677-681).",
"As previously indicated, the invention provides with two molecules linked by a common general inventive concept: SC5 and p140.The SC5 is a novel molecule fully herein described.",
"The p140 molecule has been described in the prior art as an inhibitory receptor which is expressed by sub-groups of NK cells and of CD3+ CD8+ cells from healthy (non-cancerous) patients.",
"The present invention now demonstrates that p140 is expressed by malignant T cells, and notably by CTCL cells, and provides with a novel p140 allelic form (clone 24 in the below example 3; SEQ ID No.",
"1) encoding a novel protein (SEQ ID No.",
"2).",
"The cDNA and amino acid sequences of this novel allelic form are as follow: SEQ ID No.1: CATGTCGCTCACTGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGC AGGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCC CGGCGCAGCACTGTGGTGCCTCAAGGAGGACACGTGGCTCTTCAGTGTCA CTATCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCC ACGTTCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTGATCATGGGC CCTGTGACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCC ACACTCCCTCACTGGGTGGTCGGCACCCAGCAACCCCCTGGTGATCATGG TCACAGGAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGACCCTG CTGAAATCAGGAGAGACAGTCATCGTGCAATGTTGGTCAGATGTCATGTT TGAGCACTTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCC TCGTTGGACAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGT CCCTTGATGCCTGTCCTTGCAGGAACCTACAGATGTTATGGTTCTGTTCC TCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCCCTGGACATCGTGA TCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACG GTTCAGGCAGGAGAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTA TGACATCTACCATCTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCC GTGCAGTGCCGAAGGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGG CCTGCCACCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGCCCT GCCCTGCGTGTGGTCAAACTCAAGTGACCCACTGCTTGTTTCTGTCACAG GAAACCCTTCAAGTAGTTGGCCTTCACCCACAGAACCAAGCTCCAAATCT GGTATCTGCAGACAGGTGCATGTTCTGATTGGGACGTCAGTGGTCATCTT CCTCTTCATCCTCCTCCTCTTCTTTCTCCTTTATCGCTGGTGCTCCAACA AAAAGAATGGTGCTGTAATGGACCAAGAGCCTGCGGGGGACAGAACAGTG AATAGGCAGGACTCTGATGAACAAGACCGTCAGGAGGTGACGTACGCACA GTTGGATCACTGCGTTTTCATACAGAGAAAAATCAGTCGCCCTTCTCAGA GGGCCAAGAGACCCCCAACAGATACCAGCGTGTACACGGAACTTCCAAAT GCTGAGCCCAGATCCAAAGTTGTCTCCTGCCCACGAGCACCACAGTCAGG TCTTGAGGGGGTTTTCTAGGGAGACAACAGCCCTGTCTCAAAACC SEQ ID No.2: pLMGGQDKPF LSARPSTVVP QGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPLVIMVTGN HRKPSLLAHP GTLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS ˜DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PPTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF The DNA and amino acid sequences of the previously described p140 allelic form (clone 1.1) are as follow: SEQ ID No.3: CATGTCGCTCACGGTCGTCAGCATGGCGTGCGTTGGGTTCTTCTTGCTGCA GGGGGCCTGGCCACTCATGGGTGGTCAGGACAAACCCTTCCTGTCTGCCCG GCCCAGCACTGTGGTGCCTCGAGGAGGACACGTGGCTCTTCAGTGTCACTA TCGTCGTGGGTTTAACAATTTCATGCTGTACAAAGAAGACAGAAGCGACGT TCCCATCTTCCACGGCAGAATATTCCAGGAGAGCTTCATCATGGGCCCTGT GACCCCAGCACATGCAGGGACCTACAGATGTCGGGGTTCACGCCCACACT CCCTCACTGGGTGGTCGGGACCCAGCAACCCCGTGGTGATCATGGTCACAG GAAACCACAGAAAACCTTCCCTCCTGGCCCACCCAGGGCCCCTGCTGAAA TCACGAGAGACAGTCATGCTGCAATGTTGGTCAGATGTCATGTTTGAGCAC TTCTTTCTGCACAGAGAGGGGATCTCTGAGGACCCCTCACGCCTCGTTGGA CAGATCCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTGGCTTGATG CCTGTGCTTGCAGGAACGTACAGATGTTATGGTTCTGTTCCTCACTCCGGC TATCAGTTGTCAGCTCGCAGTGACCCCCTCGACATCGTGATCACAGGTCTA TATGAGAAACCTTCTCTCTCAGCGCAGCGGGGCCCCACGGTTCAGGCAGGA GAGAACGTGACCTTGTCCTGTAGCTCCTGGAGCTCCTATGACATCTACCAT CTGTCCAGGGAAGGGGAGGCCCATGAACGTAGGCTCCGTGCAGTGCCGAA GGTCAACAGAACATTCCAGGCAGACTTTCCTCTGGGCCCTGCCACCCACGG AGGGACCTACAGATGCTTCGGCTCTTTGCGTGCCCTGCCCTGCGTGTGGTC AAACTCAAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAGTAG TTGGCCTTCACCCACAGAACCAAGCTCCAAATCTGGTATCTGCAGACACCT GCATGTTCTGATTGGGACCTCAGTGGTCATCTTCCTCTTCATCCTCCTCCT CTTCTTTCTCCTTTATCGCTGGTGCTCCAACAAAAAGAATGCTGCTGTAAT GGACCAAGAGCCTGCGGGGGACAGAACAGTGAATAGGCAGGACTCTGATG AACAAGACCCTCAGGAGGTGACGTACGCACAGTTGGATCACTGCGTTTTC ATACAGAGAAAAATCAGTCGCCCTTCTCAGAGGCCCAAGACACCCCTAAC AGATACCAGCGTGTACACGGAACTTCCAAATGCTGAGCCCAGATCCAAAG TTGTCTCCTGCCCACGAGCACCACAGTCAGGTCTTGAGGGGGTTTTCTAGG GAGACAACAGCCCTGTCTCAAAACC SEQ ID No.4: pLMGGQDKPF LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPVVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQBVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF SEQ ID No.",
"1 differs from SEQ ID No.",
"3 in five locations.",
"The resulting SEQ ID No.2 differs from the p140 disclosed in prior art (SEQ ID No.",
"4) by four amino acid substitutions (shown in bold and underlined characters): R instead of Q in position 20, V instead of L in position 92, P instead of T in position 102, and L instead of P in position 401 (see FIG.",
"11).",
"The present application encompasses any isolated compound of which sequence is, or comprises SEQ ID No.",
"1 and/or SEQ ID No.",
"2.It also encompasses any mRNA that is complementary to SEQ ID No.",
"1, and any genomic DNA coding for SEQ ID No.",
"2.Appropriate techniques for producing p140 proteins are available to the skilled persons.",
"They notably include the production of cells which have been engineered so as to produce p140 proteins in their culture medium.",
"An example of such techniques comprises inserting SEQ ID No.",
"1 or No.",
"3 in a baculo virus vector, operably transfecting an insect cell such as Sf9 cell line with this vector, recovering the p140 protein produced in the culture medium (e.g.",
"by percolation of the culture medium is through a column—e.g.",
"a Sephadex column—which protein compounds as a function of their molecular weight, and isolating the 140 kD eluate).",
"Compounds capable of binding to p140 under conditions of the physiological type (in vivo conditions, or in vitro conditions mimicking the in vivo ones) have been previously described, and may be obtained by the person of ordinary skill in the art by any appropriate available technique.",
"By “p140 binding compound”, it is herein meant any compound which is capable under said conditions of the physiological type to recognize a p140 molecule as an antigen (SEQ ID No.",
"2 and/or SEQ ID No.",
"4) when it is expressed by a cell in a transmembrane receptor location (i.e.",
"recognition of the SEQ ID No.",
"2 and/or No.",
"4 portion that is above the lipid bi-layer of said cell).",
"Such p140 binding compound notably comprise HLA-A11 and HLA-A3 molecules (natural p140 ligands), and notably fusion proteins such as Fc-HLA-A11 and Fc-HLA-A3 fusion proteins.",
"p140 binding compounds also comprise p140 antiserum (obtainable by immunizing an animal such as a rabbit against p140, and by recovering the antiserum thus produced—an additional step of immuno-purification may be performed so as to increase p140 concentration in the antiserum, e.g.",
"via percolation through a p140 column).",
"Other p140 binding compounds comprise monoclonal antibodies directed to p140 SEQ ID No.",
"4, such as e.g.",
"AZ158, Q66, Q241 described in Pende et al.",
"1996 (J. Exp.",
"Med.",
"184: 505-518).",
"Such anti-SEQ ID No.",
"4 mAbs may also recognize the new SEQ ID No.",
"2 as an antigen.",
"Any available technique for mAb production is appropriate for production of anti-p140 mAbs.",
"An example of such a technique comprise the steps of: (i) immunizing an animal against p140, (ii) producing hybridomas from the spleen cells of this animal, and cultivating them for them to produce monoclonal antibodies in their culture supernatants, (iii) evaluating the supernatants for the presence of an antibody which is capable of binding to the p140 molecule that as been used as an immunogen in step i., and which is also capable of binding resting non-malignant T cells mainly in their cytoplasmic compartment, and binding malignant CD4+ CTCL cells mainly at their cell surface, (iv) selecting and cloning hybridomas producing the desired antibody, (v) recovering the antibody from the supernatant above said clones.",
"Preferred p140 binding compounds do also comprise an anti-CD4 entity (e.g.",
"bi-specific mAb).",
"Such p140 binding compounds are useful for p140 detection on malignant T cells, such as CTCL cells, in particular CD4+ CTCL.",
"They are thus useful for CD4+ cancer detection.",
"They are also useful as polypeptidic vectors, as described below.",
"According to another aspect of the invention, there are provided polypeptidic vectors comprising at least one element selected from the group consisting of the anti-SC5 mAbs of the invention and the anti-SC5 mAb fragments of the invention.",
"Such polypeptidic vectors are useful for molecules, such as active principle molecules, to reach activated T cells, or their vicinity (e.g.",
"activated T cells observed in inflammatory diseases, or in virus-infected T cells, or malignant T cells such as malignant CTCL cells, and malignant CD4+ CTCL cells in particular).",
"It also relates to polypeptidic vectors comprising a p140 binding compound such as a mAb directed against SEQ ID No.",
"2 and/or SEQ ID No.",
"4 extra-cellular portion.",
"Such polypeptidic vectors are useful for delivering molecules to, or in the vicinity of malignant T cells, such as notably CTCL cells, and malignant CD4+ CTCL cells in particular.",
"Molecules which may be usefully delivered to malignant T cells, such as CTCL cells notably comprise molecules capable of inducing cell death or apoptosis, such as a radio-element or a toxin.",
"Such molecules also comprise any enzyme capable of transforming an antimititotic pro-drug into an active drug form, such as a carboxypeptidase.",
"A polypeptidic vector carrying such an enzyme is then advantageously used in combination with an anti-mitotic pro-drug (such as phenol mustard pro-drug).",
"The invention therefore also encompasses a medical kit comprising such a vector and such an anti-mitotic pro-drug.",
"Molecules which may be usefully delivered in the vicinity of malignant T cells such as CTCL cells notably comprise molecules capable of stimulating the immune functions of cells which are in the vicinity-of malignant T cells in an effort to induce an anti-tumor effect from these neighboring cells towards said malignant T cells.",
"Examples of such molecules notably comprise cytokines such as interferon gamma, and interleukine such as IL-2 (see e.g.",
"Xu et al.",
"2000, Cancer Research 60: 4475-4484).",
"When a polypeptide vector of the invention is intended for targeting CD4+ cells such as CD4+ CTCL cells, then this vector advantageously further comprises an anti-CD4 entity.",
"The invention also encompasses any pharmaceutical composition comprising such polypeptidic vectors, and in particular any medicament comprising such polypeptidic vectors.",
"Such medicaments are useful in the control of an inappropriate or undesired T cell proliferation and/or functional activity (including undesired IL-2 production).",
"They are particularly useful for the prevention, palliation, relieve or therapy of an inappropriate immune activity, and notably for the prevention or inhibition of a T lymphoma such as CTCL and in particular CD4+ CTCL.",
"The use of such vectors for the manufacture of a drug intended for the prevention, palliation, relieve or therapy of an inappropriate T cell proliferation and/or activity, and notably for the prevention or inhibition of a CTCL proliferation is thus also an object of the present application.",
"A compound which binds to SC5 under conditions of the physiological type may be used as a SC5 ligand agonist so as to stimulate the activation of SC5, the SC5 molecule being then regarded as a cell inhibitory receptor.",
"Such anti-SC5 compounds namely comprise anti-SC5 antisera, the anti-SC5 mAb of the invention, the Fab, F(ab′)2, fragments thereof, the humanized mAbs derived therefrom.",
"When CD4+ cells are more particularly concerned, said anti-SC5 compound advantageously further comprises an anti-CD4 entity.",
"Anti-SC5 compounds are particularly useful for modulating an inappropriate or undesired proliferation and/or functional activity of SC5+ cells.",
"They are thus useful for modulating the proliferation of T cells, CD45RO+ cells, CD3+ cells, CD4+ T cells, CD8+ T cells, or of virus-infected T cells (e.g.",
"HIV-infected CD4+ T cells), or of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.",
"They are also useful for modulating IL-2 production from T cells.",
"When the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M.",
"(which is a pentameric IgM) is used, this modulation goes in favor of an inhibition.",
"It is therefore particularly appropriate for inhibition of T lymphomas such as CTCL.",
"The application encompasses any drug comprising, as an active principle, at least one anti-SC5 compound.",
"Such a drug may be intended for the prevention, palliation, therapy of T lymphomas, of CTCL, of T infections such as viral (e.g.",
"HIV) infections, of inflammatory diseases such as those of the auto-immune type (e.g.",
"rhumatoid arthritis—e.g.",
"spondyloarthropathis—or skin immune mediated diseases such as psoriasis, eczema, atopic dermatitis).",
"Such a drug may also be intended for graft improvement (inhibition of graft rejection).",
"The invention also encompasses the use of such anti-SC5 compounds as complement recruiting agents, or as ADCC activators or stimulators.",
"The p140 binding compounds as defined above, may also be used as complement recruiting agents, or as ADCC stimulators or activators in the prevention, palliation, relieve or treatment of T lymphomas, such as CTCL and in particular CD4+ CTCL.",
"The invention therefore encompasses the use of such p140 binding compounds in the manufacture of an anti-T lymphoma drug, of an anti-CTCL drug, of an anti-CD4+ CTCL drugs, and also encompasses such drugs.",
"The invention also provides with a method for the assessment of the development level of a CTCL (staging): it enables to evaluate the proportion (e.g.",
"percentage) of malignant CD4+ CTCL cells present within a certain body compartment of a patient.",
"According to this method, the proportion of CD4+ cells expressing at their surface an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from said body compartment.",
"The proportion of CD4+ CTCL cells that are actually present in said body compartment can be considered as substantially equal to said measured proportion.",
"It usually falls within a ±10% range around this measured proportion.",
"The invention also provides with a method for CTCL diagnosis, wherein the percentage of T cells expressing an element selected from the group consisting of the SC5 proteins of the invention, the SC5 portions of the invention (notably, those extracellular portions), and the p140 molecules is measured in a biological sample collected from a patient, and is compared to the average percentage observed in non-CTCL humans (preferably in healthy humans), and in that a CTCL-positive diagnosis is decided when said measured percentage is significantly higher than said average percentage.",
"This CTCL diagnosis method particularly applies to those CTCL which are CD4+; in this case the measured T cell percentage is preferably a CD4+ percentage.",
"The average standard proportion of CD4+ T cells expressing SC5 is generally in between 1-15%, usually in between 5-10% among healthy humans.",
"When the measured percentage of SC5+ CD4+ T cells is higher than such an average percentage, then a CD4+ CTCL positive diagnosis can be concluded.",
"Of note is that, as far as SC5 protein or portions thereof are concerned, the methods of the invention more generally enables to evaluate the proportion of CD4+ activated T cells that are present in said body compartment.",
"This may e.g.",
"be useful for assessing whether a graft induces rejection reactions, and for the diagnosis and follow-up of inflammatory diseases such as those of the auto-immune type (e.g.",
"rhumatoid arthritis such as spondyloarthropathis, skin immune mediated disease such as psoriasis, eczema, atopic dermatitis).",
"Said biological sample may then be intra-synovial fluid.",
"This may be also useful for the diagnosis and monitoring of non-CTCL T lymphomas (e.g.",
"CD4+ ALL, or CD4+ T-LL).",
"The CTCL assessing/diagnostic methods of the invention are highly efficient for all kinds of CD4+ CTCL, and notably for transformed MF, for SS, for LP, as well as for CD30+ lymphomas.",
"Said biological sample may e.g.",
"then be a skin sample collected from an erythroderma (e.g; suspicion of transformed MF), and/or a PBL or total blood sample (in order to evaluate whether a transformed MF has evolved into a more aggressive form such as SS).",
"When the element detected at the surface of said CD4+ cells is a SC5 compound, then a (preliminary or ulterior) step of cytological observation (search for tumor-alike cytology) is the preferably performed to conclude with a confidence of more than 90% to an actual CD4+ CTCL.",
"When p140 is detected at the surface of said CD4+ cells, then no additional step is required: the simple fact of detecting p140 at the surface of CD4+ T cells directs to a positive diagnosis of CD4+ CTCL.",
"The methods of the invention can be implemented with any appropriate element, and notably with an element selected from the group consisting of the anti-SC5 monoclonal antibodies of the invention, the Fab, F(ab′)2 fragments thereof, the humanized mAb derived therefrom, the SC5 “extra-cellular” bank of the invention (as above-defined), the SC5 cDNA, mRNA, genomic DNA of the invention, the p140 binding compounds as herein defined, the p140 DNA, the DNA comprising SEQ ID No.",
"1 or SEQ ID No.",
"3 or encoding SEQ ID No.",
"2 or SEQ ID No.",
"4.Such an element is then used as a detection means for SC5 or p140 cell surface expression.",
"Preferably, said detection means also comprises an anti-CD4 entity so as to detect SC5+ /p140+ CD4+ cells in a one-step procedure.",
"When cDNA/mRNA material is used as a detection means, then CD4+ cells have to be lysed so as to detect those SC5 or p140 hybridizing mRNA which are actually present in said cells (detection of the transcripts).",
"Such cDNA/mRNA detection means may advantageously be placed onto or into a solid support such as a DNA chip or a DNA microsphere.",
"When mAb material or mAb-derived material is used as a detection means, then surface expression of SC5 or p140 is detected.",
"Any appropriate antibody-antigen technique is convenient.",
"Examples notably comprise flow cytometry analysis.",
"The invention also encompasses a method for the identification of a compound which is useful in the palliation, prevention, relieve, therapy of an undesired or inappropriate T cell activity, such as notably a proliferation of malignant T cells such as CTCL cells and in particular CD4+ CTCL cells.",
"This method is characterized in that it comprises the detection of a compound that is capable of binding to a SC5 protein as above-defined, or to a SC5 polypeptide compound as above-defined, or to a p140 molecule (such as SEQ ID No.",
"2 and/or SEQ ID No.",
"4).",
"Such compounds can be used as polypeptidic vectors as above-described.",
"Those compounds which, in addition to binding to SC5 molecules, are also capable of aggregating these SC5 molecules at the cell surface, are preferred for stimulating SC5 inhibitory functions.",
"An easy way to implement this identification method is to proceed with SC5 and/or p140 molecules immobilized onto a solid support such as protein A Sepharose CL-4B.",
"Candidates compounds for implementation of the method notably comprise sera immunized against SC5 or p140.The invention is illustrated by the examples, which are in no way intended to restrict the invention to the particular embodiments described below.",
"The person of ordinary skill in the art will contemplate alternative embodiments, and such alternative embodiments derived from common knowledge in the art are within the scope of the invention.",
"The skilled person will in particular appreciate that various standard immuno-techniques, and various standard molecular biology techniques are available to him (see e.g.",
"“Antibodies: a laboratory manual” Ed Harlow, David lane, Cold Spring Harbor Laboratory 1988; Maniatis 1982, “Molecular cloning: a laboratory manual”, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory; “Current protocols in Immunology” protocols on CD-ROM, John Wiley, West Sussex, England; “Immunological techniques made easy”, edited by Olivier Cochet et at.",
"; John Wiley, West Sussex, England; and the publication specifically referred to in the present application).",
"The content of such prior art documents is herein incorporated by reference.",
"It is also to be understood that experiments described herein in full details for one particular form of CD4+ CTCL can be directly transposed by the skilled person to any other form of CD4+ CTCL.",
"In these examples, reference is made to the following figures: FIGS.",
"1A, 1B and 1C illustrate cell membrane expression of SC5 molecule on PBLs from normal individual.",
"PBMC from a normal individual were stained with anti-SC5 mAb (ascites ({fraction (1/200)}) followed by FITC-conjugated isotype specific goat anti-mouse IgM second reagent and one of the following PE-conjugated mAbs: anti-CD3 (FIG.",
"1A), anti-CD56 (FIG.",
"1C) and anti-CD45RO (FIG.",
"1B).",
"The percentage of double-stained lymphocyte-gated cells is shown in the upper right quadrant (3.3 for FIG.",
"1A; 5.1 for FIG.",
"1B; 0.6 for FIG.",
"1C).",
"This experiment is representative for the 10 donors studied.",
"FIG.",
"2 illustrates cell membrane expression of SC5 molecule during activation of PBLs.",
"PBMC were stimulated with 1 μg/mL PHA and the kinetics of SC5 and CD69 expression were studied in parallel on the CD3+ cells.",
"FIG.",
"3 illustrates intracellular localization of SC5 molecule in PBL.",
"Anti-SC5, anti-CD3ζ chain mAb and anti-BY55 isotype-matched mAb were used for staining permeabilized and non-permeabilized gated lymphocytes from a normal donor.",
"The shaded histograms represent the labeling obtained with the indicated mAbs compared to an irrelevant control mAb.",
"FIG.",
"4 illustrates biochemical analysis of SC5 molecule.",
"DS6 T cell clone was surface labeled with biotin and 1% Triton X-100 lysates were immunoprecipitated, using anti SC5 mAb.",
"The samples were analyzed by SDS-10%-PAGE under reducing conditions.",
"Anti-SC5 mAb was used at two concentrations {fraction (1/200)} (lanes 2) and {fraction (1/500)} (lane 3).",
"The negative control samples were precipitated with goat anti-mouse Ig alone (lane 1) and isotype-matched irrelevant mAb (lane 4).",
"The molecular weight markers (kD), are indicated on the left.",
"FIGS.",
"5A, 5B, 5C, 5D illkustrate that anti-SC5 mAb modulates the anti-CD3-induced proliferation and cytokine secretion of normal T lymphocytes whithout affecting their cytotoxic activity: FIG.",
"5A: PBL or T cell clones LSO and GDS.3 were stimulated with immobilized anti-CD3 mAb (1 μg of mAb coated per well) or with IL-2 (50 IU/ml) in the presence of anti-SC5 mAb (1:200 final dilution of ascites) or an isotype-matched irrelevant mAb.",
"Results shown are representative of at least three separate experiments and are expressed as mean cpm±SD of triplicate wells.",
"FIG.",
"5B: GDS.3 CD4+ T cell clone was stimulated with the indicated concentrations of pre-coated anti-CD3 mAb in the presence of different concentrations of anti-SC5 mAb.",
"FIG.",
"5C: IL-2 secretion in the supernatant of anti-CD3-stimulated PBL, in the presence of anti-SC5 or control mAb, was assessed by measuring the proliferation of an IL-2 dependant T cell clone in the presence of: medium alone (a), supernatant from PBL/anti-CD3+ control mAb (b), supernatant from PBL/anti-CD3+ anti-SC5 mAb (c).",
"Data represent the mean values±SD of triplicate determinations of one out of three representative experiments.",
"FIG.",
"5D: JF1, a cytotoxic CD8+ cell clone was incubated with anti-SC5 mAb ({fraction (1/200)} final dilution of ascites) or with an isotype matched control mAb before the coculture with the FcγR+ murine tumor cells P815 at an E/T ratio of 5/1.Target cells were preincubated with the indicated final concentrations of anti-CD3 mAb.",
"Results are expressed as mean of triplicate wells.",
"FIGS.",
"6A, 6B illustrate cell membrane expression of SC5 molecule on PBL from CTCL patients.",
"Cells from peripheral blood of 8 patients with Sézary syndrome, 3 patients with Mycosis fungoides and 8 healthy donors were analysed by two-color fluorescence for the expression of SC5 molecule by CD4+ lymphocytes.",
"FIG.",
"6A: co-expression of SC5 and CD4 molecules in PBL from a patient with Sézary syndrome in comparison to a normal individual.",
"FIG.",
"6B: histograms showing the mean values of SC5 expression in the 3 indicated groups.",
"Statistically significant differences between SS patients and 2 other groups are marked, (Mann-Witney U test, p<0.001) FIGS.",
"7A, 7B, 7C illustrate surface membrane expression on malignant cell from Sézary syndrome patient.",
"FIG.",
"7A: two-color immunofluorescence analysis of PBMC from SS patient with a circulating TCRVβ22+ malignant T cell clone.",
"Cells were stained with anti-SC5 mAb followed by PE-conjugated isotype specific goat anti-mouse IgM and FITC-conjugated anti-TCRVP22 mAb.",
"FIG.",
"7B: anti-SC5-mAb labeled weakly (shaded histogram) the TCRVβ22-positive malignant cells.",
"FIG.",
"7C: cells from the same patient stained with PE-conjugated anti-CD69 mAb and FITC-conjugated anti-TCRVβ22 mAb.",
"FIG.",
"8 illustrates the modulation of anti-CD3-induced proliferation of CTCL malignant cell line by anti-SC5 mAb.",
"Pno cell line was stimulated with IL-7 (10 ng/mL), PMA alone (2 ng/mL), immobilized anti-CD3 mAb or a combination of PMA/anti-CD3 mAb.",
"SC5 mAb or an isotype-matched irrelevant mAb (1:200 final dilution of ascites) was added at the start of cultures.",
"Results shown are representative of four separate experiments and are expressed as mean cpm±SD of triplicate wells.",
"FIGS.",
"9A, 9B illustrate the detection of p140/KIR molecules on CTCL cell lines (FIG.",
"9A) and on fresh tumor lymphocytes (FIG.",
"9B).",
"Double immunostaining flow cytometric analysis was performed as described in Bagot et al.",
"1998 (Blood 91:4331-434 1) and in Poszepczynska et al.",
"2000 (Blood 96: 1056-1063).",
"FIG.",
"10 illustrates the biochemical analysis of p140 molecules: the NK clone AM61 (p140+/p70−), derived from a healthy donor, and the cell lines Pno and Cou-L were surface labeled with biotin and immunoprecipitated with anti-p140 and anti-CD8 mAbs.",
"Samples were treated (+) or not (−) with N-glycosidase F and analyzed in an 8% (left panel) or 11% (right panel) SDS-PAGE under reducing conditions.",
"Sepharose Protein-A alone represents the negative control.",
"Molecular weight markers (kD) are indicated on the right.",
"FIG.",
"11 shows the amino acid sequence alignment of KIR3D cl.",
"24 (SEQ ID No.",
"2) and cl.",
"1.1 (SEQ ID No.",
"4) encoded proteins.",
"Amino acids corresponding to the signal peptide are in small case letters, while transmembrane region is underlined in the consensus sequence.",
"Amino acids identical to the consensus sequence are indicated by dots.",
"EXAMPLE 1 SC5 Isolation, SC5 Biochemical and Functional Characterization, Production of Anti-SC5 mAbs T lymphocyte immune responses are regulated by functional cell surface molecules providing positive signals that lead to the expansion of antigen-specific clones, and negative signals which prevent excessive stimulation or responsiveness to self antigens.",
"The activation of resting T lymphocytes requires two independent signals.",
"The signal provided by the engagement of T-cell receptor (TCR) must be accompanied by a second positive signal in order to result in optimal T cell response.",
"CD28 is considered as the major co-stimulatory receptor inducing T lymphocyte activation and IL-2 synthesis and preventing cell-death.",
"Recent studies demonstrated that CD3/TCR stimulation leads to a redistribution of the detergent-insoluble glycolipid-enriched membrane fraction (DIG) or raft, which results in the aggregation of TCR/CD3 and DIG-associated signal-transducing molecules.",
"CD28 was reported to enhance raft redistribution to the site of TCR engagement.",
"Further, a series of other molecules may provide co-stimulatory signals to T cells, acting at different time points, affecting particular subsets or promoting distinct effector functions.",
"Negative regulation of T lymphocyte responses can be mediated by CD152-induced signals or by apoptosis through the members of TNF receptor superfamily.",
"Recently, a novel type of negative regulation has been reported for subsets of NK cells and CD8+ T lymphocytes (Lan998, Annu.",
"Rev.",
"Immunol.",
"16: 359-393).",
"The immunoglobulin-like (KIRs) or C-type lectin structures responsible for these signals prevent unwanted effector functions such as activation of CTL activity or cytokine production.",
"We now describe a novel 96 kD transmembrane receptor, SC5, which delineates a minor subset of peripheral blood lymphocytes in normal individuals.",
"Anti-SC5 mAb stained mainly CD45RO+ lymphocytes, from both CD8 and CD4 subsets, as well as NK lineage cells.",
"We found that SC5 was predominantly located in the intracellular compartment of resting T lymphocytes and its surface membrane expression increased rapidly after cell activation.",
"SC5 molecule engagement inhibited the anti-CD3 mAb-induced proliferation of resting T lymphocytes or T cell clones, whereas it had no effect on the cytokine-induced proliferation of these cells.",
"At the same time, the effector cytotoxic activity mediated by CD8+ T cell clones was not altered by anti-SC5 mAb.",
"Further on, we observed that the percentage of SC5+CD4+ circulating lymphocytes was significantly increased in Sézary syndrome (SS) patients as compared to peripheral blood lymphocytes of normal individuals.",
"Importantly, we were able to clearly demonstrate that Sézary cells express SC5 molecules.",
"In addition, we found that ligation of SC5 molecules in a cutaneous T cell lymphoma (CTCL) cell line resulted in a strong inhibition of the malignant cell proliferation induced by anti-CD3 mAbs.",
"Thus, SC5 molecule expression can serve to detect the presence of circulating malignant CD4+ cells in SS patients.",
"Moreover, the identification of a cell surface molecule providing negative signals upon ligation constitutes a new tool to investigate the mechanisms of pathological T cell growth and could be used to prevent it.",
"Material and Methods Production of Anti-CS mAb SC5 mAb was obtained by immunizing 6 wk old Balb/c mice with the NK cell line Ytindi, using an established protocol (David et al.",
"1990; J. Immunol.",
"144: 1-6).",
"Hybridoma supernatants were screened for selective reactivity with the immunizing cell line and a CTCL cell line termed Pno (Poszcepczynska et al.",
"2000; Blood 96: 1056-1063) by indirect immunofluorescence and flow cytometry.",
"Pno cell line derives from CTCL cells from a patient which had a CD3+ Vβ22+ CD4+ CD8αα+ CD25-phenotype, and which proliferates in response to IL-7 and to anti-CD3 mAb stimulation.",
"Any CTCL cell line is appropriate for this screening.",
"Examples of other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).",
"The reactive supernatants were further tested for their reactivity with peripheral blood lymphocytes (PBL).",
"A hybridoma supernatant was selected for its reactivity with both tumoral cell lines and with a minor PBL sub-population.",
"The hybridoma was cloned twice and cloned hybridomas were passaged into pristane-primed Balb/c mice to produce ascites.",
"The antibody isotype was determined as IgM and termed anti-SC5 mAb.",
"The ascites was dialysed against PBS, sterilized by ultrafiltration and further utilized at final dilution {fraction (1/200)}.",
"This hybridoma has been deposited on Oct. 30, 2000 at the C.N.C.M.",
"(Collection Nationale de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France) in accordance with the Budapest Treaty; the C.N.C.M.",
"deposit number is I-2575.In this example, “anti-SC5 mAb” refers to the mAb produced by hybridoma I-2575.Patients After informed consent and approval by an ethic committee (CCPPRB, Hôpital Henri Mondor, Creteil, France), we obtained blood samples from eleven patients with CTCL.",
"Eight patients had a Sézary syndrome with 10 to 80% circulating CD3+, CD4+, CD8-Sézary cells, whereas three patients had a transformed mycosis fungoides (MF) and presented with disseminated skin tumors with a CD3+, CD4+, CD8-phenotype.",
"All patients were not previously treated with chemotherapy.",
"Cells and Cell Lines Peripheral blood mononuclear cells (PBMC) were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.) density gradient centrifuigation.",
"Human T cell clones GDS.3 (CD3+TCRαβ+CD4+CD8−), DS6 (CD3+TCRγδ+CD4−CD8−), LSO (CD3+TCRγδ+CD4−CD8−) and JF1 (CD3+TCRαβ+CD4−CD8+), described in Bensussan et al.",
"1984 (J. Exp.",
"Med.",
"159: 947-952) and Yssel et al 1984.(J.",
"Immunol.",
"Methods 72: 219), were fed each 8 days with irradiated allogenic PBMC in culture medium consisting of RPMI 1640 (GIBCO, Paisley, UK), 2 mmol/L L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml), 10% heat-inactivated human serum, 50 IU/ml recombinant interleukin-2 (rIL-2, Sanofi Synthélabo, Labège, France) and 1 μg/ml phytohemagglutinin (PHA), (Wellcome, Beckenham, UK).",
"Functional studies were performed at day 7 after the feeding.",
"Standard human leukemic cell lines were mycoplasma-free and maintained in logarithmic growth in complete RPMI medium supplemented with 10% fetal calf serum (FCS) and antibiotics.",
"The human CTCL cell clone, Pno, was established from peripheral blood of a Sézary patient and maintained in culture as previously described (Poszcepczynska et al.",
"2000; see supra).",
"Pno cell line has a CD3+Vβ22+CD4+CD8αα+CD25-phenotype and proliferates in response to IL-7 and to anti-CD3 mAb stimulation.",
"Other CTCL cell lines include the HUT78 cell line (ATCC TIB-161).",
"Monoclonal Antibodies and Flow Cytometry Studies Indirect immuno-fluorescent staining was performed with hybridoma supernatants or ascites fluid using FITC-conjugated goat anti-mouse Ig from Caltag laboratories (San Francisco, Calif.).",
"For two-color analysis the immuno-fluorescent staining was performed by incubating 3×105 cells with anti-SC5 mAb for 30 min at 4° C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgM (Caltag laboratories) followed by a second PE-, ECD- or TRI-conjugated specific mAb of IgG isotype.",
"Stained cells were analyzed using a single argon flow cytometer analyser (Epics XL, Bekman-Coulter, Miami, Fla.) as described in Schiavon V et al.",
"1999; Tissue Antigens 53: 23-32.For intracellular labeling, cells were fixed in PBS 4% p-formaldehyde for 20 min at 4° C., washed, and then permeabilized with staining buffer containing 0.1% saponin.",
"Conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD69, and anti-TCRVβ22 mAbs were purchased from Immunotech (Marseille, France).",
"Other mAbs are available from Beckman Coulter (see Cell Analysis 2000 catalogue): anti-CD3 (PE) Cell Analysis 2000 catalogue reference IM1282; purified anti-CD3 Cell Analysis 2000 catalogue reference IM0178; Cell Analysis 2000 catalogue reference anti-CD4 IM0449; anti-CD8 Cell Analysis 2000 catalogue reference IM0452; anti-CD45RO Cell Analysis 2000 catalogue reference IM1307; anti-CD69 Cell Analysis 2000 catalogue reference IM1943; anti-CD94 Cell Analysis 2000 catalogue reference IM2276; anti-CD158a Cell Analysis 2000 catalogue reference IM2277; anti-CD158b Cell Analysis 2000 catalogue reference IM2278; anti-NKG2a Cell Analysis 2000 catalogue reference IM3291; anti-BY55 Cell Analysis 2000 catalogue reference IM2745.Immunoprecipitation of Biotinylated Surface Molecules.",
"2×107 cells were washed twice in PBS and surface-labeled with Sulfo-NHS-LC biotin (Pierce Europe, Interchim, Montlucon, France) (2 mg/mL in PBS) for 20 min at 4° C. After quenching for 20 min with RPMI 1640, cells were washed twice in PBS and re-suspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 1 h at 4° C. Postnuclear supernatant was then incubated for 2 h at 4° C. in a 96-well plate (Maxisorp Nunc immuno-plate) pre-coated with goat anti-mouse IgG+IgM (Caltag laboratories) followed by anti-SC5 mAb or anti-TCRβ (anti-TCRβ C305 isotype matched mAb, Cell Analysis 2000 catalogue reference IM1466; anti-TCRVbeta22, Cell Analysis 2000 catalogue reference IM2051; anti-TCRzeta, Cell Analysis 2000 catalogue reference IM3169; all from Beckman Coulter).",
"Immuno-precipitates were washed 4 times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na Vanadate, 10 mM NaF, 1 mM PMSF) and precipitated proteins were subjected to SDS-PAGE.",
"Western blot analysis was performed using streptavidin-peroxidase (Immunotech) and the ECL detection system according to manufacturers' recommendations (Amersham Pharmacia, Orsay, France).",
"Proliferation Assays For lymphocyte activation, PBMC were cultured in medium containing 15% human serum in the presence of 1 μg/ml PHA (Wellcome, Beckenham, UK).",
"For proliferation assays, 5×104 cells were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium.",
"When needed, the plates were pre-coated with anti-CD3 mAb at the indicated concentrations as previously described (Poszcepczynska et al.",
"2000, see supra).",
"For stimulation of CTCL cell line Pno, pre-coated anti-CD3 mAb was used in combination with 2 ng/ml phorbol 12 β-myristate 13 α-acetate (PMA, Sigma Biochemicals).",
"Cells were cultured for 4 days and were pulsed with 1 μCi of 3H[TdR] during the last 8-16 h of culture.",
"3H[TdR] incorporation was measured in a liquid scintillation counter (Topcount; Packard Instrument Co, Meriden, Conn.).",
"For the determination of IL-2 production, 105 PBL were stimulated with immobilized anti-CD3 mAb in the presence of anti-SC5 or control mAb.",
"After 32 hours of culture, 100 μL of supernatant was added to 105 cells from an IL-2-dependent T cell clone.",
"CD3-Induced Redirected Cytotoxicity Assays The redirected cytotoxicity assay was carried out as described in Le Cleach et al.",
"2000, Clin.",
"Exp.",
"Immunol.",
"119: 225-230.The target murine mastocytoma P815 tumor cells were loaded with 100 μl 51Cr (2.5 mCi/mL) for 90 min at 37° C., washed and incubated with anti-CD3 mAb for 15 min at room temperature.",
"Effector cells were likewise pre-incubated with anti-SC5 mAb (1:200 final dilution of ascites) and added to target cells in ratio 5:1, in a final volume of 150 μl in 96 well V-bottomed microtiter plates for 4 h. After centrifugation 100 μl aliquots were counted in a gamma-counter to determine 51Cr release.",
"The spontaneous release was always less than 20% from the maximum release (target cells with 1% NP40).",
"Percentage of specific 51Cr release was calculated as described in David et al.",
"1987, J. Immunol.",
"138: 2831-2836.Statistical Analysis Statistical analysis of the results was performed with Statistica software vers.",
"5.0 (StatSoft, Los Angeles, Calif.).",
"Significant differences between SC5 expression in patients and control group were evaluated by the Mann-Whitney U test, and the correlation between the percentage of SC5+CD4+ cells and the percentage of malignant cells in patients peripheral blood was evaluated by the Spearman Rank Order Correlations test.",
"Results The results herein reported have been obtained with the CTCL cell line Pno, and with CTCL cells collected from patients.",
"Equivalent experiments and results can be performed and obtained with any CTCL cell line, such as e.g.",
"HUT 78 (ATCC TIB-161).",
"Expression of SC5 Molecules in Normal Resting and Activated Peripheral Blood Lymphocytes Monoclonal antibodies raised against the functional tumor cell line Ytindi were analyzed for their simultaneous reactivity with the CTCL cell line Pno (Poszcepczynska et al.",
"2000, see supra) and normal peripheral blood T cells.",
"The SC5-specific mAb, of IgM isotype, stained both the immunizing and CTCL cell lines.",
"Among peripheral blood lymphocytes, anti-SC5 mAb delineated a small sub-population (see FIGS.",
"1A, 1B, 1C).",
"The SC5 mAb reactive molecule was expressed by a variable percentage of CD3+ T cells, never exceeding 20% (mean=10.5%, SD±5.6).",
"Both CD4+ and CD8+ lymphocytes were stained by anti-SC5 mAb.",
"A sub-population of peripheral blood CD56+ NK cells was also reactive with anti-SC5 mAb, though with more important inter-individual variations (mean=19.4%, SD±11.4); see the below Table 1.TABLE 1 expression of SC5 in peripheral blood lymphocyte sub-populations Cell population N tested mean (%) SD Total Ly 15 14.1 6.8 CD3+ Ly 10 10.5 5.6 CD4+ Ly 8 9.4 5.1 CD8+ Ly 8 14.8 5.1 CD56+ Ly 10 19.4 11.4 A representative example of SC5 co-expression with CD3 or CD56 molecules on gated lymphocytes from PBL of normal donors is shown in FIG.",
"1A.",
"It should be noted that most SC5+ lymphocytes belonged to the activation/memory pool, as demonstrated in FIG.",
"1B.",
"Over 70% of the SC5-positive cells co-expressed CD45RO.",
"As SC5 molecule expression was detected in all IL-2 dependent T lymphocyte clones tested, we verified whether its cell surface expression was induced during T lymphocyte activation.",
"We found that stimulation of PBL with PHA promptly increased the expression of SC5 on CD3+ lymphocytes.",
"Both the percentage of SC5+ T cells and the level of SC5 expression rose very rapidly after stimulation (see FIG.",
"2).",
"As early as 4 hours after stimulation with PHA, the percentage of SC5+CD3+ cells increased two to threefold and reached a maximum after 24 to 48 hours.",
"In the course of 5 to 7 days of in vitro stimulation SC5 expression returned to the basal level.",
"In parallel we studied the expression of the very early activation antigen CD69 and we observed similar kinetic profiles of CD69 and SC5 on CD3+ lymphocytes.",
"It should be noted, that unlike CD69 molecules, low levels of SC5 were already present on circulating PBL before stimulation (FIG.",
"2) Activation-induced antigen expression results either from de novo protein synthesis or cell surface export of preexisting intracellular molecules.",
"Therefore, we studied the expression of SC5 in non-activated permeabilized PBL.",
"As shown in FIG.",
"3, anti-SC5 mAb stained over 90% of permeabilized lymphocytes vs. 16% of the same non-permeabilized cells.",
"The specificity of anti-SC5 mAb staining was confirmed by using the isotype-matched mAb BY55 which detects a GPI-anchored cell surface structure expressed by a subset of circulating lymphocytes (see Agrawal et al.",
"1999, J. Immunol.",
"162: 1223-1226).",
"A comparable percentage of BY55+ cells was found in permeabilized and non-permeabilized lymphocytes.",
"As a positive control for the detection of an intracellular epitope, we used an anti-CD3ζ chain mAb which only labeled permeabilized T lymphocytes.",
"Thus, the surface expression of SC5 is dynamically regulated by the activation status of cells and its induction during T cell activation is due to the enhanced transport of the molecule from an intracellular pool to the surface membrane.",
"Biochemical Characterization of SC5 Antigen In order to characterize the molecular weight of the structure identified by anti-SC5 mAb, an IL-2 dependent TCRγδ+ clone, termed DS6, was surface labeled with biotin and the cell lysates were immun-precipitated with either anti-SC5 mAb or an isotype-matched anti-TCRβ mAb (as negative control).",
"Anti-SC5 mAb recognized a single molecule with apparent molecular weight of 96 kD under reducing conditions (see FIG.",
"4).",
"As tumor cell lines (the immunizing Ytindi cells) are often characterized by aberrant expression of carbohydrate epitopes (20, 22), we studied the reactivity of anti-SC5 mAb with the SC5+ NK cell line NK3.3 and with the CTCL cell line HUT78 (ATCC TIB-161) after treatment with neuraminidase or sodium periodate in concentrations destroying known sialylated epitopes (digestion of CD75 epitope on Raji cells was used as a positive control).",
"Expression of SC5 was not affected by neuraminidase or sodium periodate treatment.",
"Thus, we excluded a possible carbohydrate nature of the epitope.",
"Inhibition of Tile Anti-CD3 mAb-Induced T Lymphocyte Proliferation by Anti-SC5 mAb In order to examine the possible function of SC5 molecule in normal T cells, we studied the effect of anti-SC5 mAb alone or during the proliferative responses induced by immobilized anti-CD3 mAb.",
"We found that anti-SC5 mAb alone or in combination with PMA did not induce the proliferation of peripheral blood T lymphocytes.",
"Interestingly, when soluble anti-SC5 mAb was present together with immobilized anti-CD3 mAb, a significant inhibition of lymphocyte proliferation was observed.",
"The inhibition varied between 33 and 66% at day 4 depending on the donor as compared to the effect of an isotype-matched irrelevant control mAb (see FIG.",
"5A, upper panel).",
"As SC5 molecule is expressed on monocytes, it was important to determine whether the inhibitory effect was monocyte-independent.",
"We studied the proliferation of two T cell clones, GDS.3 and LSO, stimulated by immobilized anti-CD3 mAb in the presence of anti-SC5 mAb or an isotype control mAb.",
"The results obtained with both T cell clones indicated that engagement of SC5 molecules resulted in an inhibition of the proliferation to anti-CD3 mAb (see FIG.",
"5A, lower panel).",
"It should be noted that the inhibitory effect of anti-SC5 mAb on T cell proliferation depended on its concentration, as well as on the concentration of the agonistic anti-CD3 mAb.",
"The inhibition obtained with anti-SC5 mAb was significant only when T cells were stimulated with optimally diluted anti-CD3 mAb (see FIG.",
"5B).",
"Such inhibitory behaviour was also reported for anti-KIRs mAbs (Cambiaggi et al.",
"1999, Blood 94: 2396-2402).",
"The decrease of T cell proliferation following SC5 engagement could be due to a direct induction of cell death, a perturbation of the IL-2R expression, or the inhibition of cytokine synthesis.",
"We found that anti-SC5 mAb did not inhibit the IL-2-dependent proliferation of PBL or T cell clones (FIG.",
"5A).",
"Furthermore, the addition of rIL-2 to T cells pre-incubated with anti-SC5 mAb (ascites diluted to {fraction (1/200)}) in combination with anti-CD3 for 48 h restored their proliferation.",
"This would not be the case if the specific mAb inhibited IL-2R expression or caused cell death.",
"Finally, in order to prove that SC5 triggering may inhibit cytokine production, we compared the amount of IL-2 secreted by anti CD3-activated PBL in the presence or absence of anti-SC5 mAb, using an IL-2 dependant T cell line.",
"The quantity of IL-2 produced in the presence of anti-SC5 mAb represented 40 to 65% of the control values obtained with an isotype-matched antibody (see FIG.",
"5C).",
"Thus, the simultaneous engagement of SC5 and CD3 molecules in T cells results in the decrease of cytokine production.",
"We next studied the effect of anti-SC5 mnAb on T cell effector cytotoxic activity using an anti-CD3 mAb redirected killing assay against the FcgR+ murine cell line P815.FIG.",
"5D shows a representative experiment performed with a cytotoxic T cell clone at an E/T ratio of 5/1.The CD8+ T cell clone JF1, expressing high amount of SC5 molecules, was induced to kill murine target cells following anti-CD3 mAb is stimulation.",
"The simultaneous targeting of SC5 by its specific mAb did not influence JF1 cytotoxic activity, regardless of the E/T ratio or the concentration of stimulating anti-CD3 mAb.",
"Expression of SC5 Molecules on Malignant Lymphocytes Obtainedfrom Patients with Sézary Syndrome As we initially screened anti-SC5 mAb for its reactivity with a CTCL cell line, we further studied the expression of SC5 molecule in the periphcral blood lymphocytes isolated from 8 patients with Sézary syndrome.",
"Two-color flow cytometry analysis showed that expression of SC5 was significantly increased in the CD4+ subset (see representative results obtained with one patient in FIG.",
"6A).",
"On the average, 35.5% of the CD4+ lymphocytes in SS patient blood expressed SC5 as compared to 9.4% in healthy controls (p<0.001), (FIG.",
"6B).",
"In the 8 SS patients studied, the expression of SC5 on CD4+ cells correlated with the percentage of circulating malignant cells detected by cytomorphology (r=0.91, p=0.0012).",
"It should be noted that in these patients the percentage of total CD4+ cells (mean=88%, SD±8.8) was higher than that of Sézary cells (mean=26%, SD±12.8).",
"We also studied SC5 molecule expression in peripheral blood samples from 3 MF patients without blood involvement.",
"No significant difference in the levels of SC5 expression on peripheral blood CD4+ cells was observed between MF patients and healthy donors (FIG.",
"6B).",
"Still, an increased percentage of SC5+CD4+ was detected in the skin samples from two of these patients, that we were able to to test (26% and 19% respectively).",
"In one SS patient we had previously demonstrated that the circulating malignant cells were clonal lymphocytes with a CD4+TCRVβ22+ phenotype (Poszcepczynska et al.",
"2000, see supra).",
"Here we demonstrated that SC5 was co-expressed on the TCRVβ22+ cells, i.e.—by the malignant cell population (FIG.",
"7A).",
"It should be noted that the profile of SC5-mAb binding to TCRVβ22+ cells corresponded to a weak homogeneous expression on the whole malignant clonal cell population (FIG.",
"7B).",
"Interestingly, the malignant TCRVβ22+ cells did not co-express common activation markers as CD69 (FIG.",
"7C), CD25 or CD30 as previously described (Poszcepczynska et al.",
"2000, see supra).",
"Thus, the increased levels of SC5 in SS PBL were mostly due to the expression of the antigen on circulating malignant T cells.",
"Inhibition of the Anti-CD3mAb-Induced CTCL Cell Line Proliferation by Anti-SC5 mAb We next investigated whether SC5 molecules were able to provide a negative signal in malignant CTCL cells.",
"We used the long-term cultured Pno CTCL tumor T cell line which strongly proliferates in the presence of IL-7.As previously reported that Pno cell line expresses functional T cell receptors, since immobilized anti-CD3 mAbs or the combination of soluble anti-CD3 mAbs and PMA induced significantly their proliferation (Poszcepczynska et al.",
"2000, see supra).",
"We examined the effect of SC5 molecule engagement on the proliferation of Pno cells.",
"FIG.",
"8 shows that anti-SC5 mAb strongly inhibited the anti-CD3 mAb-induced proliferation of the CTCL cell line.",
"In contrast, PMA alone (2 ng/mL) produced a low level of proliferation which was not significantly affected by anti-SC5.Notably, anti-SC5 mAb had no effect on the IL-7-dependent proliferation, since Pno cells incubated with IL-7 in the presence of anti-SC5 mAb proliferated as vigorously as in the presence of the isotype control mAb.",
"Thus, we concluded that SC5 antigen is functional in CTCL malignant cells and may specifically and profoundly inhibit their proliferation triggered through the TCR/CD3 signaling pathway.",
"A CTCL tumor was then successfully grafted onto transgenic mice having a substantial deficiency in functionally active NK cells and T lymphocytes (SCID mice) as described in Charley et al.",
"(1990) “Establishment of a human cutaneous T cell lymphoma in CB-17 SCID mice” J.",
"Invest.",
"Dermatol.",
"94: 381-384 (see also U.S. Pat.",
"No.",
"5,530,179 Cornelius P. Terhorst and Baoping Wang for obtention of SCID mice).",
"Anti-SC5 mAb was intraveneously or intracutaneously administered at different times to half of the mice at various concentrations of purified mAb per gram of mouse, whereas the other half received the same amount of purified CD56 (isotype IgM) as a control.",
"PBL and cutaneous cells were collected for standard cytological observations of infiltrating large T cells, and the percentage of CTCL cells was assessed for each mouse of the experiment.",
"This procedure is an example of procedure that enables the is skilled person to check that anti-SC5 compounds, such as the mAb produced by the hybridoma deposited as deposit number I-2575 at the C.N.C.M., are capable of inhibiting the in vivo proliferation of human CTCL.",
"Discussion A number of receptors with dynamic surface expression mediate T cell interactions with other immune cells or cytokines and, consequently modulate T cell immune responses.",
"In this study, we identified a novel 96 kD functional molecule expressed by a minor subset of PBL whose surface expression is induced rapidly during in vitro T-cell activation.",
"SC5 is distinct from the established molecules with similar molecular weight and/or T cell modulatory functions.",
"First, SC5 was induced very early upon T cell activation, with important increase of its surface expression detected after 4 h. A similar kinetics has been observed for the very early activation antigen CD69, which is detected on the cell surface between 2 and 4 h after stimulation.",
"Most established T-cell specific inducible receptors such as 4-1BB, OX-40 LIGHT or CD152 (CTLA-4) appear on T-cell surface between 10 to 24 h following stimulation.",
"Further, SC5 expression is not T cell restricted.",
"The antigen was detected on sub-populations of NK and B lymphocytes, monocytes and granulocytes.",
"Such a wide distribution pattern is typical of certain killer cell Ig-like receptors (KIRs), as the recently described AIRM1 and IRp60, which are found on NK cells, T and B lymphocytes, myeloid and other antigen-presenting cells.",
"The family of immunoglobulin—like transcript (ILT) inhibitory receptors has also a pan-leukocyte type of expression, the individual receptors being detected on different NK, T, B and myeloid sub-populations.",
"Moreover, ILT4 has a molecular weight very close to SC5.However, neither the expression of AIRM1 and IRp60, nor of ILTRs depends on the activation status of cells.",
"The prompt induction of SC5 molecule at the surface of activated T cells followed by its down-regulation may result from trafficking of intracellular molecules to the cell membrane and vice versa, as we detected SC5 in most non-activated T lymphocytes after cell permeabilization.",
"Such a regulatory mechanism has been already reported for another T cell receptor, CD152.Further on, a heterogeneous family of lysosome associated membrane proteins (LAMP) exists, including molecules involved in endocytosis, lysosomal trafficking and secretion from intracellular granules and that may be induced on the surface of monocytes, neutrophiles, NK, B or T cells upon activation.",
"Additional studies on the intracellular localization and transport of SC5 would precise its proper role in T cell activation and functions.",
"Our studies rcvealed that triggering of SC5 by its specific mAb inhibited the anti-CD3 mAb-induced T cell proliferation.",
"This inhibitory effect required an optimal anti-CD3 mAb stimulation, which is necessary to induce maximal SC5 surface expression in PBL (see FIG.",
"5B).",
"A 1:200 final dilution of anti-SC5 ascites significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram par well and this effect of inhibition increases with lower anti-CD3 mAb concentrations −1 microgram par well; 0.5 microgram per well—A 1:400 final dilution of anti-SC5 ascites do not significantly inhibits the proliferation induced by anti-CD3 mAb at 2 microgram per well, but inhibition is significant when a lower concentration of anti-CD3 is used −1 microgram per well; 0.5 microgram per well—The person of ordinary skill in the art can proceed with such adjustments by standard interpolation from the particular results described herein, or from his own trials.",
"At the same time, the inhibitory.",
"threshold posed by SC5 engagement could be bypassed by excessive anti-CD3 mAb stimulation.",
"It should be noted that the inhibitory functional effects obtained by ligation of SC5 molecules were observed in an APC-independent system.",
"Further experiments are needed to determine whether SC5 stimulation may have different effects on the proliferation of APC or allo-stimulated T cells.",
"Recently, the negative regulation of immune responses has been extensively investigated, in particular in T lymphocytes and NK cells.",
"The best studied T cell specific inhibitory receptor, CD152 (CTLA-4), is a typical activation-induced antigen, functionally coupled to CD28 co-stimulatory receptor.",
"Indeed, CD152 may overcome CD28 positive signals at low antigenic concentrations and prevent immune response.",
"Alternatively, it may downregulate and terminate T-cell proliferation induced through CD28 by affecting IL-2R expression and IL-2 synthesis.",
"Similarly to CD152, SC5 is an intracellular molecule whose surface expression is regulated by the activation status of cells.",
"Both receptors mediate their effects by affecting IL-2 synthesis.",
"The identification of SC5 specific ligands and signaling pathways will precise the temporal and spatial correlation between these negative T-cell regulators.",
"In consideration to its wide cell distribution, SC5 functional effects are probably not restricted to T cells.",
"In several aspects, SC5 is comparable to NK cell inhibitory receptors, equally expressed by T cells and other lymphocytes sub-populations.",
"Both KIRs and CD94/NKG2 receptors were shown to inhibit antigen-specific and superantigen-driven activation of TCRαβ and TCRγδ T cells, including cytotoxicity, proliferation and cytokine secretion.",
"The KIR-induced inhibition depends on the activation status of T cells and the potency of T cell stimulation and may be bypassed under specific in vivo conditions.",
"However, SC5 is definitely distinct from KIRs, since we demonstrated that it selectively inhibited the proliferation of anti-CD3 mAb stimulated T cells without affecting their effector cytotoxic functions.",
"An important characteristic of SC5 molecule is its significantly increased expression in peripheral blood lymphocytes of patients with SS in comparison to healthy subjects.",
"SS is characterized by the generalization of a primary epidermotropic malignant T cell clone with mature phenotype.",
"Actually, none of the established T cell functional receptors conclusively identifies peripheral blood SS tumor cells.",
"MF/SS has been considered as the expansion of a T cell subset with a particular phenotype, CD4+CD7−, corresponding to a normally existing T memory subset.",
"However, a recent study demonstrated that the CD4+CD7− population does not always represent the dominant T cell clone in SS patients (Dummer et al.",
"1999, Arch.",
"Dermatol.",
"Res.",
"291: 307-311).",
"SS is regarded as a proliferation of specifically activated T cells although the nature of the stimulus is not known (Fargnoli et al.",
"1997, Leukemia 11: 1338-1346).",
"For this reason the distinct expression of activation induced antigens on SS and normal T cells is of special interest.",
"Established activation antigens such as CD69, CD25, CD30 are not consistently detected on Sézary cells, as confirmed by our own investigations.",
"Expression of CD25 rather correlates with the progression of the disease and the transformation of MF/SS cells into a large cell variant in a minority of the cases.",
"An increased rate of expression of T cell activation molecules as HLA-DR, CD25 or CD71 in SS has been primarily associated with reactive tumor-infiltrating T cells.",
"A 78 kD very late activation antigen, BE2, was reported to be specifically increased in SS peripheral blood, while not detected on normal T cells.",
"In fact, BE2 was not consistently detected in all SS patients studied, a comparatively low percentage of peripheral blood cells in relation to the reported percentage of malignant cells were BE2-positive, and BE2 was equally detected in patients without clinical evidence of blood involvement.",
"In contrast, SC5 was detected in 8/8 SS cases studied.",
"Although the percentage of malignant cells determined by cytomorphology varied considerably from patient to patient (10-80%), the expression of SC5 on CD4+ cells correlated with this percentage.",
"Finally, no elevated expression of SC5 on CD4+ cells was detected in MF patients without blood involvement.",
"Further, we demonstrated that Sézary cells identified by the expression of the clonotypic TCRVβ chain co-expressed SC5 molecule, confirming that increased levels of SC5 in the peripheral blood of SS patients were due to its expression on the malignant T cell clone.",
"A very important observation was that SC5 triggering on Sézary cells induced down-modulation of the CD3-mediated proliferation of tumor cells, analogous to the inhibition observed in normal T cells.",
"It has been already demonstrated that malignant CTCL lymphocytes preserve certain functional properties as they may be activated through basic signaling pathways although producing aberrant profiles of cytokines.",
"We now demonstrate that an inhibitory pathway is preserved in CTCL tumor cells, that may permit to counterbalance their proliferation.",
"Notably, the inhibitory effect observed after anti-SC5 mAb binding on anti-CD3 mAb-stimulated CTCL Pno cells was much more important than in normal T cells.",
"Anti-SC5 mAb does not seem to directly mediate cell death of neither normal nor Sézary T cells, since it does not modulate the rIL-induced proliferation.",
"Moreover, T-cells that had been cultivated in the presence of anti-SC5 mAb can be re-stimulated by addition of rIL-2.At the same time, we observed a diminished IL-2 production by anti-SC5 mAb-treated T cells.",
"which should account at least partially for the inhibition of their proliferation.",
"Inhibitory effects of functional receptors on the proliferation of malignant cells have been already observed.",
"It has been recently reported the inhibition of the proliferation of normal and leukemic myeloid cells mediated by the homologous NK cell sialoadhesin receptor AIRMI and CD33 myeloid-specific antigen (Vitale et al.",
"1999, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 96: 15091-15096).",
"Unlike SC5, these receptors inhibited proliferation in the presence of growvth factors and most probably by induction of apoptosis.",
"Interestingly, another T cell activation antigen, CD30, expressed by a subset of normal CD45RO+ T cells, is likely to mediate the regression of primary CTCL other than MF/SS.",
"This effect is related to the natural ligand of CD30 (CD30L) which is co-located within the cytoplasm of malignant cells and may induce cytolytic cell death independently of Fas/FasL system (Mori et al.",
"1999, Blood 94: 3077-3083).",
"In this aspect, the identification of SC5 ligand will enlighten the mechanism of its inhibitory action.",
"In conclusion, we have identified an early activation antigen which is distinct, by phenotypic, biochemical and functional criteria, from the established T lymphocyte receptors.",
"As it was underlined, SC5 molecule is expressed by a sub-population of the post-activation/memory peripheral blood T cell subset.",
"Likewise, continuous expression of KIRs is detected on minor T cell clones, and this expression seems to depend on TCR-occupancy by specific antigens.",
"It was recently proposed that such clones might be auto-reactive and maintained tolerant by KIRs signaling.",
"We may speculate that SC5 surface expression is also preserved on auto-reactive T cells, suggesting that Sézary syndrome tumor cells possibly originate from such clones.",
"Importantly, the expression of SC5 on CTCL malignant cells will serve to better define their origin and the nature of the signal driving mature T cells to uncontrolled proliferation.",
"In addition, the inhibitory effect of anti-SC5 mAb on tumor T cells constitutes a novel therapeutic approach in patients with advanced CTCL.",
"Furthermore, expression of SC5 has also been observed at the surface of CD8+ CTCL, such as CD8+ transformed MF.",
"EXAMPLE 2 P140 is Expressed by CD4+ CTCL Cells, and Identification of a Novel P140 Allelic Form Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin.",
"Mycosis fungoides (MF) is characterized by skin invasion of clonally-derived malignant CD4+ T lymphocytes that phenotypically resemble mature T helper cells.",
"A more aggressive form of CTCL develops when the is malignant cells become non-epidermotropic and is associated with extra-cutaneous involvement.",
"Sézary syndrome (SS) is a more aggressive form of CTCL that is characterized by a clonal expansion of CD4+/CD45RO+ T cells and the appearance of these malignant T cells in the blood.",
"The biology of the disease remains poorly understood, as it is difficult to identify the malignant cell, due to the lack of specific cell surface markers.",
"Thus, in cutaneous lesions it is difficult to distinguish CD4+ CTCL cells from reactive infiltrating CD4+ T lymphocytes.",
"Bagot et al.",
"previously described a unique CD4+ T cell line derived from CTCL lesions (Bagot et al.",
"1998, Blood 91: 4331-4341); this cell line and the in vivo tumor cells expressed an identical size of the complementarily-determining region 3 of TCR-Vβ transcripts.",
"More recently, Poszepczynska et al.",
"(Blood 2000, 96: 1056-1063) functionally characterized an IL-7-dependent CD4+CD8αα+ tumor T cell line isolated from the blood of another patient with a cutaneous erythrodermic CTCL.",
"This tumor T-cell line was identical to the major circulating T cell populations, as demonstrated by the expression of TCR-Vβ22 and the identity of the TCRβ-VDJ sequences.",
"Here, we show that these two different CTCL lines express the p140/KIR inhibitory receptor for HLA-A alleles.",
"Importantly, this receptor is detected on freshly isolated tumor cells derived from the same patients.",
"Moreover, the p140/KIR was also co-expressed by a major subset of CD4+ lymphocytes in seven other patients with SS, and by tumor skin CD4+ cells in two additional patients with advanced MF.",
"The p140/KIR is detected in normal individuals on a minor NK cell-subset and on rare peripheral blood CD3+CD8+ cells.",
"Moreover, T cells obtained from skin in other dermatological diseases such as inflammatory skin diseases and toxic epidermal necrolysis did not express this receptor (Le Cleach et al.",
"2000, Clin.",
"Exp.",
"Immunol.",
"119: 225-230).",
"Thus, our present findings demonstrate that the p140/KIR represents a suitable marker on CD4+ cells for the identification of CTCL.",
"Materials and Methods Patients After informed consent and approval by an ethic committee, we obtained skin and blood samples from eleven patients with a CTCL.",
"Eight patients had a Sézary syndrome with 10 to 45% circulating Sézary cells in the blood.",
"In seven cases, the phenotype of tumor cells was CD3+,CD4+,CD8−.",
"In one case, the phenotype was CD3+,CD4+,CD8αα+ (patient Pno).",
"Three patients had a transformed mycosis fungoides (Lez, Cou, Bic) and presented with disseminated skin tumors with a CD3+,CD4+,CD8− phenotype.",
"All patients were not previously treated with chemotherapy.",
"Isolation of Tumoral Lymphocytes Fresh CTCL tumor cells were obtained from tumor fragments mechanically dispersed into single-cell suspensions (Bagot et al.",
"1998, Blood 91: 4331-4341).",
"The mononuclear cells were then washed and frozen in human serum plus 10% dimethyl sulfoxide for later use.",
"For Sézary syndrome patients, the mononuclear blood cells were isolated by the technique of Ficoll-Isopaque (Pharmacia fine Chemicals, Piscataway, N.J.).",
"Long Term Culture of Tumor Cell Lines We established the long term culture of Pno cell line (TCRVβ22+,CD3+, CD4+,CD8αα+,MHC class I+,MHC class II−) in vitro from the peripheral blood of the patient as previously described (Bagot et al.",
"1998, Blood 91: 4331-4341).",
"We demonstrated that both the malignant clone circulating in the patient blood and the derived cultured T-cell line were identical for their cell surface phenotype and for their size and sequence of the TCRβ VDJ region (Poszepczynska et al.",
"2000, cf.",
"supra).",
"Other sources of SS cell lines may be found at the ATCC (e.g.",
"HUT 78 ATCC TIB-161).",
"The Cou-L cell line (TCRVβ13+) was cultured in vitro with rIL-2 for more than three years.",
"It corresponds to a subelone of the CD4+ Cou-LS CTCL line previously described (Bagot et al.",
"1998, cf supra).",
"The Cou-L cell line, the original TCRVβ13+, CD4+ Cou-LS CTCL, and the tumor cells freshly isolated from the skin shared the same size and sequence of the TCRβ VDJ region (Bagot et al.",
"1998, cf.",
"supra).",
"Monoclonal Antibodies (mAbs) and Flow Cytometry Studies One- and two-color immunofluorescence analysis was performed as previously described (Bagot et al.",
"1998, cf.",
"supra).",
"Anti-CD3 (Cell Analysis 2000 catalogue reference 6604629), anti-CD4 (Cell Analysis 2000 catalogue reference 6602138), anti-MHC Class I (Cell Analysis 2000 catalogue reference IM0107) and II (Cell Analysis 2000 catalogue reference IM0108) are obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue).",
"The anti-TCRVβ13+ mAb was purchased from Bioadvance (Emerainville, France, catalogue reference TCR 1738), and the anti-TCRVβ22+ mAb was obtained from Beckman-Coulter (Marseille, France, Cell Analysis 2000 catalogue reference IM 1484/IM1365).",
"The anti-CD8 αβ 2ST8.5H7 mAb is obtainable from Beckman Coulter (Cell Analysis 2000 catalogue reference IM 2014.As anti-p140 mAbs, Q66 (IgM, anti-p140) and AZ158 (IgG2a, recognizing both p70/NKB1 and p140) which have been described in Pende et al.",
"1996 (J. Exp.",
"Med.",
"184: 505-518) have been used; however any anti-140 binding compound is appropriate (e.g.",
"anti-p140 antiserum, Fc-HLA-A11, Fc-HLA-A3 fuision proteins).",
"Z27 (IgG1, anti-p70/NKB1), EB6 (IgG1, anti-p58.1/S50.1), GL183 (IgG1, anti-p58.2/p50.2), XA185 (IgG1, anti-CD94), Z199 and Z270 (IgG2b and IgG2a respectively, anti-NKG2A), and B9.4 (IgG2b, anti-CD8) mAbs are all obtainable from Beckman Coulter (see Cell Analysis 2000 catalogue references IM2748, IM2277, IM2278, IM1610, IM2750, IM0102).",
"Goat anti-mouse IgG FITC and goat anti-mouse IgM PE are also obtainable from Beckman Coulter (see Cell Analysis Catalogue references IM08 and IM0555).",
"Biochemical Characterization 20×106 cells were incubated (15′ at 20° C.) in 1 ml PBS pH8 containing 250 μg of EZ-LINK SULFO-NHS-LC-LC-BIOTIN (Pierce, Rockford, Ill.) and washed three times in Washing Buffer (10 mM TRIS pH 8, 0.14 M NaCl).",
"Cells were then lysed in 1% NP-40 and immuno-precipitated with Sepharose-PA (Pharmacia Biotech Inc. Piscataway, N.J.)-coupled AZ158 (IgG2a, antip70/140) or B9.4 ([gG2b, anti-CD8) mAbs.",
"Samples were analyzed by discontinuous SDS-PAGE either undigested or digested with N-glycosidase F (Boehringer Mannheim, GmbH, Germany) and transferred to Immobilon P (Millipore Corp, Bedform, Mass.).",
"After staining with Neutravidin (Pierce) the Renaissance Chemiluminescence Kit (NEN, Boston, Mass.)",
"was used for detection.",
"NK cell clones were obtained by limiting dilution as described in Pende et al.",
"1996 (J. Exp.",
"Med.",
"184: 505-518).",
"RT-PCR Analysis Total RNA was extracted from CTCL cell lines Pno and Cou-LCD8 αα using RNA-Clean System (AGS GmbH, Heidelberg, Germany).",
"cDNA synthesis was performed using oligodT priming.",
"Primers used for cDNA amplification of the complete ORF (open reading frame) of KIR displaying three Ig-like domains (1395 bp) were the following: 5′CATGT(CT)GCTCA(CT)GGTCGTC (Ig3 UP; SEQ ID No.",
"5) and 5′ GGTTTTGAGACAGGGCTG (Ig3 DOWN; SEQ ID No.",
"6).",
"Amplification was performed for 30 cycles (30 sec.",
"at 94° C., 30 sec.",
"at 55° C., 30 sec.",
"at 72° C.), followed by a 7 min.",
"incubation at 72° C., utilizing AmpliTAQ (Perkin Elmer-Applied Biosystems, Foster City, Calif.).",
"PCR products were sub-cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, Calif.).",
"DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer (Perkin Elmer-Applied Biosystems).",
"Transient Transfections COS-7 cells were transfected with pcDNA3.1 TOPO-KIR3D cl.24 or with pCR3-cl.1.1 (Pende et al.",
"1996, J. Exp.",
"Med.",
"184: 505-518) utilizing Fugene 6 (Roche, Monza Italy).",
"Briefly, cells were seeded at 5×105/plate; 24 hr later they were incubated with 6 μg plasmid and 10 μl of Fugene-6 reagent in DMEM/10% FCS.",
"After 48 or 72 hrs, transfected cells were used for cytofluorimetric analysis.",
"Cell transfectants were stained with Q66 and AZ158 mAbs, followed by a phycoerythrin-conjugated goat antibody to mouse IgG2a or IgM and analyzed by flow cytometry using a FACSort (Becton Dickinson, San Jose, Calif.).",
"Results CTCL Cell Lines are Stained by mAbs to the p140/KIR Two long-term CTCL tumor lines Pno (labeled with anti-TCR-Vβ22 mAb) (Poszepczynska et al.",
"2000, cf supra) and Cou-L (labeled with anti-CD3 mAb) (Bagot et al.",
"1998, cf.",
"supra) were analyzed for reactivity with different anti-KIR mAbs.",
"We found that both cell lines were reactive with mAbs Q66 (see FIG.",
"9A) and AZ158, both recognizing the p140/KIR.",
"In contrast, these cell lines did not express other inhibitory receptors specific for HLA class I molecules, including p58.1, p58.2, p70 KIRs and the CD94/NKG2A lectin-like receptor (Poszepczynska et al.",
"2000, cf supra).",
"Tumor T Lymph Ocytes Freshly Isolated from CTCL Patients are Stained by Anti-p140/KIR mAbs To determine whether p140/KIR was expressed by freshly isolated tumor cells, we tested the reactivity of Q66 mAb with uncultured tumor cells isolated from the blood in SS patient Pno and from tumoral skin fragments of MF patient Cou.",
"We found that the majority of tumor cells were stained by this mAb (FIG.",
"9B).",
"In particular, we observed that most TCRVβ22+ tumor lymphocytes isolated from the blood of patient Pno were reactive with Q66 antibody, and that most of the TCRVβ13+ tumor lymphocytes isolated from the skin of patient Cou were stained by the same antibody (FIG.",
"9B).",
"Next, we studied the phenotype of tumor T lymphocytes from the blood of seven additional patients with a Sézary syndrome, with malignant cells representing 10 to 45% of circulating CD4+ lymphocytes, and tumor T lymphocytes isolated from skin tumors of two other patients with a MF.",
"Remarkably, all patients tested exhibited a significant population co-expressing CD4 and p140/KIR (see Table 2 below).",
"TABLE 2 Anti-Q66 mAb stained CD4+ lymphocytes isolated from the skin of patients with transformed mycosis fungoides and from blood of patients with Sezary syndrome.",
"Percentage of positive cells with Patient anti- anti-CD4 mAb + samples CD4 mAb anti-Q66 mAb MF Lez 65 29 Bic 53 44 SS Bri 85 35 Bar 90 18 Att 95 35 Ros 98 15 Can 78 36 Pet 45 9 Riv 98 19 It should be noted that all Q66+ cells were included in the CD4+ cell population.",
"Thus, the expression of p140/KIR, which in normal individuals is restricted to subsets of lymphocytes from the NK and CD8+ populations, appears to be a characteristic of CTCL tumor CD4+ T lymphocytes, both in the skin and in the blood.",
"As control, skin T lymphocytes derived from another dermatological disease, toxic epidermal necrolysis, which were shown to contain small percentages of various KIR expressing T lymphocytes (Le Cleach et al.",
"2000, Clin.",
"Exp.",
"Immunol.",
"119: 225-230), failed to express p140/KIR.",
"Molecular Characterization of the p140 Receptor Expressed by CTCL.",
"The Pno and Cou-L cell lines were surface labeled with biotin and cell lysates were immuno-precipitated with an anti-p140 (AZ158 mAb).",
"As shown in FIG.",
"10, this mAb immuno-precipitated from a NK clone and from the Pno and Cou-L cell lines a molecule with a molecular mass of approximately 70 kD under reducing conditions.",
"Treatment with N-glycosidase revealed a protein backbone of approximately 50 kD with a slightly higher mobility for Pno and Cou-L compared to NK clone.",
"These data suggested that the p140 inhibitory receptor expressed by these CTCL tumor cell lines could be almost similar to that previously detected on normal NK cells (Pende et al.",
"1996, J. Exp.",
"Med.",
"184: 505-518).",
"Next, we determined whether the cDNA encoding the molecule recognized by Q66 and AZ158 mAbs on Pno and Cou-L CTCL cell lines corresponded to one of the already described cDNA encoding p140/KIR (SEQ ID No.",
"3).",
"To this end, RT-PCR was performed on RNA derived from these cell lines using a set of primers able to amplify all cDNA encoding KIR with three Ig-like domains.",
"From the Pno cell line, we isolated a full-length cDNA, termed KIR3D cl.24 (SEQ ID No.",
"1).",
"Comparison of its nucleotide sequence with DNA sequences coding for all KIR characterized by three extra-cellular Ig-like domains revealed that KIR3D cl.24 represents a novel allelic form of p140 receptor.",
"In particular, its nucleotide sequence displays five differences compared to the previously described cl.",
"1.1 cDNA (Pende et al.",
"1996, J. Exp.",
"Med.",
"184: 505-518), resulting in four amino acid substitutions in the mature protein (see FIG.",
"11; SEQ ID No.",
"2: clone 24 protein; SEQ ID No.",
"4: clone 1.1 protein).",
"Three of the four substitutions are found in the extra-cellular Ig-like domain (pos.",
"20, 92 and 111 of the mature protein), whereas the other is located in the cytoplasmic region (pos.401).",
"RT-PCR performed on Cou-L CTCL cell line revealed two different allelic fonns of p140/KIR one corresponding to cl.",
"1.1 cDNA and the other identical to KIR3D cl.24 (isolated from Pno cell line).",
"The cDNA derived from the CTCL cell lines were then transiently transfected in COS-7 cells.",
"As expected, all cell transfectants were brightly stained by Q66 and AZ158 mAb, while they were unreactive with p70/KIR-specific Z27 mAb used as a negative control.",
"Finally, RT-PCR was performed on RNA extracted from PBL derived from three SS patients (including patient Pno) and from skin-derived T cells of one additional MF patients.",
"Also in these samples one or another allelic isoform of p140/KIR described above could be identified.",
"The same results were obtained on malignant CTCL cells collected from patients.",
"Discussion Skin lesions in CTCL contain a heterogeneous lymphocytic infiltrate composed of malignant T cells, which are most often CD4+, and non-neoplastic tumor infiltrating T lymphocytes.",
"We previously reported CD4+ cytotoxic tumor infiltrating lymphocytes specifically directed against autologous malignant CTCL CD4+ cell line.",
"However, as no tumor restricted cell surface structure, besides the clonotypic TCR expressed by tumor cells, has been identified on CTCL, it is difficult using standard methods to distinguish malignant from non-malignant reactive CD4+ lymphocytes.",
"In the present study, we report for the first time that tumor cells from MF and SS patients express the p140/KIR.",
"This receptor has been identified in both skin and blood tumor cells from CTCL patients as well as in two long-term culture CTCL lines.",
"Two color fluorescence analysis indicated that p140/KIR expression is restricted to T cells characterized by a given TCRβ-VDJ previously identified on tumor cells.",
"Thus, p140/KIR allows rapid identification of tumor cells from tumor reactive cells among the T lymphocyte CD4+ population.",
"This could be particularly useful in SS patients, in which the tumor T cell clone is not always easily identified within the peripheral blood CD4+ lymphocyte population.",
"Moreover, during the course of the disease or after treatment, it is crucial to assess whether an increase of the CD4+ population is due to an expansion of the tumor cell population or of reactive T lymphocytes.",
"Since the anti-p140/KIR mAbs appear to recognize tumor cells in all CTCL patients analyzed, they may be considered as a suitable tool for the direct identification of CTCLs.",
"In addition, the use of p140/KIR co-expression on CD4+ CTCL cells provides a unique tool to distinguish malignant cells from normal cells in every patient.",
"This is in contrast to TCR determinations which is unique to an individual patient.",
"Moreover, the p140/KIR represents a possible target for the development of novel specific immuno-therapy of CTCL.",
"Previous studies indicated that this receptor was able to generate inhibitory signals upon recognition of HLA.A3 and HLA.A11 alleles.",
"It is of note, however, that p140/KIR expression in the various patients analyzed appears apparently independent from their own HLA Class I haplotype.",
"Nevertheless, the actual role of p140/KIR in the patho-physiology of CTCL is an important feature that remains to be studied by taking into account the potential role of this receptor in the tolerance to self.",
"In conclusion, the present study demonstrates for the first time the expression of the p140/KIR in CD4+ CTCL cells and the isolation of a novel allelic form of this receptor in tumor cells.",
"This finding is an important new issue, both for the patho-physiology and for the clinical management of CTCL patients.",
"Furthermore, p140 expression has also been observed at the surface of CD8+ CTCL such as CD8+ transformed MF.",
"EXAMPLE 3 Production of Anti-CTCL Drugs The person of ordinary skill in the art is enabled to produce anti-CTCL drugs pursuant to the following outlines, given for illustration purposes: use of anti-SC5 or anti-p140 mAb with a cytokine such as Interferon gamma or IL-2 for stimulating the immune system of cells in the vicinity of CTCL cells, use of these anti-SC5 and anti-p140 mAb as vectors for delivering anti-CTCL ingredients onto CTCL cells; examples of anti-CTCL ingredients comprise standard compounds used in chimiotherapy such as radioelements, toxines.",
"mAb vectors for radioelements can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.",
"Press O W et al.",
"“Phase two trial of iode131-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphoma”, Lancet 1995 vol.",
"346 pp336-340, of which content is herein incorporated by reference).",
"mAb vectors for toxine can be produced following standard techniques applied to an anti-SC5 mAb of the invention (see e.g.",
"Ghetie U et al.",
"“Large scale preparation of an immunoconjuguate constructed with human recombinant CD4 and deglycosylated ricin A chain”, J. Immunol.",
"Methods 1990, vol.",
"126 pp 135-141, of which content is herein incorporated by reference).",
"combined use of an antimitotic pro-drug and of anti-SC5 mAb and/or anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form (mitotic agent in a functional form).",
"Anti-SC5 mAb and anti-p140 mAb vectors delivering an enzyme capable of transforming an antimitotic pro-drug to the active drug form can be produced following standard techniques applied to an anti-SC5 mAb of the invention and to an anti-p140 mAb (see e.g.",
"the ADEPT technique, for Antibody Directed Enzymre Prodrug Therapy, described in Plakey T C et al.",
"“Anti-tumor effects of an antibody-carboxypeptidase G2 conjuguate in combination with phenol mustard prodrugs”, British J.",
"Cancer 1995 vol.",
"72 pp 1083-1088, of which content is herein incorporated by reference).",
"use of anti-SC5 or anti-p140 mAb as ligand agonists (SC5 stimulation or p140 stimulation) use of anti-SC5 or anti-p140 mAb as complement recruiting agent For the above-mentioned uses, mAbs with a double anti-SC5/anti-CD4 or anti-p140/anti-CD4 specificity are preferred.",
"use of anti-SC5 or anti-p140 (no CD4+ specificity needed) as activators of ADCC (Antibody Dependent Cell Cytotoxicity): polynuclear cells, macrophages, NK cells bear Fc receptors which are activated by the Fc portion of the anti-SC5 and anti-p140 mAb, these mAb therefore enable the ADCC activation in the vicinity of CTCL cells.",
"Abbreviations: APC: Antigen Presenting Cell, CTCL: cutaneous T cell lymphoma, DIG: detergent-insoluble glycolipid-enriched fraction, ILT: immunoglobulin-like transcript, KIR: killer cell immunoglobulin receptor, mAb: monoclonal antibody, MF: mycosis fungoides, PBL: peripheral blood lymphocytes, PBMC: peripheral blood mononuclear cells, PHA: phytohemagglutinin, PMA: phorbo112 beta-myristate13 alpha-acetate, SS: Sézary syndrome, TCR: T cell receptor"
]
] | Patent_10450818 |
[
[
"Anti-Cd28 Antibody",
"The invention concerns an antibody directed against the CD28 receptor and capable of blocking CD28/B7 interaction, and proteins derived from said antibody, for use in particular to block CD28-dependent activation of lymphocytes."
],
[
"1.A protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.2.The protein as claimed in claim 1, selected from the group consisting of: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) Fv, Fab, Fab′2 or scFv fragments of the antibody CD28.3; c) chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) Fv, Fab, Fab′2 or scFv fragments of an antibody b); and e) recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.",
"3.A nucleic acid molecule encoding a protein as claimed claim 1.4.An expression vector comprising a nucleic acid molecule as claimed in claim 3.5.The expression vector as claimed in claim 4, wherein the expression vector is the plasmid CNCM I-2762.6.A cell expressing a protein as claimed in claim 1.7.The cell as claimed in claim 6, wherein the cell is the hybridoma CNCM I-2582.8.A cell transformed with a nucleic acid molecule as claimed in claim 3, and expressing a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CO28/B7 interaction and comprising at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3, produced by the hybridoma CNCM I-2582.9.A method for preparing a protein as claimed in claim 1, comprising culturing at least one cell as claimed in claim 6, and recovering said protein from said culture.",
"10.A medicinal product comprising the protein as claimed in claim 1.11.The medicinal product as claimed in claim 10, wherein in that said protein has a single CD28 receptor-binding site, and said medicinal product is an immunosuppressor which selectively blocks T cell activation via the CD28 receptor.",
"12.A method of treating a pathological condition selected from the group consisting of transplant rejection, graft-versus-host disease, T-lymphocyte-mediated autoimmune diseases, allergic phenomena and chronic inflammatory diseases wherein the method comprises administering to a patient in need thereof an effective amount of the medicinal product of claim 11.13.A nucleic acid molecule encoding a protein as claimed in claim 2.14.A cell expressing a protein as claimed in claim 2."
],
[
"<SOH> Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment <EOH>"
],
[
"The invention relates to antibodies directed against the CD28 lymphocyte receptor and to their fragments, and to their therapeutic uses, in particular in the context of regulating T cell activation.",
"Abnormal activation of T cells is involved in the pathogenesis of many autoimmune diseases, and also in transplant rejection phenomena, where they cause an immune response directed against the transplanted organ to develop.",
"T lymphocyte activation requires an activating signal, induced by the recognition, by T receptors (TCRs) of the antigen associated with the class II major histocompatibility complex (MHC) and presented by antigen-presenting cells (APCs).",
"However, this activation only causes proliferation of T cells and secretion of specific immunomodulatory cytokines (such as interleukin 2, gamma interferon or interleukin 4) if other T co-stimulation systems are also activated.",
"One of the most important systems for regulating T lymphocyte activation is the molecular system B7/CD28/CTLA4.This system plays, for example, an essential role in the mechanisms of transplant rejection [Woodward et al., Transplantation, 66, 14-20, (1998)].",
"The molecules B7.1 (CD80) and B7.2 (CD86) borne by the APCs can activate the CD28 receptor and also the CTLA4 receptor of T lymphocytes.",
"The activation of CD28 sends the T lymphocyte a positive signal which stimulates the cell; on the other hand, the activation of CTLA4 sends a negative signal which leads to a non-response (anergy) [FALLARINO et al., J. Exp.",
"Med., 188, 205-210, (1998)].",
"Dormant T lymphocytes express a large amount of CD28 and very little CTLA4.When there is a first cognitive contact between an APC and a T lymphocyte, the CD28/B7 interaction is favored, which activates the cell.",
"It is only several hours after the initiation of activation that, due to the increase in membrane expression of CTLA4, the affinity of which for B7 is 5 to 10 times greater than that of CD28, the B7/CD28 interaction shifts in favor of a B7/CTLA4 interaction.",
"Currently, cyclosporin is mainly used to block T lymphocyte activation, in particular in the context of organ transplants.",
"Despite the effectiveness of this medicinal product, it does not, however, confer absolute protection.",
"In addition, it acts by blocking all the calcium-dependent cell activation pathways, and therefore has a biological activity which is not strictly T lyphocyte specific and leads to a considerable number of side effects.",
"It is therefore desirable to develop new immunosuppressants which have a defined method of action and greater specificity.",
"It has been postulated that selective inhibition of the agonist signal given to the T cell by CD28, leaving the antagonist system consisting of the pair CTLA4/B7 intact, via specific blocking of the CD28/B7 interaction, would make it possible to prevent T lymphocyte activation.",
"Such specific blocking of the CD28/B7 interaction can be obtained using an antibody directed against CD28.Anti-CD28 antibodies capable of preventing CD28 binding to B7 are known.",
"However, they have the drawback, when they are used in their divalent native form, of bringing about the dimerization and the activation of CD28 via their binding with this receptor.",
"However, monovalent fragments derived from these antibodies are capable of blocking the CD28 receptor without activating it [DAMLE et al., J. Immunol.",
"140, 1753-1761, (1988); NUNES et al., Int.",
"Immunol., 5, 311-315 (1993); PAGES et al., J. Biol.",
"Chem., 271, 9403, (1996)].",
"It has thus been reported [PERRIN et al., J. Immunol.",
"163, 1704-1710, (1999)] that Fab fragments derived from an anti-CD28 antibody can curb the clinical symptoms of experimental autoimmune encephalitis induced in mice by the administration of myelin or the transfer of T cells from an affected animal.",
"Monovalent Fab or scFv fragments derived from an anti-CD28 antibody can potentially be used to prevent T lymphocyte activation via specific blocking of the CD28/B7 interaction.",
"Fab fragments result from the action of papain on an immunoglobulin molecule, and each contain a light chain and the first half of a heavy chain; scfv fragments consist of the variable portions of the heavy and light chains of an antibody, connected to one another via a flexible linker [CLACKSON et al., Nature, 352, 624-628, (1991)], thus forming a single-chain protein.",
"These monovalent fragments frequently exhibit less affinity for the antigen than the native antibodies, which can limit their possibilities for use in diagnostic or therapeutic applications.",
"The inventors have succeeded in selecting, among various antibodies which recognize the CD28 antigen, an antibody capable of blocking the CD28/B7 interaction, the monovalent fragments of which exhibit sufficient affinity for the antigen so that they can be used, in vitro or in vivo, to block the CD28 receptor without activation of this receptor.",
"This antibody, called CD28.3, is produced by the hybridoma deposited, according to the terms of the Treaty of Budapest, on Nov. 28, 2000, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2582.A subject of the present invention is a protein capable of binding specifically to the CD28 lymphocyte receptor and of blocking the CD28/B7 interaction, characterized in that it comprises at least the CDRs of the heavy chain and of the light chain of the immunoglobulin CD28.3.CDRs (complementarity determining regions) are the portions of the variable regions of an immunoglobulin which are involved in antigen recognition specificity.",
"Proteins in accordance with the invention thus encompass in particular: a) the antibody CD28.3 produced by the hybridoma CNCM I-2582; b) the Fv, Fab, Fab′2 or scfv fragments of the antibody CD28.3; c) the chimeric or humanized antibodies obtained from the variable regions of CD28.3; d) the fragments of the antibodies b) above comprising the CDRs of the antibody CD28.3, with a monovalent Fv, Fab or scfv fragments, or divalent Fab′2 fragments; e) the recombinant proteins comprising a fragment b) or d) and a heterologous polypeptide.",
"They may, for example, be: divalent or plurivalent derivatives of scfv fragments, such as “diabodies” or “triabodies”, resulting from the association of 2 or 3 scFv fragments; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with at least one antibody fragment comprising the CDRs of an antibody of different specificity; by way of examples, mention will be made of bispecific immunoglobulins, conjugates of an Fv or Fab fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity, and “bispecific diabodies” resulting from the association of an scFv fragment containing the CDRs of CD28.3 with an Fv or Fab fragment of an antibody of different specificity; proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule having pharmacological activity (for example a toxin) or effector properties (for example an Fc fragment); proteins combining at least one antibody fragment comprising the CDRs of the antibody CD28.3, with a molecule which makes it possible to prolong its plasma halflife when it is administered in vivo; it is, for example, possible to combine said antibody fragment with a water-soluble polypeptide of sufficient molecular mass for the molecular mass of the fusion polypeptide thus obtained to be greater than the renal filtration threshold.",
"In this case, a polypeptide will be chosen which, unlike Fc fragments, cannot associate as dimers, and which does not have its own effector activity liable to cause unfortunate side effects.",
"Polypeptides which have these properties can advantageously be obtained from water-soluble serum proteins, namely, in particular, serum albumin, haptoglobulin, ITIH2 (inter-alpha (globulin) inhibitor, H2 polypeptide), transferrin, CBG (corticosteroid binding globulin), α1-antitrypsin, ITIH4 (inter-alpha (globulin) inhibitor, H4 polypeptide), AACT (alpha-1-antichymotrypsin), TBG (thyroxine binding globulin), fibrinogen and prothrombin, in order to prepare fusion proteins with scFv fragments derived from anti-CD28 antibodies.",
"It is also possible to conjugate a protein in accordance with the invention with a polyol, for example polyethylene glycol, as described, for example, in U.S. Pat.",
"No.",
"4,179,337.An example of a protein in accordance with the invention is illustrated in FIG.",
"1, which represents an scFv fragment derived from the antibody CD28.3.The sequences of the CDRs of the antibody CD28.3 are boxed in the sequence represented in FIG.",
"1.The nucleotide sequence encoding this scFv fragment is represented in the attached sequence listing under the number SEQ ID No.",
"1, and the corresponding peptide sequence is represented under the number SEQ ID No.",
"2.Fv, Fab or Fab′2 fragments in accordance with the invention can be obtained by the conventional techniques of enzyme digestion, from the antibody CD28.3.A plasmid containing a polynucleotide encoding an scFv fragment of CD28.3, fused to a polynucleotide encoding amino acids 53 to 425 of α1-antitrypsin was deposited, according to the terms of the Treaty of Budapest, on Dec. 11, 2001, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Cultures and Microorganisms], 25 rue du Docteur Roux, 75724 PARIS CEDEX 15), under the number I-2762.Proteins in accordance with the invention such as chimeric or recombinant antibodies, scFv fragments and their derivatives, etc., can be obtained by conventional genetic engineering techniques, such as those described by SAMBROOK et al.",
"[MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989)].",
"Polynucleotides encoding the variable regions of the anti-CD28.3 antibody can, for example, be obtained by cloning said variable regions from a cDNA library of the hybridoma CD28.3, or from the plasmid CNCM I-2762.They can also be prepared, completely or partially, by nucleic acid synthesis, based on the nucleotide sequences of said variable regions.",
"It is, for example, possible to synthesize polynucleotides encoding the CDRs of CD28.3, and to incorporate them into the framework regions (FRs) of another antibody, in particular of an antibody of human origin, using techniques, known in themselves, of CDR grafting, such as those described by ROUTLEDGE et al.",
"[“Reshaping antibodies for therapy”, in Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, 13-44, Academic Titles, Nottingham, England (1993)] or by ROGUSKA et al.",
"Protein Engineering, 9(10), 895-904, (1996)].",
"A subject of the present invention is also any nucleic acid molecule encoding a protein in accordance with the invention comprising the CDRs of the antibody CD28.3, and also any recombinant vector, in particular any expression vector, comprising said nucleic acid molecule.",
"A subject of the present invention is also any cell expressing a protein in accordance with the invention comprising the CDRs of the antibody CD28.3.This encompasses in particular the hybridoma CNCM I-2582, and also the host cells transformed with a nucleic acid molecule in accordance with the invention.",
"Nucleic acid molecules in accordance with the invention may advantageously comprise, besides a sequence encoding a protein in accordance with the invention, a sequence encoding a signal peptide allowing secretion of said protein; they may also comprise one or more sequence(s) encoding one or more marker peptide(s) for detecting, and/or facilitating the purification of, said protein.",
"Expression vectors in accordance with the invention comprise at least one nucleic acid sequence encoding a protein in accordance with the invention, associated with transcription- and translation-controlling elements which are active in the host cell chosen.",
"Vectors which can be used to construct expression vectors in accordance with the invention are known in themselves, and will be chosen in particular as a function of the host cell intended to be used.",
"Host cells which can be used in the context of the present invention can be prokaryotic or eukaryotic cells.",
"Among the eukaryotic cells which can be used, mention will in particular be made of plant cells, cells from yeast, such as Saccharomyces, insect cells, such as Drosophila or Spodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc., cells.",
"The construction of expression vectors in accordance with the invention and the transformation of the host cells can be carried out by the conventional techniques of molecular biology.",
"A subject of the invention is also a method for producing a protein in accordance with the invention, characterized in that it comprises culturing at least one cell in accordance with the invention, and recovering said protein from said culture.",
"If the protein is secreted, it can be recovered directly from the culture medium; if not, cell lysis will be carried out beforehand.",
"The protein can then be purified from the culture medium or from the cell lysate, by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular precipitation with ammonium sulfate, electrophoresis, gel filtration, affinity chromatography, etc.",
"The proteins in accordance with the invention can be used, in vitro, to study the proliferative response or the differentiation of T lymphocytes responding to an antigenic, viral, allogenic or xenogenic stimulation.",
"They can also be used, in vitro, to induce the differentiation of T lymphocytes taken from a patient, for example the induction of tolerance with respect to an antigen or to an alloantigen, intended to be subsequently re-administered in vivo.",
"They may also be used to obtain medicinal products, or diagnostic reagents.",
"Proteins in accordance with the invention which are divalent, i.e.",
"which have 2 CD28 receptor-binding sites, and thus capable of inducing dimerization of this receptor, can be used in all situations where it is desired to activate this CD28 receptor, i.e.",
"to increase the response of a T lymphocyte with respect to an antigen.",
"Proteins in accordance with the invention which are monovalent, i.e.",
"which have a single CD28 receptor-binding site, can be used in all situations where it is desired to selectively block this receptor without activating it, in order to induce immunosuppression.",
"A protein in accordance with the invention, comprising a monovalent fragment derived from an anti-CD28 antibody, can in particular be used to obtain an immunosuppressant medicinal product which selectively blocks T cell activation phenomena involving the CD28 receptor, and which does not have the drawbacks of known immunosuppressants such as cyclosporin.",
"The T immunosuppression by selective blocking of CD28 with protein in accordance with the invention has applications in all T lymphocyte-dependent pathological conditions.",
"These are essentially transplant rejection, graft-versus-host disease, T lymphocyte-mediated autoimmune diseases, such as type I diabetes or multiple sclerosis, and type IV hypersensitivity, which is involved in allergic phenomena and also in the pathogenesis of chronic inflammatory diseases following infection with a pathogenic agent (in particular leprosy, tuberculosis, leishmaniasis, listeriosis, etc.).",
"The present invention will be understood more clearly from the further description which follows, which refers to nonlimiting examples of preparation and of use of antibodies in accordance with the invention.",
"EXAMPLE 1 Choice of an Antibody Producing Monovalent Fragments, Properties of Monovalent Fab Fragments Derived from CD28.3 Some of the properties of several anti-CD28 antibodies (CD28.1, CD28.2, CD28.3, CD28.4, CD28.5 and CD28.6) are described in the publication by NUNES et al.",
"[Int.",
"Immunol., 5, 311, (1993)].",
"These various antibodies, which are not accessible to the public, were provided by the laboratory of Daniel OLIVE (INSERM).",
"The antigen-binding properties of the monovalent Fab fragments of these various antibodies were compared.",
"5 mg of Fab fragments of each of these antibodies were prepared by digestion with papain (papain/antibody molar ratio= 1/100) for 24 hours at 37° C., followed by inactivation of the enzyme with 0.03M iodoacetamide, and dialysis against PBS to remove the iodoacetamide.",
"1) Binding of the Fab Fragments to CD28+ Jurkat T Cells: 100,000 CD28+ Jurkat cells in 100 μl are incubated in PBS-1% BSA-0.1% NaN3 at 4° C. for 30 minutes with increasing concentrations of anti-CD28 antibodies or of their Fab fragments.",
"After washing, the cells are incubated in a similar way with an FITC-conjugated anti-mouse IgG goat antibody, washed, and analyzed by cytofluorometry.",
"The results are given in FIG.",
"2: Legend of FIG.",
"2: X-axis: antibody or Fab fragments concentration Y-axis: mean fluorescence intensity (MFI) —⋄—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —Δ—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —◯—: F6=Fab fragments of the antibody CD28.6 ····: W132 whole antibody CD28.1 ··-··: W2=whole antibody CD28.2 : W3=whole antibody CD28.3 ——: W5=whole antibody CD28.5 —*—: W6=whole antibody CD28.6 —▴—: Mara-1=negative control (mouse IgG1).",
"These results show that, among the Fab fragments, only those derived from CD28.3 are capable of significantly binding to the CD28+ Jurkat cells at concentrations of less than 10 μg/ml.",
"2) Effect of the Fab Fragments on the Adhesion of CD28+ Jurkat T Cells to Transfected Murine L Cells Expressing the B7-1 Molecule: 4×105 human T cells (Jurkat, CD28-positive) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.",
"Chem., 271, 9403, (1996)] have been seeded 24 hours beforehand.",
"These incubations are carried out in the presence of the Fab fragments derived from the antibodies CD28.1 to CD28.6, or of the antibody CD28.3, diluted to various dilutions in PBS buffer without Ca2+ or Mg2+.",
"The adherent cells after washing are quantified by reading the residual radioactivity in a beta counter (PACKARD TOPCOUNT).",
"The results are given in FIG.",
"3: Legend of FIG.",
"3: X-axis: percentage of adherent cells Y-axis: antibody concentration —♦—: F1=Fab fragments of the antibody CD28.1 —▪—: F2=Fab fragments of the antibody CD28.2 —▴—: F3=Fab fragments of the antibody CD28.3 —x—: F5=Fab fragments of the antibody CD28.5 —*—: F6=Fab fragments of the antibody CD28.6 ····: whole antibody CD28.3 ◯: no antibody.",
"These results show that the Fab fragments derived from CD28.3 are the most effective for inhibiting CD28/B7 interactions.",
"They give 90% inhibition of adhesion at a concentration of 3 μg/ml, and with an effectiveness comparable to that of the whole antibody CD28.3, whereas, at this concentration, the Fab fragments derived from the other antibodies give no more than 50% inhibition of adhesion.",
"3) Effect of the Fab Fragments on Proliferation in a Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells (PBMCs) are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, in the presence of varying concentrations of the antibodies CD28.1 to CD28.6 or of the Fab fragments derived from these antibodies.",
"The proliferative response in these cultures is evaluated after 3 days, by incorporation of (3H) thymidine for a period of 16 hours.",
"The results are given in FIG.",
"4: Legend of FIG.",
"4: X-axis: antibody concentration Y-axis: proliferative response (cpm) Basal level of proliferation=6 500 cpm.",
"—♦—: 28.1=antibody CD28.1 —▪—: 28.2=antibody CD28.2 —▴—: 28.3=antibody CD28.3 —x—: 28.5=antibody CD28.5 ···*···: 28.6=antibody CD28.6 ——: Fab.",
"1=Fab fragments of the antibody CD28.1 ··+··: Fab.",
"2=Fab fragments of the antibody CD28.2 ——: Fab.",
"3=Fab fragments of the antibody CD28.3 —+—: Fab.",
"5=Fab fragment of the antibody CD28.5 —⋄—: Fab.",
"6=Fab fragment of the antibody CD28.6.These results show that the Fab fragments derived from CD28.3 or from CD28.6 are the most effective for inhibiting mononuclear cell proliferation.",
"The whole antibodies CD28.1 to CD28.6, tested in parallel, have no inhibitory effect or indeed stimulate the proliferation by virtue of their stimulator action on CD28.Effect of the Fab Fragments Derived from CD28.3 on Proliferation Induced by a Superantigen For this experiment, responder CD4+ T cells were mixed with irradiated isogenic PBMCs, in the presence of 50 ng/ml of toxic shock syndrome toxin-1 (TSST-1), which specifically stimulates the vβ2+ T cell, either in the absence of antibody or in the presence of anti-B7-1 (1 μg/ml), of anti-B7-2 (0.5 μg/ml), of CTLA4Ig (10 μg/ml), or of Fab fragments derived from CD28.3 (10 μg/ml).",
"The proliferative response in these cultures is evaluated after 1, 3, 6 and 8 days, by incorporation of (3H) thymidine for a period of 16 hours.",
"The results are given in FIG.",
"5: Legend of FIG.",
"5: X-axis: culturing time Y-axis: proliferation index=PI P I = cpm mixed lumphocyte reaction - cpm irradiated stimulating cells only cpm unstimulated responder cells —♦—: anti-CD28.3 Fab —X—: anti-B7-2 —▪—: CTLA-4 Ig ··*··: anti-B7-1+2 —▴—: anti-B7-1 —◯—: no antibody TSST-1 induces considerable proliferation of CD4+ T cells.",
"In the presence of anti-B7, of CTLA4Ig or of the Fab fragments of CD28.3, 70% inhibition of this proliferation is observed after 6 days.",
"Effect of the Fab Fragments Derived from CD28.3 on Cytokine Production In order to determine whether the Fab fragments derived from CD28.3 could induce an immune deviation in vitro, a mixed lymphocyte reaction (PBMCs derived from a donor A/irradiated PBMCs derived from a donor B) was carried out, in the presence of Fab fragments derived from CD28.3.105 peripheral blood mononuclear cells from a donor are mixed with 105 allogenic mononuclear cells irradiated at 35 Gy, and cultured for 5 days in the presence or absence of 10 μg/ml of Fab derived from the antibody CD28.3.The RNA of the responder cells was extracted, and the amount of cytokine mRNA was evaluated by quantitative measurement of the number of transcripts, related to the amount of HPRT, using a TaqMan (Perkin Elmer).",
"In the presence of Fab fragments derived from CD28.3, a decrease in the production of γIFN and of IL2, and an increase in the production of IL10 are observed.",
"This deviation in the immune response suggests an orientation toward a Th2-type response.",
"This result is unexpected insofar as it has been reported that the involvement of CTLA4 (which is supposed to intervene in the blocking of CD28 alone) leads to a Th1-type response.",
"In Vitro Processing of the Antibody CD28.3 and of the Fab Fragments Derived Therefrom, by Human T Cells A possible internalization of the Fab fragments of the antibody CD28.3 in human T cells was investigated, in comparison with the whole antibody CD28.3.Jurkat T cells were incubated in culture medium with 100 μg/ml of antibody CD28.3, at 37° C. or at 0° C. At various times, the cells were washed with cold PBS buffer containing 0.1% of bovine serum albumin, and NaN3, in order to block membrane motility.",
"The bound antibodies were revealed with a fluorescein-labeled goat anti-mouse secondary antibody.",
"The cells were mounted in MOVIOL and analyzed by confocal microscopy.",
"It is thus observed that the whole CD28.3 antibodies which bind to the Jurkat T cells are captured and disappear from the cell surface at 37° C., but not at 0° C. On the other hand, the Fab fragments remain attached at the surface of the cell.",
"This indicates that the attachment of the divalent antibodies CD28.3 leads to dimerization of CD28, which brings about their entry into the cell, whereas the monovalent Fab fragments, which do not induce this dimerization, remain at the surface.",
"EXAMPLE 2 Properties of an scFv Fragment Derived from the Antibody CD28.3 FIG.",
"1 represents the nucleotide sequence and the deduced polypeptide sequence of an scFv fragment derived from the antibody CD28.3.The portions of this sequence corresponding to the variable fragment of the heavy chain and of the light chain are represented in capital letters.",
"The sequence corresponding to the variable fragment of the light chain is also underlined.",
"The sequence of the linker is represented in lower case letters.",
"The sequences of the CDRs of the heavy chain and of the light chain are boxed in.",
"The nucleotide sequence encoding this scFv fragment is also represented in the attached sequence listing, under the number SEQ ID No.1.The cDNA encoding this scFv fragment was inserted into the vector pIG6 (Biochemisches Institut, University of Zurich).",
"This vector comprises in particular an ampicillin resistance marker and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.",
"The cDNA encoding the scFv fragment described above was introduced between the EcoRI and EcoRV sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.",
"The construct obtained is called pIg6-28.3.Production in Prokaryotic Cells The vector pIg6-28.3 was used to transform E.coli JM83 cells.",
"The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the scFv fragment is produced in soluble form in the periplasma.",
"After electrophoresis and Western blotting, it appears in the form of a band at approximately 30 kDa.",
"It is purified from the periplasmic extracts of the bacteria, obtained after osmotic shock in 50 mM Tris-Cl, and ultracentrifugation of the insoluble material, by chromatography on an Ni-NTA matrix and ion exchange on DEAE-Sepharose.",
"The binding of the scFv fragments present in the eluate of the NiNTA column, to CD28+ Jurkat cells, is comparable to that obtained with Fab fragments obtained from the antibody CD28.3 by digestion with papain.",
"Production in Eukaryotic Cells The vector pSec-28.3 was used to transfect Cos cells.",
"The cells are cultured at 37° C. for 3 days.",
"The scFv fragment is produced in soluble form in the supernatant.",
"This supernatant inhibits the mixed lymphocyte reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, healthy allogenic donor.",
"The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.",
"Considerable inhibition of the incorporation, dependent on the supernatant dilution used, is observed.",
"A control supernatant exhibits no proliferation-inhibiting activity.",
"EXAMPLE 3 Production of a Fusion Protein Comprising an scFv Fragment of CD28.3 The nucleotide sequence encoding the scfv fragment described in example 2 was linked to the 5′ end of a portion of the cDNA of human α1-antitrypsin (GENBANK accession number K01396) corresponding to amino acids 53 to 425, via a hinged peptide of sequence VAAPS.",
"The resulting sequence is represented in the attached sequence listing under the number SEQ ID No.",
"3, and the corresponding polypeptide under the number SEQ ID No.",
"4.EXAMPLE 4 Construction of Expression Vectors Comprising the Sequence Encoding α1-Antitrypsin and Allowing the Introduction of a Sequence Encoding an scFv Fragment Prokaryotic Expression Vector: The vector pIG6 was used (Biochemisches Institut, University of Zurich).",
"This vector comprises in particular an ampicillin resistance marker, and an expression cassette which comprises an inducible lac promoter under the control of which are placed: a sequence encoding an ompA signal peptide, a sequence encoding a marker peptide of sequence (1-letter code) DYKD, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-5 marker.",
"The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the EcoRI and EcoRV sites of pIg6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.",
"FIG.",
"1 illustrates diagrammatically the construct obtained, called pIg6-Haat.",
"Eukaryotic Expression Vector: The vector pSECTagB (Invitrogen, De Schelp, The Netherlands) was used.",
"This vector comprises in particular an ampicillin resistance marker, a zeocin resistance marker, and an expression cassette which comprises a CMV promoter under the control of which are placed: a sequence encoding a signal peptide of the IgG kappa light chain, a sequence encoding a c-myc marker peptide, and a sequence encoding a polyhistidine-6 marker.",
"The cDNA encoding a fragment of human α1-antitrypsin corresponding to amino acids 53 to 425 was introduced between the BamHI and EcoRI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.",
"FIG.",
"2 illustrates diagrammatically the construct obtained, called pSecHaat.",
"EXAMPLE 5 Construction of Expression Vectors Integrating the Sequence Encoding the CD28.3 scFv/α1-Antitrypsin Fusion Protein Prokaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the EcoRI and XhoI sites of pIG6, downstream of the sequence encoding the peptide DYKD and upstream of the sequence encoding the c-myc marker.",
"FIG.",
"3 illustrates diagrammatically the construct obtained, called pIg6-28.3Haat.",
"Eukaryotic Expression Vector: The cDNA encoding the CD28.3 ScFv/α1-antitrypsin fusion protein described in example 3 above was introduced between the BamHI and XhoI sites of the vector PSEC B Tag, upstream of the sequence encoding the c-myc marker.",
"FIG.",
"4 illustrates diagrammatically the construct obtained, called pSec-28.3Haat.",
"This vector, harbored by E.coli DH5α, was deposited with the CNCM on Dec. 11, 2001, under the number I-2762.EXAMPLE 6 Expression and Purification of the Fusion Proteins In Prokaryotic Cells: The vector pIg6-28.3Haat was used to transform E. coli JM83 cells.",
"The cells are cultured at 25° C., up to an OD550 of 0.5.After induction with IPTG, the protein is produced in soluble form in the periplasm.",
"After electrophoresis and Western blotting, it appears in the form of a band at approximately 74 kDa.",
"It can be purified from the periplasmic extracts using an NI-NTA affinity chromatography matrix and/or an anti-c-myc affinity chromatography matrix.",
"It can also be purified using an anti-α1-antitrypsin affinity column.",
"In Eukaryotic Cells: The vector pSec-28.3Haat was used to transfect CHO cells by lipofection.",
"The cells are cultured in the presence of 200 μg/ml on zeocin in MEM medium containing 10% of fetal calf serum.",
"The protein is secreted into the culture medium.",
"After separation by electrophoresis, Western blotting, and revelation with an anti-c-myc antibody, it appears in the form of a band at approximately 80 kDa.",
"EXAMPLE 7 Assays for Activity of an scFv/α1-Antitrypsin Fusion Protein The anti-CD28 activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above was evaluated by its binding to the CD28 molecule, or to cells expressing CD28 on their membrane, and its lack of binding to cells which do not express CD28.The immunosuppressor activity of the CD28.3 scFv/α1-antitrypsin fusion protein obtained in example 6 above is evaluated by the inhibition of adhesion to B7, and the inhibition of the induced activation of the T lymphocyte.",
"These anti-CD28 and immunosuppresor activities were measured using the following assays: Anti-CD28 Activity Biosensor Measurement of the CD28-Binding Parameters: Recombinant human CD28 was immobilized on the biosensor (BIACORE) detector.",
"A CD28.3 scFv/α1-antitrypsin fusion protein obtained as described in example 6 above was brought into contact with the detector.",
"The binding parameters are: KA (1/M)2.86e9; KD (M): 3.49e-10.In comparison, these parameters measured for the Fab fragment of the antibody CD28.3 are: KA (1/M): 9.69e8; KD (M): 1.03e-9.The affinity for CD28, of the Fab fragment of the antibody CD28.3 and of the fusion protein, are therefore comparable.",
"Cytofluorometry Assay for Specific Recognition of CD28: 105 Jurkat (Cd28+) and U937 (CD28−) cells are incubated in PBS-1% BSA-0.1% NaN3, at 4° C., for 1 hour with increasing concentrations of the CD28.3 scFv/α1-antitrypsin fusion protein.",
"After washing, the cells are incubated with an anti-alpha-1-antitrypsin rabbit antibody and then with an FITC-conjugated goat anti-rabbit antibody, washed, and analyzed by cytofluorometry.",
"Binding dependent on the dose of the Jurkat cells (CD28+), and no binding to the U937 (CD28−) cells, were observed.",
"This shows the specificity of the fusion protein for the CD28 molecule and its lack of reactivity toward other molecules expressed by human hematopoietic cells.",
"Immunosuppressor Activity CD28/B7-Dependent Adhesion Assay: 4×105 human T cells (CD28-positive Jurkat cells) labeled with 51Cr are incubated for 2 hours in a microtitration plate in which 105 adherent LTK− or LB7+ cells (murine fibroblasts transfected with human B7.1 [PAGES et al., J. Biol.",
"Chem., 271, 9403 (1996)] had been seeded 24 hours beforehand.",
"These incubations are carried out in the absence or in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, diluted to various concentrations in PBS buffer without Ca2+ or Mg2+.",
"The adherent cells after washing are quantified by reading the residual radioactivity using a beta counter (PACKARD TOPCOUNT).",
"Inhibition of the adhesion in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein is observed, which inhibition is directly dependent on the dose of fusion protein used.",
"Inhibition of the Activation: 5×104 T cells (human polyclonal cells depleted of CD11b cells) are stimulated with 1×104 irradiated OKT3 hybridoma cells (anti-CD3), or with allogenic CD28− B cells (depleted of CD28+ cells), in the absence or in the presence of varying amounts of the CD28.3 scFv/α1-antitrypsin fusion protein.",
"The proliferative response in these cultures is evaluated after 3 days when the stimulation is performed with anti-CD3s, or after 7 days when the stimulation is performed with allogenic cells, by incorporation of (3H) thymidine for a period of 16 hours.",
"Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used.",
"Inhibition of the Mixed Lymphocyte Reaction: 105 peripheral blood mononuclear cells from a healthy donor are mixed with 105 peripheral blood mononuclear cells from another, allogenic, healthy donor.",
"The proliferative response in these cultures is evaluated after 5 days by incorporation of (3H) thymidine for a period of 16 hours.",
"Considerable inhibition of the incorporation is observed in the presence of the CD28.3 scFv/α1-antitrypsin fusion protein, which inhibition is directly dependent on the dose of fusion protein used."
]
] | Patent_10450832 |
[
[
"Deflector Devices",
"A deflector device (22) for use with a tow line between a seismic survey vessel and a tow, in particular a seismic streamer or streamer array, in the water behind the vessel comprises a vertically oriented wing-shaped body (28) shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel.",
"The wing-shaped body (28) includes one or more buoyancy elements, and a rearwardly extending boom (32).",
"A pivotable control surface (54) extends sideways from the boom (32), and is shaped to produce in use a force having a substantial vertical component.",
"The angle of the control surface is remotely controllable, in order to control the depth of the deflector device."
],
[
"1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.",
"2.A deflector device as claimed in claim 1, wherein the one or more buoyancy elements have a buoyancy selected to give the complete device a small positive buoyancy.",
"3.A deflector device as claimed in claim 1, wherein the remotely-operable means comprises a telescopic member connected to pivot the control surface.",
"4.A deflector device as claimed in claim 3, wherein the telescopic member is hydraulically operated.",
"5.A deflector device as claimed in claim 1, further comprising an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.",
"6.A deflector device as claimed in claim 5, further comprising additional remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.",
"7.A deflector device as claimed in claim 6, wherein the additional remotely-operable adjusting means comprises a further telescopic member connected to the auxiliary wing-shaped body.",
"8.A deflector device as claimed in claim 7, wherein the further telescopic member is hydraulically operated.",
"9.A deflector device as claimed in claim 5, wherein the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.",
"10.A method of performing a marine seismic survey, comprising: towing a plurality of laterally-spaced seismic streamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device comprising: a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel; one or more buoyancy elements disposed within and/or secured to the upper end of the principal wing-shaped body; a boom extending rearwardly from the principal wing-shaped body; a pivotable control surface extending sideways from the boom and shaped to produce in use a force having a substantial vertical component; and remotely-operable means for pivoting the control surface, thereby to control the depth of the deflector device substantially independently of the lateral position of the deflector device.",
"11.The method of claim 10, further comprising selecting a buoyancy of the one or more buoyancy elements to give the complete device a small positive buoyancy.",
"12.The method of claim 10, wherein the remotely-operable means comprises a telescopic member connected to the control surface, and further comprising pivoting the control surface using the telescopic member.",
"13.The method of claim 10, wherein the deflector device includes an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in: use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and further comprising varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body."
],
[
"This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.",
"A deflector device of this kind is described in detail in our U.S. Pat.",
"No.",
"5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.",
"In use, the wing-shaped body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.",
"As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.",
"This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.",
"The deflector device of U.S. Pat.",
"No.",
"5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.",
"In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.",
"The MONOWING deflector devices in current use are very large, typically 7.5 m high by 2.5 m wide, and weigh several tonnes.",
"They are usually suspended around 2 m to 8 m below the float by means such as a fibre rope, and are also provided with a safety chain intended to prevent separation of the float and wing-shaped body in the event that the rope breaks.",
"In rough weather, the upper part of the wing-shaped body may rise up out of the water, allowing the rope connecting the wing-shaped body and the float to go slack.",
"If the wing-shaped body then drops abruptly, the rope, and possibly even the safety chain, may break, and/or their attachment points on the wing-shaped body may be badly damaged.",
"Additionally, the depth at which the current deflector device operates is effectively determined by the length of the rope connecting it to the float.",
"As a result of this, the operating depth of the deflector device cannot readily be varied while the device is deployed in the water.",
"And since the normal operating depth of the current deflector device is typically a few meters, in the event of the onset of bad weather during a survey, the device and all the streamers and other equipment directly or indirectly attached to it have to be recovered onto the towing vessel, and then re-deployed when the bad weather has passed, both of which operations are very time consuming.",
"It is an object of the present invention to alleviate the drawbacks arising from the connection of the deflector device to the float.",
"According to the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, one or more buoyancy elements disposed within and/or secured to the upper end of the wing-shaped body, a boom extending rearwardly from the wing-shaped body, and a remotely-operable pivotable control surface extending sideways from the boom and shaped to produce in use a force having a vertical component, whereby to control the depth of the deflector device.",
"It will be appreciated that since the deflector device of the invention can generate a controllable vertical force, this force, together with the buoyancy of the one or more buoyancy elements, can be selected and adjusted so that the separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.",
"In particular, at the onset of bad weather, the deflector device and its tow can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the weather improves.",
"Advantageously, the one or more buoyancy elements has or have a buoyancy selected to give the complete device a small positive buoyancy.",
"In a preferred embodiment of the invention, the deflector device further comprises an auxiliary wing-shaped body, smaller than the firstmentioned (or principal) wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.",
"Advantageously, this embodiment further includes remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.",
"The pivotable control surface and the remotely-operable means are preferably both hydraulically operated.",
"Advantageously, the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.",
"The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic steamers over an area to be surveyed, wherein the lateral position and the depth of at least one of the streamers are controlled by a deflector device in accordance with any one of the preceding statements of invention.",
"The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.",
"1 is a somewhat schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.",
"2 is a somewhat schematic part-sectional view of a first embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.",
"1; and FIGS.",
"3A and 3B are respective perspective view of the deflector device of FIG.",
"2.The seismic survey vessel shown in FIG.",
"1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.",
"PCT/GB98/01832 (WO 99/00295).",
"The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.",
"No.",
"4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.",
"PCT/IB98/01435 (WO 99/15913).",
"The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.",
"The deflector devices 22 act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.",
"One of the deflector devices 22 is shown in more detail in FIGS.",
"2, 3A and 3B.",
"The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.",
"No.",
"5,357,892, but is a much improved version of it.",
"In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.",
"No.",
"5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 29, both of which features enhance lift.",
"Also, the main wing-shaped body 28 is provided with vortex controlling end plates 30 (see FIGS.",
"3A and 3B) of the kind described in our PCT Patent Application No.",
"PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight, while the towing bridle 27 comprises a pair of titanium chains 52 (see FIGS.",
"3A and 3B).",
"Additionally, the angle lever 10 of U.S. Pat.",
"No.",
"5,357,892 is replaced by a rearwardly extending fixed angle boom 32, which is detachably connected at one end 34 to the low pressure side 36 of the body 28 near the trailing edge flap 29, at a mounting bracket 38.The boom 32 is of sandwich construction, and is made from two similarly shaped plates 39 which are bolted together at intervals along their length and which sandwich between them the mounting bracket 38.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 22 is on the vessel 10, for ease of stowage.",
"The other end 40 of the boom 32 has a towing eye 42, coupled in use to the forward end 24 of a respective one of the two outermost streamers 18.An auxiliary wing-shaped body 44, which is much smaller than the body 28 in length, thickness and chord, is pivotally secured as will be explained hereinafter to the end 40 of the boom 32, with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.",
"2) extending parallel to the longitudinal axis of the body 28.The shape of the body 44 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because as will become apparent, the direction of the force varies in use).",
"This sideways force is increased by providing the body 44 with a fixed trailing edge flap 46, angled away from the boom 32 at an angle of about 35°.",
"As best seen in FIGS.",
"3A and 3B, the auxiliary wing-shaped body 44 is implemented in two symmetrical halves 44a and 44b, which each have vortex-reducing end plates 45 and which are disposed on opposite sides of the boom 32.These two halves 44a, 44b are rotatable in unison about a common axis perpendicular to the plane of the boom 32, so as to vary the angle of the chord of the auxiliary wing-shaped body 44 with respect to the boom.",
"Rotation of the auxiliary wing-shaped body 44 is effected by a telescopic actuator 48 pivotally mounted between the plates 39 of the boom 32, the actuator being pivotally connected to a lever arm or eccentric 50 secured to each of the two halves 44a, 44b of the auxiliary wing-shaped body.",
"The telescopic actuator 48 is hydraulically operated by a remotely controllable electro-hydraulic control pack 52 also mounted between the plates 39 of the boom 32.It will be appreciated that varying the angle of the auxiliary wing-shaped body 44 of the deflector device 22 changes the angle of the main wing-shaped body 28 with respect to the direction of tow, and so changes the lift produced by the main wing-shaped body.",
"This in turn changes the lateral offset produced by the deflector device 22.In accordance with the present invention, the deflector device 22 is made approximately neutrally buoyant, by including gas-filled pipe-like buoyancy elements 58 extending longitudinally within it from top to bottom, and/or by providing an integral buoyancy element at its upper end similar to but smaller than that described in our co-pending United Kingdom Patent Applications Nos.",
"0023775.0, 0025719.6 and 0029451.2.In practice, the deflector device 22 is preferably designed to be slightly positively buoyant, so that in the event of a malfunction, it tends to float rather than to sink.",
"Additionally, the deflector device 22 is provided with a pivotable control surface (or flap) 54, which is secured to the boom 32 in the region of the auxiliary wing-shaped body 44 by a generally triangular bracket 56, and which is pivotable about an axis perpendicular to both the pivot axis of the body 44 and the direction of tow (indicated by the arrow 58 in FIG.",
"2).",
"The flap 54 and the bracket 56 are both made from titanium.",
"The angular position of the flap 54 is controlled by a further telescopic actuator 60, which is connected to a lever arm 62 provided on the flap, and which is hydraulically operated by the electro-hydraulic control pack 52.It will be appreciated that rotation of the flap 54 about its pivot axis produces in use an upward or downward force at the end 40 of the boom 32, and thus enables the depth of the deflector device 22 to be controlled.",
"It will be appreciated that as a result of making the deflector device 22 approximately neutrally buoyant and capable of generating a remotely-controllable vertical force, a separate surface float is no longer required, and the operating depth of the device can be remotely controlled while the device is deployed in the water.",
"In particular, in the event of the onset of bad weather, the deflector device 22 and the streamers 18 attached to it can be caused to dive to a greater depth, where the effects of the bad weather are much reduced, until the bad weather passes.",
"Many modifications can be made to the described embodiment of the invention.",
"In particular, the flap 54 and the auxiliary wing-shaped body 44 can be made from a plastics material reinforced with high strength fibres, eg Kevlar fibres, and can be electrically actuated rather than hydraulically actuated, Additionally, the devices 22 and 60 can be used with tows other than streamers, for example seismic sources, and the tow need not be connected to the end 40 of the boom 32 (it could instead be connected to the lead-in 20, at a point near where the bridle 24 is connected to the lead-in).",
"Also, the invention can if desired be used with a deflector device in which the auxiliary wing-shaped body 44 is fixed, and the boom 32 is pivotable towards and away from the main deflector body 28, as described in our United Kingdom Patent Applications Nos.",
"0023755.2, 0025711.3 and 0029452.0.Indeed, the invention can even be used with a deflector device like that described in our U.S. Pat.",
"No.",
"5,357,892, ie a deflector device without the auxiliary wing-shaped body 44, by mounting a pivotable flap analogous to the flap 54 on a pivotable boom analogous to the angle lever 10 of the deflector device of the US patent.",
"Finally, although the invention has been described in relation to deflector devices whose lift can be varied by varying the angle of the device with respect to the direction of tow, it is also applicable in its broadest aspect to a fixed angle deflector device, eg of the kind referred to as a “door”."
]
] | Patent_10450862 |
[
[
"Methods for the production of multimeric proteins and related compositions",
"Improved methods for the production of multimeric-protein-complexes, such as redox proteins and immunoglobins, in association with oil bodies are described.",
"The redox protein is enzymatically active when prepared in association with the oil bodies.",
"Also provided are related nucleic acids, proteins, cells, plants, and compositions."
],
[
"1-266.",
"(canceled) 267.A method of producing an oil body associated with a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.",
"268.A method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.",
"269.A method of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.",
"270.A chimeric nucleic acid sequence encoding a multimeric-fusion-protein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"271.A recombinant multimeric-fusion-protein comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.",
"272.Isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"273.Isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"274.A cell comprising oil bodies and (i) an oil-body-targeting-protein, (ii) a first recombinant polypeptide and (iii) a second recombinant polypeptide wherein (1) said first recombinant polypeptide is capable of associating with said oil-body-targeting-protein; and (2) said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form a multimeric-protein-complex.",
"275.A composition comprising isolated oil bodies, thioredoxin and thioredoxin-reductase.",
"276.A food product, personal care product or pharmaceutical composition comprising the composition of claim 275.277.A method of reducing allergenicity of a food comprising the steps of: providing the isolated oil bodies of claim 273; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.",
"278.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing the recombinant fusion polypeptide of claim 269; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.",
"279.A method for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.",
"280.A method for preparing a redox protein associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide.",
"281.A chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.",
"282.A transgenic plant comprising the chimeric nucleic acid sequence of claim 281.283.A safflower plant comprising the chimeric nucleic acid of anyone of claim 281.284.A plant seed comprising the chimeric nucleic acid of claim 281.285.A safflower seed comprising the chimeric nucleic acid of claim 281.286.An oil body preparation obtained by the method of claim 279.287.A food product comprising an oil body preparation of claim 286.288.A composition comprising an oil body preparation of claim 286 (New).",
"289.A personal care product comprising an oil body preparation of claim 286.290.A product capable of treating oxidative stress in a target comprising an oil body preparation of claim 286.291.A product capable of chemically reducing a target comprising an oil body preparation of claim 286.292.A detergent composition comprising the product of claim 286.293.A method of cleansing an item, comprising administering the product of claim 290 to said item under conditions that promote cleansing.",
"294.An emulsion formulation prepared by the method of claim 279.295.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.",
"296.The seed of the plant of claim 281.297.An extract of the seed of claim 296, wherein the extract comprises an activity of a thioredoxin-related protein.",
"298.An oil body from the seed of claim 296.299.Oil produced from the seed of claim 296.300.A method of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; obtaining seeds from the plant; and recovering the fusion protein by isolating oil bodies from the seeds.",
"301.Oil bodies in association with a fusion protein, obtained by the method of claim 300.302.A method of reducing allergenicity of a food comprising the steps of: providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.",
"303.A composition comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.",
"304.A cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in an acceptable carrier.",
"305.A method of treating or protecting a target against oxidative stress, comprising the steps of: providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.",
"306.A nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one recombinant polypeptide and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides."
],
[
"<SOH> BACKGROUND <EOH>Multimeric proteins (i.e.",
"proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.",
"Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.",
"However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.",
"Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.",
"For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.",
"(1996) J. Cardiov.",
"Pharmacol.",
"(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.",
"(1999) J. Allerg.",
"Vlin.",
"Immunol.",
"103: 690-697) and wheat (Buchanan et al.",
"(1997) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 94: 5372-5377).",
"However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.",
"The present invention satisfies this need and provides related advantages as well."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.",
"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.",
"The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.",
"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.",
"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.",
"The oil-body-targeting-protein can be an oleosin or caleosin.",
"When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.",
"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.",
"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.",
"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.",
"In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.",
"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.",
"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.",
"Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.",
"This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.",
"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.",
"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.",
"The oil-body-targeting-protein can be an oleosin or caleosin.",
"When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.",
"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.",
"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.",
"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.",
"In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.",
"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.",
"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.",
"Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.",
"The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.",
"The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.",
"The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.",
"The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.",
"Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.",
"The oil-body-protein can be an oleosin or caleosin.",
"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.",
"Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.",
"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.",
"Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.",
"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.",
"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.",
"In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.",
"These formulations can further comprise the addition of NADP or NADPH.",
"The food product can be a milk or wheat based food product.",
"The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.",
"The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).",
"Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.",
"In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.",
"5 .",
"Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.",
"The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.",
"The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.",
"Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.",
"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.",
"Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.",
"The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.",
"Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.",
"For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.",
"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.",
"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.",
"In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.",
"The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.",
"In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis .",
"In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.",
"Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.",
"Accordingly, an emulsion formulation composition is provided.",
"Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.",
"The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.",
"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.",
"Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.",
"The chimeric nucleic acids can be contained within a plastid.",
"Accordingly, isolated plastids are provided having chimeric nucleic acids therein.",
"Also provided are plant seeds comprising the chimeric nucleic acids provided herein.",
"Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.",
"The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.",
"Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.",
"Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.",
"In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.",
"In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.",
"The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.",
"The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.",
"The second region can encode a thioredoxin and thioredoxin-reductase.",
"In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae .",
"In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.",
"The first region can precede, in a 5′ to 3′ direction, the second region.",
"Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.",
"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.",
"A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.",
"In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.",
"In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.",
"In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.",
"Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.",
"The thioredoxin-related protein can be thioredoxin.",
"The nucleic acid construct can be contained within a plastid.",
"In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.",
"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.",
"The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.",
"Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.",
"In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.",
"Also provided herein is an extract comprising an activity of a thioredoxin-related protein.",
"Also provided are oil bodies and/or oil obtained from various seeds.",
"Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.",
"In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.",
"The oil bodies can be in association with a fusion protein.",
"The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.",
"The cleaving step can make use of a protease or chemical proteolysis.",
"Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.",
"The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.",
"In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.",
"Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.",
"The oil bodies can be associated with the fusion protein.",
"Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.",
"Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.",
"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.",
"Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.",
"Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.",
"These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.",
"The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.",
"For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.",
"Other features and advantages of the present invention will become readily apparent from the following detailed description.",
"It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only."
],
[
"RELATED APPLICATIONS Benefit of priority under 35 U.S.C.",
"§119(e) is claimed to U.S. provisional application Ser.",
"No.",
"60/302,885, filed Jul.",
"5, 2001, to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”.",
"This application is also a continuation-in-part of U.S. utility application Ser.",
"No.",
"10/006,038, filed Dec. 4, 2001 to van Rooijen, et al., entitled “METHODS FOR THE PRODUCTION OF REDOX PROTEINS”; which is a continuation-in-part of U.S. utility application Ser.",
"No.",
"09/742,900, filed Dec. 19, 2000 to Heifetz, et al., entitled “METHOD OF PRODUCTION AND DELIVERY OF THIOREDOXIN”.",
"This application is also a continuation-in-part of U.S. utility application Ser.",
"No.",
"09/742,900.The subject matter of each of the provisional and utility applications is incorporated herein by reference in its entirety.",
"FIELD OF THE INVENTION The present invention relates to multimeric-protein-complexes, redox proteins, and recombinant polypeptides; and improved methods for their production.",
"BACKGROUND Multimeric proteins (i.e.",
"proteins comprising multiple polypeptide chains) are a biologically and commercially important class of proteins.",
"Antibodies for example are multimeric proteins which are used to treat a wide range of disease conditions.",
"However in view of their complexity, multimeric proteins frequently represent significant manufacturing challenges.",
"Redox proteins are also a commercially important class of proteins with applications in a variety of different industries including the pharmaceutical, personal care and food industry.",
"For example, the redox protein thioredoxin may be used in the manufacture of personal care products (Japanese Patent Applications JP9012471A2, JP103743A2, JP1129785A2), pharmaceutical compositions/products (Aota et al.",
"(1996) J. Cardiov.",
"Pharmacol.",
"(1996) 27: 727-732) as well as to reduce protein allergens present in food products such as milk (del Val et al.",
"(1999) J. Allerg.",
"Vlin.",
"Immunol.",
"103: 690-697) and wheat (Buchanan et al.",
"(1997) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 94: 5372-5377).",
"However, there is a need in the art to further improve the methods for the recombinant expression of multimeric proteins, including redox proteins.",
"The present invention satisfies this need and provides related advantages as well.",
"SUMMARY OF THE INVENTION The present invention relates to novel and improved methods of producing a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and/or thioredoxin-related proteins; in association with oil bodies.",
"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.",
"The method further contemplates isolating the oil bodies associated with said recombinant multimeric-protein-complex.",
"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and said second recombinant polypeptide.",
"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.",
"The oil-body-targeting-protein can be an oleosin or caleosin.",
"When the oil-body-targeting-protein can be an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.",
"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.",
"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.",
"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.",
"In one embodiment, the first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell.",
"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.",
"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.",
"Also provided herein is a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex; and (d) associating said first recombinant polypeptide with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first recombinant polypeptide.",
"This method further contemplates isolating from the progeny cell, oil bodies comprising the multimeric-protein-complex.",
"The second recombinant polypeptide can be associated with a second oil-body-targeting-protein capable of associating with an oil body and second recombinant polypeptide.",
"Each of said oil-body-targeting-proteins can be an oil-body-protein or an immunoglobulin.",
"The oil-body-targeting-protein can be an oleosin or caleosin.",
"When the oil-body-targeting-protein is an oleosin or caleosin, the first recombinant polypeptide can be fused to said oleosin or caleosin.",
"Likewise, the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.",
"The first and second recombinant polypeptides can be produced as a multimereic-fusion-protein comprising said first and second polypeptide, and can form a multimeric-protein-complex.",
"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.",
"In one embodiment, the first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex in said progeny cell.",
"In another embodiment, the first recombinant polypeptide can be a thioredoxin and the second recombinant polypeptide can be a thioredoxin-reductase.",
"In particular embodiments, the thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 60 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The cell can be a plant cell, such as a safflower cell, and the like.",
"Also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.",
"The second recombinant polypeptide can be associated with oil bodies through a second oil-body-targeting-protein in the second plant.",
"The oil bodies can be isolated from the progeny plant comprising said multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, wherein the oil-body-protein can be an oleosin or caleosin.",
"The first recombinant polypeptide can be fused to the oleosin or caleosin; and the second recombinant polypeptide can be fused to a second oleosin or second caleosin capable of associating with an oil body.",
"The first and second recombinant polypeptide can form a multimeric-protein-complex, such as a heteromultimeric-protein-complex, wherein the heteromultimeric-protein-complex can be an enzymatically active redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. The plant can be a safflower plant.",
"Also provided herein are chimeric nucleic acids encoding a multimeric-fusion-protein as described herein, said nucleic acid comprising: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin.",
"The oil-body-protein can be an oleosin or caleosin.",
"The multimeric-protein-complex can be a heteromultimeric-protein-complex, and the first and second recombinant polypeptide can form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence that is also a non-proteolytic linker and said sequence encoding the first recombinant polypeptide.",
"Also provided herein are recombinant multimeric-fusion-proteins comprising (i) an oil-body-targeting-protein, or fragment thereof, (ii) a first recombinant polypeptide and a (iii) second recombinant polypeptide, wherein said first and second recombinant polypeptides are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.",
"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In yet another embodiment, positioned between the nucleic acid sequence encoding an oil-body-targeting-protein and the nucleic acid sequence encoding a first recombinant polypeptide can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said sequence encoding the first recombinant polypeptide.",
"Also provided herein are isolated oil bodies comprising a multimeric-protein-complex comprising (i) an oil-body-targeting-protein and (ii) a first recombinant polypeptide, said oil bodies further comprising a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.",
"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are isolated oil bodies comprising (a) a first fusion protein comprising a first oil-body-targeting-protein fused to a first recombinant polypeptide; and (b) a second fusion protein comprising a second oil-body-targeting-protein fused to a second recombinant polypeptide, wherein said first and second recombinant polypeptide are capable of forming a multimeric-protein-complex.",
"The oil-body-targeting-protein can be selected from an oil-body-protein or an immunoglobulin, and the oil-body-protein can be an oleosin or a caleosin.",
"The multimeric-fusion-protein can be a heteromultimeric-fusion-protein, wherein said first and second recombinant polypeptide form an enzymatically active heteromultimeric redox complex or an immunoglobulin.",
"In a particular embodiment, the first recombinant polypeptide is a thioredoxin and the second recombinant polypeptide is a thioredoxin-reductase.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194; and the thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.In another embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are cells and transgenic plants comprising oil bodies, multimeric-protein-complexes, and multimeric-fusion-proteins, set forth herein.",
"In one embodiment, the first recombinant polypeptide can be an immunoglobulin-polypeptide-chain.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. In embodiments, wherein said first recombinant polypeptide is a thioredoxin and said second recombinant polypeptide is a thioredoxin-reductase, the methods described herein can be used to formulate the oil bodies for use in the preparation of a food product, personal care product or pharmaceutical composition.",
"These formulations can further comprise the addition of NADP or NADPH.",
"The food product can be a milk or wheat based food product.",
"The personal care product can reduce the oxidative stress to the surface area of the human body or can be used to lighten the skin.",
"The pharmaceutical composition can be used to treat chronic obstructive pulmonary disease (COPD), cataracts, diabetes, envenomation, bronchiopulmonary disease, malignancies, psoriasis, reperfusion injury, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, GERD (gastro esophageal reflux disease).",
"Also provided herein are compositions comprising isolated oil bodies, thioredoxin and thioredoxin-reductase, wherein said thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194, and said thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313.The composition can further comprise NADP or NADPH.",
"In another embodiment, the composition comprises a first recombinant polypeptide that can be an immunoglobulin-polypeptide-chain and a second recombinant polypeptide.",
"For example, the first recombinant polypeptide can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second recombinant polypeptide can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided are multimeric-fusion-proteins, wherein the fusion-protein contains two or more polypeptide chains selected from the group of proteins set forth in FIG.",
"5.Methods are also provided of reducing allergenicity of a food comprising the steps of providing the isolated oil bodies set forth herein; and adding the isolated oil bodies to the food, whereby allergenicity of the food is reduced.",
"The food can be selected from the group consisting of wheat flour, wheat dough, milk, cheese, yogurt and ice cream.",
"The various methods of treating food can further comprise providing NADH as a co-factor in the substantial absence of NADPH.",
"Also provided herein are methods of treating or protecting a target against oxidative stress, comprising the steps of providing the recombinant redox fusion polypeptide comprising thioredoxin and thioredoxin-reductase; and contacting the recombinant fusion polypeptide with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.",
"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.",
"Also provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.",
"The first redox protein can be a thioredoxin and the second redox protein can be a thioredoxin-reductase.",
"Also, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.",
"For example, the first immunoglobulin-polypeptide-chain can be an immunoglobulin light chain, or an immunologically active portion thereof, and the second immunoglobulin-polypeptide-chain can be an immunoglobulin heavy chain, or an immunologically active portion thereof.",
"In this embodiment, the oil-body-targeting-protein can comprise protein A, protein L or protein G. Also provided herein are methods for preparing a redox protein or an immunoglobulin associated with oil bodies comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein, or a nucleic acid sequence encoding a immunoglobulin comprising a first immunoglobulin-polypeptide-chain linked to a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide or immunoglobulin in a progeny cell comprising oil bodies; and c) isolating from said progeny cell said oil bodies comprising said redox fusion polypeptide or immunoglobulin.",
"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide or immunoglobulin can be a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged or have a molecular weight of at least 35 kd.",
"Optionally, the gene fusion further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.",
"In this optional embodiment, also contemplated is the introduction of an enzyme or chemical that cleaves said redox fusion polypeptide from said oil body, thereby obtaining isolated redox fusion polypeptide.",
"The first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.",
"In one embodiment, the thioredoxin and thioredoxin-reductase can be obtained from Arabidopsis.",
"In another embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide as compared to the production of the first redox protein without the second redox protein.",
"Also provided herein, for use with the various methods set forth herein is the formulation of an emulsion of the oil bodies associated with the redox fusion polypeptide for use in the preparation of a product capable of treating oxidative stress in a target, a product capable of chemically reducing a target, pharmaceutical composition, a personal care product or a food product.",
"Accordingly, an emulsion formulation composition is provided.",
"Also provided herein is a chimeric nucleic acid comprising: 1) a first nucleic acid sequence capable of regulating transcription in a host cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked to (ii) a nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.",
"The oil-body-protein can be an oleosin or a caleosin, the first redox protein can be a thioredoxin and said second redox protein can be a thioredoxin-reductase.",
"In certain embodiments, positioned between said nucleic acid sequence encoding a sufficient portion of an oil-body-protein and said nucleic acid sequence encoding a redox fusion polypeptide is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"In one embodiment, the gene fusion optionally further comprises a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and said nucleic acid sequence encoding a redox fusion polypeptide.",
"Also provided herein are transgenic plants, e.g., safflower plants, comprising any of the chimeric nucleic acid sequences and constructs described herein.",
"The chimeric nucleic acids can be contained within a plastid.",
"Accordingly, isolated plastids are provided having chimeric nucleic acids therein.",
"Also provided are plant seeds comprising the chimeric nucleic acids provided herein.",
"Also provided are oil body preparations obtained using any of the methods provided herein, and food products, pharmaceutical compositions, and personal care products containing the oil body preparations.",
"The products and/or compositions provided herein are capable of treating oxidative stress in a target, capable of chemically reducing a target.",
"Also provided is a detergent composition comprising an oil body preparation capable of chemically reducing a target, and related methods of cleansing an item, comprising administering such product to the item under conditions that promote cleansing.",
"Also provided herein are nucleic acid constructs comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one thioredoxin-related protein or an active fragment thereof.",
"In one embodiment, the at least one thioredoxin-related protein can be thioredoxin.",
"The thioredoxin can be selected from the group consisting of SEQ ID NOs:38, 42, 46, 50 and SEQ ID NOs:52-194.The thioredoxin can be obtained from Arabidopsis or wheat.",
"In another embodiment, the at least one thioredoxin-related protein can be thioredoxin-reductase.",
"The thioredoxin-reductase can be selected from the group consisting of those set forth in SEQ ID NOs:8, 9, 10, 40, 44, 48, 50 and SEQ ID NOs:195-313 and/or derived from Arabidopsis or wheat.",
"The thioredoxin-reductase can be an NADPH-dependent thioredoxin-reductase.",
"The second region can encode a thioredoxin and thioredoxin-reductase.",
"In one embodiment, the thioredoxin and thioredoxin-reductase is obtained from Mycobacterium leprae.",
"In another embodiment, the at least one thioredoxin-related protein can be an engineered fusion protein.",
"The first region can precede, in a 5′ to 3′ direction, the second region.",
"Alternatively, the first region follows, in a 5′ to 3′ direction, the second region.",
"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.",
"A seed-specific promoter, such as a phaseolin promoter, can be operably linked to the gene fusion.",
"In one embodiment, at least one thioredoxin-related protein is derived from a plant species selected from the group consisting of Arabidopsis and wheat.",
"In another embodiment, at least one thioredoxin-related protein can be derived from E. coli.",
"In one embodiment, the gene fusion further comprises a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the linker amino acid sequence is positioned between the first region and the second region.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"In addition, the gene fusion can further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the second region.",
"Also provided herein are transgenic plants containing a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof.",
"The thioredoxin-related protein can be thioredoxin.",
"The nucleic acid construct can be contained within a plastid.",
"In one embodiment, when the first thioredoxin-related protein is thioredoxin and the construct can further comprise a region encoding a thioredoxin-reductase.",
"The gene fusion can optionally further comprise a third region encoding a second thioredoxin-related protein or an active fragment thereof, operably linked to the first region, or to the second region, or to both.",
"The gene fusion can optionally further comprise a nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence, wherein the nucleic acid encoding the linker amino acid sequence is positioned between the region encoding an oil-body-protein and the region encoding a first thioredoxin-related protein.",
"The oil-body-surface-avoiding linker amino acid sequence can be substantially negatively charged, or have a molecular weight of at least 35 kd.",
"The gene fusion can optionally further comprise a linker nucleic acid sequence encoding an amino acid sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the oil-body-surface-avoiding linker amino acid sequence and the region encoding a first thioredoxin-related protein.",
"Also provided is a transgenic plant comprising a nucleic acid construct, a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, wherein a fusion protein comprising activities of oleosin and the thioredoxin-related protein is produced in a seed of the plant.",
"In another embodiment, a thioredoxin-related protein having concentration of at least about 0.5% of total cellular seed protein is provided.",
"Also provided herein is an extract comprising an activity of a thioredoxin-related protein.",
"Also provided are oil bodies and/or oil obtained from various seeds.",
"Also provided herein are methods of making a fusion protein comprising a thioredoxin-related activity, the method comprising the steps of: a) providing a transgenic plant comprising a nucleic acid construct comprising a seed-specific promoter operably linked to a gene fusion, wherein the gene fusion comprises a region encoding an oil-body-protein or an active fragment thereof, operably linked to a region encoding a first thioredoxin-related protein or an active fragment thereof, the gene fusion encoding a fusion protein comprising a thioredoxin-related activity; b) obtaining seeds from the plant; and c) recovering the fusion protein by isolating oil bodies from the seeds.",
"In one embodiment, the oil bodies are fractionated to achieve partial purification of the fusion protein.",
"The oil bodies can be in association with a fusion protein.",
"The oil-body-protein can be cleaved from the thioredoxin-related protein after fractionation of the oil bodies.",
"The cleaving step can make use of a protease or chemical proteolysis.",
"Also provided herein are methods of reducing allergenicity of a food comprising the steps of: a) providing a preparation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) adding the preparation to the food, whereby allergenicity of the food is reduced due to activity of the thioredoxin-related protein or fragment.",
"The food can be wheat flour, wheat dough, milk, cheese, yogurt and ice cream.",
"In one embodiment, NADH is used as a co-factor in the substantial absence of NADPH.",
"Also provided herein are pharmaceutical compositions comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.",
"The oil bodies can be associated with the fusion protein.",
"Also provided is a cosmetic formulation comprising oil bodies associated with a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof, in a pharmaceutically acceptable carrier.",
"Also provided are methods of treating or protecting a target against oxidative stress, comprising the steps of: a) providing a preparation comprising a fusion protein, the fusion protein comprising an oil-body-protein or an active fragment thereof and a thioredoxin-related protein or an active fragment thereof; and b) contacting the preparation with a target, wherein the target is susceptible to oxidative stress, thereby treating or protecting against the stress.",
"The target can be selected from the group consisting of a molecule, a molecular complex, a cell, a tissue, and an organ.",
"Also provided is a nucleic acid construct comprising a gene fusion, wherein the gene fusion comprises a first region encoding an oil-body-protein or an active fragment thereof, operably linked to a second region encoding at least one polypeptide or an active fragment thereof, and an oil-body-surface-avoiding linker in frame between the first and second region polypeptides.",
"Also provided are methods of expressing this construct into the encoded amino acid sequence; and oil bodies, formulations, emulsions, cells, and plants comprising the construct and encoded amino acid sequence.",
"These particular constructs, oil bodies, formulations, emulsions, cells, and plants can be produced according to the methods described herein.",
"The second region can encode any polypeptide, for example, a therapeutically, nutritionally, industrially or cosmetically useful peptide as set forth herein.",
"For example, the second region can encode a redox protein, an immunoglobulin, a thioredoxin-related protein or any one or more recombinant polypeptides of a multimeric-protein-complex.",
"Other features and advantages of the present invention will become readily apparent from the following detailed description.",
"It should be understood however that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of illustration only.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a ClustalW Formatted Alignment comparison of the published NADPH thioredoxin-reductase nucleic acid sequence (SEQ ID NO:9) (ATTHIREDB-Jacquot et al.",
"J. Mol.",
"Biol.",
"(1994) 235 (4):1357-63.)",
"with the sequence isolated herein in Example 1 (TR; SEQ ID NO:8).",
"FIG.",
"2 shows a ClustalW Formatted Alignment comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence (SEQ ID NO:12) (ATTHIREDB Jacquot et al.",
"J. Mol.",
"Biol.",
"(1994) 235 (4):1357-63.)",
"with the sequence isolated herein in Example 1 (TR; SEQ ID NO:13).",
"FIG.",
"3 shows a clustal alignment comparing the amino acid sequence of the Arabidopsis thaliana thioredoxin-reductase-linker-thioredoxin synthetic fusion (Arab TR-link-Trxh; SEQ ID NO:37) to the Mycobacterium leprae thioredoxin-reductase-thioredoxin natural fusion (M. lep TR/Trxh; SEQ ID NO:36) natural fusion.",
"Overall, the proteins are approximately 50% identical at the amino acid level.",
"FIG.",
"4 is a bar graph showing the thioredoxin/thioredoxin-reductase activity measurements for the various transgenic Arabidopsis seed fractions.",
"Relative specific activity is expressed as a percentage of the E. coli thioredoxin and thioredoxin-reductase activities.",
"The numbered bars in the graph correspond to the following: 1.W.T.+oleosin-thioredoxin 2.W.T.+thioredoxin-oleosin 3.W.T.+thioredoxin 4.W.T.+oleosin-thioredoxin-reductase 5.W.T.+thioredoxin-reductase-oleosin 6.W.T.+thioredoxin-reductase 7.thioredoxin+oleosin-thioredoxin-reductase 8.thioredoxin+thioredoxin-reductase-oleosin 9.thioredoxin+thioredoxin-reductase 10.thioredoxin-reductase+oleosin-thioredoxin 11.thioredoxin-reductase+thioredoxin-oleosin 12.oleosin-M. lep TR/Trxh 13.E.",
"coli thioredoxin-reductase+thioredoxin FIG.",
"5 provides a listing of exemplary proteins for use in the heteromultimeric-fusion-proteins and heteromultimeric-protein-complexes provided herein.",
"DETAILED DESCRIPTION As hereinbefore mentioned, the present invention relates to novel and improved methods for the production of multimeric proteins, including a first and second recombinant polypeptide, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulin-polypeptide-chains, immunoglobulins, redox-fusion-polypeptides, and a first and second thioredoxin-related protein; and related products.",
"These methods permit the production of active multimeric-protein-complexes in association with oil bodies.",
"The oil bodies in association with the multimeric-protein-complex may be used to prepare various useful emulsions.",
"Accordingly, provided herein are methods of producing a recombinant multimeric-protein-complex associated with an oil body, said method comprising: (a) producing in a cell comprising oil bodies, a first recombinant polypeptide and a second recombinant polypeptide wherein said first recombinant polypeptide is capable of associating with said second recombinant polypeptide in the cell to form said multimeric-protein-complex; and (b) associating said multimeric-protein-complex with an oil body through an oil-body-targeting-protein capable of associating with said oil body and said first recombinant polypeptide.",
"DEFINITIONS AND TERMS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.",
"Where permitted, all patents, applications, published applications and other publications and sequences from GenBank, SwissPro and other data bases referred to throughout in the disclosure herein are incorporated by reference in their entirety.",
"As used herein, the phrase “multimeric-protein-complex”, refers to two or more polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.",
"It should be noted that the polypeptides may be independently biologically active without interaction or coordination to form the complex.",
"The multimeric-protein-complex may provide a biological structure, or it may be capable of facilitating a chemical or biological reaction.",
"For example, one of the protein regions within the multimeric-protein-complex can repeatedly activate or repeatedly inactivate the biological or metabolic activity of one or more of the other proteins contained within the multimeric-protein-complex.",
"In one embodiment, the first and second recombinant polypeptide contained in a multimeric-protein-complex may either associate or interact as independent non-contiguous polypeptide chains or the multimeric-protein-complex may be prepared as a fusion polypeptide (multimeric-fusion-protein) between the first and second recombinant polypeptide.",
"One example of a repeated (e.g., reoccurring) interaction or association between the two or more polypeptides of a multimeric-protein-complex provided herein is the interaction between two or more non-identical redox proteins to form a heteromultimeric-protein-complex.",
"Exemplary redox proteins for use in this regard are thioredoxin and the thioredoxin-reductase.",
"A further example is the interaction between two or more immunoglobulin-polypeptide-chains to form an immunoglobulin.",
"As used herein, the phrase “heteromultimeric-protein-complex”, refers to two or more non-identical polypeptide chains that permanently or repeatedly interact or permanently or repeatedly coordinate to form a biologically active assembly comprising said two or more polypeptide chains.",
"Other examples of multimeric-protein-complexes provided herein include a first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, first and second immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and second thioredoxin-related protein.",
"The recombinant polypeptide or multimeric-protein-complex is associated with an oil body.",
"As used herein, the phrase “oil body” or “oil bodies” refers to any oil or fat storage organelle in any cell type.",
"Accordingly, the oil bodies may be obtained from any cell comprising oil bodies, including plant cells (described in for example: Huang (1992) Ann.",
"Rev.",
"Plant Mol.",
"Biol.",
"43: 177-200), animal cells (described in for example: Murphy (1990) Prog Lipid Res 29(4): 299-324), including adipocytes, hepatocytes, steroidogenic cells, mammary epithelial cells, macrophages, algae cells (described in for example: Rossler (1988) J. Physiol.",
"London, 24: 394-400) fungal cells, including yeast cells (described in for example Leber et al.",
"(1994) Yeast 10: 1421-1428) and bacterial cells (described in for example: Pieper-Furst et al.",
"(1994) J. Bacteriol.",
"176: 4328-4337).",
"Preferably the oil bodies used herein are oil bodies obtainable from plant cells and more preferably the oil bodies obtainable from plant seed cells.",
"As used herein, the phrase “is capable of associating with”, “associate” or grammatical variations thereof, refers to any interaction between two or more polypeptides, including any covalent interactions (e.g.",
"multimeric-fusion-proteins) as well as non-covalent interactions.",
"Exemplary non-covalent interactions can be between the oil-body-targeting-protein and a redox protein or immunoglobulin-polypeptide-chain, as well as between two or more different proteins contained within two or more separate oil-body-protein fusion proteins (e.g., the redox proteins in oleosin-thioredoxin and oleosin-thioredoxin-reductase).",
"As used herein, the term “recombinant” (also referred to as heterologous) in the context of recombinant proteins and amino acids, means “of different natural origin” or represents a non-natural state.",
"For example, if a host cell is transformed with a nucleotide sequence derived from another organism, particularly from another species, that nucleotide sequence and amino acid sequence encoded thereby, is recombinant (heterologous) with respect to that host cell and also with respect to descendants of the host cell which carry that gene.",
"Similarly, recombinant (or heterologous) refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.",
"A transforming nucleotide sequence may include a recombinant coding sequence, or recombinant regulatory elements.",
"Alternatively, the transforming nucleotide sequence may be completely heterologous or may include any possible combination of heterologous and endogenous nucleic acid sequences.",
"In various embodiments of the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, are produced in a cell comprising oil bodies.",
"As used herein the phrase “in a cell”, “in the cell”, or grammatical variations thereof, mean that the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, may be produced in any cellular compartment of that cell, so long as that cell comprises oil bodies therein.",
"In embodiments of the invention in which plant cells are used, the phrase is intended to include the plant apoplast.",
"In various embodiments provided herein, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, associate with an oil body through an oil-body-targeting-protein.",
"As used herein, the phrase “oil-body-targeting-protein” refers to any protein, protein fragment or peptide capable of associating with an oil body.",
"Exemplary oil-body-targeting-proteins for use herein include oil-body-proteins, such as oleosin and caleosin; immunoglobulins, such as bi-specific antibodies; and the like.",
"In embodiments described herein in which an oil-body-protein is used, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and thioredoxin-related proteins, are preferably fused to the oil-body-protein.",
"The term “oil-body-protein” refers to any protein naturally present in cells and having the capability of association with oil bodies, including any oleosin or caleosin.",
"Accordingly, provided herein a method of expressing a recombinant multimeric-protein-complex comprising a first and second recombinant polypeptide in a cell, said method comprising: (a) introducing into a cell a first chimeric nucleic acid sequence comprising: (i) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a second nucleic acid sequence encoding a first recombinant polypeptide, such as a redox protein, an immunoglobulin-polypeptide-chain or an thioredoxin-related protein, fused to an oil-body-protein; (b) introducing into said cell a second chimeric nucleic acid sequence comprising: (i) a third nucleic acid sequence capable of regulating transcription in said cell operatively linked to; (ii) a fourth nucleic acid sequence encoding a second recombinant polypeptide, such as a second redox protein, a second immunoglobulin-polypeptide-chain or a second thioredoxin-related protein; (c) growing said cell under conditions to permit expression of said first and second recombinant polypeptide in a progeny cell comprising oil bodies wherein said first recombinant polypeptide and said second recombinant polypeptide are capable of forming a multimeric-protein-complex, preferably in said progeny cell; and (d) associating said first recombinant polypeptide with an oil body through said oil-body-protein.",
"The term “nucleic acid” as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.",
"The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.",
"The nucleic acid sequences may be ribonucleic acids (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.",
"The sequences also may contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo-uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol-adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8 thiol guanine, 8-thioalkyl guanines, 8 hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.",
"Multimeric-Protein-Complexes In accordance with the methods and compositions provided herein, any two recombinant polypeptides capable of forming a multimeric-protein-complex may be used.",
"The nucleic acid sequences encoding the two recombinant polypeptides may be obtained from any biological source or may be prepared synthetically.",
"In general nucleic acid sequence encoding multimeric proteins are known to the art and readily available.",
"Known nucleic acid sequences encoding multimeric-protein-complexes may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences encoding multimeric-protein-complexes, for example, by screening cDNA or genomic libraries or using 2- or multi-hybrid systems.",
"Thus, additional nucleic acid sequences encoding multimeric-protein-complexes may be discovered and used as described herein.",
"The first and/or second recombinant polypeptides that are comprised within a multimeric-protein-complex provided herein, can themselves be in the form of heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein.",
"The nucleic acid sequence encoding the first and second recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein may be obtained from separate sources or may be obtained from the same source.",
"In general however, such nucleic acid sequence is obtained from the same or a similar biological source.",
"In certain embodiments wherein the nucleic acid sequence encoding the first and second recombinant polypeptide protein are obtained from the same source, the nucleic acid sequence encoding the first recombinant polypeptide and second recombinant polypeptide may be naturally fused.",
"In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second recombinant polypeptide are obtained from a plant source.",
"Oil-Body-Surface-Avoiding Linkers Polypeptide spacers or linkers of variable length and/or negative charge can be used herein to separate the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins from the in-frame oil-body-targeting-protein, to improve activity of and/or the accessibility of the polypeptide or complex.",
"For example, in one embodiment set forth herein, positioned between a nucleic acid sequence encoding a sufficient portion of an oil-body-protein and a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and the first and/or second thioredoxin-related proteins; is a linker nucleic acid sequence encoding an oil-body-surface-avoiding linker amino acid sequence.",
"Oil-body-surface-avoiding linkers are positioned between the oil-body targeting sequence and an in-frame recombinant polypeptide of interest, e.g., the multimeric-protein-complexes provided herein, serve to increase the distance and or decrease the interaction between the negatively charged oil body surface and the recombinant polypeptide of interest.",
"A negatively charged linker is repelled by the negatively charged oil body surface, in turn increasing the distance or decreasing the interaction of its attached recombinant polypeptide with the oil body surface.",
"As a consequence of the increased distance from the oil body surface, the recombinant polypeptide will be more accessible, e.g.",
"to its target(s) substrate, protein substrate, protein partner, and less affected by the charged oil body surface.",
"Exemplary linker sequences for use herein can be either a negatively charged linker, or a linker having a molecular weight of at least about 35 kd or more.",
"As used herein, a “negatively charged linker” sequence, refers to any amino acid segment, or nucleic acid encoding such, that has a pI less than or equal to the pI of an oil body.",
"In certain embodiments, the pI of the negatively charged linker is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, down to about 25% or more, below that of the pI of an oil body in the particular plant or cell system being used.",
"Exemplary negatively charged linkers can be prepared comprising any combination of the negatively charged amino acid residues.",
"For example, in one embodiment, a negatively charged linker comprises either a poly-glutamate or poly-aspartate sequence, or any combination of both amino acid residues.",
"The negatively charged linker is typically at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.",
"The negatively charged linkers are preferably non-proteolytic (e.g., non-proteolytic linkers), having no site for efficient proteolysis.",
"When linker size rather than charge is used to minimize interaction of the recombinant polypeptide of interest with the oil body surface, then the linker is non-proteolytic and ranges in molecular weight from about 35 kd up to about 100 kd.",
"The upper size limit is chosen such that the expression of, the activity of, the conformation of, and/or the access to target of, the recombinant polypeptide of interest is not significantly affected by the linker.",
"In certain embodiments, described herein where a non-proteolytic linker amino acid sequence is employed, the gene-fusion or protein fusion (multimeric-fusion-protein) can optionally further comprise a linker nucleic or amino acid sequence encoding a sequence that is specifically cleavable by an enzyme or a chemical, wherein the linker sequence is positioned between the non-proteolytic linker sequence and sequence encoding the desired recombinant protein region, e.g., the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins set forth herein.",
"When a cleavable linker sequence is used herein, in a particular embodiment, it is further downstream than the non-proteolytic linker sequence from the oil-body-targeting-protein region of the fusion protein.",
"By virtue of cleavable linker, the recombinant fusion polypeptides provided herein, such as the multimeric-fusion-proteins and redox fusion polypeptides, can be isolated and purified by introducing an enzyme or chemical that cleaves said multimeric-fusion-protein and/or redox fusion polypeptide from said oil body, thereby obtaining and/or isolating the desired protein.",
"It is contemplated herein that the use of cleavable linker sequence downstream of the non-proteolytic linker/spacer sequence will improve the yield of protein recovery when isolating or purifying proteins using the methods provided herein.",
"The nucleic acid sequences encoding the first or second recombinant polypeptide may be altered to improve expression levels for example, by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the first and second recombinant polypeptide, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).",
"Comparison of the codon usage of the first and second recombinant polypeptide with codon usage of the host will enable the identification of codons that may be changed.",
"For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.",
"By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.",
"Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The first and second recombinant polypeptide can be altered using for example targeted mutagenesis, random mutagenesis (Shiraishi et al.",
"(1998) Arch.",
"Biochem.",
"Biophys.",
"358: 104-115; Galkin et al.",
"(1997) Protein Eng.",
"10: 687-690; Carugo et al.",
"(1997) Proteins 28: 10-28; Hurley et al.",
"(1996) Biochemistry 35: 5670-5678), gene shuffling, and/or by the addition of organic solvent (Holmberg et al.",
"(1999) Protein Eng.",
"12: 851-856).",
"Any polypeptide spacers that are used in accordance with the methods and products provided herein may be altered in similar ways.",
"In particular embodiments provided herein, the recombinant polypeptides or thioredoxin-related proteins capable of forming a multimeric-protein-complex are capable of forming a heteromultimeric-protein-complex.",
"Examples of heteromultimeric-protein-complexes that contain polypeptide chains that repeatedly interact, either to activate, inactivate, oxidize, reduce, stabilize, etc., with one another, that can be produced in association with oil bodies using the methods provided herein include those set forth in FIG.",
"5.Accordingly, exemplary proteins for use in the heteromultimeric-protein-complexes and nucleic acid constructs encoding such, provided herein include, among others described herein, those set forth in FIG.",
"5.Other polypeptide regions that can be used in the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, provided herein include, among other, those immunoglobulin regions set forth in Table 1.TABLE 1 ANTIBODY HETERODIMERS Class or molecule Subunits Fab Variable region and first constant region of heavy chain and complete light chain Fv Variable regions of heavy and light antibody chains IgA heavy chains, light chains and J (joining) chain IgG, IgD, IgE heavy and light chains IgM heavy chains, light chains and J (joining) chain Antibody chain(s) and a toxin Antibody chain(s) and a toxin Autoantigens, allergens and Autoantigens, allergens and transplant transplant antigens with an antigens with an adjuvant or tolerogen adjuvant or tolerogen Chimeras using antibody Fc Receptor subunits fused to the constant domain region of antibody heavy chains As set forth above, in one embodiment, exemplary heteromultimeric-protein-complexes and exemplary heteromultimeric-fusion-proteins provided herein comprise redox proteins, such as the thioredoxins and thioredoxin-reductases and immunoglobulins.",
"Oil-Body-Targeting-Proteins The nucleic acid sequence encoding the oil-body-targeting-protein that may be used in the methods and compositions provided herein may be any nucleic acid sequence encoding an oil-body-targeting-protein, protein fragment or peptide capable of association with first recombinant polypeptide, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or a first and/or second thioredoxin-related protein and the oil bodies.",
"The nucleic acid sequence encoding the oil body targeting peptide may be synthesized or obtained from any biological source.",
"For example, in one embodiment the oil-body-targeting-protein is an immunoglobulin or an immunoglobulin derived molecule, for example, a bispecific single chain antibody.",
"The generation of single chain antibodies and bi-specific single chain antibodies is known to the art (see, e.g., U.S. Pat.",
"No.",
"5,763,733, U.S. Pat.",
"No.",
"5,767,260 and U.S. Pat.",
"No.",
"5,260,203).",
"Nucleic acid sequences encoding single chain antibodies functioning as oil-body-targeting-proteins may be prepared from hybridoma cell lines expressing monoclonal antibodies raised against an oleosin as described by Alting-Mees et al (2000) IBC's Annual International Conference on Antibody Engineering, Poster #1.In order to attain specificity for the first recombinant polypeptide a nucleic acid sequence encoding a second single chain antibody prepared from a monoclonal raised against the first recombinant polypeptide may be prepared and linked to the anti-oleosin single chain antibody.",
"In this embodiment the oil body associates with the first recombinant polypeptide through non-covalent interactions of the oil-body-targeting-protein with the first recombinant polypeptide and the oil body.",
"Alternatively the first recombinant polypeptide may be prepared as a fusion protein with an oil-body-targeting-protein.",
"For example, a nucleic acid sequence encoding a single chain antibody raised against an oleosin may be fused to a nucleic acid sequence encoding the first recombinant polypeptide Non-immunoglobulin-based oil-body-targeting-proteins capable of association with the first recombinant polypeptide may be discovered and prepared using for example phage display techniques (Pharmacia Biotech Catalogue Number 27-9401-011 Recombinant Phage Antibody System Expression Kit).",
"Oil-body-targeting-proteins may also be chemically modified.",
"For example, oleosins may be modified by changing chemical modification of the lysine residues using chemical agents such as biotinyl-N-hydroxysuccinimide ester resulting in a process referred to as biotinylation.",
"Conveniently this is accomplished by in vitro biotinylation of the oil bodies.",
"In vivo biotinylation may be accomplished using the biotinylation domain peptide from the biotin carboxy carrier protein of E. coli acetyl-CoA carboxylase (Smith et al.",
"(1998) Nucl.",
"Acids.",
"Res.",
"26: 1414-1420).",
"Avidin or streptavidin may subsequently be used to accomplish association of the redox protein with the oil body.",
"In a particular embodiment the oil-body-targeting-protein is an oil-body-protein such as for example an oleosin or a caleosin or a sufficient portion derived thereof capable of targeting to an oil body.",
"Nucleic acid sequences encoding oleosins are known to the art.",
"These include for example the Arabidopsis oleosin (van Rooijen et al (1991) Plant Mol.",
"Bio.",
"18:1177-1179); the maize oleosin (Qu and Huang (1990) J. Biol.",
"Chem.",
"Vol.",
"265 4:2238-2243); rapeseed oleosin (Lee and Huang (1991) Plant Physiol.",
"96:1395-1397); and the carrot oleosin (Hatzopoulos et al (1990) Plant Cell Vol.",
"2, 457-467.).",
"Caleosin nucleic acid sequences are also known to the art (Naested et al (2000) Plant Mol.",
"Biol.",
"44(4):463-476; Chen et al (1999) Plant Cell Physiol.",
"40(10):1079-1086).",
"Animal cell derived oil body proteins that may be used herein include adopihilin (Brasaemle et al, (1997) J. Lipid Res., 38: 2249-2263; Heid et al.",
"(1998) Cell Tissue Research 294: 309-321), perilipin (Blanchette-Mackie et al.",
"(1995), J. Lipid Res.",
"36: 1211-1226; Servetnick et al.",
"(1995) J. Biol.",
"Chem.",
"270: 16970-16973), apolipoproteins such as apo A-I, A-II, A-IV, C-I, C-II, CIII (Segrest et al.",
"(1990), Proteins 8:103-117) and apoB (Chatterton et al.",
"(1995) J. Lipid Res.",
"36: 2027-2037; Davis, R A in: Vance D E, Vance J. editors.",
"Lipoprotein structure and secretion.",
"The Netherlands, Elsevier, 191: 403-426.In one embodiment, the first recombinant polypeptide is fused to an oil-body-protein.",
"The methodology is further described in U.S. Pat.",
"No.",
"5,650,554, which is incorporated herein by reference in its entirety.",
"The first recombinant polypeptide may be fused to the N-terminus as well as to the C-terminus of the oil-body-protein (as described in: Moloney and van Rooijen (1996) INFORM 7:107-113) and fragments of the oil-body-protein such as for example the central domain of an oleosin molecule, or modified versions of the oil-body-protein may be used.",
"In this embodiment, the second recombinant polypeptide is expressed intracellularly and then intracellularly associates with the first recombinant polypeptide to form the multimeric-protein-complex in the cell.",
"Oil bodies comprising the multimeric-protein-complex are then conveniently isolated from the cells.",
"In a further embodiment both the first and second recombinant polypeptide are separately fused to an oil-body-protein.",
"In this embodiment nucleic acid sequences encoding the first and second polypeptides may be prepared separately and introduced in separate cell lines or they may be introduced in the same cell lines.",
"Where the nucleic acid sequences are introduced in the same cell line, these nucleic acid sequence may be prepared using two separate expression vectors, or they may be prepared using a single vector comprising nucleic acid sequences encoding both the first polypeptide fused to an oil body protein and the second polypeptide fused to an oil-body-protein.",
"Where separate cell lines are used subsequent mating of the offspring (e.g.",
"mating of plants) is used to prepare a generation of cells comprising oil bodies which comprise both the first and second recombinant polypeptide fused to an oil-body-protein.",
"In further alternate embodiment, the first and second recombinant polypeptide are fused to form a multimeric-fusion-protein comprising the multimeric-protein-complex.",
"In such an embodiment, the first and second polypeptide is associated with the oil body through an oil-body-targeting-protein capable of associating with both the fusion protein and with the oil body.",
"In a particular embodiment, the fusion protein comprising the multimeric-protein-complex is fused to an oil-body-protein, for example, an oleosin or caleosin.",
"In embodiments provided herein in which the multimeric-protein-complex is an immunoglobulin (e.g., a multimeric-immunoglobulin-complex), a particularly preferred oil body targeting protein is an oleosin or caleosin associated with an immunoglobulin binding protein, such as for example protein A (U.S. Pat.",
"No.",
"5,151,350), protein L (U.S. Pat.",
"No.",
"5,965,390) and protein G (U.S. Pat.",
"No.",
"4,954,618), or active fragments of such immunoglobulin binding proteins.",
"New oil-body-proteins may be discovered for example by preparing oil bodies (described in further detail below) and identifying proteins in these preparations using for example SDS gel electrophoresis.",
"Polyclonal antibodies may be raised against these proteins and used to screen cDNA libraries in order to identify nucleic acid sequences encoding oil-body-proteins.",
"The methodologies are familiar to the skilled artisan (Huynh et al.",
"(1985) in DNA Cloning Vol.",
"1.a Practical Approach ed.",
"D M Glover, IRL Press, pp 49-78).",
"New oil-body-proteins may further be discovered using known nucleic acid sequences encoding oil-body-proteins (e.g.",
"the Arabidopsis, rapeseed, carrot and corn nucleic acid sequences) to probe for example cDNA and genomic libraries for the presence of nucleic acid sequences encoding oil-body-proteins.",
"In one embodiment, the first and second polypeptide are a first and second redox protein.",
"Accordingly, one embodiment provided herein relates to novel and improved methods for the production of redox proteins.",
"It has unexpectedly been found that a redox protein when prepared as a fusion protein with a second redox protein is fully enzymatically active when produced in association with an oil body.",
"In contrast, when the redox protein is prepared without the second redox protein it has reduced enzymatic activity.",
"In one embodiment, the first redox protein is at least 5 times more active when produced as a redox fusion polypeptide relative to production as a non-fusion polypeptide.",
"Accordingly, provided herein are methods for producing an oil body associated with a heteromultimeric redox protein complex, said method comprising: (a) producing in a cell comprising oil bodies, a first redox protein and a second redox protein wherein said first redox protein is capable of interacting with said second redox protein, preferably in the cell, to form said heteromultimeric redox protein complex; and (b) associating said heteromultimeric redox protein complex with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said heteromultimeric redox protein complex.",
"In a particular embodiment the first and second redox protein are prepared as a fusion protein to form a redox fusion polypeptide.",
"Accordingly, provided herein are methods for preparing an enzymatically active redox protein associated with oil bodies comprising: a) producing in a cell a redox fusion polypeptide comprising a first redox protein linked to a second redox protein; b) associating said redox fusion polypeptide with oil bodies through an oil-body-targeting-protein capable of associating with said redox fusion polypeptide and said oil bodies; and c) isolating said oil bodies associated with said redox fusion polypeptide.",
"The oil bodies in association with the redox protein may be used to prepare a variety of useful emulsions.",
"As used herein the phrase “redox proteins” or grammatical variations thereof, refers to any protein or active protein fragment capable of participating in electron transport.",
"For example, redox proteins are capable of catalyzing the transfer of an electron from an electron donor (also frequently referred to as the reducing agent) to an electron acceptor (also frequently referred to as the oxidizing agent).",
"In the process of electron transfer, the reducing agent (electron donor) is oxidized and the oxidizing agent (electron acceptor) is reduced.",
"Exemplary redox proteins for use herein include iron-sulfur proteins, cytochromes, redox active thiol proteins and redox-active flavoproteins.",
"To carry out their function as conduits for electrons, redox proteins, such as thioredoxin and thioredoxin-reductase for example, are known to function by interacting or associating with one another in multimeric-protein-complexes (e.g., heteromultimeric-protein-complexes).",
"The term “redox fusion polypeptide” as used herein refers to any fusion polypeptide comprising a first redox protein linked to a second redox protein (e.g., an in-frame translational fusion).",
"The redox proteins that may be used with the methods and compositions provided herein may be any redox protein.",
"In one embodiment the first and second redox proteins are a pair of redox proteins that would normally occur together from the same source, in nature.",
"In a particular embodiment, the first redox protein is a thioredoxin and the second redox protein is a thioredoxin-reductase.",
"The redox fusion polypeptide may be produced in any cell comprising oil bodies, including any animal cell, plant cell, algae cell, fungal cell or bacterial cell.",
"In certain embodiments the redox fusion polypeptide is produced in a plant cell and in particular embodiments the redox fusion polypeptide is produced in the seed cells of a seed plant.",
"In particular embodiments the oil-body-targeting-protein that is used is an oil-body-protein.",
"In embodiments of the present invention in which an oil-body-protein is used, the first and second redox protein are preferably covalently fused to the oil-body-protein.",
"Accordingly, provided herein are methods for the preparation of a redox protein In association with an oil body comprising: a) introducing into a cell a chimeric nucleic acid sequence comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a first nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a second nucleic acid sequence encoding a redox fusion polypeptide comprising a first redox protein linked to a second redox protein operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell; b) growing said cell under conditions to permit expression of said redox fusion polypeptide in a progeny cell comprising oil bodies; and c) isolating said oil bodies comprising said redox fusion polypeptide from said progeny cell.",
"Redox Proteins In accordance with various methods and compositions provided herein, any nucleic acid sequence encoding a redox protein may be used.",
"The nucleic acid sequence encoding the first and/or second redox protein may be obtained from any biological source or may be prepared synthetically.",
"In general, nucleic acid sequences encoding redox proteins are well known in the art and readily available.",
"See, for example: Cristiano et al.",
"(1993) Genomics 17: (2) 348-354, Doyama et al.",
"(1998) Plant Sci.",
"137: 53-62, Hoeoeg et al.",
"(1984) Biosci.",
"Rep. 4: 917-923; as well as the Swiss Protein sequences set forth in Table 5.Known nucleic acid sequences encoding redox proteins may be used to design and construct nucleic acid sequence based probes in order to uncover and identify previously undiscovered nucleic acid sequences Encoding redox proteins, for example by screening cDNA or genomic libraries.",
"Thus, additional nucleic acid sequences may be discovered and used in accordance with the present invention.",
"The nucleic acid sequence encoding the first and/or second redox protein may be obtained from separate sources or may be obtained from the same source.",
"In general however, the nucleic acid sequence encoding a redox-fusion polypeptide comprises nucleic acid sequences encoding a first and a second redox protein obtained from the same or a similar biological source.",
"In certain embodiments provided herein, wherein the nucleic acid sequence encoding the first and second redox protein is obtained from the same source, the nucleic acid sequence encoding the first redox protein and second redox protein may be naturally fused.",
"In accordance with a particular embodiment, the nucleic acid sequences encoding the first and second redox protein are preferably obtained from a plant source.",
"As set forth above, a polypeptide spacer or linker of variable length may separate the first and second redox proteins from each other and/or from the oil-body-targeting-protein; and additional redox proteins (e.g., one or more) may be fused to the first and/or second redox protein.",
"The nucleic acid sequences encoding the redox proteins may be altered to improve expression levels for example by optimizing the nucleic acids sequence in accordance with the preferred codon usage for the particular cell type which is selected for expression of the redox proteins, or by altering of motifs known to destabilize mRNAs (see for example: PCT Patent Application 97/02352).",
"Comparison of the codon usage of the redox protein with codon usage of the host will enable the identification of codons that may be changed.",
"For example, typically plant evolution has tended towards a preference for CG rich nucleotide sequences while bacterial evolution has resulted in bias towards AT rich nucleotide sequences.",
"By modifying the nucleic acid sequences to incorporate nucleic acid sequences preferred by the host cell, expression may be optimized.",
"Construction of synthetic genes by altering codon usage is described in for example PCT patent Application 93/07278.The redox proteins may be altered using for example, targeted mutagenesis, random mutagenesis (Shiraishi et al.",
"(1998) Arch.",
"Biochem.",
"Biophys.",
"358: 104-115; Galkin et al.",
"(1997) Protein Eng.",
"10: 687-690; Carugo et al.",
"(1997) Proteins 28: 10-28; Hurley et al.",
"(1996) Biochemistry 35: 5670-5678) (and/or by the addition of organic solvent (Holmberg et al.",
"(1999) Protein Eng.",
"12: 851-856).",
"The polypeptide spacer between the first and second redox protein may be altered in similar ways.",
"The first and second redox protein may be selected by developing a two-dimensional matrix and determining which combination of first and second redox protein is most effective in electron transport using for example, a colorometric reduction assay (Johnson et al (1984) J. of Bact.",
"Vol.",
"158 3:1061-1069, Luthman et al (1982) Biochemistry Vol 21 26:6628-2233).",
"Combinations of thioredoxin and thioredoxin-reductase may be tested by determining the reduction of wheat storage proteins and milk storage protein beta-lactoglobulin in vitro (Del Val et al.",
"(1999) J. Allerg.",
"Clin.",
"Immunol.",
"103: 690-697).",
"Using the same strategy polypeptide spacers between the first and second redox proteins may be evaluated for their efficiency.",
"First and second redox proteins that may be used herein include without limitation any first redox protein and second redox protein selected from the group of redox proteins consisting of cytochromes, such as cytochrome a, cytochrome b and cytochrome c; porphyrin containing proteins, for example hemoglobin; iron-sulfur proteins, such as ferredoxin; flavoproteins such as thioredoxin-reductase, NADH dehydrogenase, succinate dehydrogenase, dihydrolipoyl dehydrogenase, acyl-CoA dehydrogenase, D-amino acid oxidase, xanthine oxidase, orotate reductase and aldehyde oxidase; pyridine-linked dehydrogenases, for example, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and beta-hydroxy-butarate dehydrogenase; and redox active thiol containing proteins such as thioredoxin.",
"In particular embodiments, the redox proteins provided herein are thioredoxin and its reductant thioredoxin-reductase (which are jointly also referred to herein as “thioredoxin-related” protein(s)).",
"As used herein, the term “thioredoxin” refers to relatively small proteins (typically approximately 12 kDa) that belong to the family of thioltransferases which catalyze oxido-reductions via the formation or hydrolysis of disulfide bonds and are widely, if not universally, distributed throughout the animal plant and bacterial kingdom.",
"The reduces form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.",
"In order to reduce the oxidized thioredoxin, two cellular reductants provide the reduction equivalents: reduced ferredoxin and NADPH.",
"These reduction equivalents are supplied to thioredoxin via interaction or association with different thioredoxin-reductases including the NADPH thioredoxin-reductase and ferredoxin thioredoxin-reductase.",
"The supply of these reduction equivalents requires the formation of a heteromultimeric-protein-complex comprising thioredoxin and thioredoxin-reductase.",
"Ferredoxin thioredoxin-reductase is involved in the reduction of plant thioredoxins designated as Trxf and Trxm, both of which are involved in the regulation of photosynthetic processes in the chloroplast.",
"The NADPH/thioredoxin active in plant seeds is designated Trxh (also referred to herein as thioredoxin h-type) and is capable of the reduction of a wide range of proteins thereby functioning as an important cellular redox buffer.",
"Generally, only one kind of thioredoxin, which analogous to the plant Trxh type, is found in bacterial or animal cells.",
"The h-type thioredoxins are capable of being reduced by NADPH and NADPH-thioredoxin reductase.",
"Exemplary thioredoxins are further characterized as a protein having a core of 5 beta-sheets surrounded by 4 to 6 alpha helixes.",
"Exemplary thioredoxins are further characterized by having an active site containing the consensus amino acid sequence: XCYYCZ, wherein Y is any amino acid, such as hydrophobic or non-polar amino acids, wherein X can be any of the 20 amino acids, preferably a hydrophobic amino acid, such as a tryptophan, and Z can be any amino acid, preferably polar amino acids.",
"In certain embodiments, the thioredoxins for use herein comprise an active site having the amino acid sequence XCGPCZ.",
"When the cysteines in the active site of thioredoxin or thioredoxin-like proteins are oxidized, they form an intramolecular disulfide bond.",
"In the reduced state, the same active sites are capable of participating in redox reactions through the reversible oxidation of its active site dithiol, to a disulfide and catalyzes dithioldisulfide exchange reactions.",
"Exemplary thioredoxins are well-known in the art and can be obtained from several organisms including Arabidopsis thaliana (Riveira Madrid et al.",
"(1995) Proc.",
"Natl.",
"Acad.",
"Sci.",
"92: 5620-5624), wheat (Gautier et al.",
"(1998) Eur.",
"J. Biochem.",
"252: 314-324); Escherichia coli (Hoeoeg et al (1984) Biosci.",
"Rep. 4: 917-923) and thermophylic microorganisms such as Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126).",
"Thioredoxins have also been recombinantly expressed in several host systems including bacteria (Gautier et al.",
"(1998) Eur J. Biochem.",
"252: 314-324) and plants (PCT Patent Application WO 00/58453) Commercial preparations of E. coli sourced Thioredoxins are readily available from for example: Sigma Cat No.",
"T 0910 Thioredoxin (E. coli, recombinant; expressed in E. coli).",
"Exemplary nucleic acid sequences encoding thioredoxin polypeptides for use herein are readily available from a variety of diverse biological sources including E. coli (Hoeoeg et al.",
"(1984) Biosci.",
"Rep.: 4 917-923); Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126); Arabidopsis thaliana (Rivera-Madrid (1995) Proc.",
"Natl.",
"Acad.",
"Sci.",
"92: 5620-5624); wheat (Gautier et al (1998) Eur.",
"J. Biochem.",
"252(2): 314-324); tobacco (Marty et al.",
"(1991) Plant Mol.",
"Biol.",
"17: 143-148); barley (PCT Patent Application 00/58352); rice (Ishiwatari et al.",
"(1995) Planta 195: 456-463); soybean (Shi et al.",
"(1996) Plant Mol.",
"Biol.",
"32: 653-662); rapeseed (Bower et al.",
"Plant Cell 8: 1641-1650) and calf (Terashima et al.",
"(1999) DNA Seq.",
"10(3): 203-205); and the like.",
"In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth as SEQ ID NOs:38, 42, 46 and 50; and those encoding the thioredoxin and thioredoxin-like polypeptide chains set forth in Table 5 as SEQ ID NOs:52-194.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:52-194 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).",
"As used herein, the term “thioredoxin-reductase” refers to a protein that complexes with a flavin, such as FAD.",
"The flavin compound serves as an electron donor for the thioredoxin-reductase protein active site.",
"Thioredoxin reductases have a redox active, disulfide bond site capable of reducing thioredoxin.",
"The active site of thioredoxin-reductase contains 2 cysteines.",
"The type of amino acids surrounding the 2 cysteine residues forming the active site can vary as hydrophobic, non-polar or polar.",
"An exemplary thioredoxin-reductase is NADPH-thioredoxin-reductase (NTR), which is a cytosolic homodimeric enzyme comprising typically 300-500 amino acids.",
"Crystal structures of both E. coli and plant thioredoxin-reductase have been obtained (Waksman et al.",
"(1994) J. Mol.",
"Biol.",
"236: 800-816; Dai et al.",
"(1996) J. Mol.",
"Biol.",
"264:1044-1057).",
"NADPH-thioredoxin-reductases have been expressed in heterologous hosts, for example the Arabidopsis NADPH-thioredoxin-reductase has been expressed in E. coli (Jacquot et al.",
"(1994) J. Mol.",
"Biol.",
"235: 1357-1363) and wheat (PCT Patent Application 00/58453).",
"Exemplary nucleic acid sequences encoding thioredoxin-reductase proteins can readily be obtained from a variety of sources, such as from the sequence set forth in Table 5 and the Sequence Listing provide herein, from Arabidopsis (Riveira Madrid et al.",
"(1995) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 92: 5620-5624), E. coli (Russel et al.",
"(1988) J. Biol.",
"Chem.",
"263: 9015-9019); barley (PCT Patent Application 00158352 and wheat (Gautier et al., (1998) Eur.",
"J. Biochem.",
"252: 314-324); and the like.",
"In yet other embodiments, exemplary nucleic acids for use herein include those encoding the thioredoxin-reductase polypeptide chains set forth as SEQ ID NOs:8, 9, 10, 40, 44, 48 and 50; and those encoding the thioredoxin-reductase polypeptide chains set forth in Table 5 as SEQ ID NOs:195-313.The respective nucleic acid sequences encoding the amino acids set forth in SEQ ID NOs:195-313 can be readily identified via the Swiss Protein identifier (accession) numbers provided in Table 5 (in parenthesis).",
"Also contemplated for use in the methods and compositions provided herein are nucleic acid and amino acid homologs that are “substantially homologous” to the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein, which includes thioredoxin and thioredoxin-reductase polypeptides encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides encoding the thioredoxin and thioredoxin-reductase nucleic and amino acids set forth herein (e.g., in the Examples, Sequence Listing and/or Table 5).",
"As used herein, a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.",
"By the term “substantially homologous” is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.",
"The terms “homology” and “identity” are often used interchangeably.",
"In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program.",
"The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol.",
"Biol.",
"48:443 (1970), as revised by Smith and Waterman (Adv.",
"Appl.",
"Math.",
"2:482 (1981).",
"Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.",
"The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl.",
"Acids Res.",
"14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.",
"353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.",
"By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier.",
"Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest.",
"Preferably the two molecules will hybridize under conditions of high stringency.",
"Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.",
"Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 85:2444 (1988).",
"Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.",
"In general, sequences are aligned so that the highest order match is obtained.",
"“Identity” per se has an art-recognized meaning and can be calculated using published techniques.",
"(See, e.g.",
": Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).",
"While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).",
"Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).",
"Methods to determine identity and similarity are codified in computer programs.",
"Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).",
"Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide.",
"For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.",
"As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides.",
"Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared.",
"No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides.",
"Similar comparisons may be made between a test and reference polynucleotides.",
"Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g.",
"10/100 amino acid difference (approximately 90% identity).",
"Differences are defined as nucleic acid or amino acid substitutions, or deletions.",
"As used herein: stringency of hybridization in determining percentage mismatch is as follows: 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C. 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. Those of skill in this art know that the washing step selects for stable hybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), vol.",
"3, p. B.",
"13, see, also, numerous catalogs that describe commonly used laboratory solutions).",
"SSPE is pH 7.4 phosphate-buffered 0.18 NaCl.",
"Further, those of skill in the art recognize that the stability of hybrids is determined by Tm, which is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6(log10[Na+])+0.41 (% G+C)−600/l)), so that the only parameters in the wash conditions critical to hybrid stability are sodium ion concentration in the SSPE (or SSC) and temperature.",
"It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.",
"By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 78:6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate, adjusted to a pH of 7).",
"In a particular embodiment, a heteromultimeric-protein-complex is produced as a fusion polypeptide between the first and second redox protein, wherein the first redox protein is thioredoxin and the second redox protein is a thioredoxin-reductase.",
"In one embodiment, the second recombinant polypeptide, e.g., the thioredoxin-reductase is positioned N-terminal relative to the first recombinant polypeptide, e.g., the thioredoxin.",
"Accordingly, any protein which is classified as thioredoxin, such as the thioredoxin component of the NADPH thioredoxin system and the thioredoxin present in the ferredoxin/thioredoxin system also known as TRx and TRm may be used in combination with any thioredoxin-reductase such as the NADPH thioredoxin-reductase and the ferredoxin-thioredoxin-reductase and any other proteins having the capability of reducing thioredoxin.",
"In particular embodiments the thioredoxin and thioredoxin-reductase are plant derived.",
"In an alternate embodiment, the naturally occurring nucleic acid sequence encoding the thioredoxin/thioredoxin-reductase protein fusion obtainable from Mycobacterium leprae (Wieles et al.",
"(1995) J. Biol.",
"Chem.",
"27:25604-25606) is used, as set forth in the Examples herein.",
"Immunoglobulins In another embodiment of the present invention, the multimeric-protein-complexes are immunoglobulins.",
"As used herein “immunoglobulin-polypeptide-chain” refers to a first polypeptide that is capable of associating with a second polypeptide to form an immunologically active (i.e.",
"capable of antigen binding multimeric-protein-complex.",
"The types of immunoglobulins and immunoglobulin-polypeptide-chains contemplated for use herein include the immunologically active (i.e.",
"antigen binding) portions of a light and heavy chain.",
"Exemplary immunoglobulins and immunoglobulin-polypeptide-chains for use herein include substantially intact immunoglobulins, including any IgG, IgA, IgD, IgE and IgM, as well as any portion of an immunoglobulin, including those portions well-known as Fab fragments, Fab′ fragments, F(ab′).sub2.fragments and Fv fragments.",
"In this embodiment, the first recombinant polypeptide may be any immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain.",
"Accordingly, provided herein are methods of producing an immunoglobulin, said method comprising: (a) producing in a cell comprising oil bodies, a first immunoglobulin-polypeptide-chain and a second immunoglobulin-polypeptide-chain wherein said first immunoglobulin-polypeptide-chain is capable of associating with said second immunoglobulin-polypeptide-chain to form said immunoglobulin; and (b) associating said immunoglobulin with an oil body through an oil-body-targeting-protein capable of associating with said oil bodies and said first immunoglobulin-polypeptide-chain.",
"As set forth herein, the multimeric immunoglobulin is associated with an oil body through an oil-body-targeting-protein.",
"In particular embodiments, the oil-body-targeting-protein may be a fusion polypeptide comprising an oil-body-protein and an immunoglobulin binding protein, such as for example protein A, protein L, and protein G. In yet another embodiment involving immunoglobulins, the first and second recombinant polypeptides (immunoglobulins) are separately fused to an oil body protein, for example an oleosin or caleosin.",
"For example, a) the first recombinant polypeptide may be an immunoglobulin heavy chain, including any IgG, IgA, IgD, IgE or IgM heavy chain, and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or b) the first recombinant polypeptide may be the variable and first constant domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be a kappa or lambda immunoglobulin light chain; or c) the first recombinant polypeptide may be the variable domain from an immunoglobulin heavy chain and the second recombinant polypeptide may be the variable domain from a kappa or lambda immunoglobulin light chain.",
"In certain embodiments, the fusion polypeptides are designed or selected to allow the heteromultimeric-protein-complex formation between immunoglobulin light and heavy chain sequences on the oil bodies within the cell comprising oil bodies.",
"Preparation of Expression Vectors Comprising Oil-Body-Targeting-Proteins and the First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, or the First and/or Second Thioredoxin-Related Proteins.",
"In accordance with the present invention, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are conveniently produced in a cell.",
"In order to produce the recombinant polypeptides or multimeric-protein-complexes, a nucleic acid sequence encoding either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein are incorporated in a recombinant expression vector.",
"Accordingly, provided herein are recombinant expression vectors comprising the chimeric nucleic acids provided herein suitable for expression of the oil-body-targeting-protein and the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, suitable for the selected cell.",
"The term “suitable for expression in the selected cell” means that the recombinant expression vector contains all nucleic acid sequences required to ensure expression in the selected cell.",
"Accordingly, the recombinant expression vectors further contain regulatory nucleic acid sequences selected on the basis of the cell which is used for expression and ensuring initiation and termination of transcription operatively linked to the nucleic acid sequence encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein.",
"Regulatory nucleic acid sequences include promoters, enhancers, silencing elements, ribosome binding sites, Shine-Dalgarno sequences, introns and other expression elements.",
"“Operatively linked” is intended to mean that the nucleic acid sequences comprising the regulatory regions linked to the nucleic acid sequences encoding the recombinant polypeptide or multimeric-protein-complex and/or the oil-body-targeting-protein allow expression in the cell.",
"A typical nucleic acid construct comprises in the 5′ to 3′ direction a promoter region capable of directing expression, a coding region comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or an oil-body-targeting-protein and a termination region functional in the selected cell.",
"The selection of regulatory sequences will depend on the organism and the cell type in which the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and/or the oil-body-targeting-protein is expressed, and may influence the expression levels of the polypeptide.",
"Regulatory sequences are art-recognized and selected to direct expression of the oil-body-targeting-protein and the recombinant polypeptides or multimeric-protein-complexes in the cell.",
"Promoters that may be used in bacterial cells include the lac promoter (Blackman et al.",
"(1978) Cell: 13: 65-71), the trp promoter (Masuda et al.",
"(1996) Protein Eng: 9: 101-106) and the T7 promoters (Studier et al.",
"(1986) J. Mol.",
"Biol.",
"189: 113-130).",
"Promoters functional in plant cells that may be used herein include constitutive promoters such as the 35S CaMV promoter (Rothstein et al.",
"(1987) Gene: 53: 153-161) the actin promoter (McElroy et al.",
"(1990) Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).",
"Other promoters are specific to certain tissues or organs (for example, roots, leaves, flowers or seeds) or cell types (for example, leaf epidermal cells, mesophyll cells or root cortex cells) and or to certain stages of plant development.",
"Timing of expression may be controlled by selecting an inducible promoter, for example the PR-a promoter described in U.S. Pat.",
"No.",
"5,614,395.Selection of the promoter therefore depends on the desired location and timing of the accumulation of the desired polypeptide.",
"In a particular embodiment, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins; and the oil-body-targeting-protein are expressed in a seed cell and seed specific promoters are utilized.",
"Seed specific promoters that may be used herein include for example the phaseolin promoter (Sengupta-Gopalan et al.",
"(1985) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA: 82: 3320-3324), and the Arabidopsis 18 kDa oleosin promoter (van Rooijen et al.",
"(1992) Plant.",
"Mol.",
"Biol.",
"18: 1177-1179).",
"New promoters useful in various plant cell types are constantly discovered.",
"Numerous examples of plant promoters may be found in Ohamuro et al.",
"(Biochem of Pl.",
"(1989) 15: 1-82).",
"Genetic elements capable of enhancing expression of the polypeptide may be included in the expression vectors.",
"In plant cells these include for example, the untranslated leader sequences from viruses such as the AMV leader sequence (Jobling and Gehrke (1987) Nature: 325: 622-625) and the intron associated with the maize ubiquitin promoter (See: U.S. Pat.",
"No.",
"5,504,200).",
"Transcriptional terminators are generally art recognized and besides serving as a signal for transcription termination serve as a protective element serving to extend the mRNA half-life (Guarneros et al.",
"(1982) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA: 79: 238-242).",
"In nucleic acid sequences for the expression in plant cells, the transcriptional terminator typically is from about 200 nucleotide to about 1000 nucleotides in length.",
"Terminator sequences that may be used herein include for example, the nopaline synthase termination region (Bevan et al.",
"(1983) Nucl.",
"Acid.",
"Res.",
": 11: 369-385), the phaseolin terminator (van der Geest et al.",
"(1994) Plant J.: δ: 413-423), the terminator for the octopine synthase gene of Agrobacterium tumefaciens or other similarly functioning elements.",
"Transcriptional terminators can be obtained as described by An (1987) Methods in Enzym.",
"153: 292).",
"The selection of the transcriptional terminator may have an effect on the rate of transcription.",
"Accordingly, provided herein are chimeric nucleic acid sequences encoding a first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins.",
"In one embodiment, said nucleic acid comprises: (a) a first nucleic acid sequence encoding an oil-body-targeting-protein operatively linked in reading frame to; (b) a second nucleic acid sequence encoding a first recombinant polypeptide, immunoglobulin-polypeptide-chain, or redox protein; linked in reading frame to; (c) a third nucleic acid sequence encoding a second recombinant polypeptide, immunoglobulin-polypeptide-chain or redox protein, wherein said first and second recombinant polypeptides, immunoglobulin-polypeptide-chains or redox proteins are capable of forming a multimeric-protein-complex.",
"In another embodiment, provided herein is an expression vector comprising: 1) a first nucleic acid sequence capable of regulating transcription in said cell operatively linked to; 2) a second nucleic acid sequence encoding a recombinant fusion polypeptide comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil-body-protein to provide targeting of said recombinant fusion polypeptide to an oil body linked in reading frame to (ii) a nucleic acid sequence encoding a multimeric-fusion-protein, such as a redox fusion polypeptide or immunoglobulin, comprising a first recombinant polypeptide, such as a redox protein or immunoglobulin-polypeptide-chain, linked to a second recombinant polypeptide, such as a second redox protein or a second immunoglobulin-polypeptide-chain, operatively linked to; 3) a third nucleic acid sequence capable of terminating transcription in said cell.",
"The recombinant expression vector further may contain a marker gene.",
"Marker genes that may be used in accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells including all selectable and screenable marker genes.",
"A marker may be a resistance marker such as an antibiotic resistance marker against for example kanamycin, ampicillin, G418, bleomycin hygromycin, chloramphenicol which allows selection of a trait by chemical means or a tolerance marker against for example a chemical agent such as the normally phytotoxic sugar mannose (Negrotto et al.",
"(2000) Plant Cell Rep. 19: 798-803).",
"In plant recombinant expression vectors herbicide resistance markers may conveniently be used for example markers conferring resistance against glyphosate (U.S. Pat.",
"Nos.",
"4,940,935 and 5,188,642) or phosphinothricin (White et al.",
"(1990) Nucl.",
"Acids Res.",
"18: 1062; Spencer et al.",
"(1990) Theor.",
"Appl.",
"Genet.",
"79: 625-631).",
"Resistance markers to a herbicide when linked in close proximity to the redox protein or oil-body-targeting-protein may be used to maintain selection pressure on a population of plant cells or plants for those plants that have not lost the protein of interest.",
"Screenable markers that may be employed to identify transformants through visual observation include beta-glucuronidase (GUS) (see U.S. Pat.",
"No.",
"5,268,463 and U.S. Pat.",
"No.",
"5,599,670) and green fluorescent protein (GFP) (Niedz et al.",
"(1995) Plant Cell Rep.: 14: 403).",
"The recombinant expression vectors further may contain nucleic acid sequences encoding targeting signals ensuring targeting to a cell compartment or organelle.",
"Suitable targeting signals that may be used herein include those that are capable of targeting polypeptides to the endomembrane system.",
"Exemplary targeting signals that may be used herein include targeting signals capable of directing the protein to the periplasm, the cytoplasm, the golgi apparatus, the apoplast (Sijmons et al., 1990, Bio/Technology, 8:217-221) the chloroplast (Comai et al.",
"(1988) J. Biol.",
"Chem.",
"263: 15104-15109), the mitochondrion, the peroxisome (Unger et al.",
"(1989) Plant Mol.",
"Biol.",
"13: 411-418), the ER, the vacuole (Shinshi et al.",
"(1990) Plant Mol.",
"Biol.",
"14: 357-368 and the oil body.",
"By the inclusion of the appropriate targeting sequences it is possible to direct the oil-body-targeting-protein or the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and/or thioredoxin-related proteins, to the desired organelle or cell compartment.",
"The recombinant expression vectors of the present invention may be prepared in accordance with methodologies well known to those of skill in the art of molecular biology (see for example: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press).",
"The preparation of these constructs may involve techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing and PCR.",
"A wide variety of cloning vectors is available to perform the necessary cloning steps resulting in a recombinant expression vector ensuring expression of the polypeptide.",
"Especially suitable for this purpose are vectors with a replication system that is functional in Escherichia coli such as pBR322, the PUC series of vectors, the M13 mp series of vectors, pBluescript etc.",
"Typically these vectors contain a marker allowing the selection of transformed cells for example by conferring antibiotic resistance.",
"Nucleic acid sequences may be introduced in these vectors and the vectors may be introduced in E. coli grown in an appropriate medium.",
"Vectors may be recovered from cells upon harvesting and lysing the cells.",
"Recombinant expression vectors suitable for the introduction of nucleic acid sequences in plant cells include Agrobacterium and Rhizobium based vectors such as the Ti and Ri plasmids.",
"Agrobacterium based vectors typically carry at least one T-DNA border sequence and include vectors such pBIN 19 (Bevan (1984) Nucl Acids Res.",
"Vol.",
"12, 22:8711-8721) and other binary vector systems (for example: U.S. Pat.",
"No.",
"4,940,838).",
"Production of Cells Comprising a First and/or Second Recombinant Polypeptides, Multimeric-Protein-Complexes, Heteromultimeric-Protein-Complexes, Multimeric-Fusion-Proteins, Heteromultimeric-Fusion-Proteins, Immunoglobulins, Immunoglobulin-Polypeptide-Chains, Redox-Fusion-Polypeptides, and/or a First and/or Second Thioredoxin-Related Protein and Oil-Body-Targeting-Proteins In accordance with the present invention, the recombinant expression vectors are introduced into the cell that is selected and the selected cells are grown to produce the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, a first and/or second thioredoxin-related protein; and the oil-body-targeting-protein either directly or in a progeny cell.",
"Methodologies to introduce recombinant expression vectors into a cell also referred to herein as “transformation” are well known to the art and vary depending on the cell type that is selected.",
"General techniques to transfer the recombinant expression vectors into the cell include electroporation; chemically mediated techniques, for example CaCl2 mediated nucleic acid uptake; particle bombardment (biolistics); the use of naturally infective nucleic acid sequences for example virally derived nucleic acid sequences or when plant cells are used Agrobacterium or Rhizobium derived nucleic acid sequences; PEG mediated nucleic acid uptake, microinjection, and the use of silicone carbide whiskers (Kaeppler et al.",
"(1990) Plant Cell Rep. 9:415-418) all of which may be used herein.",
"Introduction of the recombinant expression vector into the cell may result in integration of its whole or partial uptake into host cell genome including the chromosomal DNA or the plastid genome.",
"Alternatively the recombinant expression vector may not be integrated into the genome and replicate independently of the host cell's genomic DNA.",
"Genomic integration of the nucleic acid sequence is typically used as it will allow for stable inheritance of the introduced nucleic acid sequences by subsequent generations of cells and the creation of cell, plant or animal lines.",
"Particular embodiments involve the use of plant cells.",
"Particular plant cells used herein include cells obtainable from Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.",
"); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.",
"and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.",
"); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticum aestivum) and sunflower (Helianthus annuus).",
"Transformation methodologies for dicotelydenous plant species are well known.",
"Generally Agrobacterium mediated transformation is utilized because of its high efficiency as well as the general susceptibility by many, if not all dicotelydenous plant species.",
"Agrobacterium transformation generally involves the transfer of a binary vector (e.g.",
"pBIN19) comprising the DNA of interest to an appropriate Agrobacterium strain (e.g.",
"CIB542) by for example tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilization of the binary vector to the target Agrobacterium strain, or by DNA transformation of the Agrobacterium strain (Hofgen et al.",
"Nucl.",
"Acids.",
"Res.",
"(1988) 16: 9877.Other transformation methodologies that may be used to transform dicotelydenous plant species include biolistics (Sanford (1988) Trends in Biotechn.",
"6: 299-302); electroporation (Fromm et al.",
"(1985) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 82: 5824-5828); PEG mediated DNA uptake (Potrykus et al.",
"(1985) Mol.",
"Gen. Genetics 199: 169-177); microinjection (Reich et al.",
"Bio/Techn.",
"(1986) 4: 1001-1004) and silicone carbide whiskers (Kaeppler et al.",
"(1990) Plant Cell Rep. 9: 415-418).",
"The exact transformation methodologies typically vary somewhat depending on the plant species that is used.",
"In a particular embodiment the oil bodies are obtained from safflower and the recombinant proteins are expressed in safflower.",
"Safflower transformation has been described by Baker and Dyer (Plant Cell Rep. (1996) 16: 106-110).",
"Monocotelydenous plant species may now also be transformed using a variety of methodologies including particle bombardment (Christou et al.",
"(1991) Biotechn.",
"9: 957-962; Weeks et al.",
"Plant Physiol.",
"(1993) 102: 1077-1084; Gordon-Kamm et al.",
"Plant Cell (1990) 2: 603-618) PEG mediated DNA uptake (EP 0 292 435; 0 392 225) or Agrobacterium-mediated transformation (Goto-Fumiyuki et al (1999) Nature-Biotech.",
"17 (3):282-286).",
"Plastid transformation is described in U.S. Pat.",
"Nos.",
"5,451,513; 5,545,817 and 5,545,818; and PCT Patent Applications 95/16783; 98/11235 and 00/39313) Basic chloroplast transformation involves the introduction of cloned plastid DNA flanking a selectable marker together with the nucleic acid sequence of interest into a suitable target tissue using for example biolistics or protoplast transformation.",
"Selectable markers that may be used include for example the bacterial aadA gene (Svab et al.",
"(1993) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 90: 913-917).",
"Plastid promoters that may be used include for example the tobacco clpP gene promoter (PCT Patent Application 97/06250).",
"In another embodiment, the invention chimeric nucleic acid constructs provided herein are directly transformed into the plastid genome.",
"Plastid transformation technology is described extensively in U.S. Pat.",
"Nos.",
"5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos.",
"WO 95/16783 and WO 97/32977; and in McBride et.",
"al., Proc Natl Acad Sci USA 91: 7301-7305 (1994), the entire disclosures of all of which are hereby incorporated by reference.",
"In one embodiment, plastid transformation is achieved via biolistics, first carried out in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al.",
"(1988) Science 240:1534-1537)) and then extended to Nicotiana tabacum (Svab et al.",
"(1990) Proc Natl Acad Sci USA 87:8526-8530), combined with selection for cis-acting antibiotic resistance loci (spectinomycin or streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes.",
"In another embodiment, tobacco plastid transformation is carried out by particle bombardment of leaf or callus tissue, or polyethylene glycol (PEG)-mediated uptake of plasmid DNA by protoplasts, using cloned plastid DNA flanking a selectable antibiotic resistance marker.",
"For example, 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome.",
"In one embodiment, point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin can be utilized as selectable markers for transformation (Svab et al.",
"(1990) Proc Natl Acad Sci USA 87:8526-8530; Staub et al.",
"(1992) Plant Cell 4:39-45, the entire disclosures of which are hereby incorporated by reference), resulting in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.",
"The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al.",
"(1993) EMBO J 12:601-606, the entire disclosure of which is hereby incorporated by reference).",
"In another embodiment, substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al.",
"(1993) Proc Natl Acad Sci USA 90: 913-917, the entire disclosure of which is hereby incorporated by reference).",
"This marker has also been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl Acids Res 19, 4083-4089, the entire disclosure of which is hereby incorporated by reference).",
"In other embodiments, plastid transformation of protoplasts from tobacco and the moss Physcomitrella can be attained using PEG-mediated DNA uptake (O'Neill et al.",
"(1993) Plant J 3:729-738; Koop et al.",
"(1996) Planta 199:193-201, the entire disclosures of which are hereby incorporated by reference).",
"Both particle bombardment and protoplast transformation are also contemplated for use herein.",
"Plastid transformation of oilseed plants has been successfully carried out in the genera Arabidopsis and Brassica (Sikdar et al.",
"(1998) Plant Cell Rep 18:20-24; PCT Application WO 00/39313, the entire disclosures of which are hereby incorporated by reference).",
"A chimeric nucleic sequence construct is inserted into a plastid expression cassette including a promoter capable of expressing the construct in plant plastids.",
"A particular promoter capable of expression in a plant plastid is, for example, a promoter isolated from the 5′ flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues.",
"Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (Stern et al.",
"(1997) Trends in Plant Sci 2:308-315, the entire disclosure of which is hereby incorporated by reference).",
"Plastid promoters generally contain the −35 and −10 elements typical of prokaryotic promoters, and some plastid promoters called PEP (plastid-encoded RNA polymerase) promoters are recognized by an E. coli-like RNA polymerase mostly encoded in the plastid genome, while other plastid promoters called NEP promoters are recognized by a nuclear-encoded RNA polymerase.",
"Both types of plastid promoters are suitable for use herein.",
"Examples of plastid promoters include promoters of clpP genes such as the tobacco clpP gene promoter (WO 97/06250, the entire disclosure of which is hereby incorporated by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser.",
"No.",
"09/038,878, the entire disclosure of which is hereby incorporated by reference).",
"Another promoter capable of driving expression of a chimeric nucleic acid construct in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., (1994) Microbiol Rev 58:700-754; Shinozaki et al.",
"(1986) EMBO J 5:2043-2049, the entire disclosures of both of which are hereby incorporated by reference).",
"Other examples of promoters capable of driving expression of a nucleic acid construct in plant plastids include a psbA promoter or am rbcL promoter.",
"A plastid expression cassette preferably further includes a plastid gene 3′ untranslated sequence (3′ UTR) operatively linked to a chimeric nucleic acid construct of the present invention.",
"The role of untranslated sequences is preferably to direct the 3′ processing of the transcribed RNA rather than termination of transcription.",
"An exemplary 3′ UTR is a plastid rps16 gene 3′ untranslated sequence, or the Arabidopsis plastid psbA gene 3′ untranslated sequence.",
"In a further embodiment, a plastid expression cassette includes a poly-G tract instead of a 3′ untranslated sequence.",
"A plastid expression cassette also preferably further includes a 5′ untranslated sequence (5′ UTR) functional in plant plastids, operatively linked to a chimeric nucleic acid construct provided herein.",
"A plastid expression cassette is contained in a plastid transformation vector, which preferably further includes flanking regions for integration into the plastid genome by homologous recombination.",
"The plastid transformation vector may optionally include at least one plastid origin of replication.",
"The present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the chimeric nucleic acid construct is expressible in the plant plastid.",
"Also encompassed herein is a plant or plant cell, including the progeny thereof, including this plant plastid.",
"In a particular embodiment, the plant or plant cell, including the progeny thereof, is homoplasmic for transgenic plastids.",
"Other promoters capable of driving expression of a chimeric nucleic acid construct in plant plastids include transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected.",
"In these cases, the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA.",
"The transactivator is targeted to plastids using a plastid transit peptide.",
"The transactivator and the transactivator-driven DNA molecule are brought together either by crossing a selected plastid-transformed line with and a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a chimeric nucleic acid construct encoding the transactivator supplemented with a plastid-targeting sequence operably linked to a nuclear promoter.",
"If the nuclear promoter is an inducible promoter, in particular a chemically inducible embodiment, expression of the chimeric nucleic acid construct in the plastids of plants is activated by foliar application of a chemical inducer.",
"Such an inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction.",
"A particular transactivator is, for example, viral RNA polymerase.",
"Particular promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase.",
"The gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide.",
"Promoters suitable for nuclear expression of a gene, for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described above and elsewhere in this application.",
"Expression of chimeric nucleic acid constructs in plastids can be constitutive or can be inducible, and such plastid expression can be also organ- or tissue-specific.",
"Examples of various expression systems are extensively described in WO 98/11235, the entire disclosure of which is hereby incorporated by reference.",
"Thus, in one aspect, the present invention utilizes coupled expression in the nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under the control of the chemically inducible PR-1a promoter, for example of the PR-1 promoter of tobacco, operably linked with a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences, for example as described in as in U.S. Pat.",
"No.",
"5,614,395 the entire disclosure of which is hereby incorporated by reference.",
"In another embodiment, when plastid transformants homoplasmic for the maternally inherited TR or NTR genes are pollinated by lines expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry both transgene constructs but do not express them until synthesis of large amounts of enzymatically active protein in the plastids is triggered by foliar application of the PR-1a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH).",
"In a particular embodiment, two or more genes, for example TR and NTR genes, are transcribed from the plastid genome from a single promoter in an operon-like polycistronic gene.",
"In one embodiment, the operon-like polycistronic gene includes an intervening DNA sequence between two genes in the operon-like polycistronic gene.",
"In a particular embodiment, the intervening DNA sequence is not present in the plastid genome to avoid homologous recombination with plastid sequences.",
"In another embodiment, the DNA sequence is derived from the 5′ untranslated (UTR) region of a non-eukaryotic gene, preferably from a viral 5′UTR, preferably from a 6′UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage.",
"In one embodiment, a portion of the DNA sequence may be modified to prevent the formation of RNA secondary structures in an RNA transcript of the operon-like polycistronic gene, for example between the DNA sequence and the RBS of the downstream gene.",
"Such secondary structures may inhibit or repress the expression of the downstream gene, particularly the initiation of translation.",
"Such RNA secondary structures are predicted by determining their melting temperatures using computer models and programs such a the “mfold” program version 3 (available from Zuker and Turner, Washington University School of Medicine, St-Louis, Mo.)",
"and other methods known to one skilled in the art.",
"The presence of the intervening DNA sequence in the operon-like polycistronic gene increases the accessibility of the RBS of the downstream gene, thus resulting in higher rates of expression.",
"Such strategy is applicable to any two or more genes to be transcribed from the plastid genome from a single promoter in an operon-like chimeric heteromultimeric gene.",
"Following transformation the cells are grown, typically in a selective medium allowing the identification of transformants.",
"Cells may be harvested in accordance with methodologies known to the art.",
"In order to associate the oil bodies with the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, and a first and/or second thioredoxin-related protein, the integrity of cells may be disrupted using any physical, chemical or biological methodology capable of disrupting the cells' integrity.",
"These methodologies are generally cell-type dependent and known to the skilled artisan.",
"Where plants are employed they may be regenerated into mature plants using plant tissue culture techniques generally known to the skilled artisan.",
"Seeds may be harvested from mature transformed plants and used to propagate the plant line.",
"Plants may also be crossed and in this manner, contemplated herein is the breeding of cells lines and transgenic plants that vary in genetic background.",
"It is also possible to cross a plant line comprising the first recombinant polypeptide with a plant line comprising the second recombinant polypeptide.",
"Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox protein (e.g., a thioredoxin-related protein, and the like) or an immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox protein (e.g., a thioredoxin-related protein, and the like) or a second immunoglobulin-polypeptide-chain; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.",
"The second recombinant polypeptide may also associate with the oil bodies.",
"Accordingly, also provided herein are methods of producing in a plant a recombinant multimeric-protein-complex, said method comprising: (a) preparing a first plant comprising cells, said cells comprising oil bodies and a first recombinant polypeptide, such as a redox (or thioredoxin-related) protein or immunoglobulin-polypeptide-chain, wherein said first recombinant polypeptide is capable of associating with said oil bodies through an oil-body-targeting-protein; (b) preparing a second plant comprising cells, said cells comprising oil bodies and a second recombinant polypeptide, such as a second redox (thioredoxin-related) protein or a second immunoglobulin-polypeptide-chain, wherein said second recombinant polypeptide is capable of associating with said oil bodies through an oil body targeting protein; and (c) sexually crossing said first plant with said second plant to produce a progeny plant comprising cells, said cells comprising oil bodies, wherein said oil bodies are capable of associating with said first recombinant polypeptide, and said first recombinant recombinant polypeptide is capable of associating with said second recombinant polypeptide to form said recombinant multimeric-protein-complex.",
"Isolation of Oil Bodies The oil bodies provided herein may be obtained from any cell containing oil bodies, including any animal cell; plant cell; fungal cell; for example a yeast cell, algae cell; or bacterial cell.",
"Any process suitable for the isolation oil bodies from cells may be used herein.",
"Processes for the isolation of oil bodies from plant seed cells have been described in U.S. Pat.",
"Nos.",
"(6,146,645 and 6,183,762) and the isolation of oil bodies from yeast cells has been described by Ting et al.",
"(1997) J. Biol.",
"Chem.",
"272: 3699-3706).",
"In certain embodiments, the oil bodies are obtained from a plant cell such as for example a pollen cell; a fruit cell; a spore cell; a nut cell; mesocarp cell; for example the mesocarp cells obtainable from olive (Olea europaea) or avocado (Persea americana); or a seed cell.",
"In particular embodiments the oil bodies are obtained from a plant seed cell.",
"The seeds can be obtained from a transgenic plant according to the present invention.",
"In particular embodiments, a seed of a transgenic plant according to the present invention contains the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or first and/or second thioredoxin-related proteins in a concentration of at least about 0.5% of total cellular seed protein.",
"In further embodiments, a seed of a transgenic plant provided herein contains a recombinant polypeptide or multimeric-protein-complex in a concentration of at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, of total cellular seed protein.",
"The upper limits of the recombinant polypeptide or multimeric-protein-complex concentration can be up to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.",
"Thus, the ranges at least about 0.5% up to about 15%; at least about 1.0% up to about 10%; and at least about 5% up to about 8% are among the various ranges contemplated herein.",
"Among the plant seeds useful in this regard are plant seeds obtainable from the group of plant species consisting of Brazil nut (Betholletia excelsa); castor (Riccinus communis); coconut (Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.",
"); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zea mays); mustard (Brassica spp.",
"and Sinapis alba); oil palm (Elaeis guineeis); olive (Olea europaea); rapeseed (Brassica spp.",
"); safflower (Carthamus tinctorius); soybean (Glycine max); squash (Cucurbita maxima); sunflower (Helianthus annuus); barley (Hordeum vulgare); wheat (Traeticum aestivum) and mixtures thereof.",
"In a particular embodiment, oil bodies are obtainable from the seeds obtainable from safflower (Carthamus tinctorius).",
"In order to prepare oil bodies from plant seeds, plants are grown and allowed to set seed in accordance with common agricultural practices.",
"Thus, the present invention also provides seeds comprising oil bodies, wherein said oil bodies further comprise invention multimeric-protein-complexes described herein.",
"Upon harvesting the seed and, if necessary the removal of large insoluble materials such as stones or seed hulls, by for example sieving or rinsing, any process suitable for the isolation of oil bodies from seeds may be used herein.",
"A typical process involves grinding of the seeds followed by an aqueous extraction process.",
"Seed grinding may be accomplished by any comminuting process resulting in a substantial disruption of the seed cell membrane and cell walls without compromising the structural integrity of the oil bodies present in the seed cell.",
"Suitable grinding processes in this regard include mechanical pressing and milling of the seed.",
"Wet milling processes such as described for cotton (Lawhon et al.",
"(1977) J.",
"Am.",
"Oil Chem.",
"Soc.",
"63: 533-534) and soybean (U.S. Pat.",
"No.",
"3,971,856; Carter et al.",
"(1974) J.",
"Am.",
"Oil Chem.",
"Soc.",
"51: 137-141) are particularly useful in this regard.",
"Suitable milling equipment capable of industrial scale seed milling include colloid mills, disc mills, pin mills, orbital mills, IKA mills and industrial scale homogenizers.",
"The selection of the milling equipment will depend on the seed, which is selected, as well as the throughput requirement.",
"Solid contaminants such as seed hulls, fibrous materials, undissolved carbohydrates, proteins and other insoluble contaminants are subsequently preferably removed from the ground seed fraction using size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.",
"Centrifugation may be accomplished using for example a decantation centrifuge such as a HASCO 200 2-phase decantation centrifuge or an NX310B (Alpha Laval).",
"Operating conditions are selected such that a substantial portion of the insoluble contaminants and sediments and may be separated from the soluble fraction.",
"Following the removal of insolubles the oil body fraction may be separated from the aqueous fraction.",
"Gravitational based methods as well as size exclusion based technologies may be used.",
"Gravitational based methods that may be used include centrifugation using for example a tubular bowl centrifuge such as a Sharples AS-16 or AS-46 (Alpha Laval), a disc stack centrifuge or a hydrocyclone, or separation of the phases under natural gravitation.",
"Size exclusion methodologies that may be used include membrane ultra filtration and crossflow microfiltration.",
"Separation of solids and separation of the oil body phase from the aqueous phase may also be carried out concomitantly using gravity based separation methods or size exclusion based methods.",
"The oil body preparations obtained at this stage in the process are generally relatively crude and depending on the application of the oil bodies, it may be desirable to remove additional contaminants.",
"Any process capable of removing additional seed contaminants may be used in this regard.",
"Conveniently the removal of these contaminants from the oil body preparation may be accomplished by resuspending the oil body preparation in an aqueous phase and re-centrifuging the resuspended fraction, a process referred to herein as “washing the oil bodies”.",
"The washing conditions selected may vary depending on the desired purity of the oil body fractions.",
"For example where oil bodies are used in pharmaceutical compositions, generally a higher degree of purity may be desirable than when the oil bodies are used in food preparations.",
"The oil bodies may be washed one or more times depending on the desired purity and the ionic strength, pH and temperature may all be varied.",
"Analytical techniques may be used to monitor the removal of contaminants.",
"For example SDS gel electrophoresis may be employed to monitor the removal of seed proteins.",
"The entire oil body isolation process may be performed in a batch wise fashion or continuous flow.",
"In a particular embodiment, industrial scale continuous flow processes are utilized.",
"Through the application of these and similar techniques the skilled artisan is able to obtain oil bodies from any cell comprising oil bodies.",
"The skilled artisan will recognize that generally the process will vary somewhat depending on the cell type that is selected.",
"However, such variations may be made without departing from the scope and spirit of the present invention.",
"Association of the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins with oil bodies.",
"In accordance with the present invention, the oil bodies are associated with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, the first and/or second thioredoxin-related proteins through association with an oil-body-targeting-protein capable of association with these multimeric-protein-complexes and the oil bodies.",
"As used herein the phrase “associating the oil bodies with the multimeric-protein-complex” means that the oil bodies are brought in proximity of the multimeric-protein-complexes in a manner that allows the association of the oil bodies with either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.",
"The association of the oil bodies with the multimeric-protein-complexes is accomplished by association of the oil-body-targeting-protein with both the oil body and with the multimeric-protein-complex.",
"In particular embodiments, the cells expressing the multimeric-protein-complex associate with the oil bodies that are obtainable from these same cells, which permits the convenient production and isolation of the multimeric-protein-complex, including the first and/or second recombinant polypeptides, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, in an oil body-comprising host cell system.",
"Accordingly, in one embodiment, the association of the oil body with the multimeric-protein-complex is accomplished intracellularly during the growth of the cell.",
"For example, a redox fusion polypeptide may be fused to an oil-body-protein and the chimeric protein may be expressed in oil body-containing plant seeds.",
"Isolation of the oil bodies from the seeds in this case results in isolation of oil bodies comprising either the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins.",
"In another embodiment, in which the multimeric-protein-complex associates with oil bodies obtainable from the same cells in which the complex is produced, the association of the oil bodies with the multimeric-protein-complex is accomplished upon disrupting the cell's integrity.",
"For example, the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins may be expressed in such a manner that it is targeted to the endomembrane system of the seed cells.",
"Oil bodies present in the same seed cells comprising an oil-body-targeting-protein capable of association with these multimeric-protein-complexes, for example an oleosin linked to a single chain antibody capable of association with a recombinant polypeptide or multimeric-protein-complex, may then associate with the recombinant polypeptide or multimeric-protein-complex upon grinding of the seed.",
"In accordance with this embodiment, plant seed cells comprising a light and heavy chain of an immunoglobulin targeted to the plant apoplast can be prepared.",
"These particular seed cells are prepared to further comprise oil bodies associated with an oil-body-targeting-protein capable of association with the immunoglobulin, such as for example, an oleosin-protein A fusion protein, and the like.",
"Upon grinding of the seed, the oil bodies comprising protein A associate with the immunoglobulin through binding.",
"In yet another embodiment, the oil bodies used to associate with the multimeric-protein-complex are obtained from a cellular source different from the cell comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, such as from a separate plant line.",
"For example, oil bodies associated with protein A may be prepared from one plant line.",
"These oil bodies may then be mixed with ground seeds comprising an apoplastically expressed light and heavy chain constituting an immunoglobulin.",
"Alternatively, a plant line comprising oil bodies associated with protein A may be crossed with a plant line comprising an immunoglobulin.",
"The first recombinant polypeptide, second recombinant polypeptide and oil-body-targeting-protein may also be prepared in separate cellular compartments.",
"Association of the first polypeptide, second polypeptide, and oil body then may occur upon disruption of the cell's integrity.",
"For example, various mechanisms for targeting gene products are known to exist in plants, and the sequences controlling the functioning of these mechanisms have been characterized in some detail.",
"For example, the targeting of gene products to the chloroplast is controlled by a transit sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (Comai et al.",
"(1988) J Biol Chem 263: 15104-15109).",
"Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (Unger et al.",
"(1989) Plant Mol Biol 13:411-418).",
"The cDNAs encoding these products can be manipulated to target heterologous gene products to these organelles.",
"In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments.",
"Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho (1990) Plant Cell 2:769-783).",
"Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., (1990) Plant Mol Biol 14:357-368).",
"By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to the desired organelle or cell compartment.",
"As hereinbefore mentioned, the redox protein obtained using the methods provided herein is enzymatically active while associated with the oil body.",
"Preferably the redox protein is at least 5 times more active when produced as a redox fusion polypeptide with a second redox protein relative to its production in association with an oil body as a non-fusion polypeptide (i.e.",
"without the second redox protein).",
"More preferably the redox protein is at least 10 times more active when produced as a redox fusion polypeptide.",
"The activity of the redox fusion polypeptide may be determined in accordance with methodologies generally known to the art (see for example: Johnson et al (1984) J. of Bact.",
"Vol.",
"158 3:1061-1069) and may be optimized by for example the addition of detergents, including ionic and non-ionic detergents.",
"Formulation of Oil Bodies In accordance with a particular embodiment, the oil bodies comprising the first and/or second recombinant polypeptides, multimeric-protein-complexes, heteromultimeric-protein-complexes, multimeric-fusion-proteins, heteromultimeric-fusion-proteins, immunoglobulins, immunoglobulin-polypeptide-chains, redox-fusion-polypeptides, or the first and/or second thioredoxin-related proteins, are preferably formulated into an emulsion.",
"The emulsion is preferably used in the preparation of a pharmaceutical composition, personal care or a food product.",
"In emulsified form, the oil body offers certain desirable properties, such as for example excellent compatibility with the human skin.",
"It particular embodiments, the oil body formulation is stabilized so that a final product may be obtained which may be stored and preserved for longer periods of time.",
"As used herein, the term “stabilized oil body preparation” refers to an oil body preparation that is prepared so that the formulation does not undergo undesirable physical or chemical alterations when the oil body preparation is stored.",
"The stabilization requirements may vary depending on the final product.",
"For example personal care products are preferably stable for at least one year at room temperature while additionally being able to withstand short temperature fluctuations.",
"Pharmaceutical formulations may in some cases be less stable as they may be stored at lower temperatures thereby preventing the occurrence of undesirable reactions.",
"In general, stabilization techniques that may be used herein include any and all methods for the preservation of biological material including the addition of chemical agents, temperature modulation based methodologies, radiation-based technologies and combinations thereof.",
"In particular embodiments small amounts of stabilizing chemical agents are mixed with the oil body formulation to achieve stabilization.",
"These chemical agents include inter alia preservatives, antioxidants, acids, salts, bases, viscosity modifying agents, emulsifiers, gelling agents and mixtures thereof and may all be used to stabilize the oil body preparation.",
"In view of the presence of the redox fusion polypeptide the stabilizing agent is generally selected to be compatible with and resulting in good enzymatic function of the redox fusion polypeptide.",
"Diagnostic parameters to assess the stability of the oil body preparation may be as desired and include all parameters indicative of undesirable qualitative or quantitative changes with respect to chemical or physical stability.",
"Typical parameters to assess the oil body preparation over time include color, odor, viscosity, texture, pH and microbial growth, and enzymatic activity.",
"In particular embodiments, the oil body formulation is stabilized prior to the addition of further ingredients that may be used to prepare the final product.",
"However, in other embodiments, it is nevertheless possible to formulate the final formulation using non-stabilized oil bodies and stabilize the final formulation.",
"The final preparations may be obtained using one or more additional ingredients and any formulation process suitable for the preparation of a formulation comprising oil bodies.",
"Ingredients and processes employed will generally vary depending on the desired use of the final product, will be art recognized and may be as desired.",
"Ingredients and processes that may be used herein include those described in (U.S. Pat.",
"Nos.",
"6,146,645 and 6,183,762) which are incorporated by reference herein.",
"In particular embodiments, the redox fusion polypeptide comprises a thioredoxin and a thioredoxin-reductase.",
"Accordingly, provided herein are oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.",
"Also provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of treating or protecting a target against oxidative stress.",
"The stress of the target is treated or prevented by contacting the target with the formulation.",
"The target may be any substance susceptible to oxidative stress, including any molecule, molecular complex, cell, tissue or organ.",
"In another embodiment, provided herein is a formulation containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion capable of chemically reducing a target.",
"Contacting the target with the formulation reduces the target.",
"The target may be any substance susceptible to reduction, including any molecule or molecular complex.",
"Particularly susceptible targets in this regard are the disulfide bonds present in proteins.",
"The oil bodies comprising thioredoxin/thioredoxin-reductase may be used to prepare formulations used to reduce the allergenicity of food or increase the digestibility of food.",
"Preferably, the method of reducing the food allergenicity is practiced by mixing the thioredoxin/thioredoxin-reductase comprising oil bodies with food or food ingredients selected from a variety of sources including for example wheat flour, wheat dough, milk, cheese, soya, yogurt and ice cream.",
"The thioredoxin/thioredoxin-reductase comprising oil bodies may also be used to increase the digestibility of milk as well as other disulfide containing proteins (Jiao, J. et al.",
"(1992) J. Agric.",
"Food Chem 40: 2333-2336).",
"Further food applications include the use of the oil thioredoxin/thioredoxin-reductase comprising oil bodies as a food additive to enhance dough strength and bread quality properties (Wong et al., (1993) J. Cereal Chem.",
"70: 113-114; Kobrehel et al.",
"(1994) Gluten Proteins: Association of Cereal Research; Detmold, Germany).",
"Also provided herein are pharmaceutical compositions comprising, in a pharmaceutically active carrier: oil bodies comprising a thioredoxin/thioredoxin-reductase; oil bodies comprising multimeric-protein-complexes, such as heteromultimeric-protein-complexes; isolated thioredoxin/thioredoxin-reductase fusion proteins; or isolated multimeric-protein-complexes.",
"These pharmaceutical compositions may be used for the treatment of reperfusion injury (Aota et al.",
"(1996) J. Cardiov.",
"Pharmacol.",
"(1996) 27: 727-732), cataracts (U.S. Pat.",
"No.",
"4,771,036), chronic obstructive pulmonary disease (COPD) (MacNee et al.",
"(1999) Am.",
"J. Respir.",
"Crit.",
"Care Med.",
"160:S58-S65), diabetes (Hotta et al.",
"J. Exp.",
"Med.",
"188: 1445-1451), envenomation (PCT Patent Application 99/20122; U.S. Pat.",
"No.",
"5,792,506), bronchiopulmonary disease (MacNee (2000) Chest 117:3035-3175); malignancies (PCT Patent Application 91/04320) and the alleviation of the allergenic potential of airborne, for example pollen-derived, and contact allergens (PCT Patent Application 00/44781).",
"Other diseases or conditions that may be treated with the pharmaceutical compositions provided herein include: psoriasis, wound healing, sepsis, GI bleeding, intestinal bowel disease (IBD), ulcers, transplantation, GERD (gastro esophageal reflux disease).",
"In another embodiment, the pharmaceutical compositions provided herein, particularly those comprising one or more redox proteins alone or in combination with oil bodies, can be used in the treatment of inflammatory and viral diseases by reductively inactivating phospholipase A2, one of the contributing factors in inflammatory diseases.",
"Additionally, the redox fusion polypeptide system has been found to function as a self-defense mechanism in response to environmental stimuli, including oxidative stress caused by UV-generated free radicals.",
"Consequently, redox proteins, e.g., oleosin-thioredoxin, oleosin-thioredoxin-reductase, the various redox fusion polypeptides described herein, provide beneficial effects in certain skin conditions such as psoriasis, skin cancer, dandruff, diaper rash, dermatitis, acne, sun damage, aging, inflammation, and the like.",
"In another embodiment, oil-body-thioredoxin-related fusion proteins, e.g., oleosin-Thioredoxin-reductase, can also be used as a venom antidote.",
"Many animal venoms and other toxins contain disulfide bonds, including all snake venom neurotoxins, some bacterial neurotoxins including tetanus and botulinum A, bee venom phospholipase A2, and scorpion venom.",
"In a further embodiment, the redox protein related pharmaceutical compositions provided herein can be used to inactivate venom toxins by reduction of disulfide bonds.",
"A method of treating an individual suffering from the effects of a venom or toxin can include the step of administering an effective dose of a pharmaceutical composition, in a pharmaceutically effective carrier in an amount sufficient to relieve or reverse the effects of the venom toxin on the individual.",
"The pharmaceutical compositions provided herein are preferably formulated for single dosage administration.",
"The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment.",
"Typically, the compositions are formulated for single dosage administration.",
"To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.",
"Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.",
"In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.",
"Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.",
"These may be prepared according to methods known to those skilled in the art.",
"For example, liposome formulations may be prepared as described in U.S. Pat.",
"No.",
"4,522,811.The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.",
"The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.",
"The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.",
"Typically a therapeutically effective dosage is contemplated.",
"The amounts administered may be on the order of 0.001 to 1 mg/ml, preferably about 0.005-0.05 mg/ml, more preferably about 0.01 mg/ml, of blood volume.",
"Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg, more preferably about 25-75 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form.",
"The precise dosage can be empirically determined.",
"The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.",
"It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.",
"It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.",
"It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of the claimed compositions and combinations containing them.",
"Preferred pharmaceutically acceptable derivatives include acids, salts, esters, hydrates, solvates and prodrug forms.",
"The derivative is typically selected such that its pharmacokinetic properties are superior to the corresponding neutral compound.",
"Thus, effective concentrations or amounts of one or more of the compounds provided herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.",
"Compounds are included in an amount effective for ameliorating or treating the disorder for which treatment is contemplated.",
"The concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.",
"Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.",
"Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.",
"In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used.",
"Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as Tween®, or dissolution in aqueous sodium bicarbonate.",
"Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.",
"For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable carrier.",
"For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred.",
"Time release formulations are also desirable.",
"Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.",
"Upon mixing or addition of the compound with the vehicle, the resulting mixture may be a solution, suspension, emulsion or other composition.",
"The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.",
"If necessary, pharmaceutically acceptable salts or other derivatives of the compounds are prepared.",
"The compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.",
"It is understood that number and degree of side effects depends upon the condition for which the compounds are administered.",
"For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses that would not be tolerated when treating disorders of lesser consequence.",
"The compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action known to those of skill in the art.",
"The formulations of the compounds and agents for use herein include those suitable for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration or any route.",
"The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.",
"The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.",
"The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.",
"Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.",
"Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier, vehicle or diluent.",
"Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules.",
"Unit-dose forms may be administered in fractions or multiples thereof.",
"A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form.",
"Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.",
"Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.",
"The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polivinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.",
"Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.",
"If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.",
"Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).",
"The composition or formulation to be administered will contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject.",
"Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.",
"For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).",
"The tablets may be coated by methods well-known in the art.",
"The pharmaceutical preparation may also be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.",
"Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).",
"Formulations suitable for rectal administration are preferably presented as unit dose suppositories.",
"These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.",
"Formulations suitable for topical application to the skin or to the eye preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol and oil.",
"Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof.",
"The topical formulations may further advantageously contain 0.05 to 15 percent by weight of thickeners selected from among hydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly(alkylene glycols), poly/hydroxyalkyl, (meth)acrylates or poly(meth)acrylamides.",
"A topical formulation is often applied by instillation or as an ointment into the conjunctival sac.",
"It can also be used for irrigation or lubrication of the eye, facial sinuses, and external auditory meatus.",
"It may also be injected into the anterior eye chamber and other places.",
"The topical formulations in the liquid state may be also present in a hydrophilic three-dimensional polymer matrix in the form of a strip, contact lens, and the like from which the active components are released.",
"For administration by inhalation, the compounds for use herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.",
"In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.",
"Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.",
"Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.",
"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.",
"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.",
"The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.",
"Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use.",
"Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.",
"Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 to 0.2 M concentration with respect to the active compound.",
"Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.",
"The pharmaceutical compositions may also be administered by controlled release means and/or delivery devices (see, e.g., in U.S. Pat.",
"Nos.",
"3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).",
"Desirable blood levels may be maintained by a continuous infusion of the active agent as ascertained by plasma levels.",
"It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions.",
"Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).",
"The efficacy and/or toxicity of the pharmaceutical compositions provided herein, alone or in combination with other agents can also be assessed by the methods known in the art (See generally, O'Reilly, Investigational New Drugs, 15:5-13 (1997)).",
"The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.",
"Kits containing the compositions and/or the combinations with instructions for administration thereof are provided.",
"The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad.",
"Instructions are optionally included for administration of the active agent by a clinician or by the patient.",
"Finally, the pharmaceutical compositions provided herein containing any of the preceding agents may be packaged as articles of manufacture containing packaging material, a compound or suitable derivative thereof provided herein, which is effective for treatment of a diseases or disorders contemplated herein, within the packaging material, and a label that indicates that the compound or a suitable derivative thereof is for treating the diseases or disorders contemplated herein.",
"The label can optionally include the disorders for which the therapy is warranted.",
"Also provided herein are personal care formulations containing oil bodies comprising a thioredoxin/thioredoxin-reductase fusion polypeptide.",
"Personal care products comprising thioredoxin and thioredoxin-reductase are disclosed in for example Japanese Patent Applications JP9012471A2, JP103743A2, and JP1129785A2 Personal care formulations that may be prepared in accordance with the present invention include formulations capable of improving the physical appearance of skin exposed to detrimental environmental stimuli resulting in oxidative stress for example oxidative stress caused by UV-generated free-radicals.",
"The oil bodies comprising thioredoxin/thioredoxin-reductase may also be used to prepare hair care products as described in U.S. Pat.",
"Nos.",
"4,935,231 and 4,973,475 (incorporated herein by reference in their entirety).",
"The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.",
"Example 1 Isolation of Thioredoxin and NADPH Thioredoxin-Reductase Genes An Arabidopsis silique cDNA library CD4-12 was obtained from the Arabidopsis Biological Resource Centre (ABRC, http://aims.cps.msu.edu) Arabidopsis stock centre and used as a template for the isolation of the thioredoxin h (Trxh) and thioredoxin-reductase genes from Arabidopsis.",
"For the isolation of the Trxh gene the following primers were synthesized: GVR833: 5′ TACCATGGCTTCGGAAGAAGGA 3′ (SEQ ID NO:1) The sequence identical to the 5′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.",
"Underlined is an NcoI restriction site to facilitate cloning.",
"GVR834: 5′ GAAAGCTTAAGCCAAGTGTTTG 3′ (SEQ ID NO:2) The sequence complementary to the 3′ end of the Trxh gene as published in Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328, is indicated in bold.",
"Underlined is an HindIII restriction site to facilitate cloning.",
"A Polymerase Chain Reaction (PCR) was carried out using GVR833 and GVR834 as primers and the cDNA library CD4-12 as a template.",
"The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.",
"The isolated sequence encoding Trxh was identical to the published Trxh gene sequence (Rivera-Madrid et al, (1993) Plant Physiol 102: 327-328).",
"The pBluescript vector containing the Trxh gene is called pSBS2500.For the isolation of the thioredoxin-reductase gene the following primers were synthesized: GVR836: 5′ GGCCAGCACACTACCATGAATGGTCTCGAAACTCAC 3′ (SEQ ID NO:3).",
"The sequence identical to the 5′ end of the thioredoxin-reductase gene as published (Jacquot et al, J Mol Biol.",
"(1994) 235 (4):1357-63), is indicated in bold).",
"GVR837: 5′ TTAAGCTTCAATCACTCTTACCTTGCTG 3′.",
"(SEQ ID NO:4) A Polymerase Chain Reaction (PCR) was carried out using GVR836 and GVR837 as primers and the cDNA library CD4-12 as a template.",
"The resulted PCR fragment was isolated, cloned into pBluescript and sequenced.",
"The pBluescript vector containing the thioredoxin-reductase gene is called pSBS2502.A total of three clones were sequenced, the sequence of each of the three clones were identical to each other.",
"However, as depicted in FIG.",
"1 this sequence indicated several nucleotide differences compared to the published thioredoxin-reductase gene sequence published (Jacquot et al, J Mol Biol.",
"(1994) 235 (4):1357-63.).",
"The complete coding sequence and its deduced amino acid sequence is shown in SEQ ID NO:10.As a result of the nucleotide differences between the published sequence and the sequence isolated in Example 1, several amino acid changes are also predicted.",
"A comparison of the deduced amino acid sequence of the published NADPH thioredoxin-reductase sequence thioredoxin-reductase (ATTHIREDB, Jacquot et al, J Mol Biol.",
"(1994) 235 (4):1357-63.)",
"with the sequence isolated in Example 1 (TR) is shown in FIG.",
"3.Example 2 Construction of Plant Expression Vectors Expression vectors were constructed to allow for the seed specific over-expression of thioredoxin and NADPH thioredoxin-reductase in seeds.",
"Vectors were constructed to allow for over-expression in its natural subcellular location and for accumulation on oil bodies.",
"Construction of Plant Transformation Vector pSBS2520.The Arabidopsis thioredoxin h gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.",
"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324)).",
"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with Pst I and HindIII/KpnI sites respectively (see SEQ ID NO:14).",
"Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the Trxh gene.",
"The PstI-phaseolin promoter-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 (pSBS3000 is a derivative from the Agrobacterium binary plasmid pPZP221 (Hajdukiewicz et al., 1994, Plant Molec.",
"Biol.",
"25: 989-994).",
"In pSBS3000, the CaMV35S promoter-gentamycin resistance gene-CAMV 35S terminator of pPZP221 was replaced with parsley ubiquitin promoter-phosphinothricin acetyl transferase gene-parsley ubiquitin termination sequence to confer resistance to the herbicide glufosinate ammonium.)",
"The resulting plasmid is called pSBS2520.The sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence is shown in SEQ ID NO:14.Construction of Plant Transformation Vector pSBS2510.The 3′ coding sequence of an Arabidopsis oleosin gene (van Rooijen et al (1992) Plant Mol.",
"Biol.",
"18: 1177-1179) was altered to contain an NcoI site.",
"The NcoI-HindIII fragment from vector pSBS2500 (Example 1) containing the Trxh was ligated to the coding sequence of this Arabidopsis oleosin utilizing this NcoI restriction site.",
"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.",
"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) containing a synthetic PstI site (see construction of pSBS2520) to the coding sequence of the Arabidopsis oleosin.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the Trxh gene.",
"The PstI-phaseolin promoter-oleosin-Trxh-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2510.The sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence is shown in SEQ ID NO:16.Construction of Plant Transformation Vector pSBS2521.This vector contains the same genetic elements as the insert of pSBS2510 except the Trxh gene is fused to the 5′ end of the oleosin gene.",
"The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al (1992) Plant Mol.",
"Biol.",
"18: 1177-1179) was furnished with a HindIII cloning site.",
"Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the Trxh gene and to fuse the Trxh gene to the oleosin sequence.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.",
"The PstI-phaseolin promoter-Trxh oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2521.The sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence is shown in SEQ ID NO:19.Construction of Plant Transformation Vector pSBS2527.The Arabidopsis NADPH thioredoxin-reductase gene as described in example 1 was placed under the regulatory control of the phaseolin promoter and the phaseolin terminator derived from the common bean Phaseolus vulgaris (Slightom et al (1983) Proc.",
"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324).",
"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were used to furnish the phaseolin promoter and terminator with PstI and HindIII/KpnI sites respectively (see SEQ ID NO:14).",
"Standard molecular biology laboratory techniques were also used to place the phaseolin terminator downstream from the thioredoxin-reductase gene.",
"The PstI-phaseolin promoter-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000 The resulting plasmid is called pSBS2527.The sequence of the phaseolin promoter-Arabidopsis thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:22.Construction of Plant Transformation Vector pSBS2531.A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.",
"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.",
"The same gene splicing technique was used to fuse the oleosin gene to the thioredoxin-reductase coding sequence.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin downstream of the thioredoxin-reductase gene.",
"The PstI-phaseolin promoter-oleosin-thioredoxin-reductase-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2531.The sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence is shown in SEQ ID NO:24.Construction of Plant Transformation Vector pSBS2529 This vector contains the same genetic elements as the insert of pSBS2531 except the thioredoxin-reductase gene is fused to the 5′ end of the oleosin gene.",
"The 3′ oleosin coding sequence including its native stopcodon (van Rooijen et al.",
"(1992) Plant Mol.",
"Biol, 18: 1177-1179) was furnished with a HindIII cloning site.",
"Again a gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter to the thioredoxin-reductase gene and to fuse the thioredoxin-reductase gene to the oleosin sequence.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were again used to clone the HindIII KpnI fragment containing the phaseolin terminator (see construction of pSBS2520) downstream of the oleosin gene.",
"The PstI-phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2529.The sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence is shown in SEQ ID NO:27.Construction of Plant Transformation Vector pSBS2530.A plant transformation was constructed containing the Mycobacterium Leprae thioredoxin-reductase /thioredoxin gene (Mlep TR/Trxh).",
"A construct called pHIS/TR/Trxh (Wieles et al (1995) J Biol Chem 270:25604-25606) was obtained from the department of Immunohematology and Blood bank, Leiden University, The Netherlands and use as a template for PCR to generate pSBS2530.The construction of pSBS2530 was identical to the construction of pSBS2531 except that the Mlep TR/Trxh gene was used instead of the Arabidopsis thioredoxin-reductase gene.",
"A gene splicing by overlap extension technique (Horton et al (1989) 15: 61-68) was used to fuse the phaseolin promoter (Slightom et al (1983) Proc.",
"Natl Acad Sc USA 80: 1897-1901; Sengupta-Gopalan et al., (1985) PNAS USA 82: 3320-3324) to the coding sequence of the Arabidopsis oleosin.",
"The same gene splicing technique was used to fuse the oleosin gene to the Mlep TR/Trxh coding sequence.",
"Standard molecular biology laboratory techniques (see eg: Sambrook et al.",
"(1990) Molecular Cloning, 2nd ed.",
"Cold Spring Harbor Press) were again used to clone the HindIII-KpnI fragment containing the phaseolin downstream of the Mlep TR/Trxh gene.",
"The PstI-phaseolin promoter-oleosin-Mlep TR/Trxh -phaseolin terminator-KpnI insert sequence was cloned into the PstI-KpnI sites of pSBS3000.The resulting plasmid is called pSBS2530.The sequence of the phaseolin promoter-oleosin Mlep TR/Trxh -phaseolin terminator sequence is shown in SEQ ID NO:30.Construction of Plant Transformation Vector pSBS2542.From initial activity assays (FIG.",
"4), it was apparent that oil bodies expressing the oleosin-M. lep TR/Trxh fusion protein contained considerable reducing activity.",
"It was anticipated that a similar oleosin fusion construct encoding the Arabidopsis thioredoxin-reductase and thioredoxin proteins would behave in an analogous manner.",
"Molecular modeling was used to aid in the design of such a construct.",
"Primers were designed (thioredoxin link-L: 5′-ACTGGAGATGTTGACTCGACGGATACTACGGATTGGTCGACGG CTATGGAAGAAGGACAAGTGATCGCCTGC-3′; (SEQ ID NO:5), and thioredoxin link-R: 5′-ATCCGTCGAGTCAACATCTCCAGTTTCCTCGGTGGTCTCGTTAGCCTTCGAT CCAGCAATCTCTTGTAAGAATGCTCTGC-3′; (SEQ ID NO:6) to code for a synthetic linker peptide between the thioredoxin-reductase and thioredoxin proteins.",
"These primers were used in conjunction with primers GVR 873 (5′-GTGGAAGCT TATGGAGATGGAG-3′; SEQ ID NO:7) and GVR834 (5′-GAAAGCTTAAGCCAAGTGTTTG-3′; SEQ ID NO:2) to amplify a region coding for a thioredoxin-reductase-linker region-thioredoxin utilizing a gene splicing by overlap extension technique (Horton et al (1989) 15:61-68).",
"The thioredoxin-reductase-linker-thioredoxin encoding sequence was then cloned into a pre-existing pSBS3000 vector using standard molecular biology techniques (Sambrook et al (1990) Molecular Cloning 2nd Edition Cold Spring Harbour Press).",
"The resulting plasmid was called pSBS2542.The sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region is shown in SEQ ID NO:33.An amino acid sequence comparison between this Arabidopsis thioredoxin-reductase-linker-thioredoxin and the M. leprae TR/Trxh protein is shown in FIG.",
"12.Plasmids pSBS2510, pSBS2520, pSBS2521, pSBS2527, pSBS2529, pSBS2530, pSBS2531 and pSBS2542 were electroporated into Agrobacterium strain EHA101.These Agrobacterium strains were used to transform Arabidopsis.",
"Arabidopsis transformation was done essentially as described in “Arabidopsis Protocols; Methods in molecular biology Vol 82.Edited by Martinez-Zapater J M and Salinas J. ISBN 0-89603-391-0 pg 259-266 (1998) except the putative transgenic plants were selected on agarose plates containing 80 μM L-phosphinothricine, after they were transplanted to soil and allowed to set seed.",
"Example 3 Polyacrylamide Gelelectrophoresis and Immunoblotting of Transgenic Seed Extracts Source of Arabidopsis Thioredoxin, Thioredoxin-Reductase and Oleosin Antibodies.",
"The Arabidopsis thioredoxin and thioredoxin-reductase genes were cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression of Arabidopsis thioredoxin and thioredoxin-reductase proteins.",
"These proteins were purified using standard protocols (see eg Invitrogen protocol) and used to raise antibodies in rabbits using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).",
"The Arabidopsis oleosin gene genes was cloned in frame in bacterial expression vector pRSETB (Invitrogen) to allow for the overexpression Arabidopsis oleosin protein.",
"This protein was purified using standard protocols (see eg Invitrogen protocol) and used to prepare mouse monoclonal antibodies using standard biochemical techniques (See eg Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).",
"Preparation of total Arabidopsis seed extracts for PAGE.",
"Arabidopsis seeds were ground in approximately 20 volumes of 2% SDS, 50 mM Tris-Cl, this extract was boiled, spun and the supernatant was prepared for polyacrylamide gelelectrophoresis (PAGE) using standard protocols.",
"Preparation of Arabidopsis Oil-Body-Protein Extracts.",
"Arabidopsis seeds were ground in approximately 20 volumes of water and spun in a microfuge.",
"The oil bodies were recovered and washed sequentially with approximately 20 volumes of water, a high stringency wash buffer, containing 8M urea and 100 mM sodiumcarbonate and water.",
"After this last wash the oil bodies are prepared for poly acrylamide gelelectrophoresis (PAGE) using standard protocols.",
"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2510 Total seed and oil body protein extracts from plants transformed with pSBS2510 were loaded onto polyacrylamide gels and either stained with coomassie brilliant blue or electroblotted onto PVDF membranes.",
"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.",
"Expression of the oleosin-thioredoxin results in an additional band of 31.2 kDa.",
"The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).",
"This indicates that oleosin-thioredoxin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.",
"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2521 Total seed and oil body protein extracts from plants transformed with pSBS25121 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.",
"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.",
"Expression of the thioredoxin-oleosin results in an additional band of 31.2 kDa.",
"The results indicate that the thioredoxin antibodies are immunologically reactive with a band of the right predicted molecular weight (31.2 kDa), and the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight for the fusion protein (31.2 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).",
"This indicates that thioredoxin-oleosin is expressed in Arabidopsis seeds and is correctly targeted to oil bodies.",
"Analysis of seed extracts from plants transformed with pSBS2520 Total seed extracts from plants transformed with pSBS2520 were loaded onto polyacrylamide gels and either stained with Coomassie brilliant blue or electroblotted onto PVDF membranes.",
"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin and visualized using alkaline phosphatase.",
"The results indicated that the thioredoxin antibodies are immunologically reactive with a band of approximately the right predicted molecular weight (12 kDa).",
"Untransformed seeds do not show a detectable thioredoxin band.",
"Analysis of Seed and Oil Body Extracts from Plants Transformed with pSBS2529 Total seed and oil body protein extracts from plants transformed with pSBS2529 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.",
"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase, or a monoclonal antibody raised against the Arabidopsis 18.5 kDa oleosin and visualized using alkaline phosphatase.",
"Expression of the thioredoxin-reductase -oleosin results in an additional band of 53.8 kDa.",
"The results indicate that the thioredoxin-reductase antibodies are immunologically reactive with a band of the right predicted molecular weight for the fusion protein (53.8 kDa), the oleosin antibodies are also immunologically reactive with a band of the right predicted molecular weight (53.8 kDa) in addition to a band corresponding to the native Arabidopsis oleosin (18.5 kDa).",
"This indicates that thioredoxin-reductase-oleosin is expressed in Arabidopsis seeds.",
"Analysis of seed extracts from plants transformed with pSBS2527 Total seed extracts from plants transformed with pSBS2527 were loaded onto polyacrylamide gels and electroblotted onto PVDF membranes.",
"The membranes were challenged with a polyclonal antibody raised against Arabidopsis thioredoxin-reductase and visualized using alkaline phosphatase.",
"The thioredoxin-reductase antibodies are immunologically reactive with a band of approximately the right predicted molecular weight for the (35.3 kDa).",
"Untransformed seeds do not show a detectable thioredoxin band.",
"Analysis of seed extracts from plants transformed with pSBS2531 A protein gel and immunoblot was prepared assaying the expression of oleosin-DMSR in Arabidopsis T2 seeds and correct targeting to Arabidopsis oil bodies.",
"The expected molecular weight based on the deduced amino acid sequence is calculated to be 53,817 Da.",
"In the oil body extract of the transgenic oleosin-thioredoxin-reductase sample an extra band of approximately 54 kDa was observed.",
"This band was confirmed to be oleosin-thioredoxin-reductase by immunoblotting.",
"From the polyacrylamide gel it was observed that the expression of the oleosin-Thioredoxin-reductase is about double compared to the expression of the major 18.5 kDa Arabidopsis oleosin.",
"This represents approximately 2-4% of total seed protein.",
"Analysis of seed extracts from plants transformed with pSBS2530 A protein gel and immunoblot was prepared assaying the expression of oleosin-M.lep TR/Trxh in Arabidopsis T2 seeds and the correct targeting to Arabidopsis oil bodies.",
"The expected molecular weight based on the deduced amino acid sequence is calculated to be 67,550 Da.",
"In the oil body extract of the transgenic oleosin-M.lep TR/Trxh sample an extra band of approximately 68 kDa was observed.",
"This band was confirmed to be oleosin-M.lep TR/Trxh by immunoblotting.",
"From the polyacrylamide gel it was observed that the expression of the oleosin-M.lep TR/Trxh is similar to the expression of the major 18.5 kDa Arabidopsis oleosin.",
"This represents approximately 1-2% of total seed protein.",
"Analysis of seed extracts from plants transformed with pSBS2542 Crude oil body extracts from pSBS2542 lines were prepared by grinding 100 μg of seed in 1 mL of 100 mM Tris buffer at pH 7.5.The samples were then centrifuged in order to isolate the oil body fraction.",
"The oil body fraction was then loaded on an SDS polyacrylamide gel for expression analysis.",
"A Coomassie stained gel revealed that the synthetic fusion accumulated to high levels in crude oil body extracts from 3 of the 4 lines tested.",
"It was estimated that the fusion protein represented approximately 2-5% of total seed protein.",
"Furthermore, western blots utilizing either anti-thioredoxin or anti-thioredoxin-reductase antibodies confirmed that the over expressed 70 kDa protein was indeed oleosin-thioredoxin-reductase-linker-thioredoxin.",
"Example 4 Biological Activity of Thioredoxin and Thioredoxin-Reductase Transformants Initial Reduction Assays: DTNB Assay The activity of the thioredoxin and thioredoxin reductase was determined using a calorimetric DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] assay.",
"The assay was performed in a 700 μL reaction volume containing 100 mM Tris-Cl pH 8.0, 5 mM EDTA, 200 μM DTNB [5,5′-dithiolbis(2-nitrobenzoic acid)] and 200 μM NADPH.",
"If thioredoxin-reductase and thioredoxin are added, NADPH will reduce the thioredoxin-reductase, which will then reduce thioredoxin, which will, in turn, reduce DTNB (see equations below).",
"NADPH2+thioredoxin-reductaseox→thioredoxin-reductasered+NADP+thioredoxin-reductasered+thioredoxinox→thioredoxinred+thioredoxin-reductaseox thioredoxinred+DTNBox→2(2-nitro-5-mercaptobenzoic acid)+thioredoxinox The formation of the yellow product was monitored by measuring the OD412 in a spectrophotometer after a set period of time (usually 0.5-2 hours).",
"The results of initial activity assays are shown in the bar graph in FIG.",
"4 and described below.",
"Initially, 100 μg of total seed proteins were added from each of the Arabidopsis transgenic lines, pSBS2520 (cytosolic thioredoxin) and pSBS2527 (cytosolic thioredoxin-reductase), which corresponds to approximately 1 μg of cytosolic thioredoxin and thioredoxin-reductase used in the assay.",
"In this case, the amount of DTNB reduced was comparable to the reduction caused by 1 μg each of E. coli thioredoxin and thioredoxin-reductase.",
"In these plant seed samples, background readings were very low when only one of the 2 extracts (either cytosolic thioredoxin or cytosolic thioredoxin-reductase; FIG.",
"4, bars 3 and 6, respectively) was added to the reaction, along with wild type oil bodies.",
"Analysis with oil body fractions from transgenic seeds revealed that Arabidopsis thioredoxin and thioredoxin-reductase were substantially less active when fused to oleosins on oil bodies.",
"Approximately 300 μg of crude, unwashed oil-body-protein was used in the assay (which corresponds to 10-30 μg of thioredoxin-oleosin (pSBS 2521; FIG.",
"4, bar 2), oleosin-thioredoxin (pSBS 2510, FIG.",
"4, bar 1), thioredoxin-reductase-oleosin (pSBS 2529, FIG.",
"4, bar 5), or oleosin-thioredoxin-reductase (pSBS 2531, FIG.",
"4, bar 4).",
"The oil-body-proteins were tested in conjunction with 100 μg of total seed protein containing approximately 1 μg of cytosolic thioredoxin (pSBS 2520) or thioredoxin-reductase (pSBS 2527).",
"In such assays, pSBS2529 (thioredoxin-reductase-oleosin) and pSBS2531 (oleosin-thioredoxin-reductase) do contain reductase activity when combined with cytosolic thioredoxin from pSBS2520 (see FIG.",
"4, bars 7 and 8, respectively).",
"Experiments estimated that the reductase activity of oleosin-thioredoxin-reductase was about 10-15% that of the cytosolic thioredoxin-reductase.",
"The addition of tween at a final concentration of 0.4% could enhance this activity 2 or 3 fold.",
"Interestingly, oleosin-thioredoxin-reductase (pSBS 2531) appears to be capable of reducing DTNB in the absence of added thioredoxin, although added thioredoxin causes significantly more DTNB reduction (see FIG.",
"4; compare bar 4 W.T.+oleosin-thioredoxin-reductase to bar 7 thioredoxin+oleosin-thioredoxin-reductase).",
"Experiments with pSBS2521 (thioredoxin-oleosin) or pSBS2510 (oleosin-thioredoxin) combined with cytosolic thioredoxin-reductase from pSBS2527 (see FIG.",
"4, bars 10 and 11, respectively) indicate that thioredoxin activity of these fusions is undetectable at these concentrations.",
"Oil bodies from the transgenic Arabidopsis line, pSBS2530 (oleosin-M.lep TR/Trxh) contain significant thioredoxin/thioredoxin-reductase activity (see FIG.",
"4, bar 12).",
"One hundred micrograms of crude oil body protein for pSBS2530 was tested (corresponding to approximately 5 μg of oleosin-M.lep TR/trxh fusion) in the assay.",
"Based on the assay, it was estimated that this fusion is about 25-40% as active as cytosolic Arabidopsis thioredoxin and thioredoxin-reductase (FIG.",
"4, bar 9) when comparing specific activity.",
"Insulin Reduction Assay The results from the DTNB assays were confirmed with insulin reduction assays.",
"This assay contained insulin at a final concentration of 1 mg/mL in 100 mM KH2PO4 pH 7.0+5 mM EDTA.",
"In the presence of NADPH (500 μM), thioredoxin, and thioredoxin-reductase, insulin is reduced and precipitates from the solution.",
"Normally, insulin reduction is followed by measuring turbidity at OD 650.Alternatively, one can measure the conversion of NADPH2 to NADP+by monitoring the decrease in absorbance at 340 nm.",
"Both of the assays are difficult to measure when oil bodies are present, due to interference with the spectrophotometer readings.",
"However, qualitative data could be obtained by centrifuging the tubes after a set period of time, and determining if an insulin pellet was present (oil bodies float to the top, while the insulin precipitate pellets out).",
"Alternatively, samples could be filtered after a set period of time, and the change in absorbance at 340 nm could be measured.",
"As mentioned previously, the results of the insulin reduction assays agreed with those of the DTNB assay, with the exception of the observation that pSBS2531 (oleosin-thioredoxin-reductase) only reduced insulin in the presence of free thioredoxin from pSBS2520.Assays on Seeds from Arabidopsis Crosses that Co-Express Oleosin-Thioredoxin and Oleosin-Thioredoxin-Reductase.",
"Based upon initial DTNB and insulin reduction assays, it was apparent that mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase transgenic seeds resulted in very limited reducing activity (Note: the <-> indicates both configurations of oleosin fusions; ie.",
"oleosin<->thioredoxin would represent oleosin-thioredoxin and thioredoxin-oleosin fusions).",
"To determine whether having oleosin<->thioredoxin and oleosin<->thioredoxin-reductase proteins present on the same oil body would have a positive effect on the reducing activity of these proteins, crosses were set up to generate double transgenic Arabidopsis lines.",
"The crosses are illustrated in Table 2.TABLE 2 Confirmed double transgenic lines (PCR and Western Male Female Blot) oleo-thioredoxin X oleo-thioredoxin-reductase 4 oleo-thioredoxin X thioredoxin-reductase-oleo 1 thioredoxin-oleo X oleo-thioredoxin-reductase 0 thioredoxin-oleo X thioredoxin-reductase-oleo 4 oleo-thioredoxin- X oleo-thioredoxin 2 reductase oleo-thioredoxin- X thioredoxin-oleo 0 reductase thioredoxin- X oleo-thioredoxin 7 reductase-oleo thioredoxin- X thioredoxin-oleo 0 reductase-oleo Seeds from Arabidopsis crosses were germinated on PPT plates and the seedlings were transferred to soil after approximately 2 weeks.",
"PCR experiments on DNA isolated from the seedlings identified a number of plants which contain both an oleosin<->thioredoxin and an oleosin<->thioredoxin-reductase gene construct within their genome.",
"Seeds were harvested from these plants for expression and activity assays.",
"Western blots were carried out to confirm expression of both oleosin<->thioredoxin and oleosin<->thioredoxin-reductase in the lines.",
"DTNB and insulin reduction assays were also performed to compare activity between single transgenic parent lines and the double transgenic offspring and results are summarized in Table 3.Table 3 summarizes DTNB reducing activity of various transgenic lines.",
"The last 2 rows compare mixing oil bodies from single transgenic parent lines to using oil bodies from double transgenic offspring.",
"Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.",
"TABLE 3 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + 100 thioredoxin-reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Based on DTNB and insulin reduction assays, it is evident that double transgenic plants co-expressing oleosin<->thioredoxin and oleosin<->thioredoxin-reductase on the same, single oil body contained significantly more reducing activity compared to mixing oil bodies from single transgenic oleosin<->thioredoxin and oleosin<->thioredoxin-reductase lines.",
"It was additionally apparent that oil body extracts from co-expressing lines contained more reducing activity compared to line pSBS2530 (oleosin-M. lep TR/Trxh), which was previously identified as the line containing the highest reducing activity from oil bodies.",
"These results suggest that the creation of double transgenic lines (either through crossing or by co-transforming 2 expression constructs into plants) may represent one means by which we could solve our initial problem of not being able to generate reducing activity by mixing oil bodies from oleosin<->thioredoxin and oleosin<->thioredoxin-reductase single transgenic lines.",
"Assays on Seeds from Arabidopsis pSBS2542 Transgenic Lines that Express Oleosin-Thioredoxin-Reductase-Linker-Thioredoxin.",
"Oil body extracts from four pSBS2542 lines were tested for reducing activity in DTNB and insulin reduction assays, using standard protocols described previously.",
"Again, oil body extracts containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein possessed significant reducing activity.",
"Based on such assays, it was revealed that the oleosin-thioredoxin-reductase-linker-thioredoxin synthetic fusion protein was more active than the oleosin-M. lep TR/Trxh fusion.",
"Furthermore, oil bodies containing the oleosin-thioredoxin-reductase-linker-thioredoxin protein appeared to have more reducing activity compared to oil bodies from double transgenic lines that co-expressed oleosin<->thioredoxin and oleosin<->thioredoxin-reductase.",
"The results comparing reducing activity for the various thioredoxin-reductase/thioredoxin constructs is summarized in Table 4.Table 4 summarizes DTNB reducing activity of various transgenic lines.",
"The pSBS2542 line expressing oleosin-thioredoxin-reductase-linker-thioredoxin contains significant reducing activity, comparable to the “free” forms of Arabidopsis thioredoxin and thioredoxin-reductase and the equivalent E. coli proteins.",
"Relative activity for the E. coli thioredoxin and thioredoxin mixture is set at 100 percent.",
"TABLE 4 Relative Source Material Activity (%) E. coli trx + NTR 100 Arabidopsis “free” thioredoxin + thioredoxin- 100 reductase (pSBS2520 + pSBS2527) oleosin-M. lep TR/Trxh ~30 (pSBS2530) Oleosin<->thioredoxin-reductase + ~3 oleosin<->thioredoxin (mixing oil bodies from single-transgenic parents) Oleosin<->thioredoxin-reductase × ~50 oleosin<->thioredoxin (various double transgenic lines) Oleosin-thioredoxin-reductase-linker-thioredoxin ~75-100 (pSBS2542) Reduction Assays Comparing the Utilization of NADH Vs. NADPH as a Cofactor (Electron Donor) for the Thioredoxin-Reductase/Thioredoxin System.",
"DTNB and insulin reduction assays were conducted as described previously, except that NADH was substituted for NADPH as an electron donor in the system utilizing E. coli thioredoxin-reductase and thioredoxin.",
"Thus, a comparison was conducted of the utilization of NADH versus NADPH as a cofactor for the E. coli thioredoxin-reductase/thioredoxin system.",
"For the DTNB assay, the reaction mixture consisted of 400 μM DTNB, 10 μg/mL E. coli thioredoxin, and 10 μg/mL E. coli thioredoxin-reductase in 100 mM Tris-Cl buffer pH 8.0.Either NADH or NADPH was then added to the DTNB reaction as follows: Reaction A.",
"200 μM NADPH (Sigma) Reaction B.",
"800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 800 μM NADH (no TR or Trxh).",
"For the insulin reduction assay, the reaction mixture consisted of 1 mg/mL bovine pancreatic insulin, 20 μg/mL E. coli thioredoxin, and 20 μg/mL E. coli thioredoxin-reductase in 100 mM potassium phosphate buffer at pH 7.0.Either NADH or NADPH was then added to the reaction as follows: Reaction A.",
"800 μM NADPH (Sigma) Reaction B.",
"800 μM NADH (Sigma) Reaction C. 800 μM NADH (Roche) Reaction D. (−) cofactor Reaction E. 2 mM NADH (no TR or Trxh).",
"The results indicate that NADH, purchased from either Sigma or Roche, could act as an electron donor in both the DTNB and insulin reduction assays.",
"However, the rate of reduction was lower than the rate observed with NADPH as a cofactor.",
"It was estimated that the rate of insulin reduction utilizing NADH as an electron donor was approximately 25-50% when compared to the maximum rate using NADPH.",
"Furthermore, it was estimated that the rate of DTNB reduction utilizing NADH as an electron donor was approximately 5-10% of the maximum rate using NADPH.",
"Similar results were observed using the oleosin-thioredoxin-reductase-linker thioredoxin fusion protein on Arabidopsis oil bodies instead of the E. coli thioredoxin-reductase and thioredoxin.",
"Example 5 Production of Multimeric Immunoglobulin Protein in Plant Seed Cells and Capture on Oil Bodies Using Protein A-Oleosin Fusion Proteins 1—Production of Multimeric Immunoglobulin Protein in Plant Seed Cells For expression of multimeric-protein-complexes containing multimeric-immunoglobulin-complexes, the cDNA sequences encoding individual light and heavy chains can be isolated from; 1) cell lines expressing a particular antibody, such as clonal B cell lines, or a hybridoma cell line, or 2) may be a recombinant antibody, assembled by combining select light and heavy chain variable domains and available light and heavy chain constant domain sequences, respectively.",
"Variable domains with specific binding properties may be isolated from screening populations of such sequences, usually in the form of a single-chain Fv phage display library.",
"Starting from known nucleic acid sequences and a source of light and heavy chains, the mature polypeptide coding sequences of each chain is isolated with a secretion signal sequence.",
"The signal sequence can be the native antibody sequence or derived from a known secreted plant sequence (e.g.",
"a PR sequence from Arabidopsis or tobacco).",
"The addition of a plant secretion signal sequence to both light and heavy chain mature coding sequences is carried out by standard molecular biology techniques.",
"PCR fusion is used routinely to make such modifications.",
"Secretion signal sequences are included to target the light and heavy immunoglobulin polypeptides for secretion from the cell and further assembly of the two chains into a multimeric-immunoglobulin-complex.",
"For expression in transgenic plant seeds, an expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—light chain sequence, and 3) a regulatory sequence to terminate transcription.",
"A second expression cassette is assembled comprising: 1) a regulatory promoter sequence to provide expression in plant seeds, 2) the secretion signal—heavy chain sequence, and 3) a regulatory sequence to terminate transcription.",
"Each of the antibody chain expression cassettes is cloned individually into an Agrobacterium plant transformation vector or is combined into a single transformation vector with both expression cassettes.",
"In both cases, the expression cassettes are cloned into plant transformation vectors, between the left and right delineating border sequences, and adjacent to a plant selectable marker cassette.",
"Each plant transformation vector is transformed into Agrobacterium.",
"The resulting Agrobacterium strains are used to infect plant tissues.",
"Transgenic plant material is regenerated and viable transgenic plants are selected.",
"When individual transformation vectors are used, the transgenic plant lines that are produced, expressing either light or heavy chain sequences, are crossed to generate a single plant line expressing both chains in the same plant cell.",
"When a single transformation vector, containing both light and heavy expression cassettes, is used, the initial transgenic plant line produces both light and heavy chain sequences in the same plant cell.",
"2—Production of Transgenic Oil Bodies which Display Protein a for the Capture of Immunoglobulins To capture and display immunoglobulin protein on oil bodies, oil bodies are engineered to display an immunoglobulin binding protein.",
"In this example, the well-known antibody-binding domains from Protein A are used.",
"Based on the known sequence for Protein A from Staphylococcus aureus, PCR primers are designed to isolate the five consecutive Ig-binding domains from the bacterial Protein A sequence.",
"Primers are designed to allow cloning of the Protein A sequence as either an N-terminal or C-terminal fusion to an oleosin sequence for targeting to oil bodies.",
"The sequence that encodes an in-frame translational fusion between Protein A and oleosin is cloned into a plant expression cassette for seed-specific expression.",
"The final cassette consists of a regulatory promoter sequence that provides expression in seeds, the Protein A-oleosin fusion sequence, and a regulatory sequence to terminate transcription.",
"The Protein A-oleosin expression cassette is cloned into a plant transformation vector compatible with Agrobacterium-mediated plant transformation.",
"The transformation vector comprises left and right border sequences flanking the Protein A-oleosin expression cassette and an adjacent plant selectable marker cassette.",
"The Agrobacterium strain containing this vector is used to infect plant tissues and subsequent regeneration and selection from transgenic plant material to create transgenic plants.",
"3—Capture and Display of Multimeric-Immunoglobulins on Oil Bodies Displaying Protein A Having produced light and heavy chain multimeric immunoglobulin complexes in one transgenic plant line and the display of Protein A on oil bodies through the oil body targeting of a Protein A-oleosin fusion protein in a second plant line, at least two embodiments can be used to capture the immunoglobulin complex on the Protein A oil bodies.",
"In the first embodiment, transgenic seed from both the immunoglobulin and the Protein A-oleosin expression lines is combined in an optimum ratio and then ground together such that the disrupted material from both seed lines would be combined in the same extract.",
"The combined seed extracts are mixed and/or incubated under conditions that allow maximum recovery of the immunoglobulin by Protein A.",
"The oil body fraction is separated using standard phase separation techniques (e.g.",
"centrifugation).",
"The recovered oil body fraction contains both native oil bodies, from the immunoglobulin expression line, and transgenic Protein A oil bodies from the Protein A-oleosin expression line.",
"In a second embodiment, the plant lines expressing the immunoglobulin complex and the Protein A-oleosin fusion are crossed and individual plant lines expressing both components are identified and propagated.",
"In this approach, the immunoglobulin complex and the Protein A-oleosin fusion are produced in different cellular compartments of the same plant seed cell.",
"Seed from the double transgenic line is ground to disrupt the cellular material and mix the contents of all cellular compartments, including combining the immunoglobulin in the extracellular compartment and the Protein A-oleosin on the oil body in the cytosolic compartment.",
"The material is mixed and/or incubated under conditions to allow maximum recovery of the immunoglobulin by Protein A, and the oil body fraction is separated by phase separation techniques.",
"The recovered oil body fraction contains the displayed Protein A and the capture immunoglobulin complex.",
"Example 6 Production of Assembled Multimeric-Immunoglobulin-Complexes as Fusions with Oil Body Targeting Domains Individual polypeptides are produced as a fusion protein with oil body targeting sequences (e.g.",
"oleosin) for display on oil bodies.",
"It has been found that the individual subunits of naturally associating heterodimeric proteins can be co-produced as individual oleosin fusions and still associate as an active heterodimer on the surface of the oil body.",
"In this example, the heterodimer is the light and heavy chain subunits, or derived portions thereof, of an immunoglobulin complex.",
"Production of an Immunoglobulin Fab Complex on Oil Bodies.",
"The mature light chain sequence, lacking the secretion signal sequence, is attached as an in-frame N-terminal fusion to an oleosin sequence.",
"This fusion sequence is assembled into a seed-specific expression cassette consisting of a seed-specific promoter sequence, the light chain-oleosin fusion sequence, and a transcriptional terminator sequence.",
"The expression cassette is inserted between the left and right border markers, adjacent to a plant selectable marker cassette, of a transformation vector.",
"The transformation vector, in Agrobacterium, is used to infect plants and generate transgenic plants.",
"An equivalent construct for the heavy chain subunit, comprising the variable and constant heavy chain domains, is also attached as an in-frame fusion to oleosin and assembled into an expression cassette for seed-specific expression.",
"The expression cassette can be a part of a separate transformation vector for the generation of a separate transgenic line, or the heavy chain expression cassette can be combined together with the light chain cassette into a single transformation vector.",
"If light and heavy chain expression cassettes are transformed into plants on separate transformation vectors, the individual plant lines are crossed to create a single line expressing both heterodimer subunit-oleosin fusions in the same plant cell.",
"Seed from the double transgenic line, or a single transgenic line generated from the dual expression vector, is extracted to isolate oil bodies.",
"The seed material is ground to release the cellular contents and oil bodies are isolated by phase separation.",
"The targeting of both light and heavy chain sequence to oil bodies, as oleosin fusions, allows the association of the immunoglobulin complex on the surface of the oil body.",
"Similar configurations, using the entire heavy chain sequence in combination with the entire light chain sequence, or using the variable domains from both the light and heavy chain sequences, are constructed to assemble different types of heteromultimeric-immunoglobulin-complexes (e.g., heterodimers) on the surface of oil bodies.",
"The present invention should therefore not be seen as limited to the particular embodiments described herein, but rather, it should be understood that the present invention has wide applicability with respect to protein expression generally.",
"Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.",
"SUMMARY OF SEQUENCES SEQ ID NOs:1-4 set forth primers which were synthesized for the isolation of the thioredoxin h (Trxh) and thioredoxin reductase genes from Arabidopsis, as described in Example 1.SEQ ID NOs:5-7 set forth primers which were designed to code for a specific linker peptide between thioredoxin reductase and thioredoxin proteins, as described in Example 2.SEQ ID NOs:8, 10 and 11 set forth the nucleotide sequence and the deduced amino acid sequence of the NADPH thioredoxin reductase sequence isolated herein as described in Example 1.SEQ ID NOs:9 and 11, respectively, set forth the nucleotide sequence of the published NADPH thioredoxin reductase sequence (ATTHIREDB) and the deduced amino acid sequence.",
"SEQ ID NO:12 sets forth the deduced amino acid sequence of the published NADPH thioredoxin reductase sequence.",
"SEQ ID NO:13 sets forth the deduced amino acid sequence of the NADPH reductase sequence isolated in this report.",
"SEQ ID NOs:14 and 15 set forth the nucleotide sequence of the phaseolin promoter-Arabidopsis Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequence.",
"The Trxh coding sequence and its deduced amino acid sequence is indicated.",
"The phaseolin promoter corresponds to nucleotide 6-1554, and the phaseolin terminator corresponds to nucleotide sequence 1905-3124.The promoter was furnished with a PstI site (nt 1-6) and the terminator was furnished with a HindIII site (nt 1898-1903) and a KpnI site (nt 3124-3129) to facilitate cloning.",
"SEQ ID NOs:16, 17 and 18 set forth the nucleotide sequence of the phaseolin promoter-oleosin Trxh-phaseolin terminator sequence described in Example 2, and the deduced amino acid sequences.",
"The oleosin-Trxh coding sequence and the deduced amino acid sequences are indicated in SEQ ID NO:16.As in SEQ ID NO:14, the phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1908-2147) is indicated in italics.",
"The Trxh coding sequence corresponds to nt 2314-2658.The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:19, 20 and 21 set forth the nucleotide sequence of the phaseolin promoter-Trxh oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.",
"The Trxh oleosin-coding sequence and its deduced amino acid sequences are indicated in SEQ ID NO:19.As in SEQ ID NOs:14 and 16, the phaseolin promoter corresponds to nucleotide 6-1554.The Trxh coding sequence corresponds to nt 1555-1896.The sequence encoding oleosin corresponds to nt 1897-2658, the intron in this sequence (nt 2250-2489) is indicated in italics.",
"The phaseolin terminator corresponds to nucleotide sequence 2664-3884.SEQ ID NO:22 and 23 set forth the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequence.",
"The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated in SEQ ID NO:22.The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2556 and the deduced amino acid is set forth in SEQ ID NO:23.The phaseolin terminator corresponds to nucleotide sequence 2563-3782.SEQ ID NOs:24, 25 and 26 show the nucleotide sequence of the phaseolin promoter-oleosin thioredoxin-reductase-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.",
"The oleosin-thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.",
"The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt 1980-2147) is indicated in italics.",
"The thioredoxin-reductase coding sequence corresponds to nt 2314-3315.The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NOs:27, 28 and 29 show the nucleotide sequence of the phaseolin promoter-thioredoxin-reductase oleosin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.",
"The thioredoxin-reductase coding sequence and its deduced amino acid sequence is indicated.",
"The phaseolin promoter corresponds to nucleotide 6-1554.The thioredoxin-reductase coding sequence corresponds to nt 1555-2553.The sequence encoding oleosin corresponds to nt 2554-3315, the intron in this sequence (nt 2751-3146) is indicated in italics.",
"The phaseolin terminator corresponds to nucleotide sequence 3321-4540.SEQ ID NO:30, 31 and 32 show the sequence of the phaseolin promoter-oleosin-Mlep thioredoxin-reductase/thioredoxin-phaseolin terminator sequence as described in Example 2, and the deduced amino acid sequences.",
"The oleosin-Mlep thioredoxin-reductase/thioredoxin coding sequence and its deduced amino acid sequence is indicated.",
"The phaseolin promoter corresponds to nucleotide 6-1554.The sequence encoding oleosin corresponds to nt 1555-2313, the intron in this sequence (nt) is indicated in italics.",
"The Mlep thioredoxin-reductase/thioredoxin coding sequence corresponds to nt 2314-3690.The phaseolin terminator corresponds to nucleotide sequence 3698-4917.SEQ ID NOs:33, 34 and 35 set forth the nucleotide sequence of the phaseolin promoter-oleosin-thioredoxin-reductase-linker-thioredoxin-phaseolin terminator region of pSBS2542, and the deduced amino acid sequences.",
"The deduced amino acid sequence of oleosin-thioredoxin-reductase-linker-thioredoxin is also shown in SEQ ID NO:33.Amino acids representing oleosin are set forth at positions 1-173, those amino acids representing thioredoxin-reductase are set forth at positions 174-501, those amino acids representing the linker or spacer peptide are set forth at positions 501-524, and those representing thioredoxin are set forth at positions 525-636.SEQ ID NOs:38 and 39 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin h (Trx h 1) and the encoded protein, respectively.",
"SEQ ID NOs:40 and 41 set forth the nucleotide sequence of Arabidopsis Thaliana Thioredoxin Reductase (NTR1) and the encoded protein, respectively.",
"SEQ ID NOs:42 and 43 set forth the nucleotide sequence of E. Coli Thioredoxin (TrxA) and the encoded protein, respectively.",
"SEQ ID NOs:44 and 45, set forth the nucleotide sequence of E. Coli Thioredoxin Reductase and the encoded protein, respectively.",
"SEQ ID NOs:46 and 47 set forth the nucleotide sequence of Human Thioredoxin and the encoded protein, respectively.",
"SEQ ID NOs:48 and 49, set forth the nucleotide sequence of Human Thioredoxin Reductase and the encoded protein, respectively.",
"SEQ ID NOs:50 and 51, respectively, set forth the nucleotide sequence of Mycobacterium leprae Thioredoxin-Thioredoxin Reductase and the encoded protein, respectively.",
"SEQ ID NOs:52-313 are described in Table 5.TABLE 5 EXAMPLES OF REDOX PROTEINS SWISS PROTEIN IDENTIFIER SEQ.",
"ID NO.",
"(in parenthesis) PLANT THIOREDOXINS Thioredoxin f-type 52 (Q9XFH8) Thioredoxin F-type 1, chloroplast precursor (TRX- F1).",
"- Arabidopsis thaliana (Mouse-ear cress) 53 (Q9XFH9) Thioredoxin F-type 2, chloroplast precursor (TRX- F2).",
"{GENE: AT5G16400 OR MQK4.13} - Arabidopsis thaliana (Mouse-ear cress) 54 (O48897) Thioredoxin F-type, chloroplast precursor (TRX-F).",
"{GENE: TRXF} - Brassica napus (Rape) 55 (O81332) Thioredoxin F-type, chloroplast precursor (TRX-F).",
"- Mesembryanthemum crystallinum (Common ice plant) 56 (P29450) Thioredoxin F-type, chloroplast precursor (TRX-F).",
"- Pisum sativum (Garden pea) 57 (P09856) Thioredoxin F-type, chloroplast precursor (TRX-F).",
"- Spinacia oleracea (Spinach) Thioredoxin m-type 58 (P06544) Thioredoxin 1 (TRX-1) (Thioredoxin M).",
"{GENE: TRXA} - Anabaena sp.",
"(strain PCC 7119) 59 (O48737) Thioredoxin M-type 1, chloroplast precursor (TRX- M1).",
"{GENE: AT1G03680 OR F21B7_7 OR F21B7.28} - Arabidopsis thaliana (Mouse-ear cress) 60 (Q9SEU8) Thioredoxin M-type 2, chloroplast precursor (TRX- M2).",
"{GENE: AT4G03520 OR F9H3.15 OR T5L23.1} - Arabidopsis thaliana (Mouse-ear cress) 61 (Q9SEU7) Thioredoxin M-type 3, chloroplast precursor (TRX- M3).",
"{GENE: AT2G15570 OR F9O13.12} - Arabidopsis thaliana (Mouse-ear cress) 62 (Q9SEU6) Thioredoxin M-type 4, chloroplast precursor (TRX- M4).",
"- Arabidopsis thaliana (Mouse-ear cress) 63 (Q9XGS0) Thioredoxin M-type, chloroplast precursor (TRX-M).",
"- Brassica napus (Rape) 64 (P23400) Thioredoxin M-type, chloroplast precursor (TRX-M) (Thioredoxin CH2).",
"{GENE: TRXM} - Chlamydomonas reinhardtii 65 (Q41864) Thioredoxin M-type, chloroplast precursor (TRX-M).",
"{GENE: TRM1} - Zea mays (Maize) 66 (Q9ZP20) Thioredoxin M-type, chloroplast precursor (TRX-M).",
"- Oryza sativa (Rice) 67 (P48384) Thioredoxin M-type, chloroplast precursor (TRX-M).",
"- Pisum sativum (Garden pea) 68 (P07591) Thioredoxin M-type, chloroplast precursor (TRX-M).",
"- Spinacia oleracea (Spinach) 69 (Q9ZP21) Thioredoxin M-type, chloroplast precursor (TRX-M).",
"- Triticum aestivum (Wheat) 70 (P12243) Thioredoxin 1 (TRX-1) (Thioredoxin M).",
"{GENE: TRXA OR TRXM} - Synechococcus sp.",
"(strain PCC 7942) (Anacystis nidulans R2) 71 (P37395) Thioredoxin.",
"{GENE: TRXA OR TRX} - Cyanidium caldarium [Chloroplast] 72 (O22022) Thioredoxin.",
"{GENE: TRXA OR TRXM} - Cyanidioschyzon merolae [Chloroplast] 73 (P50338) Thioredoxin.",
"{GENE: TRXA} - Griffithsia pacifica [Chloroplast] 74 (P50254) Thioredoxin.",
"{GENE: TRXA} - Porphyra yezoensis [Chloroplast] 75 (P51225) Thioredoxin.",
"{GENE: TRXA} - Porphyra purpurea [Chloroplast] Thioredoxin h-type 76 (P29448) Thioredoxin H-type 1 (TRX-H-1).",
"{GENE: TRX1 OR AT3G51030 OR F24M12.70} - Arabidopsis thaliana (Mouse- ear cress) 77 (P20857) Thioredoxin 2 (TRX-2).",
"{GENE: TRXB} - Anabaena sp.",
"(strain PCC 7120) 78 (Q42388) Thioredoxin H-type 1 (TRX-H-1) (Pollen coat protein).",
"{GENE: THL-1 OR BOPC17} - Brassica napus (Rape), Brassica oleracea (Cauliflower) 79 (P29449) Thioredoxin H-type 1 (TRX-H1).",
"- Nicotiana tabacum (Common tobacco) 80 (Q38879) Thioredoxin H-type 2 (TRX-H-2).",
"{GENE: TRX2 OR AT5G39950 OR MYH19.14} - Arabidopsis thaliana (Mouse-ear cress) 81 (Q39362) Thioredoxin H-type 2 (TRX-H-2).",
"{GENE: THL-2} - Brassica napus (Rape) 82 (Q07090) Thioredoxin H-type 2 (TRX-H2).",
"- Nicotiana tabacum (Common tobacco) 83 (Q42403) Thioredoxin H-type 3 (TRX-H-3).",
"{GENE: TRX3 OR AT5G42980 OR MBD2.18} - Arabidopsis thaliana (Mouse-ear cress) 84 (Q39239) Thioredoxin H-type 4 (TRX-H-4).",
"{GENE: TRX4} - Arabidopsis thaliana (Mouse-ear cress) 85 (Q39241) Thioredoxin H-type 5 (TRX-H-5).",
"{GENE: TRX5} - Arabidopsis thaliana (Mouse-ear cress) 86 (O64432) Thioredoxin H-type (TRX-H).",
"{GENE: PEC-2} - Brassica rapa (Turnip) 87 (P80028) Thioredoxin H-type (TRX-H) (Thioredoxin CH1).",
"{GENE: TRXH} - Chlamydomonas reinhardtii 88 (Q96419) Thioredoxin H-type (TRX-H).",
"- Fagopyrum esculentum (Common buckwheat) 89 (Q42443) Thioredoxin H-type (TRX-H) (Phloem sap 13 kDa protein-1).",
"- Oryza sativa (Rice) 90 (O65049) Thioredoxin H-type (TRX-H).",
"{GENE: SB09} - Picea mariana (Black spruce) 91 (Q43636) Thioredoxin H-type (TRX-H).",
"- Ricinus communis (Castor bean) 92 (O64394) Thioredoxin H-type (TRX-H) (TrxTa).",
"- Triticum aestivum (Wheat) 93 (P29429) Thioredoxin.",
"- Emericella nidulans (Aspergillus nidulans) VIRUSES, BACTERIA AND FUNGI THIOREDOXINS 94 (P80579) Thioredoxin (TRX).",
"{GENE: TRXA} - Alicyclobacillus acidocaldarius (Bacillus acidocaldarius) 95 (O28137) Thioredoxin.",
"{GENE: AF2145} - Archaeoglobus fulgidus 96 (P14949) Thioredoxin (TRX).",
"{GENE: TRXA OR TRX} - Bacillus subtilis 97 (P00276) Thioredoxin.",
"{GENE: NRDC} - Bacteriophage T4 98 (O51088) Thioredoxin (TRX).",
"{GENE: TRXA OR BB0061} - Borrelia burgdorferi (Lyme disease spirochete) 99 (P57653) Thioredoxin (TRX).",
"{GENE: TRXA OR BU597} - Buchnera aphidicola (subsp.",
"Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 100 (O51890) Thioredoxin (TRX).",
"{GENE: TRXA} - Buchnera aphidicola (subsp.",
"Schizaphis graminum) 101 (P10472) Thioredoxin (TRX).",
"{GENE: TRXA} - Chlorobium limicola f.sp.",
"thiosulfatophilum 102 (Q9PJK3) Thioredoxin (TRX).",
"{GENE: TRXA OR TC0826} - Chlamydia muridarum 103 (Q9Z7P5) Thioredoxin (TRX).",
"{GENE: TRXA OR CPN0659 OR CP0088} - Chlamydia pneumoniae (Chlamydophila pneumoniae) 104 (P52227) Thioredoxin (TRX).",
"{GENE: TRXA} - Chlamydia psittaci (Chlamydophila psittaci) 105 (O84544) Thioredoxin (TRX).",
"{GENE: TRXA OR CT539} - Chlamydia trachomatis 106 (P00275) Thioredoxin C-1.- Corynebacterium nephridii 107 (P07887) Thioredoxin C-2.- Corynebacterium nephridii 108 (P52228) Thioredoxin C-3.- Corynebacterium nephridii 109 (P09857) Thioredoxin (TRX).",
"{GENE: TRXA} - Chromatium vinosum 110 (P21609) Thioredoxin (TRX).",
"{GENE: TRXA} - Clostridium litorale (Bacterium W6) 111 (P81108) Thioredoxin (TRX) (Fragment).",
"{GENE: TRXA} - Clostridium sporogenes 112 (P81109) Thioredoxin (TRX) (Fragment).",
"{GENE: TRXA} - Clostridium sticklandii 113 (Q9UW02) Thioredoxin (Allergen Cop c 2).",
"- Coprinus comatus (Shaggy mane) 114 (P29445) Thioredoxin 1.",
"{GENE: TRXA OR TRX1} - Dictyostelium discoideum (Slime mold) 115 (P29446) Thioredoxin 2 (Fragment).",
"{GENE: TRXB OR TRX2} - Dictyostelium discoideum (Slime mold) 116 (P29447) Thioredoxin 3.",
"{GENE: TRXC OR TRX3} - Dictyostelium discoideum (Slime mold) 117 (P00274) Thioredoxin 1 (TRX1) (TRX).",
"{GENE: TRXA OR TSNC OR FIPA OR B3781} - Escherichia coli, Salmonella typhimurium 118 (P52232) Thioredoxin-like protein SLR0233.",
"{GENE: SLR0233} - Synechocystis sp.",
"(strain PCC 6803) 119 (P33636) Thioredoxin 2 (Trx2).",
"{GENE: TRXC OR B2582 OR Z3867 OR ECS3448} - Escherichia coli, Escherichia coli O157:H7 120 (P21610) Thioredoxin (TRX).",
"{GENE: TRXA} - Eubacterium acidaminophilum 121 (P43785) Thioredoxin (TRX).",
"{GENE: TRXA OR TRXM OR HI0084} - Haemophilus influenzae 122 (P43787) Thioredoxin-like protein HI1115.",
"{GENE: HI1115} - Haemophilus influenzae 123 (P56430) Thioredoxin (TRX).",
"{GENE: TRXA OR HP0824 OR JHP0763} - Helicobacter pylori (Campylobacter pylori), Helicobacter pylori J99 (Campylobacter pylori J99) 124 (Q9S386) Thioredoxin (EC 1.6.4.5) {GENE: TRXA} - Listeria monocytogenes 125 (Q57755) Thioredoxin.",
"{GENE: TRX OR MJ0307} - Methanococcus jannaschii 126 (P47370) Thioredoxin (TRX).",
"{GENE: TRXA OR TRX OR MG124} - Mycoplasma genitalium 127 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].",
"{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 128 (P75512) Thioredoxin (TRX).",
"{GENE: TRXA OR TRX OR MPN263 OR MP570} - Mycoplasma pneumoniae 129 (O30974) Thioredoxin (TRX).",
"{GENE: TRXA} - Mycobacterium smegmatis 130 (P52229) Thioredoxin (TRX) (MPT46).",
"{GENE: TRXA OR TRX OR TRXC OR RV3914 OR MT4033 OR MTV028.05} - Mycobacterium tuberculosis 131 (P42115) Thioredoxin.",
"{GENE: TRX} - Neurospora crassa 132 (P34723) Thioredoxin.",
"{GENE: TRXA} - Penicillium chrysogenum 133 (Q9X2T1) Thioredoxin (TRX).",
"{GENE: TRXA OR TRX OR PA5240} - Pseudomonas aeruginosa 134 (P10473) Thioredoxin (TRX).",
"{GENE: TRXA} - Rhodospirillum rubrum 135 (P08058) Thioredoxin (TRX).",
"{GENE: TRXA} - Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) 136 (Q9ZEE0) Thioredoxin (TRX).",
"{GENE: TRXA OR RP002} - Rickettsia prowazekii 137 (P33791) Thioredoxin (TRX) (Fragment).",
"{GENE: TRXA} - Streptomyces aureofaciens 138 (P52230) Thioredoxin (TRX).",
"{GENE: TRXA OR SCH24.11C} - Streptomyces coelicolor 139 (Q05739) Thioredoxin (TRX).",
"{GENE: TRXA} - Streptomyces clavuligerus 140 (P52231) Thioredoxin (TRX).",
"{GENE: TRXA OR SLR0623} - Synechocystis sp.",
"(strain PCC 6803) 141 (P73263) Thioredoxin-like protein SLR1139.",
"{GENE: SLR1139} - Synechocystis sp.",
"(strain PCC 6803) 142 (P52233) Thioredoxin (TRX).",
"{GENE: TRXA} - Thiobacillus ferrooxidans 143 (P96132) Thioredoxin (TRX) (Fragment).",
"{GENE: TRXA} - Thiocapsa roseopersicina 144 (P81110) Thioredoxin (TRX) (Fragment).",
"{GENE: TRXA} - Tissierella creatinophila 145 (O83889) Thioredoxin (TRX).",
"{GENE: TRXA OR TP0919} - Treponema pallidum ANIMAL THIOREDOXIN 146 (O97680) Thioredoxin.",
"{GENE: TXN} - Bos taurus (Bovine) 147 (Q95108) Thioredoxin, mitochondrial precursor (MT-TRX).",
"{GENE: TXN2} - Bos taurus (Bovine) 148 (Q09433) Thioredoxin.",
"{GENE: B0228.5} - Caenorhabditis elegans 149 (P99505) Thioredoxin (Fragment).",
"{GENE: TXN} - Canis familiaris (Dog 150 (P08629) Thioredoxin.",
"{GENE: TXN} - Gallus gallus (Chicken) 151 (P47938) Thioredoxin (Deadhead protein).",
"{GENE: DHD OR CG4193} - Drosophila melanogaster (Fruit fly) 152 (P10599) Thioredoxin (ATL-derived factor) (ADF) (Surface associated sulphydryl protein) (SASP).",
"{GENE: TXN OR TRDX OR TRX} - Homo sapiens (Human) 153 (Q99757) Thioredoxin, mitochondrial precursor (MT-TRX).",
"{GENE: TXN2} - Homo sapiens (Human) 154 (P29451) Thioredoxin.",
"{GENE: TXN} - Macaca mulatta (Rhesus macaque) 155 (P10639) Thioredoxin (ATL-derived factor) (ADF).",
"{GENE: TXN} - Mus musculus (Mouse) 156 (P97493) Thioredoxin, mitochondrial precursor (MT-TRX).",
"{GENE: TXN2} - Mus musculus (Mouse) 157 (P82460) Thioredoxin (Fragment).",
"{GENE: TXN} - Sus scrofa (Pig) 158 (P08628) Thioredoxin.",
"{GENE: TXN} - Oryctolagus cuniculus (Rabbit) 159 (P11232) Thioredoxin.",
"{GENE: TXN} - Rattus norvegicus (Rat) 160 (P97615) Thioredoxin, mitochondrial precursor (MT-TRX).",
"{GENE: TXN2 OR TRX2} - Rattus norvegicus (Rat) 161 (P50413) Thioredoxin.",
"{GENE: TXN} - Ovis aries (Sheep) PLANTS THIOREDOXIN-LIKE PROTEINS 162 (O23166) THIOL-DISULFIDE INTERCHANGE LIKE PROTEIN (THIOREDOXIN-LIKE PROTEIN) {GENE: C7A10.160 OR AT4G37200 OR HCF164} - Arabidopsis thaliana (Mouse-ear cress) 163 (Q9C9Y6) Thioredoxin-like protein {GENE: F17O14.18} - Arabidopsis thaliana (Mouse-ear cress) 164 (Q9FYD5) Thioredoxin-like protein {GENE: F21E1_180} - Arabidopsis thaliana (Mouse-ear cress) 165 (Q38878) THIOREDOXIN-LIKE PROTEIN {GENE: TRX6 OR T7D17.3} - Arabidopsis thaliana (Mouse-ear cress) 166 (Q9LVI2) Thioredoxin-like protein - Arabidopsis thaliana (Mouse-ear cress) 167 (Q9SCN9) Thioredoxin-like protein {GENE: T4D2.150} - Arabidopsis thaliana (Mouse-ear cress) 168 (Q9SRD7) Thioredoxin-like protein, 49720-48645 {GENE: F28O16.13} - Arabidopsis thaliana (Mouse-ear cress) 169 (Q9SU84) THIOREDOXIN-LIKE PROTEIN {GENE: T16L4.180 OR AT4G29670} - Arabidopsis thaliana (Mouse-ear cress) 170 (Q9SWG6) Thioredoxin-like protein {GENE: TRX} - Hordeum bulbosum 171 (Q9SWG4) Thioredoxin-like protein {GENE: TRX} - Lolium perenne (Perennial ryegrass) 172 (Q9AS75) Thioredoxin-like protein {GENE: P0028E10.17} - Oryza sativa (Rice) 173 (O04002) CDSP32 protein (Chloroplast Drought-induced Stress Protein of 32 kDa) - Solanum tuberosum (Potato) 174 (Q9SWG5) Thioredoxin-like protein {GENE: TRX} - Secale cereale (Rye) 175 (Q9SP36) Thioredoxin-like protein (Fragment) {GENE: TRX} - Secale cereale (Rye) 176 (Q9U515) Thioredoxin-like protein - Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-LIKE PROTEINS 177 (P43221) Thiol: disulfide interchange protein tlpA (Cytochrome c biogenesis protein tIpA).",
"{GENE: TLPA} - Bradyrhizobium japonicum 178 (P43787) Thioredoxin-like protein HI1115.",
"{GENE: HI1115} - Haemophilus influenzae 179 (Q9GUP7) Thioredoxin-like protein {GENE: TRXLP} - Leishmania major 180 (Q9UVH0) Thioredoxin-like protein - Mortierella alpina 181 (P95355) Thioredoxin-like protein - Neisseria gonorrhoeae 182 (Q98G37) Thioredoxin-like protein {GENE: MLL3505} - Rhizobium loti (Mesorhizobium loti) 183 (P36893) Thiol: disulfide interchange protein helX precursor (Cytochrome c biogenesis protein helX).",
"{GENE: HELX} - Rhodobacter capsulatus (Rhodopseudomonas capsulata) 184 (P52232) Thioredoxin-like protein SLR0233.",
"{GENE: SLR0233} - Synechocystis sp.",
"(strain PCC 6803) 185 (P73263) Thioredoxin-like protein SLR1139.",
"{GENE: SLR1139} - Synechocystis sp.",
"(strain PCC 6803) 186 (Q9USR1) Thioredoxin-like protein {GENE: SPBC577.08C} - Schizosaccharomyces pombe (Fission yeast) 187 (Q9R788) Thioredoxin {GENE: TPTRX} - Treponema pallidum ANIMALS THIOREDOXIN-LIKE PROTEINS 188 (Q9UAV4) F46E10.9 PROTEIN (THIOREDOXIN-LIKE PROTEIN DPY-11) {GENE: F46E10.9 OR DPY-11} - Caenorhabditis elegans 189 (Q9N2K6) Thioredoxin-like protein (Y54E10A.3 protein) (Thioredoxin-like protein TXL) {GENE: TXL OR Y54E10A.3} - Caenorhabditis elegans 190 (Q9VRP3) THIOREDOXIN-LIKE PROTEIN TXL (CG5495 PROTEIN) {GENE: TXL OR CG5495} - Drosophila melanogaster (Fruit fly) 191 (O43396) Thioredoxin-like protein (32 kDa thioredoxin-related protein).",
"{GENE: TXNL OR TRP32 OR TXL} - Homo sapiens (Human) 192 (O76003) Thioredoxin-like protein - Homo sapiens (Human) 193 (Q9S753) THIOREDOXIN-LIKE PROTEIN {GENE: TRX} - Phalaris coerulescens 194 (O77404) TRYPAREDOXIN - Trypanosoma brucei brucei PLANT THIOREDOXIN-REDUCTASES 195 (Q39243) Thioredoxin-reductase 1 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 1) (NTR 1).",
"{GENE: NTR1 OR AT4G35460 OR F15J1.30} - Arabidopsis thaliana (Mouse-ear cress) 196 (Q39242) Thioredoxin-reductase 2 (EC 1.6.4.5) (NADPH- dependent thioredoxin-reductase 2) (NTR 2).",
"{GENE: NTR2 OR AT2G17420 OR F5J6.18} - Arabidopsis thaliana (Mouse-ear cress) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 197 (O66790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR AQ_500} - Aquifex aeolicus 198 (P80880) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (General stress protein 35) (GSP35).",
"{GENE: TRXB} - Bacillus subtilis 199 (P94284) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR BB0515} - Borrelia burgdorferi (Lyme disease spirochete) 200 (P57399) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR BU314} - Buchnera aphidicola (subsp.",
"Acyrthosiphon pisum) (Acyrthosiphon pisum symbiotic bacterium) 201 (P81433) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB} - Buchnera aphidicola (subsp.",
"Schizaphis graminum) 202 (Q9PKT7) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR TC0375} - Chlamydia muridarum 203 (Q9Z8M4) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR CPN0314 OR CP0444} - Chiamydia pneumoniae (Chlamydophila pneumoniae) 204 (O84101) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR CT099} - Chlamydia trachomatis 205 (P52213) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB} - Clostridium litorale (Bacterium W6) 206 (P39916) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB} - Coxiella burnetii 207 (P09625) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR B0888 OR Z1232 OR ECSO973} - Escherichia coli, Escherichia coli O157: H7 208 (P50971) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB} - Eubacterium acidaminophilum 209 (P43788) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR HI1158} - Haemophilus influenzae 210 (Q9ZL18) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR JHP0764} - Helicobacter pylori J99 (Campylobacter pylori J99) 211 (P56431) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR HP0825} - Helicobacter pylori (Campylobacter pylori) 212 (O32823) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR LMO2478} - Listeria monocytogenes 213 (P47348) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR MG102} - Mycoplasma genitalium 214 (P46843) Bifunctional thioredoxin-reductase/thioredoxin [Includes: Thioredoxin-reductase (EC 1.6.4.5) (TRXR); Thioredoxin].",
"{GENE: TRXB/A OR TRX OR ML2703} - Mycobacterium leprae 215 (P75531) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR MPN240 OR MP591} - Mycoplasma pneumoniae 216 (O30973) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB} - Mycobacterium smegmatis 217 (P52214) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (TR).",
"{GENE: TRXB OR RV3913 OR MT4032 OR MTV028.04} - Mycobacterium tuberculosis 218 (P51978) Thioredoxin-reductase (EC 1.6.4.5).",
"{GENE: CYS-9} - Neurospora crassa 219 (P43496) Thioredoxin-reductase (EC 1.6.4.5).",
"{GENE: TRXB} - Penicillium chrysogenum 220 (Q9ZD97) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR RP445} - Rickettsia prowazekii 221 (Q92375) Thioredoxin-reductase (EC 1.6.4.5).",
"{GENE: SPBC3F6.03} - Schizosaccharomyces pombe (Fission yeast) 222 (Q05741) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB} - Streptomyces clavuligerus 223 (P52215) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR SCH24.12} - Streptomyces coelicolor 224 (O83790) Thioredoxin-reductase (EC 1.6.4.5) (TRXR).",
"{GENE: TRXB OR TP0814} - Treponema pallidum 225 (P80892) Thioredoxin-reductase (EC 1.6.4.5) (TRXR) (Fragment).",
"{GENE: TRXB} - Vibrio fischeri 226 (P29509) Thioredoxin-reductase 1 (EC 1.6.4.5).",
"{GENE: TRR1 OR YDR353W OR D9476.5} - Saccharomyces cerevisiae (Baker's yeast) 227 (P38816) Thioredoxin-reductase 2, mitochondrial precursor (EC 1.6.4.5).",
"{GENE: TRR2 OR YHR106W} - Saccharomyces cerevisiae (Baker's yeast) ANIMAL THIOREDOXIN-REDUCTASES 228 (O62768) Thioredoxin-reductase (EC 1.6.4.5).",
"{GENE: TXNRD1} - Bos taurus (Bovine) 229 (Q17745) Thioredoxin-reductase (EC 1.6.4.5).",
"{GENE: C06G3.7} - Caenorhabditis elegans 230 (Q16881) Thioredoxin-reductase (EC 1.6.4.5).",
"{GENE: TXNRD1} - Homo sapiens (Human) 231 (Q25861) Thioredoxin-reductase (EC 1.6.4.5) (TrxR).",
"{GENE: TR OR GR} - Plasmodium falciparum (isolate FCH-5) Other thioredoxin-reductases PLANTS THIOREDOXIN-REDUCTASES 232 (O22229) Thioredoxin-reductase {GENE: AT2G41680} - Arabidopsis thaliana (Mouse-ear cress) 233 (Q39951) NADPH thioredoxin-reductase (Fragment} - Helianthus annuus (Common sunflower) VIRUSES, BACTERIA AND FUNGI THIOREDOXIN-REDUCTASES 234 (O28718) THioredoxin-reductase (TRXB) {GENE: AF1554} - Archaeoglobus fulgidus 235 (Q9K703) Thioredoxin-reductase (NADPH) (EC 1.6.4.5) {GENE: TRXB OR BH3571} - Bacillus halodurans 236 (Q9K7F3) Thioredoxin-reductase {GENE: BH3408} - Bacillus halodurans 237 (Q9KCZ0) Thioredoxin-reductase {GENE: BH1429} - Bacillus halodurans 238 (Q9KCZ1) Thioredoxin-reductase {GENE: BH1428} - Bacillus halodurans 239 (Q9PIY1) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR CJ0146} - Campylobacter jejuni 240 (Q9A4G3) Thioredoxin-reductase {GENE: CC2871} - Caulobacter crescentus 241 (Q97EM8) Thioredoxin-reductase {GENE: CAC3082} - Clostridium acetobutylicum 242 (Q97IU2) Thioredoxin-reductase {GENE: CAC1548} - Clostridium acetobutylicum 243 (Q9EV96) Thioredoxin-reductase {GENE: TRXB} - Clostridium sticklandii 244 (Q9RSY7) THioredoxin-reductase {GENE: DR1982} - Deinococcus radiodurans 245 (O30739) Thioredoxin-reductase (Fragment} - Enterococcus faecalis (Streptococcus faecalis) 246 (O54535) Thioredoxin-reductase {GENE: TRXB OR TRXB1_2 OR VNG6452G OR TRXB1_1 OR VNG6074G} - Halobacterium sp.",
"(strain NRC-1) [Plasmid pNRC100, and Plasmid pNRC200] 247 (P82854) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Halobacterium sp.",
"(strain NRC-1) 248 (Q9HN08) Thioredoxin-reductase {GENE: TXRB3 OR VNG2301G} - Halobacterium sp.",
"(strain NRC-1) 249 (O25779) THioredoxin-reductase (TRXB) {GENE: HP1164} - Helicobacter pylori (Campylobacter pylori) 250 (O86255) Thioredoxin-reductase {GENE: TRXB} - Klebsiella oxytoca 251 (Q9AEV9) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis (subsp.",
"lactis) (Streptococcus lactis) 252 (Q9CF34) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB2} - Lactococcus lactis (subsp.",
"lactis) (Streptococcus lactis) 253 (Q9CH02) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB1} - Lactococcus lactis (subsp.",
"lactis) (Streptococcus lactis) 254 (Q9ZFC8) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Lactococcus lactis 255 (O32822) Hypothetical 39.7 kDa protein (Fragment) - Listeria monocytogenes 256 (O26804) THioredoxin-reductase {GENE: MTH708} - Methanothermobacter thermautotrophicus 257 (P94397) Homologue of thioredoxin-reductase of Mycoplama genitalium {GENE: YCGT} - Bacillus subtilis 258 (Q98PK9) THioredoxin-reductase (EC 1.6.4.5) {GENE: MYPU_7130} - Mycoplasma pulmonis 259 (Q9JU23) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB OR NMA1538} - Neisseria meningitidis (serogroup A) 260 (Q9JZ28) Thioredoxin-reductase {GENE: NMB1324} - Neisseria meningitidis (serogroup B) 261 (Q9I0M2) Thioredoxin-reductase 1 {GENE: TRXB1 OR PA2616} - Pseudomonas aeruginosa 262 (Q9I592) Thioredoxin-reductase 2 {GENE: TRXB2 OR PA0849} - Pseudomonas aeruginosa 263 (Q9V0Q8) THioredoxin-reductase (TRXB) {GENE: TRXB OR PAB0500} - Pyrococcus abyssi 264 (Q9ZD33) THioredoxin-reductase (TRXB2) {GENE: RP514} - Rickettsia prowazekii 265 (O54079) Thioredoxin-reductase (EC 1.6.4.5) {GENE: TRXB} - Staphylococcus aureus 266 (Q9RIS2) Thioredoxin-reductase {GENE: TRXB OR TRXB2} - Streptomyces coelicolor 267 (Q9K4L6) Thioredoxin-reductase {GENE: SC5F8.08C} - Streptomyces coelicolor 268 (Q97PY2) Thioredoxin-reductase {GENE: SP1458} - Streptococcus pneumoniae 269 (Q9A0B5) Thioredoxin-reductase {GENE: SPY0850} - Streptococcus pyogenes 270 (Q97V69) Thioredoxin-reductase (trxB-2) (EC 1.6.4.5) {GENE: TRXB-2} - Sulfolobus solfataricus 271 (Q97W27) Thioredoxin-reductase (trxB-3) (EC 1.6.4.5) {GENE: TRXB-3} - Sulfolobus solfataricus 272 (Q97WJ5) Thioredoxin-reductase (trxB-1) (EC 1.6.4.5) {GENE: TRXB-1} - Sulfolobus solfataricus 273 (Q98I59) Thioredoxin-reductase {GENE: MLL2552} - Rhizobium loti (Mesorhizobium loti) 274 (Q98M06) Thioredoxin-reductase {GENE: MLL0792} - Rhizobium loti (Mesorhizobium loti) 275 (Q9UR80) 35 kDa THioredoxin-reductase HOMOLOG (FRAGMENT) {GENE: TRR1 AND YDR353W} - Saccharomyces cerevisiae (Baker's yeast) 276 (Q9ZEH4) THIOREDOXIN {GENE: TRXA OR SA0992} - Staphylococcus aureus, Staphylococcus aureus subsp.",
"aureus N315 277 (Q9S1H1) Thioredoxin-reductase (Fragment) {GENE: TRXB} - Staphylococcus xylosus 278 (Q9HJI4) Thioredoxin-reductase {GENE: TA0984} - Thermoplasma acidophilum 279 (Q9WZX3) THioredoxin-reductase {GENE: TM0869} - Thermotoga maritima 280 (Q979K8) Thioredoxin-reductase {GENE: TVG1183005} - Thermoplasma volcanium 281 (Q9PR71) Thioredoxin-reductase {GENE: TRXB OR UU074} - Ureaplasma parvum (Ureaplasma urealyticum biotype 1) 282 (Q9KSS4) Thioredoxin-reductase {GENE: VC1182} - Vibrio cholerae 283 (Q9PDD1) Thioredoxin-reductase {GENE: XF1448} - Xylella fastidiosa 284 (Q9X5F7) Thioredoxin-reductase {GENE: TRXB1} - Zymomonas mobilis ANIMAL THIOREDOXIN-REDUCTASES 285 (Q9GKW9) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Bos taurus (Bovine) 286 (Q9N2I8) Thioredoxin-reductase (EC 1.6.4.5) - Bos taurus (Bovine) 287 (Q9N2K1) Thioredoxin-reductase homolog - Caenorhabditis elegans 288 (Q9NJH3) Thioredoxin-reductase - Caenorhabditis elegans 289 (Q9VNT5) CG11401 PROTEIN (THioredoxin-reductase 2) {GENE: TRXR-2 OR CG11401} - Drosophila melanogaster (Fruit fly) 290 (O95840) Thioredoxin-reductase - Homo sapiens (Human) 291 (Q9UES8) Thioredoxin-reductase GRIM-12 - Homo sapiens (Human) 292 (Q9UH79) Thioredoxin-reductase {GENE: TR} - Homo sapiens (Human) 293 (Q9UQU8) Thioredoxin-reductase - Homo sapiens (Human) 294 (Q9NNW6) Thioredoxin-reductase TR2 (Fragment) - Homo sapiens (Human) 295 (Q9NNW7) Thioredoxin-reductase TR3 - Homo sapiens (Human) 296 (Q9P101) Thioredoxin-reductase 3 (Fragment) {GENE: TRXR3} - Homo sapiens (Human) 297 (Q9P2Y0) Thioredoxin-reductase II beta (EC 1.6.4.5) - Homo sapiens (Human) 298 (Q9H2Z5) Mitochondrial thioredoxin-reductase {GENE: TRXR2A} - Homo sapiens (Human) 299 (Q99475) KM-102-DERIVED REDUCTASE-LIKE FACTOR (THioredoxin-reductase) - Homo sapiens (Human) 300 (Q99P49) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 301 (Q9CSV5) Thioredoxin-reductase 1 (Fragment) {GENE: TXNRD1} - Mus musculus (Mouse) 302 (Q9CZE5) Thioredoxin-reductase 1 {GENE: TXNRD1} - Mus musculus (Mouse) 303 (Q9JHA7) Thioredoxin-reductase TR3 {GENE: TXNRD2 OR TR3} - Mus musculus (Mouse) 304 (Q9JLT4) Thioredoxin-reductase {GENE: TXNRD2 OR TRXR2} - Mus musculus (Mouse) 305 (Q9JMH5) Thioredoxin-reductase 2 {GENE: TXNRD2 OR TXNRD2} - Mus musculus (Mouse) 306 (Q9JMH6) Thioredoxin-reductase 1 {GENE: TXNRD1 OR TXNRD1} - Mus musculus (Mouse) 307 (O89049) Thioredoxin-reductase - Rattus norvegicus (Rat) 308 (Q9JKZ3) Thioredoxin-reductase 1 (Fragment) - Rattus norvegicus (Rat) 309 (Q9JKZ4) Thioredoxin-reductase 1 - Rattus norvegicus (Rat) 310 (Q9JLE6) Thioredoxin-reductase (Fragment) - Rattus norvegicus (Rat) 311 (Q9R1I3) NADPH-dependent thioredoxin-reductase {GENE: TRR1} - Rattus norvegicus (Rat) 312 (Q9Z0J5) Thioredoxin-reductase precursor {GENE: TRXR2} - Rattus norvegicus (Rat) 313 (Q9MYY8) Redox enzyme thioredoxin-reductase - Sus scrofa (Pig)"
]
] | Patent_10450903 |
[
[
"Adaptive comparator circuit and acoustic distance sensor comprising said circuit",
"The invention relates to an adaptive comparator circuit comprising a comparator with a threshold value input which receives a threshold voltage and a signal input which receives a voltage signal.",
"The threshold value input is connected to one pole by means of a switch and connected to the other pole of a first voltage source by means of a capacitor.",
"The threshold value input is also connected to the signal input of the comparator by means of a diode or a second voltage source.",
"The switch is controlled by the control signal of a signal transmitter.",
"When the switch is in a closed position, the capacitor is charged with the voltage of the first voltage source.",
"When the switch is in an open position, the capacitor is discharged in such a way that the threshold voltage of the signal voltage is corrected at a given time interval."
],
[
"1.An adaptive comparator circuit, particularly for an acoustic distance sensor, comprising a first comparator (1), having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage (Uref1), said comparator emits a first switching signal (S1), characterized by a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).",
"2.The adaptive comparator according to claim 1, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).",
"3.The adaptive comparator according to claim 2, characterized in that the voltage supplied by the second voltage source (7) can be regulated or adjusted.",
"4.The adaptive comparator according to claim 1, characterized in that the signal transmitter (2) emits an electric control signal (SSt) at regular intervals.",
"5.The adaptive comparator according to claim 1, characterized in that the first switch (3, 103) is a switch that can be electrically or electronically controlled by the control means (3a, 103a) and the control signal (SSt) is an electric signal that is transmitted by the signal transmitter (2) via a control output (2a) and fed to the control input (3a, 103a).",
"6.The adaptive comparator according to claim 5, characterized in that the first switch (3, 103) is a transistor (103) and the control-current terminal (3a, 103a) is the base (103) or the gate terminal of the transistor (103).",
"7.The adaptive comparator according to claim 1, characterized in that a first resistor (11) is connected between the first pole (4a) and the first threshold voltage input (1a), and a second resistor (12) is connected between the first threshold voltage input (1a) and the second pole (4b), so that the first resistor (11) and the second resistor (12) form a first voltage divider (11, 12).",
"8.The adaptive comparator according to claim 7, characterized in that first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the switch output (3c, 103c).",
"9.The adaptive comparator according to claim 1, characterized by a second comparator (20) that has a second threshold voltage input (20a) and a second signal input (20b), and that emits a second switching signal S2 when a greater voltage is present at the second signal input (20b) than at the second threshold voltage input (20a), a third resistor (13) via which the first pole (4a) is connected to the second threshold voltage input (20a), a fourth resistor (14) that, on the one hand, is connected to the second threshold voltage input (20a) and, on the other hand, to the second pole (4b) of the first voltage source, so that the third resistor (13) and the fourth resistor (14) form a second voltage divider (13, 14), a second switch (23) that is connected in series to the diode (5) and that can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator (20) emits the second switching signal S2, and otherwise it is in the open state.",
"10.The adaptive comparator according to claim 9, characterized in that a fifth resistor (15) is connected between the switch output (3c, 103c) and the second threshold voltage input (20a).",
"11.The adaptive comparator according to claim 9, characterized in that the second voltage divider (13, 14) is a potentiometer.",
"12.The adaptive comparator according to claim 1, characterized in that a sixth resistor (16) is connected between the control output (2a) and the control-current terminal (3a, 103a).",
"13.The adaptive comparator according to claim 6, characterized in that a seventh resistor (17) is connected between the control-current terminal (3a, 103a) and the first pole (4a).",
"14.The adaptive comparator according to claim 1, characterized in that the diode (5) or the second voltage source (7) is connected in series to an eighth resistor (18).",
"15.The adaptive comparator according to claim 1, characterized in that, before reaching the first signal input (1b), the voltage signal Usignal passes through a voltage follower or impedance transformer (21).",
"16.The adaptive comparator according to claim 1, characterized in that the voltage supplied by the first voltage source (4a, 4b) is greater in magnitude than the voltage of the maximum of the voltage signal Usignal.",
"17.The adaptive comparator according to claim 1, characterized in that the diode (5) is connected to at least another diode codirectionally in series.",
"18.An acoustic distance sensor, comprising a control unit (31) that at times transmits signal pulses (41) to an oscillator (32) which, during the presence of a signal pulse (41), transmits an alternating voltage to a sound transducer (34) that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer (34) as an echo as a result of reflection, and of converting them into an electric received signal, and also comprising an envelope curve shaper (37) to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a voltage signal Usignal, characterized in that the voltage signal is fed to an adaptive comparator circuit comprising a first comparator (1) having a first threshold voltage input (1a) to which a first threshold voltage (Uref1) is applied and a first signal input (1b) to which a voltage signal (Usignal) is applied and, when a voltage signal (Usignal) is present that is greater than the first threshold voltage Uref1, said comparator (1) emits a first switching signal (S1), a signal transmitter (2), especially a microcontroller or microprocessor, that at times emits a control signal (SSt), a first switch (3, 103) that has a control means (3a, 103a), a switch input (3b, 103b) and a switch output (3c, 103c), and that can be regulated or activated through the effect of the control signal (SSt) on the control means (3a, 103a) in such a way that it is in the closed state when the signal transmitter (2) emits a control signal (SSt), and otherwise it is in the open state, or vice versa, whereby the switch input (3b, 103b) is connected to the positive or negative first pole (4a) of a first direct voltage source and the switch output (3c, 103c) is connected to the first threshold voltage input (1a), so that the first threshold voltage (Uref1) is equal to the voltage present at the switch output (3c, 103c), a capacitor (6) via which the switch output (3c, 103c) is connected to the negative or positive pole (4b) of the first direct voltage source, and a diode (5) via which the switch output (3c, 103c) is connected to the first signal input (1b) in such a way that the anode (5a) or the cathode (5b) of the diode (5) is connected to the switch output (3c, 103c).",
"19.The acoustic distance sensor according to claim 18, characterized in that, instead of the diode (5), a second direct voltage source (7) is used whose positive pole (7a) is in the place of the anode (5a) of the diode (5) and whose negative pole (7b) is in the place of the cathode (5b) of the diode (5).",
"20.The acoustic distance sensor according to claim 18 characterized in that the alternating voltage emitted by the oscillator (32) reaches the sound transducer (34) after passing through a driver stage (33).",
"21.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through an amplifier (36c).",
"22.The acoustic distance sensor according to claim 21, characterized in that the amplifier (36c) is a logarithmic amplifier.",
"23.The acoustic distance sensor according to claim 18, characterized in that, before entering the envelope curve shaper (37), the received signal passes through a voltage limiter (35) that limits the amplitude of the received signal to a maximum value.",
"24.The acoustic distance sensor according to claim 18, characterized in that the signal transmitter (2) is integrated into the control unit (31) or is a component of the control unit (31).",
"25.The acoustic distance sensor according to claim 18, characterized in that the first switching signal (S1) is transmitted to the control unit (31) for evaluation purposes.",
"26.The acoustic distance sensor according to claim 18, characterized in that the signal pulses (41) are synchronized with the control signals (SSt) in such a way that the control signals (SSt) each begin before the signal pulses (41).",
"27.The acoustic distance sensor according to claim 18, characterized in that the signal pulses 41 are synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses (41).",
"28.An adaptive comparator circuit for an acoustic distance sensor, comprising a first comparator (1) having a first signal input and a first threshold voltage input; a first direct voltage source (4) for delivering a threshold voltage to the first threshold voltage input, wherein the first direct voltage source comprises a first and a second pole; a first switch (3), wherein the first switch is connected to the first direct voltage source and to the first threshold voltage input; a signal transmitter (2), wherein the signal transmitter controls the first switch through an input of the switch; a capacitor (6), wherein a first plate of the capacitor is connected to the a first threshold voltage input and wherein a second plate of the capacitor is connected to second pole the first direct voltage source; a diode (5) connected the first signal input and to the first threshold voltage input of the first comparator.",
"29.The adaptive comparator circuit according to claim 28, wherein the diode is connected in series to a first resistor.",
"30.The adaptive comparator circuit according to claim 1, wherein the signal transmitter emits an electric control signal at regular intervals.",
"31.The adaptive comparator circuit according to claim 28 further comprising a second resistor (11) connected between the first pole of the first direct voltage source and the first threshold voltage input, and a third resistor (12) connected between the first threshold voltage input and the second pole of the first direct voltage source, so that the second resistor (11) and the third resistor (12) form a first voltage divider (11, 12).",
"32.The adaptive comparator circuit according to claim 31, wherein the first voltage divider (11, 12) is an adjustable potentiometer whose pick-up is connected to the first threshold voltage input.",
"33.The adaptive comparator circuit according to claim 28 further comprising a fourth resistor (17) connected between the first pole of the first direct voltage source and the input of the first switch; a fifth resistor (16) connected between the input of the first switch and the output of the signal transmitter (2)."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN <EOH>FIG.",
"1 —a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.",
"2 —a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.",
"1 , FIG.",
"3 —a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.",
"4 —a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.",
"3 , FIG.",
"5 —a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.",
"6 —a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.",
"7 and 8 —a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.",
"9 —a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8 , whereby an echo occurs during the quiescent phase, FIG.",
"10 —a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8 , whereby an echo additionally occurs during the relaxation phase, and FIGS.",
"11 to 13 —circuit diagrams of further embodiments of comparator circuits according to the invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?",
"FIGS.",
"1 to 4 serve to further illustrate the state of the art.",
"FIG.",
"1 shows a schematic block diagram of a variant of an acoustic distance sensor 30 a with which a measure according to the state of the art has been taken in order to reduce the short range.",
"The distance sensor 30 a shown in FIG.",
"1 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , an amplifier 36 a with regulatable amplification factor, an envelope curve shaper 37 , a comparator 1 as well as an amplification factor actuation stage 38 a.",
"The control unit 31 transmits short signal pulses 41 to the oscillator 32 .",
"The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.",
"to emit sound waves.",
"Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30 a.",
"During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.",
"This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.",
"In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.",
"The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36 a with a regulatable amplification factor.",
"The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36 a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.",
"The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.",
"In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.",
"The amplification factor of the amplifier 36 a is regulated by the amplification factor actuation stage 38 a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35 .",
"Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto ( FIG.",
"2 ).",
"The amplification factor actuation stage 38 a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31 .",
"In this manner a reduction of the short range can be achieved.",
"The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.",
"FIG.",
"2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36 a , c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.",
"1 .",
"The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.",
"As was already explained with reference to FIG.",
"1 , during the signal pulse 41 , the amplification factor 42 reaches a minimum 42 a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.",
"During the signal pulse 41 , the envelope curve voltage 43 reaches a maximum (envelope curve segment 43 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .",
"Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43 b ), whereby the course of the envelope curve 43 b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.",
"The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.",
"The advantageous result of this is a reduction of the short range.",
"In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43 b ).",
"The threshold voltage 44 is constant over time.",
"Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44 , in a disadvantageous manner, at the beginning of the signal pulse 41 , a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44 .",
"An echo signal (envelope curve segment 43 d ) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41 .",
"The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.",
"Further drawbacks associated with a distance sensor of the type illustrated in FIG.",
"1 were already explained above.",
"As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.",
"For purposes of further illustrating the state of the art, FIG.",
"3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30 b .",
"The distance sensor 30 b shown in FIG.",
"3 comprises a control unit 31 , an oscillator 32 , a driver 33 , a sound transducer 34 , a voltage limiter 35 , a preferably logarithmic amplifier 36 b , an envelope curve shaper 37 , a comparator 1 as well as a threshold value actuation stage 38 b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1 .",
"The essential difference from the distance sensor 30 a illustrated in FIG.",
"1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.",
"FIG.",
"4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30 b shown in FIG.",
"3 .",
"The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.",
"During a signal pulse 41 , the envelope curve voltage 53 reaches a maximum (envelope curve segment 53 a ) at which it still remains during the lag time dt after the end of the signal pulse 41 .",
"Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53 b ), since the amplifier 36 b is a logarithmic amplifier.",
"Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53 c ).",
"The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38 b in such a way that it rises before every signal pulse 41 , remains at a maximum value for a certain period of time (envelope curve segment 54 a ), then drops (curve segment 54 b ) and finally reaches a constant level (envelope curve segment 54 c ) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53 , as long as no echo is received.",
"The threshold value actuation stage 38 b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31 .",
"The advantageous result is a reduction of the short range.",
"Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41 .",
"Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53 d ) associated with an echo, so that the switching signal 56 is a useful switching signal 56 .",
"Drawbacks associated with a distance sensor of the type illustrated in FIG.",
"3 were already explained above.",
"Now reference is made to FIG.",
"5 , which illustrates a schematic circuit diagram of an embodiment 10 a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2 , a first regulatable switch 3 , a capacitor 6 , a diode 5 and a first comparator 1 , that has a first threshold value input 1 a and a first signal input 1 b.",
"At the first threshold value input 1 a , a threshold voltage U ref1 , is present that is emitted internally in the adaptive comparator circuit according to the invention.",
"At the signal input 1 b , a voltage signal U signal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.",
"The first comparator 1 emits a first switching signal S 1 when the voltage signal U signal is greater than the first threshold voltage U ref1 .",
"The first switching signal S 1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.",
"Via a control output 2 a , the signal transmitter 2 emits an electric control signal S St at times, for example, at regular intervals.",
"In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.",
"Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.",
"Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal S St having a time duration of typically, for example, 20 microseconds.",
"The first switch 3 has a control means 3 a , a switch input 3 b and a switch output 3 c .",
"The control means 3 a is connected to the control output 2 a and can be regulated by the control signal S St in such a way that it is in the closed state when the signal transmitter 2 emits a control signal S St , and otherwise it is in the open state.",
"The first switch 3 can be, for example, a transistor.",
"The switch input 3 b is connected to the positive first pole 4 a of a first direct voltage source 4 a , 4 b (not shown here).",
"The switch output 3 c is connected to the first threshold value input 1 a of the comparator 1 , so that the first threshold voltage U ref1 is equal to the voltage present at the switch output 3 c .",
"Thus, when the switch 3 is closed, the potential of the positive first pole 4 a is present at the first threshold value input 1 a of the comparator 1 .",
"The switch output 3 c is also applied via a capacitor 6 to the negative second pole 4 b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.",
"The switch output 3 c is also connected to the anode 5 a of a diode 5 .",
"The cathode 5 b of the diode 5 is connected to the first signal input 1 b of the comparator.",
"As soon as the signal transmitter 2 emits a control signal S St , the first switch 3 is in the closed state.",
"Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.",
"When the switch 3 is closed, this voltage is also present at the threshold value input 1 a.",
"After the end of the control signal S St , the switch 3 is in the open state.",
"The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first U ref1 reaches a value that corresponds to the sum of the voltage signal U signal and the diode flow voltage dU.",
"This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal U signal : the smaller the voltage signal U signal , the more the voltage and thus the threshold voltage U ref1 drop according to the invention at the capacitor.",
"Hence, a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the fundamental voltage distance dU.",
"According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage U ref1 for as long as and only until the difference of U ref1 −U signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal U signal increases.",
"If the voltage signal U signal drops, then the threshold voltage U ref1 follows the voltage signal U signal at the distance dU.",
"In contrast, if the voltage signal U signal increases, then the threshold voltage U ref1 remains constant because of the blocking effect of the diode 5 .",
"Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.",
"1 , so that the first pole 4 a is negative and the second pole 4 b is positive.",
"In this case, the diode 5 in the circuit shown in FIG.",
"1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3 c and its anode to the second pole 4 b.",
"In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7 .",
"According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5 a of the diode 5 and whose negative pole is in the place of the cathode 5 b of the diode 5 .",
"FIG.",
"6 shows an embodiment 10 b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.",
"1 .",
"The diode shown in FIG.",
"5 has been replaced by a second voltage source 7 , whereby its positive pole 7 a has been put in the place of the anode 5 a and the negative pole 7 b has been put in the place of the cathode 5 b .",
"The fundamental voltage distance dU shown in FIG.",
"1 has now been replaced by the voltage of the voltage source 7 , that is to say, the fundamental voltage distance dU between U ref1 and U signal is now defined by the voltage supplied by the second direct voltage source.",
"If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.",
"FIG.",
"7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10 a shown in FIG.",
"5 is used.",
"A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32 .",
"When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.",
"The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.",
"The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10 a shown in FIG.",
"5 .",
"In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals S St that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals S St each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.",
"9 a and 9 b .",
"In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage U ref1 is greater than the voltage signal U signal , so that no dummy switching signal is triggered.",
"In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals S St in such a way that the control signals S St each begin before or simultaneously with the end of the signal pulses 41 .",
"This ensures that, without any time lag, the threshold voltage U ref1 is supplied to the dropping voltage signal U signal at the fundamental voltage distance dU already at the beginning of the relaxation phase.",
"The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.",
"FIG.",
"8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10 a shown in FIG.",
"5 is likewise used.",
"The distance sensor shown in FIG.",
"8 —in comparison to that shown in FIG.",
"7 —additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34 .",
"Moreover, before the received signal is fed into the envelope curve shaper 37 , it is passed through a voltage limiter 35 and through a logarithmic amplifier 36 c. Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.",
"The amplifier 36 c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.",
"In FIG.",
"8 , in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31 .",
"For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31 .",
"In FIG.",
"8 , the first switching signal S 1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.",
"FIGS.",
"9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.",
"FIG.",
"9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8 , whereby an echo occurs during the quiescent phase.",
"The control unit 31 transmits short signal pulses 41 to the oscillator 32 , as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet ( FIG.",
"9 , Curve a).",
"Moreover, the signal transmitter 2 transmits a control signal S St to the switch 3 , which is synchronized with the signal pulse 41 in such a way that the control signal S St begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former ( FIG.",
"9 , Curve b).",
"FIG.",
"9 , Curve c, shows the course of the voltage signal U signal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage U ref1 (dotted curve).",
"The course of the U signal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53 , which was explained with reference to FIG.",
"4 .",
"While the control signal S St is present at the switch 3 , the latter is opened, so that a threshold voltage U ref1 is established which is identical to the voltage supplied by the first direct voltage source 4 a , 4 b ( FIG.",
"9 , Curve c).",
"Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3 .",
"In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36 c ( FIG.",
"8 ).",
"According to the invention, after the end of the control signal S St , a threshold voltage U ref1 is established that is greater than the voltage signal U signal by the voltage distance dU.",
"Therefore, the threshold voltage U ref1 drops immediately after the end of the control signal by a certain quantity.",
"From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal U signal as long as the latter does not increase.",
"A dummy switching signal is not triggered.",
"If, however, the voltage signal U signal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal U signal leads to a triggering of the comparator 1 as soon as the voltage signal U signal exceeds the threshold voltage U ref1 .",
"In this case, the comparator 1 emits a first switching signal S 1 ( FIG.",
"9 , Curve d).",
"FIG.",
"10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8 , whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.",
"The courses over time of the signal pulse 41 and of the control signal S St are identical to those shown in FIG.",
"9 .",
"During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal U signal ( FIG.",
"10 , Curve a).",
"Up until the beginning of the second echo peak 61 , the curves correspond to those shown in FIG.",
"9 , Curve c. According to the invention, however, the decrease of the threshold voltage U ref1 comes to a halt as soon as the voltage signal U signal is no longer dropping, and a first switching signal S 1a is triggered by the second echo peak 61 .",
"After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage U ref1 and the voltage signal U signal .",
"During the quiescent phase, another, third echo peak 62 of the voltage signal U signal occurs, as a result of which another switching signal S 1b is triggered.",
"Now reference will be made to FIGS.",
"11 to 13 , which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.",
"FIG.",
"11 shows a preferred embodiment 10 c of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.",
"6 as follows: a) The first switch is a transistor 103 .",
"The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.",
"b) A first resistor 11 is connected between the first pole 4 a and the first threshold value input 1 a , and a second resistor 12 is connected between the first threshold value input 1 a and the second pole 4 b , so that the first resistor 11 and the second resistor 12 form a first voltage divider 11 , 12 .",
"Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the first voltage divider.",
"Thus, through the use of the first voltage divider 11 , 12 , the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal U signal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dU a .",
"When this embodiment 10 c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.",
"This is advantageous for many applications such as, for example, for the detection of small objects.",
"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.",
"c) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.",
"The purpose of these resistors was already explained above.",
"d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage U ref1 is adapted to the voltage signal U signal .",
"The eighth resistor also serves to smooth the course of the threshold voltage U ref1 with respect to very rapid fluctuations of the voltage signal U signal .",
"FIG.",
"12 shows another preferred embodiment 10 d of the invention, which differs from the adaptive comparator circuit 10 c shown in FIG.",
"11 in that, before the voltage signal U signal reaches the first signal input 1 b , it passes through an impedance transformer.",
"This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.",
"FIG.",
"13 shows another preferred embodiment 10 e of the invention which differs from the adaptive comparator circuit 10 a shown in FIG.",
"6 as follows: a) The first switch is a transistor 103 .",
"The base 103 a of the transistor 103 serves as the control-current terminal, the emitter 103 b serves as the switch input and the collector 103 c as the switch output.",
"b) A second comparator 20 is used that has a second threshold value input 20 a and a second signal input 20 b , and that emits a second switching signal S 2 when a greater voltage is present at the second signal input 20 b than at the second threshold value input 20 a. c) The first pole 4 a of the first voltage source 4 a , 4 b is connected to the second threshold value input 20 a via a third resistor 13 .",
"The second threshold value input 20 a is connected to the second pole 4 b via a fourth resistor 14 , so that the third and the fourth resistors 13 , 14 form a second voltage divider which can be configured as a potentiometer.",
"d) The collector 103 c that serves as the switch output and the second threshold value input 20 a are connected via a fifth resistor 15 .",
"e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S 2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S 2 , and otherwise it is in the open state.",
"The second switch 23 can be a transistor.",
"f) A sixth resistor 16 is connected between the control output 2 a of the signal transmitter 2 and the base 103 a , and a seventh resistor 17 is connected between the base 103 a and the first pole 4 a , which is a plus pole in the example shown.",
"The purpose of these resistors was already explained above.",
"g) The diode 5 is connected in series to an eighth resistor 18 .",
"The purpose of this resistor was already explained with reference to FIG.",
"11 .",
"h) Before reaching the first signal input 1 b , the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.",
"12 .",
"Due to the second voltage divider 13 , 14 , a voltage U ref2 is present at the second threshold value input 20 a .",
"As long as the second switch 23 is closed, the first threshold voltage U ref1 follows the dropping voltage signal U signal , parallel-offset by the fundamental voltage distance dU, as explained above.",
"According to the invention, however, the second switch opens as soon as the voltage signal U signal exceeds the second threshold voltage U ref2 .",
"As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage U ref1 are ended.",
"Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage U ref1 has dropped to the magnitude of the second threshold voltage U ref2 supplied by the second potentiometer 13 , 14 .",
"Thus, through the use of the second voltage divider 13 , 14 , the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal U signal with respect to voltage signal U signal , from the fundamental voltage distance dU to a value dU b .",
"The second voltage divider 13 , 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20 a.",
"The fifth resistor 15 is connected between the switch output 103 c and the second threshold value input 20 a .",
"Therefore, the magnitude of the first threshold voltage U ref1 counters the voltage that is supplied by the second voltage divider 13 , 14 .",
"In particular, through the use of the fifth resistor 15 , it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal U signal strives towards a value dU b that—depending on the voltage supplied by the second voltage divider 13 , 14 —can advantageously be not only larger but also smaller than the fundamental voltage distance dU.",
"When this embodiment 10 e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13 , 14 .",
"All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10 b , 10 c , 10 d and 10 e illustrated in FIG.",
"6 and FIGS.",
"11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.",
"8 instead of the adaptive comparator circuit 10 a shown there by way of an example."
],
[
"TECHNICAL AREA The invention relates to an adaptive comparator circuit that can be used particularly for reducing the short range of an acoustic distance sensor, as well as to an acoustic distance sensor with such a circuit.",
"STATE OF THE ART Acoustic distance sensors are used to measure distances by means of the pulse-propagation time method.",
"In this process, a sound transducer emits a short sound pulse that propagates in a medium (gas, liquid or solid).",
"This pulse is reflected, for example, on an object and comes back as an echo.",
"The distance to the object can be determined on the basis of the propagation time of the sound pulse and the sound velocity in the medium.",
"As a rule, the frequency of the sound used for such measurements lies in the ultra-sound range, typically in the range between 20 kHz and 500 kHz, for example, at 400 kHz.",
"The sound transducer, which in particular can be a piezoelectric ceramic element, is normally used to transmit the sound pulse as well as to receive the echo.",
"Typically, sound pulses of about 3 to 50 oscillations periods each are emitted.",
"This results in time durations of typically about 6 to 2050 μs for the emitted sound pulses.",
"During this time, i.e.",
"the transmitting phase, the sound transducer oscillates at a high oscillation amplitude.",
"This time duration is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.",
"The relaxation time is typically, for example, 300 μs to 3 ms.",
"The time interval between the end of the relaxation time and the emission of the next sound pulse will be called “quiescent phase” below.",
"Due to noise and other interferences, however, the oscillation amplitude does not drop completely to zero even during the quiescent phase, but rather remains at a certain constant mean disturbance level.",
"Therefore, during the receiving phase that follows the transmitting phase, even in the absence of a useful echo, the oscillation amplitude that dies out during the relaxation time and the mean disturbance level consistently form a finite background signal that is very large immediately after the end of the transmitting phase, that generally decreases exponentially within the relaxation time and that finally remains at the disturbance level.",
"A useful echo signal is superimposed on this background and, as a rule, is detected by means of a comparator that emits a switching signal when the received signal is greater than a certain reference value that will be called “threshold voltage” hereinafter.",
"On the one hand, the threshold voltage should be as low as possible so that even weak useful echo signals, e.g.",
"of small objects, can be detected, which translates into high sensitivity.",
"On the other hand, the threshold voltage has to lie above the disturbance level by a certain safety margin so as to avoid erroneous triggering of the comparator during the quiescent phase.",
"When the threshold voltage is constant over time, useful echo signals can be detected as soon as the background signal has dropped below the threshold voltage as a result of the relaxation of the sound transducer.",
"Before this time, however, that is to say, during the transmitting phase and during part of the relaxation time, the background signal is greater than the threshold voltage so that the comparator—irrespective of the presence of an echo—is triggered in any case during this time.",
"Thus, during this time, called dead time, the system is fundamentally insensitive to the reception of echoes.",
"Therefore, the measurement of distances below a certain minimum distance is impossible.",
"This minimum distance is called the “short range” and is typically a few centimeters.",
"The existence of the short range often has a detrimental effect, for example, wherever due to space constraints, the sensor is to be situated as close as possible to the object to be detected.",
"Various methods are known for reducing the short range.",
"The simplest method is to increase the threshold voltage.",
"This causes the background signal, which dies out after the transmitting phase, to drop below the threshold voltage at an earlier point in time within the relaxation time.",
"However, this method entails the serious drawback that an increase in the threshold voltage translates into a reduction of the system sensitivity, as a result of which weak echoes can no longer be detected.",
"Another method lies in conveying the received signal through an amplifier whose amplification factor can be changed over time.",
"The amplification factor is minimal immediately after the transmittal pulse and is then boosted, for example, by means of a suitable electronic circuit as a function of the time.",
"In this manner, despite a constant threshold voltage over time, a reduction of the short range can be combined with a high sensitivity during the quiescent phase.",
"Of course, for this purpose, the course over time of the amplification factor has to be adapted to the relaxation time.",
"The drawback is that a one-time optimal adaptation is not sufficient here, since the relaxation time of the sound transducer is not constant but rather, it depends on the medium on the temperature, on the age of the sound transducer and on other influencing factors.",
"Therefore, in actual practice, either complex compensation measures have to be undertaken or re-adaptations have to be made repeatedly, or else a large safety margin has to be maintained right from the start for purposes of optimal adaptation, which diminishes the system sensitivity, at least during the relaxation time.",
"Another method is to provide a threshold voltage that can be changed over time.",
"In the ideal case, this threshold voltage drops at a gradient that is similar to the decaying background signal and, at the beginning of the quiescent phase, turns into a constant value, whereby the threshold voltage nevertheless always remains sufficiently greater than the background signal.",
"This method can be additionally improved by subjecting the background signal to a logarithmic amplification so that the decrease is linear instead of exponential.",
"The drawback here, however, is that a one-time adaptation of the time behavior of the threshold voltage is not sufficient to ensure optimal operation over the long run, since here, too, the individual dying-out behavior of the sound transducer is not taken into account on a case-to-case basis.",
"Another method for reducing the short range lies in the propagation time-dependent control of the length of the emitted sound pulses.",
"In the case of a short propagation time, that is to say, in the case of an object that is close, very short sound pulses are used to that the oscillation amplitude of the sound transducer does not fully develop and thus the short range is reduced.",
"Another method lies in the use of threshold voltage that is dynamically changed by means of a microprocessor and a D/A transducer.",
"The microprocessor regulates the D/A transducer in such a way that a dynamic threshold is created that always lies above the dying-out signal.",
"Here, an A/D transducer measures the decaying transmission signal.",
"These methods, however, call for considerable hardware and software resources.",
"Therefore, the invention is based on the objective of simply and inexpensively providing a comparator circuit with which, in every single case, the course over time of the threshold voltage is automatically adapted to the individual decay behavior of the sound transducer and the magnitude of the disturbance level.",
"This objective is achieved according to the invention by an adaptive comparator circuit, especially for an acoustic distance sensor, comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and having a first signal input to which a voltage signal (Usignal) is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, characterized by a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.",
"This is the first basic form of an adaptive comparator circuit according to the invention.",
"According to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.",
"This is the second basic form of an adaptive comparator circuit according to the invention.",
"These two basic forms can be augmented advantageously as will still be explained below.",
"An acoustic distance sensor according to the invention comprises a control unit that at times transmits signal pulses to an oscillator which, during the presence of a signal pulse, transmits an alternating voltage to a sound transducer that is excited by the alternating voltage to emit sound waves and that is capable of receiving sound waves coming back to the sound transducer in the form of an echo as a result of reflection, and of converting them into an electric received signal, and it also comprises an envelope curve shaper to which the received signal is fed and which forms the envelope curve of the received signal and emits it as a signal voltage, characterized in that the signal voltage is fed to an adaptive comparator circuit comprising a first comparator having a first threshold value input to which a first threshold voltage is applied and a first signal input to which a voltage signal is applied and, when a voltage signal is present that is greater than the first threshold voltage, said comparator emits a first switching signal, a signal transmitter, especially a microcontroller or microprocessor, that at times emits a control signal, a first switch that has a control means, a switch input and a switch output, and that can be regulated or activated through the effect of the control signal on the control means in such a way that it is in the closed state when the signal transmitter emits a control signal, and otherwise it is in the open state, or vice versa, whereby the switch input is connected to the positive or negative first pole of a first direct voltage source and the switch output is connected to the first threshold value input, so that the first threshold voltage is equal to the voltage present at the switch output, a capacitor via which the switch output is connected to the negative or positive pole of the first direct voltage source, and a diode via which the switch output is connected to the first signal input in such a way that the anode or the cathode of the diode is connected to the switch output.",
"An acoustic distance sensor according to the invention thus comprises the adaptive comparator circuit according to the invention.",
"Preferably, the control signal emitted by the signal transmitter is an electric control signal and the first switch is an electrically or electronically controlled switch, for example, a transistor, whose base terminal or gate terminal function as a control-current terminal or, for example, a relay.",
"In another embodiment of the invention, the control signal emitted by the signal transmitter is an optical control signal that is emitted, for example, by a light diode, and the first switch is an optically controlled switch, e.g.",
"a photoresistor.",
"As soon as the signal transmitter emits a control signal, the first switch is in the closed state, so that the capacitor is charged with the voltage supplied by the first voltage source.",
"After the end of the control signal, the switch is in the open state.",
"The capacitor voltage and thus the first threshold voltage present at the first threshold value input now assumes a value that corresponds to the sum of the voltage signal and the diode flow voltage or else to the sum of the voltage signal and the voltage of the second direct voltage source.",
"The diode flow voltage or the voltage of the second direct voltage source are referred to below as “fundamental voltage distance” dU.",
"The task of the diode or of the second direct voltage source is to allow the discharging of the capacitor and thus a drop in the threshold voltage for as long as and only until the difference of the threshold voltage minus the voltage signal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal increases.",
"Therefore, the time behavior of the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal as long as the voltage signal does not increase.",
"In contrast, if the voltage signal increases, the threshold voltage remains constant.",
"Consequently, a maximum, e.g.",
"a peak, of the voltage signal leads to the triggering of the comparator as soon as the voltage signal exceeds the threshold voltage.",
"If the voltage signal drops again after having increased, then the threshold voltage follows the voltage signal, once again parallel-offset, as soon as the fundamental, voltage distance dU has been reached again.",
"Hence, according to the invention, the threshold voltage is specified by the magnitude of the voltage signal at every point in time of the receiving phase.",
"If the course over time of the voltage signal is changed, then there is automatically a correspondingly changed course over time of the threshold voltage.",
"Thus, the course over time of the threshold voltage is automatically adapted to that of the voltage signal.",
"The diode flow voltage is temperature-dependent.",
"It decreases as the temperature rises.",
"Consequently, an acoustic distance sensor according to the invention becomes more sensitive as the temperature rises.",
"On the other hand, the attenuation of ultrasound waves generally increases as the temperature rises.",
"Therefore, the resultant sensitivity loss is advantageously compensated for partially with the use of a diode to create the fundamental voltage distance dU.",
"As already explained above, according to the invention, instead of the diode, a second direct voltage source can be used whose positive pole is in the place of the anode of the diode and whose negative pole is in the place of the cathode of the diode.",
"In this case, the fundamental voltage distance dU is defined by the voltage supplied by the second direct voltage source.",
"The basic forms described above of adaptive comparator circuits according to the invention can be advantageously augmented as will be explained below.",
"In a preferred embodiment of the invention, one of the above-described basic forms has been augmented by a first resistor that is connected between the first pole and the first threshold value input, and by a second resistor that is connected between the first threshold value input and the second pole, so that the first resistor and the second resistor form a first voltage divider.",
"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.",
"Thus, through the use of the first voltage divider, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.",
"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.",
"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.",
"Another preferred embodiment of an adaptive comparator circuit according to the invention comprises the following in addition to the basic forms described above: a second comparator that has a second threshold value input and a second signal input, and that emits a second switching signal when a greater voltage is present at the second signal input than at the second threshold value input, a third resistor via which the first pole of the first direct voltage source is connected to the second threshold value input, a fourth resistor that, on the one hand, is connected to the second threshold value input and, on the other hand, to the second pole of the first voltage source, so that the third resistor and the fourth resistor form a second voltage divider, and a second switch that is connected in series to the diode and that can be regulated or activated by the second switching signal in such a way that it is in the closed state when the second comparator emits the second switching signal, and otherwise it is in the open state.",
"Due to the second voltage divider, a voltage is present at the second threshold value input that will be called “second threshold voltage” Uref2 below.",
"As long as the second switch is closed, the first threshold voltage follows the dropping voltage signal Usignal with the fundamental voltage distance dU, as explained above.",
"According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor and thus the dropping of the first threshold voltage are ended.",
"Hence, in this embodiment of the invention, the capacitor can only discharge until the first threshold voltage Uref1 has dropped to the value of the second threshold voltage Uref2.Thus, through the use of the second voltage divider, the voltage distance by which the first threshold voltage is changed parallel-offset with respect to voltage signal Usignal after the decay of the voltage signal Usignal, advantageously by the fundamental voltage distance dU to a value dUb.",
"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor according to the invention, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be reduced.",
"The second voltage divider can be an adjustable potentiometer whose pick-up is connected to the second threshold value input.",
"In another configuration of this embodiment of the invention, a fifth resistor is connected between the switch output and the second threshold value input.",
"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider.",
"In particular, through the use of the fifth resistor, it can be additionally achieved that the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.",
"When this embodiment of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased.",
"In another advantageous embodiment of the invention, the voltage signal passes through a voltage follower or impedance transformer before reaching the first signal input.",
"In another embodiment of the invention, a sixth resistor is connected between the control output and the control-current terminal for purposes of limiting the current flow.",
"In another embodiment of the invention, the first switch is a transistor whose base functions as a control-current terminal, whereby a seventh resistor is connected between the base and the first pole of the first direct voltage source.",
"In another embodiment of the invention, the diode or the second direct voltage source is connected in series with an eighth resistor.",
"This brings about a slowing down of the discharging of the capacitor as well as a smoothing of the threshold voltage, which is advantageous for many applications.",
"The voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.",
"In this manner, it is prevented that the first comparator is triggered while the first switch is still closed, that is to say, while the threshold voltage cannot yet adapt to the voltage signal.",
"Moreover, in this manner, it is advantageously achieved that, in any case, the adaptation of the threshold voltage to the voltage signal can begin immediately after the opening of the first switch.",
"An adaptive comparator circuit according to the invention can be used especially advantageously in an acoustic distance sensor.",
"In this case, the voltage signal is advantageously formed by the envelope curve of the received signal supplied by the sound transducer.",
"This received signal consists of a superimposition of the background signal with the useful echo signal.",
"The formation of the envelope curve, for example, by means of a rectifier circuit, is known from the state of the art and will thus not be elaborated upon in greater depth here.",
"In the absence of an echo, the envelope curve dies out exponentially during the relaxation phase and, at the beginning of the quiescent phase, turns into a horizontal straight line.",
"Such an envelope curve will be called “background envelope curve” below.",
"The threshold voltage adapts according to the invention to the individual course of the background envelope curve in that it follows said background envelope curve parallel-offset by a voltage distance dU or dUa or dUb.",
"An echo causes a peak to be superimposed on the background envelope curve so that the voltage signal increases.",
"According to the invention, the threshold voltage does not follow this increase, so that the comparator is triggered and the echo can thus be detected.",
"As already mentioned above, the voltage supplied by the first voltage source is preferably selected such that it is greater in magnitude than the value of the maximum of the voltage signal.",
"Since in this case, the threshold voltage already follows the background envelope curve at the voltage distance dU at the beginning of the relaxation phase, the voltage signal in the absence of an echo is not greater than the threshold voltage at any point in time, especially not during the entire relaxation phase.",
"Therefore, throughout the entire receiving phase, the comparator can only be triggered by an echo, but not by the decay behavior of the sound transducer.",
"Thus, an acoustic distance sensor according to the invention is fundamentally able to detect an echo during the quiescent phase as well as especially during the entire relaxation phase.",
"As a result, the undesired short range of the sensor is considerably reduced according to the invention.",
"BRIEF DESCRIPTION OF THE DRAWING IN WHICH THE FOLLOWING IS SHOWN FIG.",
"1—a schematic block diagram of a variant of an acoustic distance sensor according to the state of the art, FIG.",
"2—a schematic representation of a typical course over time of a) a signal pulse, b) an amplification factor, c) an envelope curve and d) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.",
"1, FIG.",
"3—a schematic block diagram of another variant of an acoustic distance sensor according to the state of the art, FIG.",
"4—a schematic representation of a typical course over time of a) a timing pulse, b) an envelope curve and c) a comparator output signal, all of which can occur during the operation of the distance sensor shown in FIG.",
"3, FIG.",
"5—a circuit diagram of an embodiment of a comparator circuit according to the invention, FIG.",
"6—a circuit diagram of an alternative embodiment of a comparator circuit according to the invention, FIGS.",
"7 and 8—a schematic block diagram of embodiments of an acoustic distance sensor according to the invention, FIG.",
"9—a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) an envelope curve and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8, whereby an echo occurs during the quiescent phase, FIG.",
"10—a schematic representation of a typical course over time of a) an envelope curve and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8, whereby an echo additionally occurs during the relaxation phase, and FIGS.",
"11 to 13—circuit diagrams of further embodiments of comparator circuits according to the invention.",
"FIGS.",
"1 to 4 serve to further illustrate the state of the art.",
"FIG.",
"1 shows a schematic block diagram of a variant of an acoustic distance sensor 30a with which a measure according to the state of the art has been taken in order to reduce the short range.",
"The distance sensor 30a shown in FIG.",
"1 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, an amplifier 36a with regulatable amplification factor, an envelope curve shaper 37, a comparator 1 as well as an amplification factor actuation stage 38a.",
"The control unit 31 transmits short signal pulses 41 to the oscillator 32.The latter oscillates when a signal pulse is applied, thus emitting an alternating voltage of, for example, 400 kHz, which is then amplified by the driver 33 and subsequently used to excite the sound transducer 34 so that the latter is excited so as to oscillate, i.e.",
"to emit sound waves.",
"Corresponding to the short signal pulses, the sound transducer 34 emits short sound wave packets that constitute the transmittal pulse of the distance sensor 30a.",
"During the transmitting phase, the sound transducer 34 oscillates at a high oscillation amplitude.",
"This period of time is followed by a relaxation time in which the oscillation amplitude dies out—usually exponentially.",
"In the subsequent quiescent phase, the oscillation amplitude generally remains at a constant mean disturbance level.",
"The signal emitted by the sound transducer and thus also echoes returning after the distance-proportional propagation time are fed via the voltage limiter 35 to the amplifier 36a with a regulatable amplification factor.",
"The voltage limiter keeps the high voltage used for exciting the sound transducer away from the amplifier 36a Using, for example, an internal rectifier circuit, the envelope curve shaper 37 forms the envelope curve of the voltage limiter 35 on the basis of the alternating voltage signal emitted by said voltage limiter 35 and transmits to the comparator 1 an envelope curve voltage signal whose course over time corresponds to that of the envelope curve.",
"The comparator 1 transmits a switching signal to the control unit 31 for evaluation purposes when the envelope curve voltage signal is greater than a first steadily supplied threshold voltage.",
"In particular, the comparator then emits a switching signal when an echo signal is received whose magnitude exceeds that of the threshold voltage.",
"The amplification factor of the amplifier 36a is regulated by the amplification factor actuation stage 38a in a time-dependent manner such that the amplification factor is minimal during and immediately after the transmittal pulse and grows as soon as the oscillation amplitude of the sound transducer 34 has died out to such an extent that the magnitude of the received signal is no longer limited by the voltage limiter 35.Therefore, the growth of the amplification factor does not begin completely simultaneously with the end of the signal pulse, but rather delayed by a certain lag time dt with respect thereto (FIG.",
"2).",
"The amplification factor actuation stage 38a receives the time information necessary for synchronizing the time behavior of the amplification factor with the rhythm of the transmittal pulse from the control unit 31.In this manner a reduction of the short range can be achieved.",
"The course over time can advantageously be selected in such a way that the distance-dependent attenuation of the echo is countered and the system sensitivity remains constant.",
"FIG.",
"2 shows a schematic representation of a course over time of a) a signal pulse, b) an amplification factor of the amplifier 36a, c) an envelope curve and threshold voltage as well as d) a comparator output signal, that can typically occur during the operation of the distance sensor shown in FIG.",
"1.The time axes of the curves a) to d) are selected identically in order to illustrate the courses over time.",
"As was already explained with reference to FIG.",
"1, during the signal pulse 41, the amplification factor 42 reaches a minimum 42a that lasts longer than the signal pulse 41 by a certain lag time dt, and then it grows monotonously over time.",
"During the signal pulse 41, the envelope curve voltage 43 reaches a maximum (envelope curve segment 43a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 43 drops during the relaxation phase (envelope curve segment 43b), whereby the course of the envelope curve 43b results from the multiplication of, for example, the exponential decay of the sound transducer amplitude by the growing amplification factor.",
"The temporary lowering of the amplification factor during and after the signal pulse causes the envelope curve voltage 43 to fall below the threshold voltage 44 earlier than would be the case if the amplification factor were not lowered.",
"The advantageous result of this is a reduction of the short range.",
"In the quiescent phase, the envelope curve voltage 43 finally reaches a low constant level (envelope curve segment 43b).",
"The threshold voltage 44 is constant over time.",
"Since the comparator 1 emits a switching signal, as soon as the envelope curve voltage 43 exceeds the threshold voltage 44, in a disadvantageous manner, at the beginning of the signal pulse 41, a dummy switching signal 45 is always triggered that lasts until the envelope curve voltage 43 has dropped below the threshold value 44.An echo signal (envelope curve segment 43d) causes a useful switching signal 46 so that the control unit has to be capable of distinguishing a useful switching signal 46 from a dummy switching signal 45 which, without the presence of an echo signal, was caused exclusively by the signal pulse 41.The capability to make such a distinction requires complex hardware or software resources and thus entails disadvantages.",
"Further drawbacks associated with a distance sensor of the type illustrated in FIG.",
"1 were already explained above.",
"As already explained above, another method of reducing the short range lies in providing a threshold voltage that can be changed over time.",
"For purposes of further illustrating the state of the art, FIG.",
"3 shows a schematic block diagram of a corresponding variant of an acoustic distance sensor 30b.",
"The distance sensor 30b shown in FIG.",
"3 comprises a control unit 31, an oscillator 32, a driver 33, a sound transducer 34, a voltage limiter 35, a preferably logarithmic amplifier 36b, an envelope curve shaper 37, a comparator 1 as well as a threshold value actuation stage 38b which emits a threshold voltage that can be changed over time and transmits it to the comparator 1.The essential difference from the distance sensor 30a illustrated in FIG.",
"1 lies in the fact that the short range reduction is not achieved by an amplification factor that can be changed over time, but rather by a threshold voltage that can be changed over time.",
"FIG.",
"4 shows a schematic representation of a course over time of a) a signal pulse, b) an envelope curve and threshold value as well as c) a comparator output signal, that can typically occur during the operation of the distance sensor 30b shown in FIG.",
"3.The time axes of the curves a) to c) are selected identically in order to illustrate the courses over time.",
"During a signal pulse 41, the envelope curve voltage 53 reaches a maximum (envelope curve segment 53a) at which it still remains during the lag time dt after the end of the signal pulse 41.Then the envelope curve voltage 53 drops linearly during the relaxation phase (envelope curve segment 53b), since the amplifier 36b is a logarithmic amplifier.",
"Finally, in the quiescent phase, the envelope curve voltage 53 reaches a low constant level (envelope curve segment 53c).",
"The threshold voltage 54 is not constant over time but rather is regulated by the threshold value actuation stage 38b in such a way that it rises before every signal pulse 41, remains at a maximum value for a certain period of time (envelope curve segment 54a), then drops (curve segment 54b) and finally reaches a constant level (envelope curve segment 54c) during the quiescent phase, whereby the threshold voltage 54 remains greater than the envelope curve voltage 53, as long as no echo is received.",
"The threshold value actuation stage 38b receives the necessary time information for synchronizing the time behavior of the threshold voltage 54 with the rhythm of the signal pulse 41 from the control unit 31.The advantageous result is a reduction of the short range.",
"Moreover, advantageously, no dummy switching signal is triggered by the signal pulse 41.Rather, a switching signal 56 is triggered only by the rise in the envelope curve 53 (envelope curve segment 53d) associated with an echo, so that the switching signal 56 is a useful switching signal 56.Drawbacks associated with a distance sensor of the type illustrated in FIG.",
"3 were already explained above.",
"Now reference is made to FIG.",
"5, which illustrates a schematic circuit diagram of an embodiment 10a of an adaptive comparator circuit according to the invention It comprises a signal transmitter 2, a first regulatable switch 3, a capacitor 6, a diode 5 and a first comparator 1, that has a first threshold value input 1a and a first signal input 1b.",
"At the first threshold value input 1a, a threshold voltage Uref1, is present that is emitted internally in the adaptive comparator circuit according to the invention.",
"At the signal input 1b, a voltage signal Usignal is present that can be, for example, an envelope curve of the received signal of a sound transducer and that is fed via a first electric terminal 8 to the adaptive comparator circuit according to the invention.",
"The first comparator 1 emits a first switching signal S1 when the voltage signal Usignal is greater than the first threshold voltage Uref1.The first switching signal S1 can be tapped, for example; via a second electric terminal 9 and transmitted for evaluation purposes.",
"Via a control output 2a, the signal transmitter 2 emits an electric control signal SSt at times, for example, at regular intervals.",
"In the simplest case, the signal transmitter 2 can be, for instance, a manually operated signaling key.",
"Moreover, the signal transmitter can be, for example, an electronic square-wave timing generator circuit with a suitable duty cycle.",
"Preferably, the signal transmitter 2 is a microcontroller or microprocessor that is programmed in such a way that, at regular intervals, it emits an electric control signal SSt having a time duration of typically, for example, 20 microseconds.",
"The first switch 3 has a control means 3a, a switch input 3b and a switch output 3c.",
"The control means 3a is connected to the control output 2a and can be regulated by the control signal SSt in such a way that it is in the closed state when the signal transmitter 2 emits a control signal SSt, and otherwise it is in the open state.",
"The first switch 3 can be, for example, a transistor.",
"The switch input 3b is connected to the positive first pole 4a of a first direct voltage source 4a, 4b (not shown here).",
"The switch output 3c is connected to the first threshold value input 1a of the comparator 1, so that the first threshold voltage Uref1 is equal to the voltage present at the switch output 3c.",
"Thus, when the switch 3 is closed, the potential of the positive first pole 4a is present at the first threshold value input 1a of the comparator 1.The switch output 3c is also applied via a capacitor 6 to the negative second pole 4b of the first direct voltage source so that the capacitor 6 is charged with the voltage supplied by the first voltage source when the switch 3 is closed.",
"The switch output 3c is also connected to the anode 5a of a diode 5.The cathode 5b of the diode 5 is connected to the first signal input 1b of the comparator.",
"As soon as the signal transmitter 2 emits a control signal SSt, the first switch 3 is in the closed state.",
"Therefore, the capacitor 6 is charged with the voltage supplied by the first voltage source.",
"When the switch 3 is closed, this voltage is also present at the threshold value input 1a.",
"After the end of the control signal SSt, the switch 3 is in the open state.",
"The capacitor 6 can now discharge according to the invention via the diode 5 to such an extent until the first Uref1 reaches a value that corresponds to the sum of the voltage signal Usignal and the diode flow voltage dU.",
"This means that the voltage that is established at the capacitor 6 depends on the magnitude of the voltage signal Usignal: the smaller the voltage signal Usignal, the more the voltage and thus the threshold voltage Uref1 drop according to the invention at the capacitor.",
"Hence, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the fundamental voltage distance dU.",
"According to the invention, the task of the diode 5 is to allow the discharging of the capacitor 6 and thus a drop in the threshold voltage Uref1 for as long as and only until the difference of Uref1−Usignal has dropped to the fundamental voltage distance dU, but to prevent a recharging of the capacitor and thus an increase in the threshold voltage if the voltage signal Usignal increases.",
"If the voltage signal Usignal drops, then the threshold voltage Uref1 follows the voltage signal Usignal at the distance dU.",
"In contrast, if the voltage signal Usignal increases, then the threshold voltage Uref1 remains constant because of the blocking effect of the diode 5.Of course, the polarity of the first direct voltage source can also be the opposite from the one in FIG.",
"1, so that the first pole 4a is negative and the second pole 4b is positive.",
"In this case, the diode 5 in the circuit shown in FIG.",
"1 has to be arranged in the reverse direction, so that its cathode is connected to the switch output 3c and its anode to the second pole 4b.",
"In another embodiment of the invention, the fundamental voltage distance dU is augmented by a codirectional series connection of a plurality of diodes 5 or second direct voltage sources 7.According to the invention, as an alternative, the function of the diode 5 can be assumed by a second direct voltage source whose positive pole is in the place of the anode 5a of the diode 5 and whose negative pole is in the place of the cathode 5b of the diode 5.FIG.",
"6 shows an embodiment 10b of an adaptive comparator circuit according to the invention, which can be used as an alternative to that shown in FIG.",
"1.The diode shown in FIG.",
"5 has been replaced by a second voltage source 7, whereby its positive pole 7a has been put in the place of the anode 5a and the negative pole 7b has been put in the place of the cathode 5b.",
"The fundamental voltage distance dU shown in FIG.",
"1 has now been replaced by the voltage of the voltage source 7, that is to say, the fundamental voltage distance dU between Uref1 and Usignal is now defined by the voltage supplied by the second direct voltage source.",
"If the voltage supplied by the second voltage source 7 can be regulated or adjusted, then the fundamental voltage distance dU can be varied as needed.",
"FIG.",
"7 shows a schematic block diagram of an embodiment of an acoustic distance sensor according to the invention in which the adaptive comparator circuit 10a shown in FIG.",
"5 is used.",
"A control unit 31 transmits short signal pulses at times, preferably at regular intervals, each lasting, for example, 25 microseconds, to an oscillator 32.When a signal pulse is present, the oscillator 32 transmits an alternating voltage to a sound transducer 34 that is excited by the latter to emit sound waves.",
"The sound transducer 34 is capable of receiving sound waves reflected, for example, from an object and coming back to the sound transducer 34 as an echo, and of converting them into an electric received signal, whereby the propagation time of the sound waves is a measure of the distance from the object.",
"The received signal is fed to an envelope curve shaper 37 which forms the envelope curve of the received signal and transmits it via an electric terminal 8 to the adaptive comparator circuit 10a shown in FIG.",
"5.In a preferred embodiment of the invention, the signal pulses 41 that the control unit 31 transmits to the oscillator 32 are synchronized with the control signals SSt that the control unit 2 transmits to the switch 3 and this is done in such a way that the control signals SSt each begin shortly before the signal pulses 41 as will still be explained below with reference to FIGS.",
"9a and 9b.",
"In this manner, it is ensured that, already at the beginning of the transmittal phase, the threshold voltage Uref1 is greater than the voltage signal Usignal, so that no dummy switching signal is triggered.",
"In another preferred embodiment of the invention, the signal pulses 41 are advantageously synchronized with the control signals SSt in such a way that the control signals SSt each begin before or simultaneously with the end of the signal pulses 41.This ensures that, without any time lag, the threshold voltage Uref1 is supplied to the dropping voltage signal Usignal at the fundamental voltage distance dU already at the beginning of the relaxation phase.",
"The sensitivity of the distance sensor is defined by the fundamental voltage distance dU.",
"FIG.",
"8 shows a schematic block diagram of another embodiment of an acoustic distance sensor according to the invention, in which the adaptive comparator circuit 10a shown in FIG.",
"5 is likewise used.",
"The distance sensor shown in FIG.",
"8—in comparison to that shown in FIG.",
"7—additionally has a driver 33 that amplifies the alternating voltage supplied by the oscillator 32 and transmits it to the sound transducer 34.Moreover, before the received signal is fed into the envelope curve shaper 37, it is passed through a voltage limiter 35 and through a logarithmic amplifier 36c.",
"Among other things, the voltage limiter 35 counters an overmodulation of the amplifier.",
"The amplifier 36c serves to adapt the low input level of just a few microvolts to the higher input level of the subsequent evaluation circuit.",
"In FIG.",
"8, in order to facilitate the synchronization, the signal transmitter 2 is accommodated in the control unit 31.For example, the signal transmitter 2 and the control unit 31 can be combined in one single unit, for example, in a microprocessor, that concurrently fulfills the function of the signal transmitter 2 as well as that of the control unit 31.In FIG.",
"8, the first switching signal S1 of the control unit 31 is also supplied for evaluation purposes, especially for determining the propagation time and calculating the distance.",
"FIGS.",
"9 and 10 serve to further explain the advantageous effects of the invention, whereby the time axes of all of the curves have been selected identically in order to illustrate the course over time.",
"FIG.",
"9 shows a schematic representation of a typical course over time of a) a signal pulse, b) a control signal, c) a voltage signal and threshold voltage as well as d) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8, whereby an echo occurs during the quiescent phase.",
"The control unit 31 transmits short signal pulses 41 to the oscillator 32, as a result of which the sound transducer 34 is excited so as to emit a threshold voltage packet (FIG.",
"9, Curve a).",
"Moreover, the signal transmitter 2 transmits a control signal SSt to the switch 3, which is synchronized with the signal pulse 41 in such a way that the control signal SSt begins shortly before the signal pulse 41 and ends at the same time as or during the presence of the former (FIG.",
"9, Curve b).",
"FIG.",
"9, Curve c, shows the course of the voltage signal Usignal (solid curve) that is identical to the envelope curve, and the course of the threshold voltage Uref1 (dotted curve).",
"The course of the Usignal during the transmittal and relaxation phase and at the beginning of the quiescent phase corresponds essentially to that of the envelope curve 53, which was explained with reference to FIG.",
"4.While the control signal SSt is present at the switch 3, the latter is opened, so that a threshold voltage Uref1 is established which is identical to the voltage supplied by the first direct voltage source 4a, 4b (FIG.",
"9, Curve c).",
"Therefore, this has been advantageously selected in such a way that it is greater than the maximum of the voltage signal, so that the adaptation of the threshold voltage to the voltage signal begins immediately after the opening of the switch 3.In the present example, the maximum possible magnitude of the voltage signal is supplied by the voltage limiter 35 or by the overmodulation limit of the amplifier 36c (FIG.",
"8).",
"According to the invention, after the end of the control signal SSt, a threshold voltage Uref1 is established that is greater than the voltage signal Usignal by the voltage distance dU.",
"Therefore, the threshold voltage Uref1 drops immediately after the end of the control signal by a certain quantity.",
"From this point in time on, the threshold voltage proceeds parallel-offset by a fundamental voltage distance dU with respect to that of the voltage signal Usignal as long as the latter does not increase.",
"A dummy switching signal is not triggered.",
"If, however, the voltage signal Usignal increases, the threshold voltage remains constant so that a first echo peak 60 of the voltage signal Usignal leads to a triggering of the comparator 1 as soon as the voltage signal Usignal exceeds the threshold voltage Uref1.In this case, the comparator 1 emits a first switching signal S1 (FIG.",
"9, Curve d).",
"FIG.",
"10 shows a schematic representation of a typical course over time of a) a voltage signal and threshold voltage as well as b) a comparator switching signal, all of which can occur during the operation of the distance sensor according to the invention shown in FIG.",
"8, whereby an echo occurs during the relaxation phase and another echo occurs during the quiescent phase.",
"The courses over time of the signal pulse 41 and of the control signal SSt are identical to those shown in FIG.",
"9.During the relaxation time, an echo signal occurs, which manifests itself in a second echo peak 61 of the voltage signal Usignal (FIG.",
"10, Curve a).",
"Up until the beginning of the second echo peak 61, the curves correspond to those shown in FIG.",
"9, Curve c. According to the invention, however, the decrease of the threshold voltage Uref1 comes to a halt as soon as the voltage signal Usignal is no longer dropping, and a first switching signal S1a is triggered by the second echo peak 61.After the second echo peak 61 that occurs during the relaxation time, the fundamental voltage distance dU is once again established between the threshold voltage Uref1 and the voltage signal Usignal.",
"During the quiescent phase, another, third echo peak 62 of the voltage signal Usignal occurs, as a result of which another switching signal S1b is triggered.",
"Now reference will be made to FIGS.",
"11 to 13, which show additional advantageous embodiments of an adaptive comparator circuit according to the invention.",
"FIG.",
"11 shows a preferred embodiment 10c of the invention which differs from the adaptive comparator circuit 10a shown in FIG.",
"6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.",
"b) A first resistor 11 is connected between the first pole 4a and the first threshold value input 1a, and a second resistor 12 is connected between the first threshold value input 1a and the second pole 4b, so that the first resistor 11 and the second resistor 12 form a first voltage divider 11, 12.Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the first voltage divider.",
"Thus, through the use of the first voltage divider 11, 12, the voltage distance by which the threshold voltage is parallel-offset with respect to the voltage signal after the voltage signal Usignal has died out can advantageously be changed by the fundamental voltage distance dU, to a value dUa.",
"When this embodiment 10c of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be changed and, in particular increased.",
"This is advantageous for many applications such as, for example, for the detection of small objects.",
"The first voltage divider can be an adjustable potentiometer whose pick-up is connected to the switch output.",
"c) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.",
"The purpose of these resistors was already explained above.",
"d) The diode 5 is connected in series to an eighth resistor 18 which serves to slow down the discharging of the capacitor 6 and thus to reduce the speed with which the threshold voltage Uref1 is adapted to the voltage signal Usignal.",
"The eighth resistor also serves to smooth the course of the threshold voltage Uref1 with respect to very rapid fluctuations of the voltage signal Usignal.",
"FIG.",
"12 shows another preferred embodiment 10d of the invention, which differs from the adaptive comparator circuit 10c shown in FIG.",
"11 in that, before the voltage signal Usignal reaches the first signal input 1b, it passes through an impedance transformer.",
"This embodiment is especially advantageous when the voltage signal arriving at the electric terminal 8 is high-ohmic, whereby said voltage signal can be the envelope curve voltage generated, for example, by an envelope curve shaper.",
"FIG.",
"13 shows another preferred embodiment 10e of the invention which differs from the adaptive comparator circuit 10a shown in FIG.",
"6 as follows: a) The first switch is a transistor 103.The base 103a of the transistor 103 serves as the control-current terminal, the emitter 103b serves as the switch input and the collector 103c as the switch output.",
"b) A second comparator 20 is used that has a second threshold value input 20a and a second signal input 20b, and that emits a second switching signal S2 when a greater voltage is present at the second signal input 20b than at the second threshold value input 20a.",
"c) The first pole 4a of the first voltage source 4a, 4b is connected to the second threshold value input 20a via a third resistor 13.The second threshold value input 20a is connected to the second pole 4b via a fourth resistor 14, so that the third and the fourth resistors 13, 14 form a second voltage divider which can be configured as a potentiometer.",
"d) The collector 103c that serves as the switch output and the second threshold value input 20a are connected via a fifth resistor 15.e) A second switch 23 is connected in series to the diode 5 and the former can be regulated or activated by the second switching signal S2 in such a way that it is in the closed state when the second comparator 20 emits the second switching signal S2, and otherwise it is in the open state.",
"The second switch 23 can be a transistor.",
"f) A sixth resistor 16 is connected between the control output 2a of the signal transmitter 2 and the base 103a, and a seventh resistor 17 is connected between the base 103a and the first pole 4a, which is a plus pole in the example shown.",
"The purpose of these resistors was already explained above.",
"g) The diode 5 is connected in series to an eighth resistor 18.The purpose of this resistor was already explained with reference to FIG.",
"11.h) Before reaching the first signal input 1b, the voltage signal passes through an impedance transformer 21 whose purpose was already explained with reference to FIG.",
"12.Due to the second voltage divider 13, 14, a voltage Uref2 is present at the second threshold value input 20a.",
"As long as the second switch 23 is closed, the first threshold voltage Uref1 follows the dropping voltage signal Usignal, parallel-offset by the fundamental voltage distance dU, as explained above.",
"According to the invention, however, the second switch opens as soon as the voltage signal Usignal exceeds the second threshold voltage Uref2.As a result, the discharge of the capacitor 6 and thus the dropping of the first threshold voltage Uref1 are ended.",
"Hence, in this embodiment of the invention, the capacitor 6 can only discharge until the first threshold voltage Uref1 has dropped to the magnitude of the second threshold voltage Uref2 supplied by the second potentiometer 13, 14.Thus, through the use of the second voltage divider 13, 14, the voltage distance by which the first threshold voltage is advantageously changed parallel-offset after the decay of the voltage signal Usignal with respect to voltage signal Usignal, from the fundamental voltage distance dU to a value dUb.",
"The second voltage divider 13, 14 can be an adjustable potentiometer whose pick-up is connected to the second threshold value input 20a.",
"The fifth resistor 15 is connected between the switch output 103c and the second threshold value input 20a.",
"Therefore, the magnitude of the first threshold voltage Uref1 counters the voltage that is supplied by the second voltage divider 13, 14.In particular, through the use of the fifth resistor 15, it can additionally be achieved that the voltage distance by which the first threshold voltage is parallel-offset with respect to the voltage signal Usignal strives towards a value dUb that—depending on the voltage supplied by the second voltage divider 13, 14—can advantageously be not only larger but also smaller than the fundamental voltage distance dU.",
"When this embodiment 10e of an adaptive comparator circuit according to the invention is used in an acoustic distance sensor, the sensitivity of the distance sensor that is achieved during the quiescent phase can thus be either increased or decreased by means of the second voltage divider 13, 14.All of the embodiments of an adaptive comparator circuit according to the invention and thus also the embodiments 10b, 10c, 10d and 10e illustrated in FIG.",
"6 and FIGS.",
"11 to 13 can be used according to the invention in the acoustic distance sensor shown in FIG.",
"8 instead of the adaptive comparator circuit 10a shown there by way of an example.",
"Commercial Utilization The invention can be utilized commercially, for example, in distance measuring technology, in filling level measuring technology and in automation technology.",
"The main figure is FIG.",
"5.List of Reference Numerals and Letters 1 first comparator 1a first threshold value input 1b first signal input 2 signal transmitter 2a control output of 2 2b input of 2 3 first switch 3a control-current terminal of 3 3b switch input of 3 3c switch output of 3 4a, 4b first, second pole of the first direct voltage source 5 diode 5a, 5b anode, cathode of 5 6 capacitor 7a, 7b positive, negative pole of 7 7 second direct voltage source 8, 9 first, second electric terminal 10a,b,c,d,e adaptive comparator circuits 11-18 first to eighth resistor 20 second comparator 20a second threshold value input 20b second signal input 21 voltage follower 23 second switch 30a distance sensor with constant threshold voltage 30b distance sensor with variable threshold voltage 31 control unit 32 oscillator 33 driver 34 sound transducer 35 voltage limiter 36a amplifier with regulatable amplification factor 36b, c logarithmic amplifier 37 envelope curve shaper 38a amplification factor actuation stage 38b threshold value actuation stage 41 signal pulses 42 course over time of the amplification factor of 36a 42a minimum of 42 43, 53 courses over time of the envelope curve voltage 43a, 53a segments of 43, 53 during the signal pulses 41, 51 43b, 53b segments of 43, 53 during the relaxation time of 34 43c, 53c segments of 43, 53 during the quiescent phase 43d, 53d segments of 43, 53 during an echo 44, 54 threshold values of 30a, 30b 45 dummy switching signal 46, 56 useful switching signals 60 first echo peak 61 second echo peak 62 third echo peak 103 transistor 103a base of 103 103b emitter of 103 103c collector of 103 S1, S1a, S1b switching signals of 1 S2 switching signal of 20 dU fundamental voltage distance Uref1 first threshold voltage Usignal voltage signal"
]
] | Patent_10450932 |
[
[
"Fuel injector and method for installing a fuel injector in a valve seat",
"A fuel injector, which, at its inflow-side end, has an intake connector at whose outer circumference at least two sealing rings are provided, which seal the fuel injector from a valve seat.",
"Due to an intermediate ring, the two sealing rings are axially set apart from one another in a respective ring chamber.",
"The intermediate ring is embodied independently of the intake connector and is made up of two parts.",
"A flexible outer ring is locked in place on an inner extruded support ring, the outer ring only being mounted once the first sealing ring has already been moved across the support ring and has been stripped off.",
"The fuel injector is particularly suited for the use in mixture-compressing internal combustion engines having external ignition."
],
[
"1-13.",
"(canceled) 14.A fuel injector for injecting fuel into a combustion chamber of a mixture-compressing internal combustion engine having external ignition, the fuel injector having a longitudinal valve axis, the fuel injector comprising: an intake connector situated at an inflow-side end, and having at least two sealing rings provided at an outer periphery of the intake connector to seal the fuel injector from a valve seat; and an intermediate ring, wherein, via the intermediate ring, the two sealing rings are axially set apart from one another, being situated in each case in a ring chamber, the intermediate ring being embodied independently of the intake connector and including two parts.",
"15.The fuel injector of claim 14, wherein the intermediate ring includes an inner support ring and an outer ring.",
"16.The fuel injector of claim 15, wherein the inner support ring, via an inwardly projecting lip, engages in a groove of the intake connector.",
"17.The fuel injector of claim 15, wherein the inner support ring is made of plastic.",
"18.The fuel injector of claim 17, wherein the inner support ring is made of the same plastic as a plastic extrusion coat partially surrounding the fuel injector.",
"19.The fuel injector of claim 14, further comprising: an outwardly projecting locking bead, at an outer diameter of the support ring, at which the outer ring is able to lock into place.",
"20.The fuel injector of claim 19, wherein an upper locking surface of the locking bead extends largely perpendicularly to the longitudinal valve axis and a lower locking surface has a slanted arrangement.",
"21.The fuel injector of claim 15, wherein the outer ring is made of flexible plastic.",
"22.The fuel injector of claim 14, wherein the intermediate ring forms a support shoulder for at least one of the at least two sealing rings.",
"23.A method for installing a fuel injector in a valve seat, the fuel injector, at the inflow-side end, having an intake connector at whose outer circumference at least two sealing rings are provided, which are axially set apart from one another by an intermediate ring and which seal the fuel injector from the valve seat, the method comprising: affixing an inner support ring of the intermediate ring on a circumference of the intake connector; spreading open a first one of the at least two sealing rings, mounting it on the intake connector, moving it across the inner support ring and stripping it into a ring chamber, and mounting an outer ring on the inner support ring to complete the intermediate ring; spreading open a second sealing ring, mounting it on the intake connector and stripping it into an additional ring chamber; and subsequently inserting the fuel injector in the valve seat together with a sealing system thus mounted on the intake connector.",
"24.The method of claim 23, wherein the inner support ring of the intermediate ring is extruded on a circumference of the intake connector in a same extrusion procedure as a plastic extrusion coat at least partially surrounding the fuel injector.",
"25.The method of claim 23, wherein the outer ring of the intermediate ring is locked into place on a locking bead of the inner support ring.",
"26.The method of claim 23, wherein the fuel injector is secured after having been inserted in the valve seat with corresponding affixation elements provided on the valve seat and on the fuel injector."
],
[
"<SOH> BACKGROUND INFORMATION <EOH>At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.",
"For instance, German Patent Application No.",
"28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.",
"These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.",
"German Patent Application No.",
"28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.",
"Furthermore, German Patent Application No.",
"28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.",
"Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.",
"37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.",
"Moreover, German Patent Application No.",
"197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.",
"In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.",
"Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.",
"Of particular advantage may be the simple handling of the two partial components of the intermediate ring.",
"While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.",
"The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.",
"The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.",
"The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.",
"In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.",
"It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.",
"Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings."
],
[
"FIELD OF THE INVENTION The present invention relates to a fuel injector, and a method for installing a fuel injector in a valve seat.",
"BACKGROUND INFORMATION At the outer periphery of various fuel injectors, a plurality of sealing elements in the form of sealing rings may be arranged in succession so as to ensure an effective sealing of the fuel injector from a valve seat.",
"For instance, German Patent Application No.",
"28 27 789 refers to providing two sealing rings at a stepped inflow connector of the fuel injector.",
"These may be embodied as whole ring stacks, which then include a stuffing-box ring, an o-ring, an asbestos ring cord, and a plastic fusion ring arranged in immediate succession.",
"German Patent Application No.",
"28 27 878 refers to fuel injectors whose sealing is accomplished via sealing stacks, additional rings, having so-called emergency-sealing properties, being provided in addition to the actual sealing rings in o-form.",
"Furthermore, German Patent Application No.",
"28 27 850 refers to spacing two sealing rings at the periphery of the fuel injector by using a shoulder that radially projects beyond the outer circumference of the inflow-connecting piece.",
"Furthermore, a sealing system on a fuel injector is referred to in German Patent Application No.",
"37 03 615, in which two sealing rings in each case surround one of two connecting pieces independently from one another and appropriately seal it from a fuel-supply line.",
"Moreover, German Patent Application No.",
"197 35 665 refers to a fuel-injection system in which a fuel injector is connected via an intermediate piece to a valve-seat connector of a fuel-supply line and sealed therefrom.",
"In this arrangement, both the inflow connector of the fuel injector and also the intermediate pieces each have a sealing ring at the outer circumference, which spatially are thus clearly located at a distance from one another.",
"SUMMARY OF THE INVENTION The exemplary fuel injector according to the present invention provides a sealing system having two sealing rings at its outer periphery, which may be especially easy to install, avoid excessive expansion and excessive spread of the sealing rings and may therefore basically avoid damage of the sealing rings.",
"Nevertheless, it may guarantee sufficiently large support shoulders for the sealing rings and completely prevent the release of hydrocarbons into the environment, in compliance with ever stricter environmental demands.",
"Of particular advantage may be the simple handling of the two partial components of the intermediate ring.",
"While the inner support ring may be installed in a very simple manner in a groove on the intake connector of the fuel injector by way of an inwardly projecting lip, the outer ring may be snapped into place on the support ring in an uncomplicated and reliable manner.",
"The locking bead on the support ring used for locking is arranged such that the high compression forces of the one sealing ring during the installation of the fuel injector in the receiving socket are absorbed by an upper horizontal locking surface.",
"The lower axial demounting forces of the other sealing ring are sufficiently absorbed by a lower locking surface of the locking bead, which has a slanted configuration.",
"The exemplary method according to the present invention for installing a fuel injector in a valve seat may have the advantage that the overall installation may be performed in a fully automatic manner.",
"In an especially advantageous manner, the sealing rings are handled very gently during the mounting, so that damage to the sealing rings is excluded.",
"It may be especially advantageous that the sealing rings, despite being inserted in separate ring chambers, are barely spread open, so that there is no need to stretch them to a dangerous extent.",
"Because the intermediate ring is arranged or configured in two parts, it is ensured that the first sealing ring is able to be installed without being overstretched, yet sufficiently large support shoulders are subsequently available for both sealing rings.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows an inflow-side end of a fuel injector having a sealing system configured according to the present invention for sealing the fuel injector from a valve seat.",
"FIG.",
"2 shows an enlargement of the cut-out section II in FIG.",
"1.DETAILED DESCRIPTION FIG.",
"1 shows a partial representation of a fuel injector 1, which, for instance, is embodied as a fuel injector for the direct injection of fuel into the combustion chamber of a mixture-compressing internal combustion engine having external ignition.",
"By way of an inflow-side end 2, fuel injector 1 projects into a corresponding valve seat 3.Valve seat 3 is embodied, for instance, as a cup-shaped intake connecting piece 4, which constitutes part of a fuel-supply line (not shown further), such as a fuel rail.",
"The fuel-supply line usually has a plurality of intake connecting pieces 4, so that the fuel conveyed via the fuel-supply line may be distributed to a plurality of fuel injectors 1.Intake connecting piece 4 has a stepped arrangement or configuration, for example, and, at its end facing away from the fuel-supply line, has one or a plurality of affixation elements 5 projecting in the manner of a lip.",
"These affixation elements 5 engage in openings 6 of an securing clamp 7, which is used to reliably secure fuel injector 1 in valve seat 3.Securing clamp 7 is inserted, for instance, in a circumferential groove 8 at the outer periphery of a plastic extrusion coat 9 of fuel injector 1.Fuel injector 1 is largely surrounded by plastic extrusion coat 9, for example.",
"However, at inflow-side end 2 of fuel injector 1, a metal intake connector 11 for the supply of fuel to the interior of fuel injector 1 projects beyond plastic extrusion coat 9.Above plastic extrusion coat 9, two sealing rings 13, 14, configured as O-rings, are situated at the outer periphery of intake connector 11.Due to an intermediate ring 15 configured according to the exemplary embodiment of the present invention, the two sealing rings 13, 14, which follow each other closely in the axial direction, are positioned at a spatial distance from one another.",
"Forming the upstream end of fuel injector 1 is an upper retaining ring 16, which is made of plastic and mounted on intake connector 11.In this manner, both sealing rings 13, 14 are embedded between retaining ring 16 and upper end face 17 of plastic extrusion coat 9, intermediate ring 15 forming two ring chambers 18, 19 for sealing rings 13, 14.In contrast to the sealing rings having so-called emergency-sealing properties known in part from the related art, the two sealing rings 13, 14 are two “full-fledged” sealing rings made of known elastomer sealing material.",
"The necessity of two sealing rings immediately following one another at the periphery of fuel injector 1 results from the composition of certain fuels.",
"In long-term use of sealing rings it has become apparent that hydrocarbons permeate through the elastomers normally used for sealing rings and may even permeate them completely.",
"In order to fully prevent the emission of hydrocarbons into the environment in accordance with ever stricter environmental regulations, it may be advantageous to situate an additional sealing ring immediately behind a first sealing ring.",
"In this manner, the second sealing ring following a sealing ring permeated in the manner described above, offers a substantially improved sealing reliability.",
"Intermediate ring 15, which partitions the sealing chamber into two ring chambers 18, 19, is made up of two sub-components.",
"An inner support ring 22 engages, for instance, in a circumferential groove 23 at the periphery of intake connector 11.Ideally, support ring 22 is extruded (injected) on the metal valve body, together with plastic extrusion coat 9 and retaining ring 16, in one plastic-extrusion procedure.",
"On its inner side, support ring 22 has a slanted lip 24, which projects toward the inside and is able to be inserted in groove 23 with form accuracy.",
"Provided at the outer diameter of support ring 22 is a locking bead 25, which projects radially outward and at which an outer ring 26, which also belongs to intermediate ring 15, is able to be snapped into place.",
"The region of locking bead 25 is shown in FIG.",
"2 in an enlarged view.",
"Locking bead 25 at support ring 22 is arranged or configured such that the high compression forces of second sealing ring 13 during the installation of fuel injector 1 in intake connecting piece 4 are absorbed by an upper locking surface 28, which mostly extends perpendicularly to a longitudinal valve axis 30.The lower axial demounting forces of first sealing ring 14 are sufficiently absorbed by a lower locking surface 29 of locking bead 25, locking surface 29 having a slanted arrangement or configuration.",
"Outer ring 26 is made of flexible plastic.",
"When outer ring 26 is mounted on inner support ring 22, this plastic expands and then locks into place in the limit position above locking bead 25.Intermediate ring 15, made up of outer ring 26 and support ring 22, is used as support shoulder for second sealing ring 13.The mounting of the sealing system on intake connector 11 of fuel injector 1 is briefly explained in the following.",
"As already described above, plastic extrusion coat 9, inner support ring 22 of intermediate ring 15, and retaining ring 16 are, for instance, simultaneously extruded on the metal valve body in one extrusion procedure.",
"Subsequently, first sealing ring 14 is spread open, mounted on intake connector 11, moved across support ring 22 and stripped into ring chamber 19.Then, outer ring 26 is clipped onto inner support ring 22 to complete intermediate ring 15.Second sealing ring 13 is then spread open, mounted on intake connector 11 and stripped into ring chamber 18.With the aid of the sealing system mounted in this manner on intake connector 11, fuel injector 1 may be inserted in valve seat 3.The exemplary embodiment and/or method of the present invention is not limited to the described exemplary embodiment or method.",
"Thus, intake connector 11, for instance, may have a stepped arrangement or configuration in the region of sealing rings 13, 14 and/or sealing rings 13, 14 may have different cross sections or diameters."
]
] | Patent_10450961 |
[
[
"PROCESS FOR THE PRODUCTION OF GRAIN ORIENTED ELECTRICAL STEEL STRIPS",
"A process for the production of grain oriented electrical Fe—Si strips in which a Si-containing alloy is directly cast as a strip between 2.5-5.0 mm thick and cold rolled in one stage, or in more stages with intermediate annealing, to a final thickness of between 0.15-1.0 mm.",
"The strip is then continuously annealed to carry out the primary recrystallization and then annealed to carry out the oriented secondary recrystallization.",
"The process further includes that after solidification of the strip, and before its coiling, a phase transformation from Ferrite to Austenite is induced into the metal matrix for a volume fraction between 25-60%, obtained by controlling the alloy composition so that the Austenite fraction is allowed within the stability equilibrium between the two phases.",
"The strip is then deformed by rolling in-line with the casting step to obtain a deformation higher than 20% in the temperature interval 1000-1300° C."
],
[
"1-8.",
"(canceled) 9.A process for the production of electrical grain oriented Fe—Si strips in which a Si-containing molten alloy composition is directly cast as continuous strips 2.5 to 5 mm thick, cold rolled in one step or more steps with intermediate annealing to a final thickness of between 1 and 0.15 mm, the strip being then continuously annealed to carry out oriented secondary recrystallisation, characterised in that after the strip solidification and before coiling of said strip in a coiling phase, a ferrite to austenite transformation is induced in the metal matrix via deformation, by rolling said strip between two cooled rolls to obtain a deformation over 20% in a temperature range of 1000-1300° C., thereby producing a volume fraction of austenite to be between 25 and 60%, said molten alloy composition being chosen such that said volume fraction of austenite is stable in a temperature interval of between 1100 and 1200° C. 10.The process according to claim 9, in which between the rolling phase and the coiling one, the strip is held between 1100 and 1200° C. for at least 5 s. 11.The process according to claim 9, in which the as-solidified strip thickness is comprised between 1.5 and 4.0 mm and after the rolling phase the strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.",
"12.The process according to claim 9, in which before cold rolling the strip is annealed at a maximum temperature of 1200° C. 13.The process according to claim 12, in which after said annealing the strip is continuously quenched from a temperature comprised between 750 and 950° C. down to 400° C. in less than 12 s. 14.The process according to claim 9, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5 wt % Cu, less than 2 wt % Cr+Ni+Mo.",
"Less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 15.The process according to claim 9, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 16.The process according to claim 9, in which in the alloy at least an element is added chosen between Sn, Sb, P, Bi.",
"17.A process for the production of grain oriented electrical Fe—Si strip, in which a Si-containing alloy is directly cast as continuous strip 2.5 to 5 mm thick, in-line hot-rolled and then cold-rolled in one step or more steps, with intermediate annealing, to a final thickness of between 1 and 0.15 mm, the cold-rolled strip being, then continuously annealed to carry out primary recrystallisation and subsequently again annealed to carry out secondary recrystallisation, characterized in that the Si-containing alloy composition is selected to induce in the alloy, during the hot-rolling step in which a deformation rate of at least 20% is utilized in a temperature interval of between 1000 and 1300° C., a ferrite to austenite phase transformation with an austenite volume fraction of between 25 to 60% stable in a temperature interval of between 1100 to 1200° C. 18.The process according to claim 17, in which between the hot-rolling and the coiling steps the strip is held in the temperature range of 1100 to 1200° C. for at least 5 s. 19.The process according to claim 17, in which an as-cast strip 2.5 to 4 mm thick is in-line hot-rolled and the thus obtained hot-rolled strip is quenched to obtain a volume fraction of martensite comprised between 5 and 15%.",
"20.The process according to claim 17, in which before cold-rolling the strip is annealed at a maximum temperature of 1200° C. 21.The process according to claim 20, in which, after said annealing, the strip is continuously quenched from a temperature of between 750 and 950° C. down to 400° C. in less than 12 s. 22.The process according to claim 17, in which the cast alloy comprises 2.5-5.0 wt % Si, 200-1000 ppm C, 0.05-0.5 wt % Mn, 0.07-0.5% Cu, less than 2.0% Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. 23.The process according to claim 22, in which in the alloy at least an element is added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V, Co. 24.The process according to claim 22, in which in the allot at least an element is added chosen between Sn, Sb, P, Bi."
],
[
"<SOH> FIELD OF THE INVENTION <EOH>Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.",
"More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.",
"Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.",
"Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.",
"Further objects of present invention will be evident from the following description of the invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF THE INVENTION Present invention refers to the production of grain oriented electrical steel strips havig excellent magnetic characteristics, dedicated to the production of transformer cores.",
"More precisely, rhe invention refers to a process in which a Fe—Si alloy is continuously cast directly as strip and, before coiling, the strip itself is continuously deformed by rolling to induce the formation in the metal matrix of a given fraction of Austenite, controlled as amount and distribution, thus obtaining a strip micro-structure stably and uniformly recrystallised before cold rolling.",
"STATE OF THE ART Grain oriented electrical steel strips (Fe—Si) are typically industrially produced as strips having a thickness comprised between 0,18 and 0,50 mm and are characterised by magnetic properties variable according to the specific product class.",
"Said classification substantially refers to the specific power losses of the strip subjected to given electromagnetic work conditions (e.g.",
"P50 Hz at 1,7 Tesla, in W/kg), evaluated along a specific reference direction (rolling direction).",
"The main utilisation of said strips is the production of transformer cores.",
"Good magnetic properties (strongly anisotropic) are obtained controlling the final crystalline structure of the strips to obtain all, or almost all, the grains oriented to have their easiest magnetization direction (the <001> axis) aligned in the most perfect way with the rolling direction.",
"In practice, final products are obtained having the grains mean diameter generally comprised between 1 and 20 mm having an orientation centred around the Goss orientation ({110} <001>).",
"The minor the angular dispersion around the Goss one, the better the product magnetic permeability and hence the lesser the magnetic losses.",
"The final products having low magnetic losses (core losses) and high permeability have interesting advantages in terms of design, dimensions and yield of the transformers.",
"The first industrial production of the above materials was described by the U.S. Firm ARMCO at the beginning of the thirties (U.S. Pat.",
"No.",
"1,956,559).",
"Many important improvements have been since introduced in the production technology of grain oriented electrical strips, in terms both of magnetic and physical quality of products and of transformation costs and cycles rationalisation.",
"All existing technologies exploit the same metallurgical strategy to obtain a very strong Goss structure in the final products, i.e.",
"the process of oriented secondary recrystallisation guided by uniformly distributed second phases and/or segregating elements.",
"The, non metallic, second phases and the segregating elements play a fundamental role in controlling (slowing down) the movement of grain boundaries doing the final annealing which actuates the selective secondary recrystallisation process.",
"In the original ARMCO technology, utilising MnS as inhibitor of the grain boundaries movement, and in the subsequent technology developed by NSC, in which the inhibitors are mainly aluminium nitrides (AIN+MnS) (EP 8.385, EP 17.830, EP 202.339), a very important binding step common to both production processes is the heating of the continuously cast slabs (ingots, in old times), immediately before the hot rolling, at very high temperatures (around 1400° C.) for a time sufficient to guarantee a complete dissolution of sulphides and/or nitrides coarsely precipitated during the slab cooling after casting, to re-precipitate them in a very fine and uniformly distributed form throughout the metallic matrix of the hot rolled strips.",
"Such a fine re-precipitation can be started and completed, as well as the precipitates dimensions adjusted, during the process, in any case, however, before the cold rolling.",
"The slab heating to said temperatures requires using special furnaces (pushing furnaces, liquid-slag walking-beam furnaces, induction furnaces) due to the ductility at high temperatures of the Fe-3% Si alloys and to formation of liquid slags.",
"New casting technologies of the liquid steel are intended to simplify the production processes to make them more compact and flexible and to reduce costs.",
"One of said technologies is the “thin slab” casting, consisting in the continuous casting of slabs having the typical thickness of conventional already roughened slabs, apt to a direct hot rolling, through a sequence of slabs continuous casting, treating in continuous tunnel-furnaces to rise/maintain the temperature of slabs and finishing-rolling, down to coiled strip.",
"The problems connected to the utilisation of said technique for grain oriented products mainly consist in the difficulty to maintain and control the high temperatures necessary to keep in solution the elements forming the second phases, which have to be finely precipitated at the beginning of the finishing hot-rolling step, if desired best micro-structural and magnetic characteristics are to be obtained in the end products.",
"Such problems were dealt with in different ways, for instance utilising the low thickness of the cast slabs in connection to specific concentration intervals of the micro-alloying elements to stably control the second phases precipitation (grain growth inhibitors) during hot rolling, or drastically modifying the strategy of the inhibitors formation in the metal matrix.",
"The casting technique potentially offering the highest rationalisation level of the processes and the higher production flexibility is the one consisting in the direct production of strips from the liquid steel (Strip Casting), totally eliminating the hot rolling step.",
"Such an exaordinary innovation was conceived and patented long time ago, and since long time were also devised and patented process conditions to produce electrical steel strips, and more particularly grain oriented ones.",
"However, up to now there is not an industrial production in the world of grain oriented electrical steel according to the above technique, though the state of the art relating to the casting machines is ready for industrial applications, as shown by existing plants, producing only carbon steels and stainless steels.",
"The present inventors believe that to industrially produce grain oriented electrical steel strips from direct solidification of a strip (Strip Casting) it is necessary to have a strip micro-structure before cold rolling significantly different from the one obtained during the casting stage.",
"The high solidification speed of the cast strip makes it difficult to have a homogeneous and reproducible grain structure throughout the strip and between different castings, due to the high sensitivity of the solidification structure to the fluctuations of the casting conditions and to the alloy composition.",
"The micro-structure of the intermediate products starting from strip casting is much more influenced by the solidification structure, with respect to the ones derived from conventional slab casting, because of the lack of deformation in the strip during the typical hot rolling.",
"SUMMARY OF THE INVENTION The aim of present invention is to solve the inconveniences due to the quality of electrical steel strips deriving from strip casting.",
"Thus, it is an object of present invention a process for the production of electrical steel strips in which, through an in-line thickness reduction of the strip between casting and coiling stations, a significant level of recrystallisation by means of phase transformation is induced, thus normalising the crystalline structure before cold rolling, so that possible fluctuations in the process conditions are substantially non-influent with respect to the quality of the final product.",
"Another object of present invention is to make it possible to industrially produce grain oriented electrical steel strips having excellent magnetic characteristics and constant quality, the process being stable and simplyfied with respect to the conventional processes currently utilised.",
"Further objects of present invention will be evident from the following description of the invention.",
"DETAILED DESCRIPTION OF THE INVENTION A first important aspect of present invention resides in that a molten alloy containing silicon is directly solidified in the form of a strip, through the casting technology known as strip casting (casting between twin cooled and counter-rotating rolls), thus avoiding, with respect to currently utilised technologies, casting the alloy in slabs or ingots, subjecting said slabs to thermal treatment in special high-temperature furnaces for long times (to attain the necessary thermal homogeneity) and transforming said slabs into strips through hot rolling with total reductions which, according to the slab casting technologies, vary between 96 and 99%.",
"A second important aspect of present invention resides in that the chemical composition of the Silicon containing alloy is selected specifically to control the thermodynamic stability of the Austenite phase in the matrix (face-centered cubic lattice) in equilibrium with the Ferrite phase (body-centered cubic lattice).",
"More precisely, to obtain excellent final magnetic characteristics, it is convenient to adjust the chemistry of the alloy so that an Austenite fraction comprised between 25 and 60% is stable between 1100 and 1200° C. Consequently, to balance the strong tendency of silicon to stabilise the Ferrite phase, a number of elements are utilised, favouring the Austenite formation.",
"Aming those elements, Carbon is particularly important due to its intrinsic austenitising effect as well as to its particular mobility into the matrix, making it possible its easy elimination by means of solid-state decarburising processes which, in this field, are usually carried out by extraction from the strip surfaces utilising annealing atmospheres having a controlled oxidising potential.",
"The carbon is conveniently present in the steel composition in amount apt to control the desired Austenite fraction, in that in this way it is possible to rise again the stability of the Ferrite by means of a simple decarburisation process, and thus avoiding during the final secondary recrystallisation annealing important phase transition phenomena which would be detrimental for the final desired texture.",
"As known, however, in said materials itis necessary to reduce the carbon content in the final products at levels of under 50 ppm, to eliminate the adverse effect on the core losses due to formation of carbides.",
"The higher the carbon content of the alloy, the longer the time necessary to carry out the decarburisation.",
"For productivity reasons it is then convenient to keep the carbon content within a maximum of 0,1 wt %.",
"Present inventors eveluated the obtainable Austenite fractions according to different alloy compositions both experimentally and according to empirical relationships available in the literature.",
"A third aspect of the invention resides in that the Derrite to Austenite transformation in the metal matrix of the cast strip is induced, in a temperature interval centered around 1150° C., typically 1000-1300° C., by means of a sudden deformation higher than 20%, by rolling between cooled rolls, in-line with the continuous casting and before the coiling.",
"Said sudden and localised deformation imparts to the material the energy necessary to nucleation and formation of the Austenite phase in the matrix, which phase would not be obtained for kinetic reasons, though thermodynamically very stable.",
"In fact, to obtain equilibrium conditions between the two phases at the considered temperature very long times are necessary, while the working and cooling times are intrinsecally very short, particularly in the case of direct casting as strip (strip casting).",
"The phase transformation from Ferrite to Austenite is tunable, according to present invention, in quantity, according to selection of chemical composition, and consistently reproducible, as necessary in an industrial process.",
"As a consequence of the phase transformation induced in the temperature interval defined according to present invention, the grains distribution in the produced strip, in terms both of dimensions and of texture, is exrtremely homogeneous and reproducible through the whole geometrical profile of the strip.",
"This, in particular, solves the problem the drawback of microstructural etereogeneity, typical of the production of oriented grain steel strips, in that the selection process of the final texture is sensible even to small local differences in the structure and orientation of grains, and even more sensible in the case of strip-cast products.",
"In fact, in the traditional processes the strip structure before cold rolling is the result of a strong hot deformation of the cast slabs, which contributes to fragment, recrystallise and homogenise the solidification structure; on the contrary, in the strips obtained by direct solidification the structure directly depends on the solidification one, and due to the high solidification speed and to the strongly dynamic nature of the process any even small fluctuation of the casting conditions (such as strip thickness, casting speed, heat transfer to the casting rolls, etc.)",
"can induce local variations, periodic or random, in the solidification structure and therefore in the final strips micro-structure throughout its geometrical profile.",
"The process of the invention overcomes the drawbacks inherent in the directly cast steel strips, due to lack of high hot deformation levels refining and homogenising the micro-structure.",
"Said high deformation levels are typical of technologies based on conventional casting, and in present invention are very efficiently replaced by causing a controlled, as amount and distribution, phase transformation Ferrite to Austenute, able to refine and homogenise the micro-structure.",
"The high solification speeds proper of strip casting are also an important metallurgical opportunity to exploit in the best way the process according to present invention.",
"In fact, in the traditional technologies starting from slabs or ingots the Ferrite/Austenite transformation, if any, is localised in chemical segregation zones, in which austenitising elements are concentrated, particularly in the semi-products core.",
"Thus, in said zones the austenitic transformation can occur, due to local concentration of austenitising elements, even if the mean chemical composition of the steel would not consent it.",
"On the contrary, in the strip casting the high solidifation speeds strongly limit the segregating phenomena, thus making homogeneous in the matrix the distribution of austenitising elements.",
"In said conditions, by hot rolling in the prescribed temperature field, it is obtained in a stable and reproducible way the volumetric fraction of Austenite, defined by chosing the steel composition, throughout the whole geometrical profile of the strip.",
"A further element of present invention if the definition of a process utilising a controlled volumetric fraction of Austenite, induced within the strip as above defined, to obtain a controlled distribution of hard phases (Carbides, Cementite, Pearlite, Bainite) and to control the formation of some Martensite (tetragonal lattice) within the metal matrix, by quenching the strip between the in-line hot rolling and the coiling steps.",
"The presence of homogeneously distributed hard phases (quenching phases) permits the cold rolling to control the adequate deformation texture, clearly because of the different deformation models and of the higher hardening levels obtained by cold rolling when hard phases are present with reference to the case in which a quenching structure is not present.",
"This permits to reduce the thickness of the strip to be cold rolled (for the same final thickness) and consequently to reduce the thickness of the cast strip, with important advantages on the casting productivity.",
"In fact, the thinner the cast strip, the higher the casting productivity, in that the strip becomes longer in direct proportion to the thickness reduction, while the casting speed rises with the square of the thickness reduction.",
"A further element of present invention is a process in which the strip, after in-line deformation, is kept at a temperature around 1150° C., typically 1100-1200° C., for at least 5 s, utilising a continuous heating apparatus between the in-line rolling mill and the coiler.",
"This can be obtained for instance a heating chamber provided with burners, or with electric heating, or with infrared lamps, or with an induction-heating apparatus; however, any active or passive system apt to obtain the desired strip temperature in the prescribed interval and for at least 5 s. In this case, the optional quenching step will be carried out at the exit from said chamber.",
"Another aspect of present invention is a process in which the strip is annealed, before cold rolling, at temperature not exceeding 1200° C., preferably not exceeding 1170° C. Such an annealing can be advantegeous for the grain oriented electrical steel strip production process, for a number of reasons, particularly with respect to the magnetic characteristics control of the final products.",
"Some useful phenomena for the process are, for instance, the precipitation of non-metallic second phases, necessary in present products to the control of the oriented secondary recrystallisation, or the possibility to carry out a controlled surface decarburisation of the strips before the cold rolling, which can have positive effects on the texture of the cold rolled strip.",
"Moreover, this annealing can offer the possibility to shift to this process step the formation of quenching phases, instead of forming them before coiling the strip after the casting process.",
"In this case, at the end of the annealing furnace a suitable cooling device must be present able to reach the necessary cooling speed.",
"For instance, the strip cooling can be usefully obtained with respect to the teaching of present invention, by means of a group of lances provided with nuzzles to spray on the strip surface a mixture of water and steam, at a controlled pressure.",
"Typically, after the in-line rolling the strip is quanched to obtain a Martensite volume fraction comprised between 5 and 15%.",
"The quenching device operate starting from a temperature of between 750 and 950° C., to cool down the strip down to 400° C. in less than 12 s. A last element of present invention is a process in which the chemical composition requires the presence of elements chosen between two distinct classes: (i) elements useful to control the desired equilibtium bewteen Austenite anf Ferrite in the metal matrix and (ii) elements useful to control a second phases distribution, such as sulphides, selenides, nitrides, carbo-nitrides etc., necessary for the grain growth control and of grain orientation during the primary and secondary recrystallisation steps.",
"Typically, the cast steel composition comprises 2,5-5 wt % Si; 200-1000 ppm C, 0,05-0,5 wt % Mn, 0,07-0,5 wt % Cu, less than 2 wt % Cr+Ni+Mo, less than 30 ppm O, less than 500 ppm S+Se, 50-400 ppm Al, less than 100 ppm N. To this composition at least an element can be added chosen in the group consisting of Zr, Ti, Ce, B, Ta, Nb, V and Co, and at least an element chosen in the group consisting of Sn, Sb, P, Bi.",
"Many are the elements useful to the equilibrium control berween Austenite and Ferrite phases and there are no specific choice limitations, but cost and yield convenience.",
"However, and specifically in electric-furnace steel shops utilising steel scraps as raw material, it can be convenient to balance the content of silicon as well as of chromium, nickel, molybdenum, niobium, copper, manganese and tin.",
"Many are also the elements useful to control the distribution of second phases particles for the grain growth inhibition.",
"It is convenient to chose said elements among the ones able to form suphides, selenides, carbonitrides, nitrides, to obtain a mix of second phases having different composition in which co-exist compounds thermally stable, as solubility, at different temperatures.",
"As a consequence of this choice, the drag force of the grain boundaries movement due to second phases particles gradually diminishes as temperature rises, in that during the heat treatments the more soluble particles will dissolve and/or grow before the less soluble ones.",
"This permits a better control of grain growth, with respect to the utilisation of inhibitors of a single composition type chacarterised by a narrower solubilisation temperatures interval.",
"The following Examples are intended solely to illustration purposes not limiting the scope of present invention.",
"EXAMPLE 1 A number of steels having the compositions shown in Table 1 were cast as a strip 3,5 mm thick in a strip casting machine provided with twin counter-rotating rolls.",
"The cast strips were then in-line hot rolled at the temperature of 1150° C. to a 2,0 mm thickness.",
"During the casting operation of each steel composition and at about mid casting time, the cast strip thickness was reduced to 2,0 mm and the in-line rolling suspended.",
"The hot rolled strips were then annealed at 1100° C. and single-stage cold rolled to 0,30 mm.",
"TABLE 1 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 500 3.1 0.2 75 300 100 250 0.1 B 300 3.1 0.1 68 350 120 270 0.15 C 350 3.2 0.4 70 320 110 230 0.3 D 400 3.1 0.3 80 290 150 280 0.25 E 500 3.1 0.4 50 400 100 280 0.2 The cold rolled strips were then decarburised, coated with an MgO based annealing separator, box annealed with an heating rate of 15° C./h up to 1200° C., held at this temperature for 20 h, and then received an insulating and tensioning coating.",
"On the as-cast strips the austenite (γ phase) content at 1150° C. was calculated by means of dilatometric measures; data obtained are shown in Table 2.TABLE 2 Steel γ(1150) (%) A 27 B 11 C 15 D 19 E 25 The magnetic characteristics measured on the final product for the different steel composition are shown in Table 3.TABLE 3 In-line hot rolled Not in-line rolled Steel B800 (mT) B800 (Mt) A 1950 1700 B 1720 1650 C 1730 1630 D 1900 1680 E 1945 1710 EXAMPLE 2 A number of steels having different compositions as shown in Table 4 were directy cast as strips 2,1 mm thick in a strip-casting machine provided with twin counter-rotating rolls.",
"TABLE 4 C Si Mn S Steel (ppm) (%) (%) (ppm) Cr (ppm) Ni (ppm) Al (ppm) Cu (ppm) A 550 3.3 0.3 80 450 200 280 0.15 B 300 3.1 0.2 68 350 120 270 0.2 C 350 3.2 0.4 70 320 130 230 0.3 D 400 3.0 0.3 80 290 180 280 0.25 E 400 3.1 0.4 75 250 200 290 0.25 The cast strips were then in-line hot rolled at 1170° C. to a thickness of 1,0 mm, quenched by means of water and steam at high pressure down to a temperature of 150° C. and then coiled.",
"After casting about half of the steel the quenching was stopped and the strips wound at 700° C. Table 5 shows the Martensite fractions metallographically measured on the strip after coiling.",
"TABLE 5 Quenched strip Not-quenched strip Steel Martensite (%) Martensite (%) A 19 0 B 3 0 C 5 0 D 13 0 E 15 0 The strips were then divided into lesser coils, part of which were cold rolled to 0,3 mm (the casting A did show fragility problems during cold rolling and was not transformed into finished product), decarburised, coated with an MgO based annealing separator, then box annealed with a heating rate of 20° C./h up to 1200° C. and then held at this temperature for 20 h. Table 6 shows the magnetic characteristics (induction at 800 A/m) measured on the finished product.",
"TABLE 6 Quenched strip Not-quenched strip Steel B800 (mT) B800 (mT) A — 1830 B 1790 1650 C 1890 1630 D 1920 1820 E 1950 1830 EXAMPLE 3 The other lesser rolls of Example 2 without quenching and coiled at 700° C. were annealed at 1150° C. for 60 s, quenchecd by means of water and steam at high pressure down to 150° C., pickled and colied at room temperature.",
"The strips were then transformed into finished product as in preceding Example.",
"Table 7 shows the Martensite fractions measured on the coiled strips and relevant magnetic characteristics.",
"TABLE 7 Martensite B800 Steel (%) (mT) A 12 1950 B 2 1700 C 5 1740 D 8 1920 E 9 1920 EXAMPLE 4 Five different alloys of composition (in ppm) shown in Table 8 were cast directly as strips 2,2-2,4 mm thick in a casting machine with twin counter-rotating rolls.",
"TABLE 8 Si C Mn Cu Sn Cr Mo Nb Ni P Al Ce N S A 3.2 0.07 0.40 0.25 0.1 0.03 0.1 0.03 0.02 — 0.030 0.01 0.01 0.010 B 3.3 0.06 0.06 0.07 0.09 0.03 — 0.03 — 0.004 — 0.007 0.025 C 3.0 0.03 0.95 0.40 0.06 0.30 0.02 0.02 0.20 0.02 0.015 — 0.007 0.015 D 3.1 0.05 0.15 0.25 — 0.02 0.03 — 0.02 — 0.028 — 0.008 0.007 E 3.4 0.07 0.40 0.35 — 0.03 0.05 0.01 0.03 0.01 0.030 — 0.008 0.006 TABLE 9 Decarburation.",
"T (° C.) A1 B1 C1 D1 E1 A2 B2 C2 D2 E2 830 1890 1800 1920 1930 1910 1690 1520 1730 1640 1580 850 1930 1750 1940 1910 1920 1730 1540 1780 1540 1630 870 1940 1590 1890 1900 1890 1780 1530 1690 1520 1540 The cast steels were in-line hot rolled at 1150° C. to a thickness of 1,2 mm.",
"From said coiled strips were obtained lesser coils.",
"For each condition a strip was then double-stage annealed with quick heating to 1170° C., cooling at 1100° C. and quenched to room temperature with water plus steam jets (strips A1, B1, C1, D1, E1).",
"A second group of strips, similar the the previous one was annealed with a similar thermal cycle, without however the quenching step (strips A2, B2, C2, D2, E2).",
"All the strips were then single-stage cold rolled to a final thickness of 0,29 mm.",
"The strips were then treated in a continuous pilot line for primary recrystallisation, nitriding, secondary recrystallisation.",
"Each strip was then treated as follows: in the first treating zone (primary recrystallisation) the temperatures of 830, 850 and 870° C. were adopted, in a wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,60 and for 180 s (50 of which for the heating at treating temperature) in the second treating zone nitriding was carried out at 890° C. in wet Nitrogen-Hydrogen atmosphere with a pH2O/pH2 ratio of 0,09, with the addition of 30% vol of ammonia, for 50 s in the third zone, at 1100° C. in a wet Nitrogen/Hydrogen atmosphere with a pH2O/pH2 ratio of 0,01 for 50 s. After coating with an Mg/O based annealing separator the strips treated in the pilot line were then box annealed with a heating rate of about 60° C./h up to 1200° C. in a 50% Nitrogen-Hydrogen atmosphere, held at this temperature for 3 h in pure hydrogen and cooled down to 800° C. in hydrogen and subsequently to room temperature in nitrogen.",
"The magnetic characteristics measured on samples of each of said strips were measured as induction mean value B800 in mT, and are shown in Table 9."
]
] | Patent_10450969 |
[
[
"Generic architecture for adaptable software",
"This invention concerns adaptable software.",
"In particular it concerns a method for constructing an adaptable soft-ware application.",
"A first step involves instantiating a first set of knowledge elements (kitems) related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application and used to produce dynamic and flexible templates.",
"A second step involves instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.",
"In a further aspect it concerns an adaptable software application comprising at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations."
],
[
"1.A method for constructing an adaptable software application, comprising the steps of: instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application; and, instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.",
"2.A method according to claim 1, where the step of instantiating the second set of kitems occurs dynamically, during the use of the application.",
"3.A method according to claim 1, where further instantiating steps produce further sets of kitems from the immediately preceding set.",
"4.A method according to claim 1, comprising the further step of directing enquiries against kitems at any level of instantiation using a consultation process.",
"5.A method according to claim 1, comprising the further step of accessing the business objects using a consultation process during use of the application.",
"6.A method according to claim 1, comprising the further step of accessing kitems related to the type of business objects for the purposes of inspecting or managing the design of the application.",
"7.An adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.",
"8.An application comprising adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable system of claim 7.9.An application according to claim 8, further comprising adaptable meta-module to define and implement the logic between the other modules.",
"10.An application according to claim 9, where the meta-module monitors a part of, or the whole chain of modules."
],
[
"<SOH> BACKGROUND ART <EOH>An application is a software package designed for the manipulation, management and processing of business elements or objects.",
"These are often, but not exclusively, documents.",
"Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.",
"Such applications are normally hard-coded using standard programming techniques.",
"The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.",
"The consequence of this approach is that systems are difficult to design and develop.",
"They are also difficult to maintain and modify.",
"When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.",
"In many cases, the effort is so difficult and time consuming that it is not attempted.",
"Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.",
"This has the advantage that the business rules can, in principle, be changed without having to change the application.",
"In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.",
"Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.",
"The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.",
"Few applications use expert systems technology; it is a niche technology.",
"A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.",
"The contents of these three applications are incorporated into this specification by reference.",
"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.",
"The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.",
"The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.",
"A knowledge application is typically characterised by a single instantiation process for all the kitems.",
"The kitems can also be used as contexts or templates from which further kitems can be instantiated.",
"Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.",
"This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.",
"The enquiry is itself a kitem instantiated from a kitem template.",
"Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.",
"Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.",
"The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.",
"Alternatively, these templates can be activated manually.",
"The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.",
"The business objects are easily created, and can be easily manipulated.",
"Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.",
"Instantiating the second set of kitems may take place dynamically, during the use of the application.",
"Applications can be built with a greater number of cascades or instantiations larger than two.",
"Enquiries can be directed against kitems at any level of instantiation using a consultation process.",
"The business objects may be accessed using a consultation process during use of the application.",
"The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.",
"The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.",
"The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.",
"So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.",
"The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.",
"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.",
"Each adaptable module implements the knowledge about the best way to run that module.",
"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.",
"An adaptable meta-module may define and implement the logic between the other modules.",
"In addition the meta-module may monitor a part of, or the whole chain of modules.",
"This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.",
"It may also adapt dynamically to the needs of users as software is being used.",
"Adaptablability is a very important commercial feature for software.",
"It holds the promise of speeding up software development, implementation, customization and flexibility.",
"In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.",
"Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.",
"Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.",
"Using adaptable software generic applications can be quickly produced.",
"The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.",
"An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.",
"Also client specific applications can be quickly produced.",
"Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements."
],
[
"TECHNICAL FIELD This invention concerns adaptable software.",
"In particular it concerns a method for constructing an adaptable software application.",
"In a further aspect it concerns an adaptable software application.",
"BACKGROUND ART An application is a software package designed for the manipulation, management and processing of business elements or objects.",
"These are often, but not exclusively, documents.",
"Manipulation, management and processing covers operations such as definition, access, editing, display, distribution, etc.",
"Such applications are normally hard-coded using standard programming techniques.",
"The knowledge of the business requirements to be met by the application is designed into the architecture of the application and the code itself.",
"The consequence of this approach is that systems are difficult to design and develop.",
"They are also difficult to maintain and modify.",
"When change is required to software, due to changing business conditions for example, designers must carry out extensive redesign and coding.",
"In many cases, the effort is so difficult and time consuming that it is not attempted.",
"Another approach is to develop an expert system in which the business rules are separate from the hard-coded part of the application.",
"This has the advantage that the business rules can, in principle, be changed without having to change the application.",
"In practice, the process of entering and testing the rules is difficult and time consuming, and their reliability can only be ascertained through extensive testing.",
"Expert systems are used to build only these applications for which knowledge is explicit and available, and where the effort required to enter and maintain the rules is clearly offset by big savings during operations.",
"The difficulties associated with developing, implementing and maintaining expert systems are well documented in the expert system literature of the past twenty years.",
"Few applications use expert systems technology; it is a niche technology.",
"A further approach is to build knowledge applications as described in co-pending Australian patent application PR2152 entitled Generic Knowledge Agents, International patent application PCT/AU99/00501 entitled Generic Knowledge Management System, and International patent application PCT/AU01/01155 entitled Intelligent Courseware Development and Delivery.",
"The contents of these three applications are incorporated into this specification by reference.",
"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.",
"The process for building knowledge applications starts in a knowledge application development environment where the business needs of the application determine the knowledge elements, or ‘kitems’, to be instantiated, or created, from contexts and templates.",
"The outcome is an application or knowledge base in which the kitems are regions, knowledge items etc in the application—one can think of these kitems as documents.",
"A knowledge application is typically characterised by a single instantiation process for all the kitems.",
"The kitems can also be used as contexts or templates from which further kitems can be instantiated.",
"Once the knowledge application is built the kitems can be accessed and managed, that is edited, displayed, distributed, etc.",
"This entails defining an enquiry in a consultation process that retrieves the knowledge elements of interest for perusal and further manipulation.",
"The enquiry is itself a kitem instantiated from a kitem template.",
"Although designed and implemented in a radically different way from traditional expert systems, the business role of these knowledge applications and the way knowledge is accessed are similar to that of expert systems.",
"SUMMARY OF THE INVENTION The invention, in a first aspect, is a method for constructing an adaptable software application, comprising the steps of: Instantiating a first set of kitems related to the type of business objects or operations which will be handled by the adaptable software application, from a knowledge application development environment identifying the business needs of the application.",
"Instantiating a second set of kitems related to business objects, from the first set by specifying parameters to determine the features of the business objects.",
"The first level of knowledge and the corresponding instantiations in effect produce dynamic templates that work as dynamic adaptations to the needs of users.",
"Alternatively, these templates can be activated manually.",
"The business objects in an application are treated as knowledge elements that embody business knowledge, such as knowledge about events that took place, or are expected to take place, in the business.",
"The business objects are easily created, and can be easily manipulated.",
"Because business objects are knowledge elements, they can be easily managed, that is accessed, displayed, analysed, etc.",
"Instantiating the second set of kitems may take place dynamically, during the use of the application.",
"Applications can be built with a greater number of cascades or instantiations larger than two.",
"Enquiries can be directed against kitems at any level of instantiation using a consultation process.",
"The business objects may be accessed using a consultation process during use of the application.",
"The kitems related to the type of business objects may be accessed for inspecting and managing the design of the application.",
"The invention, in a second aspect, is an adaptable software application, comprising: at least two levels of instantiation, where the templates for the second level instantiations are the first level instantiations.",
"The use of a two level instantiation, or cascading, design enables the first level to define the application without hard-coding, and the second to define the documents that are handled by the application.",
"So, adaptable software transforms the definition of the application into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.",
"The application may involve adaptable, intelligent modules or agents that communicate with each other, where each module has its business logic defined and implemented using the adaptable architecture of the system.",
"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.",
"Each adaptable module implements the knowledge about the best way to run that module.",
"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.",
"An adaptable meta-module may define and implement the logic between the other modules.",
"In addition the meta-module may monitor a part of, or the whole chain of modules.",
"This software is able to adapt itself automatically using the knowledge entered into it, without programming, to determine when and how it should adapt; that is, modify its behaviour.",
"It may also adapt dynamically to the needs of users as software is being used.",
"Adaptablability is a very important commercial feature for software.",
"It holds the promise of speeding up software development, implementation, customization and flexibility.",
"In addition to RAD (Rapid Application Development), adaptable software holds the promise of software that can be modified easily during all stages of its life-cycle, without major effort or redesign.",
"Adaptable software also holds the promise of software that can adapt itself, that is customize itself and evolve as needs change while it is being used.",
"Adaptable software has the potential to save, worldwide, enormous sums in development costs, and to reduce the total cost of ownership of software solutions over their lifetimes.",
"Using adaptable software generic applications can be quickly produced.",
"The frameworks for a variety of generic applications, for a variety of domains, can be quickly produced.",
"An application framework is a knowledge system in which the knowledge elements express the knowledge about what the application is meant to do.",
"Also client specific applications can be quickly produced.",
"Knowledge in generic framework can be easily modified or added to, so as to represent the knowledge about a client's specific requirements.",
"BRIEF DESCRIPTION OF THE DRAWINGS An example of the invention will now be described with reference to the accompanying drawings, in which: FIG.",
"1 is a block diagram showing the steps in the construction and use of an adaptable software application.",
"FIG.",
"2 is a block diagram showing the architecture of an adaptable software application.",
"BEST MODES OF THE INVENTION Adaptable software differs from the knowledge applications described in the background art in that there are at least two levels of instantiation instead of one; and the templates for the second level instantiations are the first level instantiations (they can be objects and not classes in object oriented programming).",
"Adaptable software extends the architecture of known knowledge applications.",
"The use of a two level instantiation, or cascading, design enables the first level to define the application (without hard-coding) and the second to define the documents that are handled by the application.",
"Adaptable software transforms the definition of the application (typically hardcoded) into specifying a knowledge application, and treats its documents (or business objects) as knowledge items inside the application.",
"The process for building an adaptable software application will now be described with reference to FIG.",
"1.The process starts in a knowledge application development environment 10 where the business needs of the application determine the knowledge to be instantiated, or created, from contexts and templates.",
"The first step 11 is the instantiation of this knowledge.",
"The outcome is an application module dynamic framework 12 in which the kitems relate to the type of business objects or operations which will be handled by the adaptable software application.",
"This first step is distinguished from the construction of an application knowledge base since it does not produce the documents or the knowledge to be accessed via a consultation.",
"Instead these kitems describe the application as a framework that will be used to generate the dynamic templates for the objects, documents, that relate to the application.",
"A second step 13 then involves populating the adaptable software application with business objects instantiated from contexts or templates produced in the first step.",
"The process for defining these business objects is similar to defining an enquiry in a knowledge application.",
"That is, parameters are specified that determine the features of the business object which is then created.",
"The outcome is an application module 14 populated with business objects.",
"The business objects so created can be managed 15, that is accessed, edited, displayed, distributed, etc.",
"These operations are simple to implement as they correspond to manipulating kitems, and they use standard kitem manipulation methods, and other specific methods if required.",
"For example, the business objects could be accessed using a question-answer session.",
"Specific applications (or modules) are implemented by customising the generic framework produced in the first step, and this corresponds to instantiating different kitems as business objects for each application or module.",
"This customizing can take place dynamically, during the use of the application.",
"This corresponds to dynamic adaptation of the application to the needs of users.",
"Enquiries can be directed against both levels of instantiated kitems via a consultation process.",
"Enquiries directed to the upper level of documents, produced by the first step, are typically used for inspecting and managing the design of the application.",
"To users, adaptable applications do not need to look different from normal applications.",
"For example, a dialogue could take place that enquires about the needs or intentions of a user.",
"Knowing the user's needs or intentions then enables the application to adapt or customize dynamically the template that is presented to the user; this templates defines the documents, or the type of documents, that the user enters into the application.",
"The two level instantiation process can also be described as cascading knowledge items.",
"That is, some knowledge items determine which other knowledge items will be activated or instantiated to define documents.",
"Applications can be built with a greater number of cascades or instantiations larger than two.",
"Most software applications are made of modules that communicate with each other.",
"For example, a typical business management application is made of a client module, a project module, a contact module, a resource module and an accounting module.",
"FIG.",
"2 shows adaptable, intelligent modules or agents.",
"In particular there are adaptable intelligent client 21, project 22, contact 23, resource 24 and accounting 25 modules.",
"Each module is adaptable.",
"For example, in the client module, a different set of questions could be asked based on the age or location or socio-economic profile.",
"Similarly for the other modules.",
"Each module has its business logic defined and implemented using an adaptable architecture.",
"This means that each module can be quickly developed and that its functionality can evolve with the business and with experience.",
"Each adaptable module implements the knowledge about the best way to run that module.",
"These modules are used to facilitate development and to increase the functionality and adaptability of the solution.",
"Adaptable meta-module(s) 30 are used to define and implement the logic between the other modules.",
"This could include workflow or project costing for example, where a client would be treated differently based on the number of projects commissioned in the past eighteen months and the success of these projects.",
"In addition the meta-modules are adaptable.",
"In FIG.",
"2, the meta-module monitors a part of, or the whole chain of modules.",
"For example, the accounting module may detect that some projects from some clients are not profitable.",
"That means that the project module would need to be modified (using its adaptable feature) to ensure that these projects are run differently.",
"It could also mean that additional information should be asked of these clients to perhaps rejects them as clients for a certain type of project.",
"In this case the client module is modified using its adaptable feature.",
"A specific example is an access control database.",
"All the operations possible with a database that needs to be controlled are listed as the context in a GKMS type application.",
"Access is to be controlled for each group of users and for users in the groups (control is determined by the group and, in some cases, by the user).",
"In this case, the first level of instantiation corresponds to defining the template to be used to give rights to each group of users.",
"Each user, when linked to a group get an access rights document based on the template for the group it belongs to.",
"This is the second level of instantiation.",
"This document represents the right of that user.",
"The administrator can then modify each document if desired to take account of the characteristics of the user within the group.",
"It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described.",
"The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive."
]
] | Patent_10450974 |
[
[
"Generic knowledge agents",
"This invention concerns a generic knowledge software agent, or kuark, existing within a knowledge environment.",
"The kuark comprises: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark, and a destination context of information identifiable by the kuark.",
"The kuark has a defined source region, or pattern, within the and problems source context and a mapping between that source region and a defined destination region, or pattern, within the destination context.",
"The kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.",
"It also concerns systems or kitems, made up of kuarks and a process for constructing them.",
"It also concerns mult-kitem systems and searching processes for those systems.",
"Finally it also concerns software agents and a knowledge management system comprising kitems."
],
[
"1.A generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.",
"2.A software agent according to claim 1, and further comprising: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; or, links to other kuarks.",
"3.A software agent according to claim 2, where the links are: the hierarchical parent's universal id, and more than one parents could be allowed; the id of the previous version kuark; the ids of the kuarks that are part of this kuark; or, the ids of the kuarks this kuark belongs to or is associated with.",
"4.A software agent according to claim 1, operating to: create a daughter/parent kuark; adopt an existing kuark as daughter or parent; display an internal state (contexts, regions, mapping); or make a copy each time it fires.",
"5.A software agent according to claim 1 where, depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines the relevance of the environment.",
"6.A software agent according to claim 1 where, the mapping is a program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context, and.",
"the mapping executes when the environment is compatible with the source context and the region.",
"7.A software agent according to claim 1, implemented as classes and objects using an object oriented language.",
"8.A system, or kitem, comprising more than one software agent according to claim 1, where the kitem has its own properties and methods in addition to those of its components 9.A process for constructing a system, or kitem, according to claim 8 from its components, comprising the steps of: concatenating their source contexts; concatenating their destination contexts; concatenating their source regions; concatenating their destination regions; concatenating their mappings; concatenating their explanations.",
"10.A process according to claim 9, comprising the additional step of building a list of the component kuarks or kitems.",
"11.A system, or kitem, according to claim 8, where all its components (kuarks or kitems) keep their individual contexts.",
"12.A system, or kitem, according to claim 8, where all its components have their individual contexts concatenated.",
"13.A system, or kitem, according to claim 8, expressed as an object containing another object.",
"14.A multi-kitem system comprising two or more systems, or kitems, according to claim 8, where the kitems are linked to one another.",
"15.A multi-kitem system according to claim 14, comprising a serial arrangement of kitems in which the destination context of one kitem is used as at least part of the source context of another.",
"16.A multi-kitem system according to claim 14, comprising a parallel arrangement of kitems in which the kitems that have independent (or disjointed), but can also have overlapping, source contexts.",
"17.A kitem according to claim 8, including within itself a multi-kitem system and having access to all the properties and methods of kitems contained within it, in addition to the properties that belong only to that kitem.",
"18.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as its source context and region, and a has a mapping which is a program or procedure that produces material to be displayed as output when the relevance mapping fires.",
"19.A kitem according to claim 15, comprising two kuarks, where the first kuark, depending on the the state of the environment with respect to the source context and source region, fires a relevance mapping that determines the relevance of the environment, and the second kuark uses the output of the first kuark as part of its source context and its source region, and a has a mapping which is a program or procedure that produces a destination region as output when the relevance mapping fires.",
"20.A searching process for identifying those kitems in a multi-kitem system according to claim 14, that have source regions compatible with an enquiry, the process comprising the step of: determining the ‘relevance status’ of a kitem with respect to the enquiry or consultation so far, where an available status is the default status of a kitem before a search takes place, a candidate kitem is one that is not incompatible with the enquiry so far, and a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.",
"21.A searching process according to claim 20, where the source context is organised as a tree structure, and each kitem is indexed against the context tree so that each object in the context possesses a list of the kitems it is associated with, and during the search questions are context objects and all the indexed kitems to the questions asked and answered so far in the consultation have their relevance status checked and modified if appropriate.",
"22.A searching process according to claim 21, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"23.A searching process according to claim 20, where the source context is organised as a tree structure in which each element is a kitem and the search process is also a kitem which flags the kitems in the tree structurre that correspond to the questions answered so far.",
"24.A searching process according to claim 23, where context tree element determines whether all the indexed elements have taken into account the values of the context tree element, and if so, it passes the value(s) to the indexed kitems and waits until all the responses have been received before the next step in the enquiry can proceed.",
"25.A searching process according to claim 24, where the tree structure is a modified context space kitem, and the enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"26.A searching process according to claim 20, where the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.",
"27.A searching process according to claim 20, where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it also possess a set of applicability addresses that correspond to the kitems in its source region, and during processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.",
"28.A searching process according to claim 27, where each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process; and the enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.",
"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry, or enquiry step.",
"29.An agent comprising a kitem according to claim 15, and having the following structure: Beliefs, Desires and Intentions are parallel kitems and their respective components are also kitems; and the Beliefs, Desires and Intentions kitems are organised in a serial architecture, that is the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.",
"30.A knowledge management system, comprising: a context element is a display kitem according to claim 18; a source or destination context kitem is a parallel display kitem, and its members comprise the list of all the context elements defined in the domain of discourse; a context executable kitem to edit a context element, edit a context space or display a context space as a tree; a source or destination context tree as an executable kitem, where the display kitem display the elements that belong to the source and destination kitems, its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context; a knowledge editing kitem is an executable kitem that has: as source space: the source and destination contexts, as destination space: the source and destination contexts, as source region a subset of the source context that defines the applicability of the knowledge kitem, as mapping a link to a subset of the destination context, or a procedure or executable (sub-)kitem, as destination region a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure, and an explanation; a search kitem is an executable kitem with: as source context the source and destination contexts, as source region the enquiry as defined so far, as destination context the kitems in the domain of discourse (if known), as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry, as destination region the candidate and definite kitems ‘retrieved’ by the mapping; an enquiry kitem is an executable kitem with: as source space the enquiry (the consultation process, showing each step in the question-answer session), as destination space the status of the enquiry (answered, not answered, etc.",
"), the ids of the definite kitems; a display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way."
],
[
"<SOH> BACKGROUND ART <EOH>The model for the agents uses the inventions described in International Patent Application No.",
"PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.",
"A summary of these knowledge models will now be given with reference to FIGS.",
"1 to 4 .",
"A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.",
"Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.",
"1 .",
"The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.",
"2 .",
"FIGS.",
"3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.",
"FIG.",
"4 is an extension of FIG.",
"3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.",
"Once again it involves signal exchanges between servers and clients.",
"The accepted architecture for agents is summarized in FIGS.",
"5 and 6 .",
"FIG.",
"5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.",
"FIG.",
"5 shows that in each part of the BDI model, agents need knowledge to be able to operate.",
"They must have belief which is knowledge about what their role in existence is.",
"Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.",
"Similarly, each of the DBI boxes in FIG.",
"5 is made of components, each representing a kind of knowledge in its category.",
"All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.",
"In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.",
"This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.",
"It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.",
"5 , but to any knowledge that may be useful in agents.",
"FIG.",
"6 shows how an agent situated in an environment interacts with this environment.",
"Th interaction is very similar to FIGS.",
"3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.",
"Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.",
"So the kuark makes predetermined information automatically available to a user when input criteria are met.",
"The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.",
"The source region may be identicle to the source context, or it may be a subset within it.",
"A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.",
"The links implement relationships to other kuarks.",
"The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.",
"This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.",
"The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.",
"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.",
"A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.",
"The relevance can be expressed as a probability.",
"The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.",
"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.",
"Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.",
"This kuark executes its mapping when the environment is compatible with the source context and the region.",
"That is, the input to the procedure is the source region in the source context.",
"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.",
"Kuarks may be implemented as classes and objects using an object oriented language such as Java.",
"A kuark is a class in OOP and some of its properties are static, final, etc.",
"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.",
"A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.",
"In a kitem all the component kuark and kitems may keep their identities.",
"The new kitem may have its own properties and methods in addition to those of its component kitems.",
"The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.",
"A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.",
"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.",
"The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.",
"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.",
"Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.",
"A kuark or kitem can be represented as an object using an object oriented language such as Java.",
"A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.",
"This means that kitem objects can be generated without programming.",
"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.",
"Kitems can be linked.",
"A link has a source or origin kitem and a destination kitem.",
"The links can be stored inside the kitem or kuark, as a property of the kitem.",
"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.",
"A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.",
"More than two kitems can be chained.",
"Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.",
"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.",
"An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.",
"Some of the kitems may be linked in series or in parallel.",
"A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.",
"Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.",
"A display kitem is a serial kitem comprising two kuarks.",
"The first kuark is a relevance kuark.",
"It uses its region and the state of the environment to determines the relevance of the display kitem.",
"The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.",
"The material to be displayed comprises the destination region and the explanation.",
"An executable kitem is a serial kitem comprising two kuarks.",
"The first kuark is a relevance kuark.",
"It uses its region and the state of the environment to determines the relevance of the executable kitem.",
"The second kuark is a procudure kuark.",
"It has the relevance (the output of the first kuark) as region and as part of its source context.",
"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.",
"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.",
"A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.",
"Searching involves identifying those kitems that have source regions compatible with the enquiry.",
"It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.",
"Searches may be synchronous or asynchronous.",
"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.",
"The source context may be organised as a tree structure.",
"Each kitem may be indexed against the context tree.",
"This means that each object in the context possesses a list of the kitems it is associated with.",
"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.",
"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.",
"At the implementation level, the context tree is a modified context space kitem.",
"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.",
"The search process updates the status of the kitems according to the enquiry.",
"Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.",
"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.",
"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.",
"The search process waits until all the tree elements corresponding to the enquiry so far have replied.",
"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).",
"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.",
"At the implementation level, the context tree is a modified context space kitem.",
"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.",
"Each kitem updates its status according to the enquiry and informs the searc process.",
"Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.",
"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.",
"The state of the relevance region determines what happens to the application region.",
"Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.",
"During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.",
"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.",
"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.",
"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).",
"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.",
"It is up to the enquiry process to manage all the replies it gets from the network.",
"The solution outlined here is applicable to distributed processing.",
"Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).",
"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.",
"Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.",
"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.",
"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.",
"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.",
"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).",
"In a multi-kitem system, knowledge processing involves running a question-answer session.",
"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.",
"The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.",
"Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.",
"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.",
"Therefore the questions are selected dynamically.",
"Alternatively the search may have processing delegated to each kitem.",
"Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.",
"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.",
"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.",
"In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.",
"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.",
"In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.",
"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.",
"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.",
"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.",
"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.",
"Therefore they are included in the compiled kitem.",
"This is faster than the partial compilation.",
"An agent may be represented as a kitem with the following structure: Beliefs 61 , Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61 , Desires 62 and Intentions 63 kitems are organised in a serial architecture.",
"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.",
"The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).",
"A source or destination context kitem is a parallel display kitem.",
"Its members comprise the list of all the context elements defined in the domain of discourse.",
"Context executable kitems may edit a context element, edit a context space or display a context space as a tree.",
"A source or destination context tree is an executable kitem.",
"The display kitem may display the elements that belong to the source and destination kitems.",
"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.",
"A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.",
"As destination space: the source and destination contexts.",
"As source region: a subset of the source context that defines the applicability of the knowledge kitem.",
"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.",
"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.",
"An explanation.",
"A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.",
"An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.",
"), the ids of the definite kitems.",
"A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.",
"This technology supports rapid application development (RAD).",
"RAD is a very important commercial feature for software.",
"It holds the promise of speeding up software development, implementation and customization.",
"RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.",
"RAD is also a very serious technical challenge for designers.",
"Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.",
"In addition agents, with proper architecture, should scale up.",
"That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.",
"Currently agents are difficult to design and buid.",
"This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.",
"They are: control theory, cognitive psychology and artificial intelligence."
],
[
"TECHNICAL FIELD This invention concerns generic knowledge software agents, or kuarks, and their use as the building blocks for agent based applications.",
"It also concerns systems or kitems, made up of kuarks and a process for constructing them.",
"It also concerns mult-kitem systems and searching processes for those systems.",
"Finally it also cocerns software agents and a knowledge management system comprising kitems.",
"BACKGROUND ART The model for the agents uses the inventions described in International Patent Application No.",
"PCT/AU99/00501 entitled ‘Generic Knowledge Management System’ (GKMS), and Australian provisional patent application no PR0852 entitled ‘Networked Knowledge Management And Learning’ (NKML) both of which are incorporated herein by reference.",
"A summary of these knowledge models will now be given with reference to FIGS.",
"1 to 4.A computerised generic knowledge management system, comprises: a multi-dimensional global space within computer memory defined by attributes, where each attribute defines a feature of the external world or the internal state of the system, or actions that can be taken to modify them, and each attribute is a dimension of the global space; a source space, within the global space, made up of selected ones of the attributes to define a context in which to state problems; a destination space, within the global space, made of selected ones of the attributes to define a context in which to provide answers to problems stated in the source space; mappings between defined parts of the source space which each represent one or more stated problems, to defined parts of the destination space which each represent one or more answers expressing and embodying knowledge supplied by experts appropriate to the respective problems stated in the part of the source space.",
"Knowledge is mapped as regions (or patterns) in a source (or problem) space that are linked to regions (or patterns) in a destination (or solution) space, as illustrated in FIG.",
"1.The interactions for defining the problem and solution spaces, and for defining the mappings, and the interactions for accessing the knowledge take place in a network of client-servers, as illustrated in FIG.",
"2.FIGS.",
"3 and 4 show the decision-making process and the knowledge capture process, and how knowledge can be improved on an on-going basis.",
"FIG.",
"4 is an extension of FIG.",
"3 in which the consequences of the decisions taken by a GKMS are evaluated and used to qualify the knowledge in the GKMS.",
"Once again it involves signal exchanges between servers and clients.",
"The accepted architecture for agents is summarized in FIGS.",
"5 and 6.FIG.",
"5 illustrates the BDI model, that is agents have Beliefs, Desires and Intentions.",
"FIG.",
"5 shows that in each part of the BDI model, agents need knowledge to be able to operate.",
"They must have belief which is knowledge about what their role in existence is.",
"Their desires or goals represent knowledge about what they are capable of and their intentions represent the knowledge about implementing the goals.",
"Similarly, each of the DBI boxes in FIG.",
"5 is made of components, each representing a kind of knowledge in its category.",
"All these different types of knowledge can be easily and conveniently represented using the GKMS model for knowledge.",
"In addition, the use of that knowledge can be supported by the knowledge processing described in the GKMS patent.",
"This means that the advantages of the GKMS model for knowledge representation and processing can become available to agents.",
"It is also important to note that the use of the GKMS knowledge model for representation and processing is not restricted to the categories of knowledge shown in FIG.",
"5, but to any knowledge that may be useful in agents.",
"FIG.",
"6 shows how an agent situated in an environment interacts with this environment.",
"Th interaction is very similar to FIGS.",
"3 and 4 which show how interaction with the environment and learning can take place in the GKMS model.",
"Therefore the GKMS model is also suitable for dealing with agents interacting with their environments and learning in the process.",
"SUMMARY OF THE INVENTION The invention is a generic knowledge software agent, or kuark, existing within a knowledge environment, and comprising: a defined domain of discourse within the environment which determines a source context of information detectable by the kuark and a destination context of information identifiable by the kuark; the kuark having a defined source region, or pattern, within the source context and a mapping between that source region and a defined destination region, or pattern, within the destination context; and the kuark detects all environmental parameters compatible with the source region and source context so that when the source region is satisfied the mapping fires to determine the destination region in the destination context and makes it available to the environment where it can be observed by a user.",
"So the kuark makes predetermined information automatically available to a user when input criteria are met.",
"The kuark is only able to detect parameters that fall within its defined source context and when the environment satisfies the source region the mapping is triggered.",
"The source region may be identicle to the source context, or it may be a subset within it.",
"A kuark may comprise many other elements, such as: a univeral id; a title; an explanation, which may be multimedia; a time and date created; an author; a time and date when last modified; a person who last modified agent; a version number; a status to an enquiry, such as available, candidate, rejected, definite; a description of the source context at time of last firing; a description of destination context after last firing; and, links to other kuarks.",
"The links implement relationships to other kuarks.",
"The are four links: The hierarchical parent's universal id, and more than one parents could be allowed; The id of the previous version kuark; The ids of the kuarks that are part of this kuark; The ids of the kuarks this kuark belongs to or is associated with.",
"This last link may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.",
"The kuark may also operate to do the following: Create a daughter/parent kuark; Adopt an existing kuark as daughter or parent; Display an internal state (contexts, regions, mapping); and Make a copy each time it fires.",
"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.",
"A first special kuark, known as a ‘relevance kuark’, determines the relevance of the environment, and depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping that determines (calculates) the relevance of the environment.",
"The relevance can be expressed as a probability.",
"The destination context may be made of the different relevance values that the environment can take with respect to the source context and region.",
"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.",
"Another special kuark is known as a ‘procedure kuark’, and contains as a mapping a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.",
"This kuark executes its mapping when the environment is compatible with the source context and the region.",
"That is, the input to the procedure is the source region in the source context.",
"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.",
"Kuarks may be implemented as classes and objects using an object oriented language such as Java.",
"A kuark is a class in OOP and some of its properties are static, final, etc.",
"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.",
"A system made of several kuarks is still a kuark with all the allowed properties and methods, but is known as a ‘kitem’.",
"In a kitem all the component kuark and kitems may keep their identities.",
"The new kitem may have its own properties and methods in addition to those of its component kitems.",
"The new kitem may have its own title and explanation, and may also have its own source and destination contexts and regions, and its own mapping.",
"A kitem may be constructed form kuarks and kitems by the following process: concatenating the source contexts; concatenating the destination contexts; concatenating the source regions; concatenating the destination regions; concatenating the mappings; concatenating the explanations.",
"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.",
"The kitem construction process may also build the list of kuarks or kitems that are part of the new kitem.",
"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.",
"Alternatively the kitem construction process may be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.",
"A kuark or kitem can be represented as an object using an object oriented language such as Java.",
"A kitem can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.",
"This means that kitem objects can be generated without programming.",
"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.",
"Kitems can be linked.",
"A link has a source or origin kitem and a destination kitem.",
"The links can be stored inside the kitem or kuark, as a property of the kitem.",
"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.",
"A multi-kuark or multi-kitem system may comprise a serial arrangement, in which the destination context of one kitem is used as the source context (or part of the source context) of another.",
"More than two kitems can be chained.",
"Alternatively, a parallel kitem is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.",
"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.",
"An application or knowledge base may comprise a set of knowledge items, kitems, defined with respect to a domain of discourse, where the kitems are arranged in parallel.",
"Some of the kitems may be linked in series or in parallel.",
"A kitem may be a class that supports the creation of objects, or knowledge elements, where the kitem has access to all the properties and methods of other kitems contained within it, in addition to the properties that belong only to that kitem.",
"Such a kitem is referred to as a ‘meta-kitem’ and is able to express meta-knowledge.",
"A display kitem is a serial kitem comprising two kuarks.",
"The first kuark is a relevance kuark.",
"It uses its region and the state of the environment to determines the relevance of the display kitem.",
"The second kuark is a procedure kuark and has the relevance (the output of the first kuark) as source context and region.",
"The material to be displayed comprises the destination region and the explanation.",
"An executable kitem is a serial kitem comprising two kuarks.",
"The first kuark is a relevance kuark.",
"It uses its region and the state of the environment to determines the relevance of the executable kitem.",
"The second kuark is a procudure kuark.",
"It has the relevance (the output of the first kuark) as region and as part of its source context.",
"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.",
"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.",
"A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.",
"Searching involves identifying those kitems that have source regions compatible with the enquiry.",
"It means determining the ‘relevance status’(available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.",
"Searches may be synchronous or asynchronous.",
"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.",
"The source context may be organised as a tree structure.",
"Each kitem may be indexed against the context tree.",
"This means that each object in the context possesses a list of the kitems it is associated with.",
"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.",
"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.",
"At the implementation level, the context tree is a modified context space kitem.",
"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.",
"The search process updates the status of the kitems according to the enquiry.",
"Alternatively, the search process (also a kitem) may flag the elements in the context tree that correspond to the questions answered so far.",
"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.",
"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.",
"The search process waits until all the tree elements corresponding to the enquiry so far have replied.",
"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).",
"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.",
"At the implementation level, the context tree is a modified context space kitem.",
"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.",
"Each kitem updates its status according to the enquiry and informs the searc process.",
"Alternatively, in asynchronous searches, the source region may determine whether the outcome of the kitem becomes applicable.",
"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.",
"The state of the relevance region determines what happens to the application region.",
"Where each kitem has a unique ID or address that determines its location in a distributed memory space or network, it may also possess a set of applicability addresses that correspond to the kitems in its source region.",
"During processing, the enquiry process may submit each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.",
"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.",
"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.",
"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).",
"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.",
"It is up to the enquiry process to manage all the replies it gets from the network.",
"The solution outlined here is applicable to distributed processing.",
"Another model for asynchronous searches is to give even more responsibility to the distributed kitems, and each kitem has the address of each of the context objects in its context and in its region (a subset of the context).",
"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.",
"Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.",
"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.",
"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.",
"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.",
"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).",
"In a multi-kitem system, knowledge processing involves running a question-answer session.",
"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.",
"The steps involved are: identifying the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asking these questions; on the basis of the answers to the questions, determining whether any answers can be found, and the next best questions to ask.",
"Identifying the most promising questions may involve a synchronous search through a context tree with processing delegated to a search agent.",
"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.",
"Therefore the questions are selected dynamically.",
"Alternatively the search may have processing delegated to each kitem.",
"Context tree search with processing delegated to each kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.",
"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.",
"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.",
"In an asynchronous searches the enquiry process (that sends the enquiry over the network for kitems to process it) may perform several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.",
"In addition, the enquiry process may perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.",
"In situations where the domain of discourse of a body of knowledge is known, one can identify the kitems with the largest number of indexed kitems with relevance status available or candidate in order to find hidden knowledge.",
"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.",
"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.",
"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.",
"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.",
"Therefore they are included in the compiled kitem.",
"This is faster than the partial compilation.",
"An agent may be represented as a kitem with the following structure: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.",
"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.",
"The kitems needed to produce a workable implementation for knowledge management are shown below: A context element is a display kitem (a serial kitem made of two kuarks).",
"A source or destination context kitem is a parallel display kitem.",
"Its members comprise the list of all the context elements defined in the domain of discourse.",
"Context executable kitems may edit a context element, edit a context space or display a context space as a tree.",
"A source or destination context tree is an executable kitem.",
"The display kitem may display the elements that belong to the source and destination kitems.",
"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.",
"A knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.",
"As destination space: the source and destination contexts.",
"As source region: a subset of the source context that defines the applicability of the knowledge kitem.",
"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.",
"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.",
"An explanation.",
"A search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.",
"An enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.",
"), the ids of the definite kitems.",
"A display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.",
"This technology supports rapid application development (RAD).",
"RAD is a very important commercial feature for software.",
"It holds the promise of speeding up software development, implementation and customization.",
"RAD, or any significant progress in this area, has the potential to save, worldwide, enormous sums (billion of dollars) in development, and to reduce the total cost of ownership of software solutions over their lifetimes.",
"RAD is also a very serious technical challenge for designers.",
"Agents hold the promise of autonomous and intelligent software that is capable of co-operating with other agents (and human beings) towards achieving a goal.",
"In addition agents, with proper architecture, should scale up.",
"That is, they could provide a promising platform for building complex, adaptable systems that could have knoweldge and perhaps exhibit intelligent behaviour.",
"Currently agents are difficult to design and buid.",
"This is because agents draw on several disciplines, each one bringing its own theoretical and practical difficulties to design and development.",
"They are: control theory, cognitive psychology and artificial intelligence.",
"BRIEF DESCRIPTION OF THE DRAWINGS The background to the invention has been described above with reference to the following drawings: FIG.",
"1 is a diagram illustrating knowledge as mappings between a problem space and a solution space.",
"FIG.",
"2 is a diagram illustrating a network of client-servers, each able to run agents.",
"FIG.",
"3 is a diagram illustrating a knowledge capture loop in the GKMS.",
"FIG.",
"4 is a diagram illustrating an expanded knowledge capture loop in an on-the-job problem-solving system.",
"FIG.",
"5 is a diagram illustrating the agent DBI model.",
"FIG.",
"6 is a diagram illustrating an agent situated in and interacting with its environment.",
"The invention will now be described with reference to the following drawings: FIG.",
"7 is a diagram illustrating a generic elementary cognitive element (kuark).",
"FIG.",
"8 is a diagram illustrating a relevance kuark.",
"FIG.",
"9 is a diagram illustrating a procedure kuark.",
"FIG.",
"10 is a diagram illustrating a serial architecture.",
"FIG.",
"11 is a diagram illustrating a parallel architecture.",
"FIG.",
"12 is a diagram illustrating the agent implementing the DBI model as a serial kitem.",
"BEST MODES OF THE INVENTION Generic Elementary Cognitive Agent (Kuark) Referring to FIG.",
"7, the generic elementary cognitivey agent (kuark) 10 is the building block of agent-based applications.",
"The kuark knowledge model implements and extends the GKMS model described in the GKMS patent.",
"The kuark 10 monitors the environment by detecting all environment parameters compatible with the source region 11 and source context 12.When the source region 11, which determines the applicability of the mapping 13, is satisfied, the mapping 13 fires.",
"The mapping determines the output region 14 in the destination context 15 and makes the region available to the output, and hence to the environment.",
"Regions are sometimes referred to as patterns.",
"EXAMPLE 1 The source context comprises two sets of integers and the destination context one set of integers.",
"The source region is the positive integers for the two sets, the mapping a multiplication of the two inputs accepted in the source region.",
"The destination region is the result of the multiplication of the two positive numbers.",
"EXAMPLE 2 The source context is the state of a logical variable and the region the value ‘true’ of that variable.",
"The destination context is the state of the same variable and the mapping the ‘negation’ of the original value, that is ‘false’.",
"EXAMPLE 3 The source context is the temperature and pressure inside a boiler and the destination context a list of commands triggering alarms and associated processes (that can be software based).",
"The source region is the range of pressures and temperatures that trigger a ‘code red’ alert and the destination region the command triggering the ‘code red’ alarm.",
"EXAMPLE 4 The source context and region is as in the example 3.The destination context is the list of the relevance status of the kuark and the region the actual relevance status of the kuark, based on the source region.",
"That is, the ‘code red’ alert is given a relevance status (perhaps expressed as a probability) based on the environment.",
"Kuark Properties The common properties of a kuark are: univeral id; title; explanation (multimedia); time and date created; author; time and date when last modified; person who last modified agent; version number; domain of discourse source context (what it knows about) 12; destination context 15; applicability source region or pattern 11 (can be identical to source context); relevance status to an enquiry (available, candidate, rejected, definite); mapping 13 (the process that produces the outcome based on the state of source context) outcome result of the mapping between source and destination context; the mapping determines the destination region or pattern; status (enabled/disabled/archived); state of source context at time of last firing (describes source context and region); state of destination context after last firing (describes destination region); links to other kuarks the hierarchical parent's universal id (more than one parents could be allowed); the id of the previous version kuark; the ids of the kuarks that are part of this kuark (see later for multi-kuark kuarks); the ids of the kuarks this kuark belongs to or is associated with (see later for multi-kuark kuarks).",
"The property ‘links to other kuarks’ is the mechanism used to implement relationships between kuarks.",
"The fourth link mentioned may be implemented as separate kuark(s) designed to contain the IDs of the kuarks the kuark in question relates (or belongs) to.",
"Kuark Methods The main methods of a kuark are: monitor applicability (detect only what its context allows); determines whether environment fits within source region, if yes triggers/fires mapping; execute mapping (modifies destination state) mapping determines the outcome which is a subset of the destination context; create daughter/parent kuark; adopt existing kuark as daughter or parent; display internal state (contexts, regions, mapping); make copy each time it fires.",
"It is possible to implement these methods outside the current kuark, in a separate class or subroutine for example.",
"Relevance Kuark Referring now to FIG.",
"8, a relevance kuark 20 is a kuark 10 (as described previously) that determines the relevance of the environment.",
"Depending on the the state of the environment with respect to the source context and source region, the kuark fires a relevance mapping 23 that determines (calculates) the relevance of the environment.",
"The relevance can be expressed as a probability.",
"The destination context 15 is made of the different relevance values that the environment can take with respect to the source context and region.",
"For example, the values could be ‘definitely relevant’, ‘possibly relevant’, ‘relevant’, ‘likely not relevant’, ‘not relevant’.",
"Procedure Kuark Referring now to FIG.",
"9, a procedure kuark 30 is one that contains a (usually small) program or procedure that takes as input some elements of the source context and produces, as output, a pattern that fits within the destination context.",
"The kuark executes its procedure mapping 33, when the environment is compatible with the source context and the region.",
"That is, the input to the procedure is the source region in the source context.",
"The destination context describes the range of outputs the procedure can produce when run, and the destination region or pattern is the actual result of the execution of the mapping, using an instance of the environment.",
"The procedure mapping can be a trivial mapping.",
"It can simply display a fixed pattern and an explanation in a fixed destination context.",
"The context item, see later, is an implementation of the trivial procedure kuark.",
"Relevance kuarks tend to have the same destination context, whereas procedure kuarks typically have context that are strongly application dependent and that can vary within an application.",
"Kuarks and Object Oriented Programming Kuarks can be implemented as classes and objects using an object oriented language such as Java.",
"A kuark is a class in OOP and some of its properties are static, final, etc.",
"A kuark that is given values for its source and destination contexts and regions are objects or instances of the corresponding kuark class.",
"Multi-Kuark Systems or Applications This section describes how kuarks can be used together to increase the power of applications.",
"Multi-Kuark Kitems A system made of several kuarks is still a kuark with all the allowed properties and methods.",
"However, the term kuark is reserved for simple (or elementary) kuarks, as described above.",
"A multi-kuark kuark is named a kitem.",
"Kitems are constructed as follows: the designer identifies the kuarks (or kitems) to be included in the new kitem; the designer clicks ‘construct kitem’.",
"The construction process simply puts a wrapper around the kuarks and kitems to include in the new kitem.",
"All the component kuark and kitems keep their identities.",
"The new kitem can have its own properties and methods in addition to those of its component kitems.",
"The new kitem has its own title and explanation, and can also have its own source and destination contexts and regions, and its own mapping.",
"One can also view the new kuark as having undergone a construction process that carried the following operations: concatenates the source contexts; concatenates the destination contexts; concatenates the source regions; concatenates the destination regions; concatenates the mappings; concatenates the explanations.",
"Here concatenates means ‘take the union’ of the entities in question; for mappings, it is the juxtaposition of the individual mappings.",
"The kitem construction process also builds the list of kuarks or kitems that are part of the new kitem.",
"Typically, when a kitem is built, all its components (kuarks or kitems) keep their individual contexts.",
"Alternatively the kitem construction process can also be set so that all components get the same source and destination contexts, that is the concatenation of the individual contexts of the kitem components.",
"Kitems have explicit domains of discourse, the source and destination contexts, and explicit regions; kitems are ‘aware’ of their domains of discourse and when their knowledge, expressed as their mappings and destination regions, can be applied.",
"Not all kuarks can be assembled in kitems as some concatenation may not be allowed; this issue is not treated further here.",
"Refer later for different architectures for multi-kitem systems.",
"Kitem Hierarchy When defining kitems for an application, it is often convenient to organise them as a hierarchy of objects, as in a file system for example.",
"In fact, all kitems are defined within a hierarchical structure similar to that of the domain names on the Internet.",
"Each kitem has a parent, except the root kitem which is the top of the tree.",
"The hierarchical organisation of the kitems imposes no restriction on how they can be grouped (in multi-kitem systems) to achieve some purposes in an application.",
"That is, composed kitems can be assembled out of any kitem from any location in the hierarchy.",
"Kitems and Object Oriented Programming A kitem as outlined above, together with its GKMS features, mean that kitems can be implemented using an object oriented language, as objects or instances of the kuark or kitem classes.",
"As indicated above, a kuark or kitem can be represented as an object using an object oriented language such as Java.",
"A multi-kuark kitem as outlined above, can be expressed as an object containing another object (or a class with sub-classes) which preserves all the features for knowledge items disclosed in the GKMS patent.",
"This means that kitem objects can be generated without programming.",
"The other features disclosed in the GKMS patent also apply to kitems implemented as classes.",
"Rationale for Multi-Kitem Systems The advantages of building applications using kitems are very significant: the methods and properties can be used across the hierarchy (for example, a method to display the components kitems of a kitem can be re-used at all levels in the hierarchy of kitems); kitems provide a unified way for thinking and implementing applications; kitems can be fashioned as simple building blocks that offer maximum re-use; kitems implement knowledge at every level (in every kitem) that make up the application; knowledge permeates the design and the use of the application; applications can be largely soft-coded, instead of hard-coded; applications can be developed rapidly (RAD: rapid application development).",
"Links Between Kitems Kitems can be linked.",
"A link has a source or origin kitem and a destination kitem.",
"The links can be stored inside the kitem or kuark, as a property of the kitem.",
"An alternative is to store inside the kitem the address of the ‘link kitem’, that is, the kitem that is designed to contain the links that relate to a source kitem.",
"In a link kitem, the source context contain the ID of the source kitem and the type of link (see above for list of possible links), and the destination regions contains the IDs of the corresponding links (the destination context is the domain of discourse of the source kitem).",
"It is also possible, indeed often preferable, to have the source and destination contexts and regions for a kitem expressed via links.",
"That is, one or several link kitems contain the source and destination context, and the regions (their kitems and values).",
"Multi-Kitem Architectures This section discusses the architectures available for building multi-kuark or multi-kitem systems.",
"In particular, these architectures can be used to build the display and executable kitems.",
"Serial Architecture In the serial architecture 40, the destination context of one kitem 41 is used as the source context (or part of the source context) of another 42.More than two kitems can be chained, as illustrated in FIG.",
"10.The state of the environment is used as input to kitem 41 (and perhaps to other kitems).",
"Similarly, the serial kitem output is the output from kitem n 45 (and perhaps from other kitems).",
"In the most simple case, the source context is the source context of kitem 41 and the destination context the destination context of kitem n. In general, the source context of the serial kitem is a subset of the source contexts of all its ‘internal kitems’.",
"Similarly for the destination context and for the regions in these contexts.",
"The source context of an internal kitem can comprise some elements from the environment in addition to the output from the previous kitem.",
"The serial kitem is a kitem and has all the properties of kitems discussed above.",
"Parallel Architecture Referring now to FIG.",
"11, a parallel kitem 50 is made of kitems that have independent (or disjointed), but can also have overlapping, source contexts.",
"The power of parallel kitems derives from the links that can be established between its internal kitems.",
"The objective is to ensure that the information and knowledge contained in two kitems becomes linked.",
"For example, a kitem could describe clients and another projects; and a link would connect a client with a project.",
"A link has an origin and a destination.",
"Inside a parallel kitem, the component kitems are treated as peers, and links are defined between peers.",
"When the class of a parallel kitem is defined, the operator has to specify which kitems are instantiated as objects when the parallel kitem object is created.",
"In linked kitems, it is convenient, but not mandatory, to instantiate the origin kitem but not the destination kitem.",
"Links can be unidirectional or bidirectional.",
"For example, from clients to projects, or from clients to projects and from projects to clients.",
"Hierarchical Kitems The kitems that are part of a parallel (or serial kitem) keep their properties, notably the one identifying their parents.",
"Therefore the parallel kitem can, in fact, contain a hierarchy of kitems even if they are organised in a flat hierarchical (that is, parallel) way in the parallel kitem.",
"A Knowledge Application as A Parallel Architecture Kitem An application or knowledge base is a set of knowledge items (kitems) defined with respect to a domain of discourse (source and destination contexts and mappings available).",
"These kitems are arranged in parallel and some of them can be linked.",
"For example, in an application all the knowledge items are typically linked (that is refer) to the same domain of discourse, as explained above.",
"Hybrid Architecture It is possible to define a hybrid architecture, with the hybrid kitem containing some kitems linked in a serial way and others in a parallel way.",
"Meta-Kitems and Meta-Knowledge In FIGS.",
"10 and 11, each kitem is a class that supports the creation of objects, or knowledge elements.",
"It is desirable to be able to define a class that can have attributes and methods that span more than one of the kitems contained in the parallel, serial or hybrid kitem.",
"This can be achieved by giving the outside kitem (eg: the serial, parallel or hybrid kitem) access to all the properties and methods of the contained kitems, in addition to the properties that belong only to the outside kitems.",
"This means that it is possible to define meta-knowledge about the kitems contained in the outside kitems.",
"The outside kitem, when instanced as an object, can observe the behaviour of the kitems in it and take actions when necessary.",
"The outside kitems are referred to as meta-kitems able to express meta-knowledge.",
"Display and Executable Kitems In this section we consider two different types of very useful kitems: the display kitem and executable kitem.",
"We show that each can be constructed using one relevance kuark and one procedure kuark.",
"Display Kitem The display kitem is a serial kitem comprising two kuarks.",
"The first kuark is a relevance kuark.",
"It uses its region and the state of the environment to determines the relevance of the display kitem.",
"The second kuark has the relevance (the output of the first kuark) as source context and region.",
"The material to be displayed comprises the destination region and the explanation (the destination region can be the explanation.",
"Context Kitem Contexts and regions are made of elements or objects that describe the contexts and regions.",
"Each such object can have values.",
"Here we show that kitems are suitable for describing and implementing these context objects.",
"A context object is a kitem as shown below (only some properties are mentioned): kitem tile → object title source context → object values source region → instantiated values destination context → same as source context destination region → same as source region mapping → maps source to destination (contexts and regions) explanation → optional.",
"Explanation Kitem An explanation object is a kitem as shown below (only some properties are mentioned): kitem title → explanation title (can be ‘explanation’) source context → not used source region → not used destination context → not used destination region → not used mapping → not used explanation → multimedia explanation.",
"Context Element A context element is a kitem made of, typically, a context kitem and an explanation kitem (some context items can have more than one explanation).",
"To create such a kitem, the designer defines a parallel kitem using the procedure described in the GKMS patent.",
"It is also possible to have a context item made of a single context kitem, by using its explanation instead of the explanation kitem.",
"This solution does not work if more than one explanation is required.",
"For example, an explanation relating to the meaning of the context item and another relating to its use in a knowledge item.",
"Context Space (Source Or Destination) The context space is a kitem made of context items (also kitems) using the parallel architecture.",
"Typically no links are used between context items.",
"Executable Kitem The executable kitem is a serial kitem comprising two kuarks.",
"The first kuark uses its region and the state of the environment to determines the relevance of the executable kitem.",
"The second kuark has the relevance (the output of the first kuark) as region and as part of its source context.",
"When the relevance is not ‘null’, the second kuark executes its mapping to produce the destination region.",
"The inputs to the executable mapping are elements of the second kuark's source context that can be additional to the relevance context.",
"Knowledge Item and Knowledge Item Definition A knowledge item is a kitem comprising a source context, a source region, a destination context, a destination region, a mapping and an explanation.",
"Defining a knowledge item, as described in the GKMS patent, involves defining a region in the source context (and other things).",
"This is done using the context space kitem, that is made of context objects (or elements).",
"The architecture of a knowledge item is normally that of a parallel kitem.",
"It is often desirable to explain why a certain context element is part of a knowledge item or how to use or interpret this context element when it is attached to the knowledge item.",
"This can be achieved simply by giving the user defining the knowledge item the facility to add an explantion kitem to the list of kitem that are part of the knowledge item, and linking this explanation kitem to the context element (kitem) in question.",
"Alternatively, the explanation can be part of the context element kitem.",
"That is, the explanation kitem is already linked to the context element (with reference to OOP, the context element class ‘contains’ the explanation).",
"A knowledge item is defined with respect to a domain of discourse, defined by the source and destination context and the mappings available at definition time.",
"It would not be efficient to include the contexts kitems in the knowledge item.",
"Instead, the knowledge item is linked to the context space kitems.",
"This is in agreement with the definition of links in the parallel architecture kitem.",
"Kitem Hardcoding We have discussed how kitems can be implemented using other kitems.",
"However it is always possible to implement a kitem as a single hardcoded entity.",
"For example, in the GKMS patent, the context elements and the knowledge items were implemented as single entities with their specific architectures.",
"When this is done, many of the modular and flexible features of the kuark-kitem model are lost.",
"When a similar effect is desired, it is preferable to implement the kitem as a permanently compiled multi-kitem kitem (see later).",
"Knowledge Processing in Multi-Kitem Systems A multi-kitem system is a system comprising a set of kitems representing the body of knowledge about a certain domain of discourse.",
"Typically the domain of discourse is identical for all the kitems in the system, although it is not essential.",
"Such a system is identified as a knowledge module or database.",
"Knowledge processing involves the system (or server) running a question-answer session.",
"The system asks questions, get answers and, on the basis of these answers, asks further questions until the correct answers are found or no answers exist.",
"The steps involved are: identify the most important questions (the importance is determined by the ability of the answers to the question to lead to the correct answers as quickly as possible); asks these questions; on the basis of the answers to the questions, determine a) whether any answers can be found, and b) the next best questions to ask.",
"The processing refers to: the identification of the correct answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most promising provisional answers available in the knowledge base, on the basis of the information available so far in the consultation; and the identification of the most important questions to ask on the basis of the information available.",
"Searching for relevant or important knowledge Knowledge access in a multi-kitem system (a knowledge module) follows the same pattern as for a GKMS knowledge base; that is, it is a question-answer session or consultation.",
"The consultation process is managed with, and recorded in, a document.",
"This document can also be a kitem called the consultation kitem.",
"That is, the consultation kitem has all the properties and methods of the kitem class.",
"All the utilities for kuarks and kitems can be used with consultation kitems.",
"Searching involves identifying those kitems that have source regions compatible with the enquiry (see GKMS patent for meaning of compatible).",
"It means determining the ‘relevance status’ (available, candidate, rejected or definite) of a kitem with respect to the enquiry or consultation so far.",
"Searches can be synchronous or asynchronous.",
"An available status is the default status of a kitem before a search takes place; a candidate kitem is one that is not incompatible with the enquiry so far; a rejected kitem is one that is incompatible with the enquiry so far and a definite kitem is one for which the enquiry satisfies the source region.",
"Synchronous Searches Database Search The database search is a SQL search or equivalent.",
"This means that the kitems are stored as records in a database.",
"This is not discussed further in this document Full Text Search The full text search has to process each relevant field in each record where each record describes a knowledge element.",
"This process can be slow; it is not discussed further in this document.",
"Context Tree Search with Processing Delegated to A Search Agent The source context is organised as a tree structure.",
"A source region, which determines whether a kitem will fire (firing depends on the region's compatibility with an enquiry), is a subset of the source context.",
"Each kitem (eg: knowledge element) can be indexed against the context tree.",
"This means that each object in the context possesses a list of the kitems it is associated with.",
"The context tree is a formalisation of one of the kuarks'properties (see above).",
"During processing, all the indexed kitems to the questions (context objects) asked and answered so far in the consultation have their relevance status checked and modified if appropriate.",
"Kitems which become definite can be presented to the user, candidates remain contenders for definite status depending on future answers, and rejected items will not be considered any longer.",
"At the implementation level, the context tree is a modified context space kitem.",
"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.",
"The search process updates the status of the kitems according to the enquiry.",
"Context Tree Search with Processing Delegated to Each Kitem The search process (also a kitem) flags these elements in the context tree that correspond to the questions answered so far.",
"Each context tree element is itself a kitem and its role is to determine whether all the indexed elements have taken into account the values of the context tree element.",
"If they do this, it passes the value(s) to the indexed kitems and waits until all the responses have been received.",
"The search process waits until all the tree elements corresponding to the enquiry so far have replied.",
"When all have replied, the enquiry has been processed (the knowledge elements have determined their status based on the enquiry so far).",
"Synchronisation is assured and the next step in the enquiry (presenting the results to the user and waiting for its input) can proceed.",
"At the implementation level, the context tree is a modified context space kitem.",
"The enquiry process instantiates a consultation kitem at each iteration, which contains the list of the linked kitems affected by the enquiry, with their states.",
"These kitems affected are the available or candidate kitems in the knowledge base after the previous iteration, or all the kitems in the knowledge base is the enquiry searches the whole database at each iteration.",
"Each kitem updates its status according to the enquiry and informs the searc process.",
"Asynchronous Searches Processing Delegated to Each Kitem In asynchronous searches, the processing is distributed and controlled by each item.",
"There is no central authority that decides what to do when.",
"Each kitem (or agent) monitors its environment and reacts appropriately.",
"In doing so, it may change the environment and trigger new actions by other kitems.",
"This is in contrast to synchronous searches in which it is the search process that determines whether a kitem is applicable or not.",
"In particular, in asynchronous searches it is each kitem that determines whether it is applicable to the environment as it is detected via the source context and the source region.",
"In asynchronous searches, the source region determines whether the outcome of the kitem becomes applicable.",
"That is, the source region determines the state of the kitem's relevance status and, depending on its state, the mapping becomes applicable.",
"The state of the relevance region determines what happens to the application region.",
"In the case of non-executable mappings (or display mappings) the process is as illustrated in FIG.",
"8; the relevance mapping ‘releases’ the destination region to the kuark output.",
"State of Environment (or Context) Communicated to the Items As indicated above, each kitem has a unique ID or address that determines its location in a distributed memory space or network (such as the Internet for example).",
"It also possesses a set of applicability addresses that correspond to the kitems in its source region.",
"During processing, the enquiry process submits each answer to all the kitems which have, in their list of applicability addresses, the ID of the kitem that formed the question.",
"In this model, each kitem determines by itself whether it can fire or not: the enquiry process submits its answers to the network; the answers reach the kitems that have the corresponding addresses in their applicability list (the kitems that form the source region); the kitems update their relevance status with respect to this enquiry; if the relevance status is definite or candidate, the kitem sends its ID and its status to the enquiry process.",
"The enquiry process, when submitting, sends its (return) address, an enquiry number and an enquiry step number.",
"These numbers are returned by the kitems that reply and this enables the enquiry process to know what replies correspond to what enquiry (or enquiry step).",
"Each kitem can decide whether to answer more than once to an enquiry from the same origin or process.",
"It is up to the enquiry process to manage all the replies it gets from the network.",
"The solution outlined here is applicable to distributed processing.",
"The Kitems Seek to Know the Status of their Source Context Another model for asynchronous searches is to give even more responsibility to the distributed kitems than in the previous description.",
"In this model, each kitem has the address of each of the context objects in its context and in its region (a subset of the context).",
"The kitem interrogates the network at regular interval to determine whether the status of the region, and hence of the applicability of the kitem, has changed.",
"This model means that the kitem takes the intiative (this has some important applications not discussed here).",
"However during searching, the model imposes much traffic on the network that is of no benefit, unless the context object has changed since the last status check.",
"Another problem is that there may be an unacceptable delay between status checks, during which time, if the status has changed, the kitem doing the interrogating cannot determine its applicability.",
"Cascading Knowledge Cascading refers to a knowledge element having, as part of its destination region, an object that belongs to the source region of another knowledge element.",
"When the first knowledge element fires, it may change its source region in a way that impact on the applicability of the second knowledge element.",
"The second knowledge element may have to fire, not as a direct effect of the enquiry but as an indirect effect, that is, a consequence of another knowledge element having changed its state.",
"This process can extend to any arbitrary number of knowledge elements, called cascading knowledge elements.",
"In order to ensure that all the cascaded elements update their state if appropriate, it is necessary to a) link the cascaded elements, and b) have the search process update the relevance status of these cascaded knowledge elements (synchronous search) or c) to communicate the state of the environment (outputs of previously fired knowledge elements) to the linked cascaded knowledge elements (asynchronous search).",
"Validity of the Relevant Knowledge For simple knowledge bases on a single server it is a relatively simple matter to ensure that all kitems they contain have the same domain of discourse.",
"This, however, does not apply for large or distributed knowledge bases where the knowledge comes from different sources.",
"In general, therefore, there is no certainty that all the knowledge items (kitems) accessed during a search have the same source and destination contexts.",
"In fact it is unlikely.",
"This means that a user inspecting a definite kitem (with respect to an enquiry) may not know what the domain of discourse of that kitem might be.",
"This information is very important as it defines the context for the knowledge in the kitem in question.",
"A solution to this situation is to give the user the ability to inspect the source and destination context of every kitem that is provided by the search process either as a candidate or definite kitem.",
"Identifying the Most Promising Questions Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Context objects which index a large number of available or candidate kitems are potentially very important when used as questions.",
"This is because their answers have the potential to impact on the relevance status of a large number of kitems.",
"This indicates that the importance of these context objects as questions in an enquiry is determined by the number of kitems with status available or candidate they index.",
"As the context tree is updated each time a question is considered, the most important questions to ask next depend on the consultation so far; that is, on the state of the context tree after the previous enquiry has been processed.",
"Therefore the questions are selected dynamically.",
"Context Tree Search with Processing Delegated to Each Kitem As explained above, the search process waits until all the tree elements corresponding to the enquiry so far have replied.",
"It is the responsibility of the enquiry process to collect these replies and to count the number that come from each of the tree context element: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.",
"In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relerance status), this process can also be asynchronous it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.",
"Asynchronous Searches Processing Delegated to Each Kitem The enquiry process (that sends the enquiry over the network for kitems to process it) performs several tasks: it waits for answers to get back from the network; it counts the number of candidate kitems from each context tree element—the ones with the highest number of candidate kitems correspond to the best questions to ask.",
"In addition, the enquiry process can perform the following operations: it can pass the kitems it received to the display kitem (that will display them according to their relevance status), this process can be asynchronous; it can build its own operational domain of discourse (source and destination context spaces) by concatenating the context spaces of all the answers received so far.",
"Search for Hidden Knowledge Hidden knowledge refers to knowledge that may be relevant to an enquiry but that is not yet linked to any question answered in the enquiry process so far.",
"The question is how to identify this hidden knowledge and how to extract its ‘best questions’.",
"In situations where the domain of discourse of a body of knowledge is known, one can simply identify the kitems with the largest number of indexed kitems with relevance status available or candidate (see context tree above).",
"There is no simple solutions when the domain of discourse of a body of knowledge is not known.",
"Processing Speed Synchronous Searches Context Tree Search with Processing Delegated to A Search Agent Synchronous searches can be very quick as they are under the control of one process that can be optimised, based on known conditions in the server.",
"Context Tree Search with Processing Delegated to Each Kitem This processing can be distributed across a network, but it is infrequent.",
"This processing is slower than the previous one as it imposes communications overheads between kitems (those doing some processing and the enquiry process).",
"Nevertheless, the enquiry process runs on a single server and can be optimised.",
"When the process is distributed, several kitems can process simultaneously; this can partially offset the communication overheads.",
"Asynchronous Searches Processing Delegated to Each Kitem This processing is slowest unless the number of kitems doing processing is large enough to offset the communication overhead between kitems and the network delays.",
"Compilation Compilation is the process that takes a collection of kitems and organises them for the purpose of speeding up processing.",
"The result of compilation is a kitem.",
"In general, compilation is applied to any kitem.",
"In practice it is often applied to all the kitems that belong to a domain of discourse (also a kitem) on a single server.",
"That is, all the kitems involved have domains of discourse that are subset of the compiled domain of discourse.",
"Compilation is also applied to a group of kitems contained in a serial, parallel or hybrid kitem.",
"The process of producing a context tree is a compilation of the kitems in an application or domain of discourse.",
"Compilation keeps the identity of the kitems and all the data and information in these kitems that pertain to their execution.",
"Keeping the identity of these kitems enables the non-executable parts of the kitems to be retrieved at execution time on a ‘as needed’ basis.",
"The processing model for the multi-agent systems described in this document imposes, like the Java language, that kitems call other kitems that in turn may call other kitems, etc, until the processing can be completed.",
"Once this is done, the results of the processing need to travel up the hierarchy of kitems, back to the initiating process.",
"The compilation process is designed to speed up the ‘down-hierarchy’ and ‘up-hierarchy’ travelling described above.",
"The context tree compilation achieves this purpose by ‘flattening’ the hierarchy to a single branch tree, where each context element (or node) in the branch contains all the kitems that have that context element as part of their regions.",
"There are two types of compilation: Just-in-time compilation imposes the compiled kitem to take the latest version of each kitem in its process, in particular the latest version of the source and destination regions; or Permanent compilation accepts that the kitems do not change (between compilations) and that their attributes and methods can be used without checking them.",
"Therefore they are included in the compiled kitem.",
"This is faster than the partial compilation.",
"Compilation can be be carried out to different depths: 1.build context tree at runtime; 2.get regions of indexed kitems in the context tree at runtime; 3.include regions of indexed kitems in the context tree; Compilations of type 1 and 2 above are just-in-time compilations.",
"They updates the context tree each time the search process is triggered; permanent compilation produces the context tree and leaves it untouched until another compilation is carried out.",
"Agents as Kitem Systems An agent, as illustrated in FIG.",
"5, can be represented as a kitem 60 with the structure illustrated in FIG.",
"12.: Beliefs 61, Desires 62 and Intentions 63 are parallel kitems (their respective components are also kitems); the Beliefs 61, Desires 62 and Intentions 63 kitems are organised in a serial architecture.",
"That is, the agent kitem is a serial kitem; the destination and output region of the Beliefs kitem map on the source and source region of the Desires kitem; the destination and output region of the Desires kitem map on the source and source region of the Intentions kitem.",
"It is recommended but not essential that the kitems that comprise the agent operate in a synchronous mode.",
"Internal synchronous processing is possible, even if a multi-agent systems behaves asynchronously.",
"As an agent is a kitem, a multi-agent system is a multi-kitem system with a parallel architecture and continuous asynchronous processing.",
"Kitems Needed for A Workable Solutions The kitems needed to produce a workable implementation for knowledge management are shown below: Context Management Context Elements A context element is a display kitem (a serial kitem made of two kuarks).",
"Source and Destination Contexts A source or destination context kitem is a parallel display kitem.",
"Its members comprise the list of all the context elements defined in the domain of discourse.",
"Context Executable Kitems Edit a context element Edit a context space Display a context space as a tree A source or destination context tree is an executable kitem.",
"The display kitem can display the elements that belong to the source and destination kitems.",
"Its source context is the source or destination kitem and its destination context is the visual tree-like representation of the context.",
"Knowledge Definition Knowledge Editing Kitem The knowledge editing kitem is an executable kitem that has: As source space: the source and destination contexts.",
"As destination space: the source and destination contexts.",
"As source region: a subset of the source context that defines the applicability of the knowledge kitem.",
"As mapping: a link to a subset of the destination context, or a procedure or executable (sub-)kitem.",
"As destination region: a subset of the destination context that defines the outcome of the mapping, or the outcome of the procedure.",
"An explanation.",
"Knowledge Access and Display Search Kitem The search kitem is an executable kitem with: as source context: the source and destination contexts; as source region: the enquiry as defined so far; as destination context: the kitems in the domain of discourse (if known); as mapping: the search process that identifies the candidate and definite kitems with respect to the enquiry; as destination region: the candidate and definite kitems ‘retrieved’ by the mapping.",
"Enquiry or Consolation Kitem The enquiry kitem is an executable kitem with: as source space: the enquiry (the consultation process, showing each step in the question-answer session); as destination space: the status of the enquiry (answered, not answered, etc.",
"), the ids of the definite kitems.",
"Display Kitem The display kitem is an executable kitem with: as source space: the kitems to be displayed (itself another kitem); the display parameters; as destination space: the code that puts the kitems on the screen in a hierarchical way.",
"Administration of Contexts and Other Kitems The hierarchical (tree) structure of the kitems gives a very convenient way of controlling user access to kitems: the administrator can decide which branch of the hierarchy users can have access to and the type of access for the branch and each kitem in the branch; access control can also be carried out at the level of the kitem.",
"Dynamic Kitem Management The objective of dynamic kitem management is to be able to manage the integrity of the knowledge base when a kitem is edited.",
"This issue is important as kitems (eg: a context kitem) can be used in many knowledge elements and a change in such a kitem can have impacts on the validity of these knowledge elements (from a knowledge viewpoint, not operational viewpoint).",
"It is essential that the users are not left in the dark about the potential ramification of their changes and that they are helped in checking the validity of some affected kitems.",
"As much as possible, the system should ensure the integrity of the knowledge itself.",
"The process for achieving the above is to: use the enquiry process (by the system or the user) to retrieve all kitems that could be affected (the system or the user can specify the profile of the kitems affected by the change); make the changes automatically if acceptable; or ask the user to review/recertify the kitems affected (the system needs to provide guidance in this process).",
"The profile mentioned above refers to the values given to kitems in the source and destination context.",
"The system treats these values as an enquiry and retrieves all the kitems that are compatible with the enquiry.",
"Change to properties All the properties of the kitems can be changed.",
"Potentially, all the kitems that are linked to the kitem being edited are affected.",
"Some properties and the way their editing can be handled is listed below.",
"Change to Source Context and Region If a new context object is added: the system asks the user to specify the profile of the kitems that may be affected, the system retrieves these kitems and the user is asked to recertify them.",
"If an existing context object is edited or deleted: the system retrieves all the kitems that are related to this kitem (that contain it as part of a link for example) and asks the user to recertify them and to edit them if necessary before doing so.",
"Change to Destination Context and Region As for a change to the source context and region.",
"Change to Mapping The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.",
"Change to Status (Enabled/Disabled/Archived) The system retrieves all the kitems that are related to this kitem (that contain it as part of a link or region for example) and asks the user to recertify them and to edit them if necessary.",
"Change to the ‘Links to Other Kitems’ No checks required, the change is localised.",
"Consistency Issues Consistency issues may arise in a knowledge base when some kitems have the following features: kitems have overlapping (at least one common kitem with at least a common value) or identical destination regions with non-identical source regions; kitems have disjointed destination regions with overlapping or identical source regions.",
"The system can easily have an agent that checks all the kitems in a knowledge base and identifies these kitems that may be inconsistent.",
"The system presents the kitems to their authors for editing if necessary and recertification.",
"Missing Knowledge Missing knowledge refers to parts of a domain of discourse for which there is no knowledge or inadequate knowledge.",
"When the domain of discourse for a set of kitems is explicit, it is a simple matter to check that the union of the source regions in the kitems cover the entire source context.",
"If it is not the case, it means that the some additional knowledge is required.",
"The user can be informed and the location of this missing knowledge presented.",
"The location of the missing knowledge can be pointed out on each dimension (or context kitem) of the source context.",
"The user selects a context item and a value (or range of values) for which there is no region, and the system responds by showing the other context items and their values for which there is no region.",
"The user then selects a second context item and gives it some values (where there is no region), and so on for the other kitems if desired, until the user is confident of being able to add some missing knowledge.",
"New Kitems When a new knowledge element is being saved (the user has finished its definition), the system checks that it is consistent with the other knowledge elements in the knowledge base (see section 12.2).",
"The system also retrieves all kitems that overlap with the new kitem's source and/or destination regions.",
"The system informs the user that these kitems may be affected (their knowledge may be superseeded by the new kitem) and presents them to the user for checking.",
"The system can also ask the user to define the profile of the kitems that may be superseeded by the new kitem.",
"Terminology Term Explanation Applicability region The region in the source context that determines whether the kitem is applicable.",
"Application context The context for the application made of the source or space and destination contexts.",
"Available kitem The status of a kitem that is available for an enquiry (the other status are definite, candidate and rejected.",
"Candidate kitem A kitem that is compatible with the enquiry so far but for which the applicability conditions are not completely met.",
"Definite kitem A kitem that is compatible with the enquiry so far with its applicability conditions completely met.",
"Destination context The space of solutions considered by an application or a kitem.",
"Domain of discourse The space of problems and solutions (or situations and outcomes) considered by an application or a kitem; it is made of the source and destination contexts.",
"Enquriy process The agent or process that controls the question- answer session or consultation.",
"Executable mapping A mapping that takes an input from the source context and applies an algorithm to it to produce an outcome.",
"Kitem A object or element that comprises a source context and region, a destination context and region, and a mapping.",
"Knowledge item A kitem that is used to express knowledge.",
"Kuark An elementary kitem (the smallest building block) that can be used to build other kitems and/or complete applications.",
"Mapping The transformation that is applied to a source region to produce a destination region or outcome.",
"Outcome Another term for destionation region or solution.",
"Parallel kitem A kitem comprising other kitems organised in parallel.",
"Rejected kitem An kitem that is not compatible with an enquiry.",
"Relevance context The destination context that expresses the range of possible relevance for kitems in an application.",
"Relevance region A region in the relevance context.",
"Relevance status The relevance of a kitem with respect to an enquiry (see GKMS patent for details).",
"Serial kitem A kitem comprising other kitems organised in series.",
"Solution Another term for destination region or outcome.",
"Source context The space of outcomes considered by an application or a kitem."
]
] | Patent_10450975 |
[
[
"Support and guiding structure for friction element in disc brakes",
"Method and apparatus for friction element control in spot-type automotive disc brakes provides for use in a disc brake (10) of the kind comprising a fixed caliper (32) and at least one axially slidable disc (12, 14), a friction element mounting in which the friction clement (20) is supported on lengthwise guides (52) in the region of its circumferentially spaced ends (62, 64) and is further supported by an axially-extending abutment element (66) fixed to its upper edge and received in a channel (68) in the brake caliper (32).",
"The abutment element an its channel serve to provide improved support for the friction element in resisting torque applied to the friction element during braking."
],
[
"1-9.",
"(canceled) 10.A method of mounting a friction element in a spot-type automotive disc brake, the disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation means therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at least one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and said method comprising; g) providing abutment structure adapted to resist torque applied to said at least one friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and the method comprising causing same to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.",
"11.A method of mounting a friction element in a disc brake comprising an axially slidable disc, the method comprising providing guide structure adapted to act at two spaced locations on said friction element between said friction element and a non-rotatable mounting therefor, and said method comprising providing abutment structure located between said spaced locations on said friction element, and said method comprising causing said abutment structure to resist torque applied to said friction element during use of said brake.",
"12.A brake friction element comprising abutment structure located between guide structure at spaced locations of said friction element and adapted to resist torque applied to said friction element during use in a brake system.",
"13.A spot-type automotive disc brake comprising: a) at least one rotatable brake disc; b) a rotatable mounting for said brake disc to permit such rotation and which is adapted to drive said brake disc and to have exerted thereon a braking effect by said brake disc when the disc brake is actuated; c) at least one pair of friction elements adapted to frictionally engage braking surfaces on opposite sides of said brake disc to effect braking on actuation of actuation device therefor and having a line of action intersecting said friction elements; d) said brake disc being axially slidable in use with respect to said mounting therefor under the action of said friction elements and said actuation device therefor; e) a non-rotatable axially fixed mounting for said friction elements adapted to permit axial sliding movement of at least one of said friction elements into and out of frictional engagement with said disc while locating same and resisting movement of same under the action of friction forces generated by engagement of same with said at lest one disc; f) axially-extending guide structure being provided in relation to said non-rotatable mounting and adapted to act on said at least one friction element at two spaced locations thereof; and g) abutment structure adapted to resist torque applied to said at least on friction element and comprising an abutment element located intermediate said spaced locations of said friction element, and adapted to resist torque applied to said friction element during use of said brake while permitting said friction element to move towards and away from said disc.",
"14.A disc brake comprising an axially slidable disc, and guide structure adapted to act at two spaced locations on a friction element and between said friction element and a non-rotatable mounting therefor, and said brake comprising abutment structure located intermediate said spaced locations and adapted to resist torque applied to said friction element during use of said brake.",
"15.A brake friction element comprising abutment structure located between circumferentially spaced ends of said friction element and adapted to resist torque applied to said friction element during use in a brake system.",
"16.A spot-type automotive disc brake according to claim 13, wherein said abutment structure is located generally radially in-line with the line of action of said actuation device.",
"17.A spot-type automotive disc brake according to claim 13, wherein said abutment structure comprises an abutment element fixed at the upper periphery of said at least one friction element and extending lengthwise in the direction of sliding brake-actuating motion of said friction element and slidingly received in a complementary channel formed in said non-rotatable mounting or caliper.",
"18.A brake according to claim 17, wherein said abutment element having a length-to-width ratio of at least in the range of 1 to 1 to 3 to 3."
],
[
"This invention relates to friction element control in disc brakes.",
"An example of the application of the invention is to the control of friction elements in spot-type automotive disc brakes, particularly such brakes of the kind in which a fixed caliper and sliding friction elements are employed.",
"The invention may find an application outside the strict limits of the technical field of the disc brakes disclosed in the specific embodiments described below, but principally the invention is concerned with controlling the movement of friction elements in relation to the associated structures of a disc brake, particularly the disc or discs themselves (which rotate during use), and the relatively fixed or non-rotatable structures such as that of the caliper on which the friction elements are mounted.",
"Generally, the friction elements with which the invention is concerned are those which are not actually fixed to a position-defined structure such as the end plate of a fixed caliper and thus, the invention is particularly applicable to friction elements of the kind employed as the middle one of three friction elements interleaved with twin discs in a fixed caliper/sliding disc assembly as disclosed below.",
"In such a disc brake, a need arises for excellent dynamic control of the movement/position/attitude of the friction elements during brake application in order to maximise the effectiveness of the brake and minimise the extent of uneven wear during use.",
"In this connection, we have already disclosed in co-pending applications arrangements whereby the friction elements are provided with anti-tilt systems to maintain as far as possible the parallel and co-planar relationship of the friction-producing faces of the friction elements and of the brake discs themselves, and this has largely been achieved by means of spring arrangements exerting substantial resilient forces on the sliding disc assemblies and likewise on the sliding friction elements assemblies to achieve the necessary degree of control under the dynamic conditions of use.",
"Developments thus far have enabled us to provide a satisfactory degree of control in respect of those functions which may be conveniently grouped under the heading “anti-tilt control”, but we have discovered that there is a further area in relation to friction element control which requires attention in order to provide further advances in relation to the dynamic aspects of the performance of the disc brake itself.",
"These further factors which require attention concern principally the question of the response of the friction elements to the effect of the torque exerted thereon by the frictional forces during brake application.",
"Thus, it will be readily understood that upon brake application and frictional engagement of the friction pads with the rotating disc, the applied forces acting on the friction elements produce a torque due to the rotary motion of the brake discs and such torque or turning effect on the pad and thus on its backing plate and thus on the friction element as a whole can cause destabilisation of the friction elements leading to a reduction in brake efficiency, or possibly uneven wear of the friction pads, and an object of the present invention is to provide improvements in this regard in relation to friction elements for disc brakes, or indeed improvements generally.",
"According to the invention there is provided a method and apparatus as defined in the accompanying claims.",
"In embodiments of the invention described below the axially slidable friction elements are provided with the basic guide means and supporting means whereby they are able to execute supported axial movement for frictional engagement with the brake discs.",
"However, in addition, the slidable friction elements are provided with abutment or torque-transmitting means in the form of an abutment or torque-transmitting element and an associated abutment or torque-transmitting channel in which the abutment or torque-transmitting element is received.",
"These structures (provided respectively at the upper edge of the friction element and at the underside of the brake caliper) serve to provide an important abutment or torque-transmitting function for the friction elements whereby the potentially twisting effect of the braking torque applied by the rotating brake discs is effectively neutralised by the abutting engagement of the abutment or torque-transmitting element with its corresponding abutment or torque-transmitting channel.",
"Moreover, although it would be possible to provide the abutment or torque-transmitting element as merely an upstanding peg or the like structure having the same thickness as the remainder of the friction element backing plate (and some benefit in terms of torque neutralisation would undoubtably be available), in the embodiments the abutment or torque-transmitting element is constructed so as to have a length dimension (extending lengthwise in the direction of the thickness of the backing plate material) whereby its torque neutralisation effects are significantly enhanced.",
"It also potentially assists in guiding its friction element.",
"A particular feature of the embodiments is the location of the abutment or torque-transmitting element in relation to the line of action of the actuation means for the brake, such as the centre line of the actuating piston.",
"In the embodiments, the abutment or torque-transmitting element is positioned directly above the line of action of the piston, and this is the preferred position.",
"In the case where twin actuating pistons are provided, then a central position between the pistons (and thus effectively at the centre of pressure produced by the pistons) is preferred.",
"Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which: FIG.",
"1 shows, in block diagram format a spot-type automotive disc brake comprising a pair of axially slidable discs and associated friction elements, an actuating mechanism therefor and a fixed caliper or bridge structure overlying same; FIG.",
"2 shows on a somewhat larger scale an exploded perspective view of the caliper and an associated friction element with an abutment or torque-transmitting element shown at an upper edge of the friction element together with an associated abutment or torque-transmitting channel to receive same; FIG.",
"3 shows a side elevation view of the assembly of FIG.",
"2 in its assembled condition; and FIG.",
"4 shows a plan view, as seen in the direction of arrow IV in FIG.",
"3, of the assembly of FIGS.",
"2 and 3.As shown in FIG.",
"1 a spot-type automotive disc brake 10 comprises a pair of rotatable brake discs 12, 14, a rotatable mounting 16 for the brake discs to permit rotation of the discs and which is adapted to drive the brake discs and have exerted thereon the braking effect by the discs when the disc brake 10 is actuated.",
"Two pairs of friction elements 18, 20 and 22 are provided and are adapted to frictionally engage braking surfaces 24, 26 provided at opposite sides of brake discs 12, 14 to effect braking on actuating actuation means for the brake.",
"Central friction element 20 is double-sided for frictional engagement with the mutually-inwardly facing braking surfaces 24, 26 of brake discs 12, 14 and is provided with appropriately facing friction pad material accordingly.",
"Friction elements 18, 20, 22 comprise (as shown in FIGS.",
"3 and 4) in each case a generally flat metal backing plate 28 and secured thereto and standing proud thereof a body of friction material of known construction for high durability frictional engagement with the relevant braking surface of the relevant brake disc.",
"In the case of central friction element 20, the friction material is provided at both faces of the backing plate 28.Brake discs 12, 14 are axially slidable in use with respect to their rotatable mounting 16 under the action of friction elements 18, 20, 22 and the actuation means (to be described below) therefor during braking.",
"For example the brake discs may be keyed to the rotatable mounting or hub 16 at three or more locations and resilient means may act there between.",
"A non-rotatable mounting 32 for friction elements 18, 20, 22 is provided comprising a caliper or bridge structure 34 which is mounted on a fixed structure of the vehicle to be braked, for example on the wheel mounting and which straddles the brake discs 12, 14 and also provides a mounting for actuation means 36,38 (indicated diagrammatically) which applies inwardly directed braking forces to the outer friction elements 18, 22, thereby causing frictional engagement with the brake discs 12, 14, and slight sliding movement of those discs with respect to their rotatable mounting 16.In FIG.",
"1 of course it can be seen that the clearances between the structures have been greatly exaggerated for simplicity of diagrammatic illustration.",
"The actuation means 36, 38 could comprise a pair of piston and cylinder assemblies.",
"However only one such is strictly needed since the actuation means can be one-sided with a fixed structure at one side or the other of the assembly of discs and friction elements (which fixed structure could simply be a stop extending from caliper 34), and against which fixed structure the assembly is pushed by the single actuation means.",
"Further details in this regard may be found in our co-pending applications WO 98/26192 (docket 2558).",
"Non-rotatable mounting 32 for the friction elements 18 to 22 is adapted to permit sliding movement of the friction elements into and out of frictional engagement with the brake discs while resisting movement of the friction elements under the action of frictional forces generated by engagement of the friction elements with the discs 12, 14.Turning now to the details of the structures shown in FIGS.",
"2, 3 and 4, we mention first that the friction element illustrated is taken to be the central friction element 20 which (though not shown in FIG.",
"2) is provided as shown in FIGS.",
"3 and 4 with pads 40, 42 of friction material facing axially at its opposite sides.",
"Other structures seen in the drawings include the casting 44 to which caliper 34 is secured and from which the actuating piston 46 can extend under the action of the usual hydraulic and driver-controlled brake actuation system having a line of action 48 which of course intersects the friction elements, this location being shown in FIG.",
"2 at 50 which identifies the line of action of the centre of piston 46.As also shown in FIGS.",
"2 to 4 axially-extending primary abutment or torque-transmitting means 52 is provided in relation to caliper 34 and is adapted to act on the slidable friction elements 20, 22 (end friction element 18 being secured or fixed to an end plate (not shown) extending downwards from the outboard end of caliper 34).",
"Primary guide means 52 comprises axially-extending guide rails 54, 56, one at each side of caliper 34 and adapted to receive corresponding hook elements 58, 60 provided at spaced locations namely the opposite circumferentially-spaced ends 62, 64 of friction element 20.Thus, guide rails 54, 56 and hook elements 58, 60 serve to ensure that friction element 20 (and likewise sliding friction element 22 in a similar way) is able to hang from the caliper for sliding axial movement lengthwise thereof towards and away from the brake discs 12, 14.In addition to the guide rails 54, 56 and hook elements 58, 60 there is provided further abutment or torque-transmitting means comprising an abutment or torque-transmitting element 66 located intermediate spaced locations namely the opposite ends 62, 64 of the friction element and adapted to enable the friction element to resist torque from frictional forces applied to it during use of the brake, while permitting the friction element to move towards and away from the brake discs.",
"Abutment or torque-transmitting element 66 is received in an abutment or torque-transmitting channel 68 formed in the underside 70 of caliper 34.It will be noted from FIG.",
"2 that abutment or torqur-transmitting element 66 is generally radially in-line with the line of action 48 of actuating piston 46 and indeed is directly above the piston centre line point 50.FIG.",
"2 of the drawings shows the proportions of abutment or torque-transmitting element 66 in a clear manner with respect to the remainder of the friction element structure.",
"Thus, the abutment or torque-transmitting element has an axial length L and a traverse width W such that L/W lies in the range of 2 to 3.In general, the ratio L/W should preferably be at least 1 and more preferably still in the range of 1 to 3.Such a ratio has the effect of maximising the abutment or torque-transmitting influence of the abutment or torque-transmitting element in relation to frictionally-developed torque forces applied to the friction element during use, thereby minimising the tendency for taper wear to develop in the pads 40, 42 of friction material.",
"The significance of the aspect ratio L/W in terms of maximising the torque-transmitting effect of the abutment or torque-transmitting element is discussed above.",
"So far as the length aspects of the dimensions of the abutment element 66 are concerned, the following applies.",
"As shown in FIG.",
"3, there is shown at 72 an indication of the possibility of friction element tilt in an axial plane which can occur when the brake is in its off condition.",
"Such tilting is limited by engagement of abutment element 66 with the top of groove 68.The possibility of tilt in the mode indicated at 72 is decreased with increasing length of the abutment element 66.Likewise, a similar tilt-inhibiting effect is exerted by minimising clearance between the top of the abutment element 66 and the top of groove 68.In the on condition of the brake, as indicated in FIG.",
"3, the brake-applying force Fp is indicated at 74 and acts along line 48 on the friction element 20 and has a turning (or taper wear) moment Fp×xp at the element 66 where xp is the radial distance (identified at 76) of off-set of line 48 from the top of channel 68.This force is resisted by a force at the end of the abutment element 66 acting on the top of groove 68 accordingly.",
"This function likewise is enhanced by increasing the length of abutment or torque-transmitting element 66.In use, disc brake 10 operates in substantially the usual manner, which is implicit from the above description, so far as concerns the general mode of frictional engagement of the relevant surfaces.",
"So far as guidance of the friction elements during use is concerned, the friction elements are supported on guide rails 54, 56 through hook elements 58, 60 and frictionally-developed torque forces applied to the pads 40, 42 of friction material which tend to produce twisting forces arising from the rotation of the brake discs are largely taken out of the friction element structure by the abutment or torque-transmitting element 66 and the engagement of same with the side walls of abutment or torque-transmitting channel 68 in which the abutment or torque-transmitting element is a close sliding fit.",
"Indeed, in principle, the tolerances in relation to the sliding fit of abutment or torque-transmitting element 66 within abutment or torque-transmitting channel 68 are tighter (meaning a closer fit) than exists between hook elements 58, 60 and guide rails 54, 56.As a result, wear on the pads 40, 42 or friction material is greatly improved in terms of its uniformity throughout the total working area of the friction material.",
"Amongst other modifications which could be made in the above embodiment while remaining within the scope of the appended claims are changes to the shape and dimensions and exact location of the abutment or torque-transmitting element 66 in relation to caliper 34."
]
] | Patent_10451031 |
[
[
"Lugged cap forming system",
"Transfer apparatus and method are provided from first (15A, 16A) to second (15B, 16B) stations of tooling in a cap (11) making press.",
"This cap is biased against the first upper tools by a first airstream (50) introduced under the cap and moves upward with the first station punch (45).",
"As the punch approaches its top location, a transfer airstream (52) begins while the first airstream is still on, and moves the cap out through a transfer chute (18) to the second station.",
"The cap departs the chute and passes detents (67) on a pair of closed retention fingers (60) which define an extension of the transfer path from the chute into the open second station tools.",
"A vacuum (85) applied to a port in the second station punch then holds the cup against the rising upper tools.",
"When the punch clears the closing fingers and approaches its top location, an ejection airstream (87) commences to propel the finished cup via a discharge chute (19)."
],
[
"1.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed position in which said tools are closely spaced to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing further operations thereon; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station tools; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute.",
"2.A system as defined in claim 1, wherein said chute terminates adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being movably supported on opposite sides of the second station tools for movement perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open.",
"3.A system as defined in claim 2, wherein said fingers have complementary tracks formed thereon to receive and guide a part exiting said chute, said tracks having inwardly curved end sections which align with the center of the second station tools to provide a termination of the path of a cap part entering the second station tools, and cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening.",
"4.A system as defined in claim 3, wherein said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts.",
"5.A system as defined in claim 1 wherein said second station tools are constructed to form lugs on a rolled rim of the cap parts.",
"6.A system for forming container caps, comprising: a first station including first station tools comprising a blank punch tool and a cooperating compound die tool adapted for mounting in a reciprocating press for cyclic movement between an open position in which said tools are separated and a closed interface position in which said tools are closely spaced (a) to form a cap from a metal blank separated from a supply of thin sheet metal during the closing motion of said tools and (b) then to draw the blank into a cup shaped part with a top panel and a side wall extending from said top panel, said tools also form an outward rolled rim on the side wall; a second station aligned with and spaced from said first station, said second station including second station tools for receiving parts from said first station and performing a lug shaping operation on the rolled rim; a chute extending between said first and second stations at the open position of said first station tools to guide parts in transfer from the first station into the second station, said chute terminating adjacent the interface of the second station tools; a set of spaced apart guide fingers for forming a continuation of said chute, said fingers being pivotally supported on opposite sides of the second station tools for opening and closing motion perpendicular to said second station tools such as to provide for closing of the second station tools with said fingers moved apart and for closing motion of said fingers into an extension of said chute when the second station tools are open; said fingers have complementary tracks formed thereon to receive and guide a part exiting said cute, said tracks having inwardly curved end sections which align with the center of the second station tools, said finger tracks include detents spaced from said curved end sections to define a centered position of the incoming parts and to provide a termination of the path of a cap part entering the second station tools; cam means operated by the press in coordination with opening and closing of the second section tools to move the fingers apart when the tools are closing and to close the fingers into a chute extension when the tools are opening; means projecting a first airstream into the part to hold the top panel against said blank punch tool during separating motion of said first station tools upon forming of a part; means projecting a second airstream across said blank punch tool at its open position and into said chute so as to propel cap parts into said second station; and control means coordinated with the operation of the press for switching on said first and second airstream projecting means to (a) initiate the first airstream at the beginning of separation of said first station tools and maintaining the first airstream at least until said first station tools are fully separated, and (b) initiate the second airstream before said first station tools are fully separated and maintaining the second airstream sufficiently to propel the part through said chute, said control means including an input pulse generator driven in synchronism with the cyclic operation of the press to generate a train of control pulses related to the angular position of the press crankshaft as it rotates to open and close the tools, whereby the initiation and termination of said airstream projecting means is synchronized with cyclic operation of the tools.",
"7.A method for discharging and transferring a cup shaped object from a work station, having upper and lower tooling which is opened and closed to form the object, along a transfer path, the open position of the upper tooling defining a ready position, comprising the steps of: (a) locating the object within the work station in the ready position by directing a first stream of pressurized gas against the object to cause the object to remain with the upper tooling upon opening of the tooling whereby the object is supported by the first stream against the upper tooling; (b) prior to locating the object in said ready position, initiating a second flow of pressurized gas through orifice means located adjacent to and directed across said ready position, thereby causing the transfer of said object from the work station when the object is released at the ready position; and (c) discontinuing the second flow of pressurized gas through said orifice means after the object has exited the open tooling.",
"8.The method of claim 7 including the additional steps of: (d) receiving the object in a chute extending to a second work station which defines the transfer path to adjacent second upper and lower tools of the second work station, (e) directing the object into an extension of the transfer path defined by pivoting fingers located at opposite sides of the second tools, and (f) opening the fingers when the second tools are closing, and closing the fingers when the second tools are opening such as to guide the object into a centered position between the second station tools."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>U.S.",
"Pat.",
"Nos.",
"6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.",
"That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.",
"The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.",
"However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.",
"With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.",
"a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.",
"in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.",
"To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.",
"Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.",
"Nos.",
"4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.",
"Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.",
"This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.",
"Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.",
"The caps are of a type having substantial height with respect to their diameter.",
"Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.",
"Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.",
"The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.",
"This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.",
"As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.",
"This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.",
"At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.",
"As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.",
"As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute."
],
[
"BACKGROUND OF THE INVENTION U.S. Pat.",
"Nos.",
"6,082,944 and 6,015,062, assigned to the assignee of this application, disclose closure constructions for reclosable containers (e.g a can body) wherein a domed container end with a neck portion having a pour opening is provided with a reclosable lugged type of cap.",
"That invention provides a unique and versatile container for fluids, particularly for beverages, wherein various standard can bodies are provided with a two part end including a neck with a pour opening, a lug formation on the neck below the pour opening, a reclosable cap having a lug formation which can interlock with the lug formation on the neck and including a seal surrounding the pour opening, and thus capable of maintaining product under pressure.",
"The two part end is affixed to a can body by conventional double rolled seam attachment between the bottom of the neck and the rim of the can body.",
"However, it is possible to affix the domed end to a can body without a cap, fill the can though the pour opening, and then apply the cap.",
"With the possibility of expanded markets for these lugged caps, which are also useful on various jars and bottles, there is a need for a system (method and tooling) for producing lugged cap members in a single machine, e.g.",
"a reciprocating press fitted with appropriate tooling, which is capable of precise high speed (e.g.",
"in the range of 135 to 150 strokes/minute) to achieve acceptable commercial production of the cap.",
"To develop such production speeds it is desirable to divide the progressive tooling operations into more than one step, and this in turn requires a rapid and precise transfer system to move the partially completed caps from a first station to a second station, and precisely register the caps in the second station.",
"Prior art transfer systems are known for moving and registering can end shells, such as in U.S. Pat.",
"Nos.",
"4,770,022 and 4,895,012, however, the end shells are relatively flat disc-like objects with a quite small height to diameter ratio, whereas the caps made by the present invention have a substantially greater height with respect to their diameter.",
"Thus, the physical dimensions of the caps involved in this invention are quite different from can shells or easy self-opening can ends.",
"This in turn introduces needs not required or anticipated in shell transfer systems, for example with regard to tipping of the caps during high speed transfer operations.",
"Also, because of the relatively high press cycling, and need for precision in cap positioning and deceleration immediately after each transfer to another press station, there is a requirement for precise transfer of each cap from a first press station through high acceleration, very rapid transfer to the next press station, and high deceleration to a precisely defined stationary location at that next station.",
"SUMMARY OF THE INVENTION The present invention provides a transfer apparatus and method for a first to a second station of progressive tooling in a cap making press.",
"The caps are of a type having substantial height with respect to their diameter.",
"Thus a first station punch and die form a cap of generally inverted cup shape, with an outward curled rim, and a second station punch and die form lugs into that rim, requiring just two strokes of the press to sever a disc from a supply sheet, form it, and discharge a completed cap.",
"Thus, in the first station, a cap is formed except for lugs which are added to the cap in the second station.",
"The formed cap in the first station, which is in the nature of an inverted cup, has an outward curl formed on its lower edge during the initial up stoke of the press.",
"This cup is then biased against the upper forming punch, by a first airstream introduced into the cavity within the underside of the cap, and moves upward in contact with the first station punch.",
"As the punch approaches its top dead center location (tooling fully open), a second airstream begins, before the first station punch is fully raised and while the first airstream is still on.",
"This second airstream moves the cap off the raised first station punch, and into and through a transfer chute directed toward the second station.",
"At the second station, the cap departs the chute and passes detents on a pair of closed retention fingers which, with the tooling open, define an extension of the transfer path from the chute into the open second station tools.",
"As the second station tools begin to close and the partially finished cap is located, the fingers are opened and the tooling operates to form lugs into the cap rim.",
"As the second station tooling opens, a vacuum applied to a port in the second station punch begins to hold the finished cup against that punch tool, and when that punch clears the closing fingers and approaches its top dead center location, an ejection airstream commences to propel the finished cup from the tooling via a discharge chute.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a bottom view of a container cap as provided by the invention.",
"FIG.",
"2 is a transverse cross-sectional view of a typical completed cap, as shown in FIG.",
"1.FIG.",
"3 is an over all perspective view of the tooling for a four-out cap making system.",
"FIG.",
"4 is an enlarged perspective view, with the die shoe removed, showing details of the tooling, including four first station tools in the center and four related second station tools outwardly in opposite directions of the first stations.",
"FIG.",
"5 is a further enlarged perspective view showing details of one pair of first and second stations of the tooling.",
"FIG.",
"6 is a cross-section view of a first station punch or upper tool.",
"FIG.",
"7 is a cross-section view of a first station die or lower tool.",
"FIG.",
"8 is a cross-section view illustrating the first station punch and die in closed position, and showing a cap formed except for an outward curl at the lower rim of the cap.",
"FIG.",
"9 is a cross-section view showing first station and corresponding second station tools, a transfer chute between them, the fingers which provide an extension of the chute into the second stage tool, and cams which control movement of the fingers.",
"FIGS.",
"10 and 11 are detail views of the top of the second station die, showing the open and closed positions of the fingers.",
"FIG.",
"12 is an enlarged cross-section view of the second station tools, and FIG.",
"12A is a further enlargement of the circled area in the center of FIG.",
"12.FIGS.",
"13A and 13B1 & 13B2 (three sheets) together are a schematic diagram of the pneumatic supply & control system of the press and tooling.",
"FIG.",
"14 is a flow diagram of the electronic control system for the tooling package.",
"DESCRIPTION OF PREFERRED EMBODIMENT The present invention provides a transfer apparatus and method for two station progressive tooling in a cap making press and system.",
"The caps are of a type having substantial height with respect to their diameter.",
"A typical such cap is shown in FIGS.",
"1 and 2, which is also shown in FIGS.",
"2, 4A and 4B of U.S. Pat.",
"No.",
"6,015,062.The preferably integral cap 11, in the general form of an inverted cup, includes a top panel 12, a peripheral sidewall 13, and a curled rim 14.In the present drawings, FIGS.",
"3-12A depict multi-lane progressive tooling comprising four lanes for simultaneously forming four caps 11, each lane comprising pairs of first and second tooling stations 15A, 15B, 16A, 16B, 17A,17B, and 18A, 18B, with the first stations 15A-18A arranged centrally of the tooling (FIGS.",
"3, 4 & 5) and corresponding second stations 16A-16B arranged outward of the first stations toward opposite sides of the upper and lower die plates 20A, 20B which support the upper (punch) and lower (die) tooling, and mount between the bed and slide of a reciprocating press (not shown).",
"Except for their orientation in the overall tooling package, the respective first station and second station tools are alike, and the following detailed description applies to all.",
"Taking stations 15A and 15B as examples, each pair of corresponding first and second stations has an associated transfer chute 19 (FIGS.",
"4, 5 & 9) between them, and each second station has a discharge chute 19A, the four of which are directed out opposite sides of the tooling (FIGS.",
"3 & 4).",
"Sheets of metal, with an appropriate pattern of lithographed materials for each cap, are fed centrally into the first stations by sheet feeding mechanism of known construction (not shown) which moves the sheets one at a time in step wise fashion, synchronized to the press strokes, along the feed path indicated by arrow IN in FIGS.",
"3 & 4.First Station The first station tools comprise an upper or blank punch tool 45 and a compound lower die.",
"During the initial operation of the first station tools, with the lithographed patterns aligned with respect to the first station tools, a blank is cut from the material (typically aluminum or thin cold rolled steel) on the down stroke of the press by blank punch 45.On the continuation of the down stroke, the blank punch and lower die tool cooperate such that the blank is drawn into a cup shaped cap part 11P (FIG.",
"8).",
"At the bottom of the stroke the panel shape 12 is formed into the top of cap part lip by the punch 45 and cooperating die 46 (FIGS.",
"6, 7 & 8).",
"On the up stroke, the lower curl ring 48, which is under spring pressure, raises with the blank punch.",
"The bottom edge of the cap part 11P is curled outward into the cavity 49 formed by curl ring 48 and blank punch 45, completing a formed cap 11 with an outward curled rim 15.The formed cap in the first station, which is in the nature of an inverted cup, is biased against the upper forming die by a first airstream introduced through passage 50 (see FIGS.",
"8 & 9) into the cap as the first station tooling opens, and causes the cap to follow upward against the bottom of the punch 45.During the upward travel of the cap, a second airstream is initiated through a nozzle 52 directed across the upper fist station tooling toward chute 18, and is at its full power when punch 45 (with a cap 11 held thereto by the upward directed first airstream) traverses the space between nozzle 52 and the entry 18E to chute 18 (see FIG.",
"9).",
"By the time the first station tooling reaches full open at the top-dead-center of a press stroke, the cap has actually been transferred into the second station 15B; see the Press Rotation timing chart below (page 8).",
"Thus, the first and second airstreams, appropriately switched on and off, together with the associated chute, constitute a first part of an essentially passive press transfer system.",
"Second Station At the second station, a separated pair of fingers 60 reach around the sides of the second station tooling (FIGS.",
"9, 10 & 11), defining partial sides of a receiving space 62 when the second station tooling is open, and an extension of the transfer chute when closed.",
"The fingers are supported by pivots 63 inward of their rear ends 61, and are biased into their closed position (FIG.",
"10, forward ends parallel) by spring mechanism 64.Each finger has a narrow ledge-like track 65 extending part way along its forward upper edge (FIGS.",
"10-12), facing each other and ending in a curved section 66.Extending horizontally inward over tracks 65 are spring-loaded ball detents 67 which, together with the curved track sections 66, define the termination of the transfer path for the incoming caps.",
"A cap propelled from the first station traverses the adjacent transfer chute 19 and enters receiving space 62, passing across ball detents 67 and resting against the curved track sections 66 (see FIG.",
"11).",
"The fingers and their operating mechanism form the remainder of the unique transfer system.",
"As the tooling proceeds to close during the beginning of a next stroke, fingers 60 are swung outward by descending cams 48 pressing against followers 49 on the rear ends 61 of the fingers to move the followers 49 inward and the forward section of the fingers outward (FIGS.",
"10 & 12) before the second station tools close.",
"The elongated cams 48 are mounted on the upper (die) tooling base (FIG.",
"12), and this action centers a cap (FIG.",
"11) and then opens the fingers associated with the second station tooling, before that tooling closes to form lugs on rim 15 of a cap 11.This closing action of fingers 60 is momentary, and they are then opened substantially before the second station tooling closes; see Press Rotation timing chart below.",
"In the second station 15B in the press, the tooling includes an upper punch 70 and a lower die 72.The punch tool 70 includes an annular knock out ring 71 with an inner shape conforming to a cap exterior, and a headed knock out pin 73 which is spring loaded toward a position flush with the lower edge of ring 71 (FIGS.",
"12 & 12A) when the tooling is opened.",
"In the lower die 72 there is a vertically extending tapered cam 74 mounted onto a base plate 75 and surrounded by a plurality of die pins 76 also mounted onto plate 75.The cam 74 and pins 76 project through a vertically movable, upwardly biased ring 78 which contains a plurality of lug forming dies 80, equal in number to pins 76 and movable laterally outward through forces from cam 74 as it is made to enter ring 78.The top of ring 78 has an upper surface 82 contoured to fit within a cap 11.Thus, when the second station tooling closes a cap is positioned with the curled rim 15 between the upper ends of pins 76 and the radially outward moving forming dies 80, to for the predetermined number of lugs in the rim (FIG.",
"1).",
"Then, the completed cap is held to the knockout pin by means of a vacuum created in passage 85 though the head of that pin 73, such that the cap is carried upward past an air nozzle 87 directed across the path of the rising upper tool toward an associated discharge chute 19A.",
"The vacuum is created by air flow through a small venturi (not shown) so the vacuum is relieved immediately upon cessation of air flow through that venturi.",
"A discharge air stream is started from nozzle 87 and moves the cap from the upper knock out ring and pin, into and through the associated discharge chute 19.Press Rotation Relation to Tooling Function 1st Station 0° Top of the Stroke; Top Dead Center 140° Material is Blanked 180° Form & draw complete; Bottom Dead Center; Cap Overall Height ˜0.810 inch 190° Cap Curl complete; Overall Height is ˜0.625 inch 190° 1st Air turned on; Blows cap against punch tool 220° Blank punch exits Die tool, cap against its face 230° 2nd Transfer Air on; Blows cap into transfer chute 330° 1st Cap arrives into catch fingers at 2nd Station.",
"2nd Station 0° Top of a stroke; Top Dead Center 60° Upper vacuum is turned on 136° Catch fingers start open, upper vacuum holds 1st cap against upper knockout and tools close 180° Lugs formed in 1st cap; Bottom Dead Center 181° 1st cap (completed) and tools move up together 224° Catch fingers close completely after tooling passes 270° Upper vacuum to knockout turned off 280° Discharge air valve to nozzle is turned on 290° Knock out air to nozzle 87 is turned on 330° 2nd Cap arrives into catch fingers 336° 1st Cap actually moves to discharge position 335° Vacuum actually turns off on Cap #1 350° 1st Cap actually leaves press via chute 19 FIGS.",
"13A and 13B comprise a pneumatic diagram for the pneumatic portion of the press control.",
"A “shop air” source of compressed air is supplied to the various electrically controlled valves (which are all labeled) to direct air under pressure to the above mentioned parts of the tooling, under management control of the electronics control (FIG.",
"14) of the system.",
"The details of such controls are apparent to persons skilled in this art from these diagrams FIG.",
"14 is a block diagram of electrical/electronics controls for the system, which selectively turn on and off the compressed air to the nozzles providing lift or “blow up” air to hold the cap part against the rising upper tools in the first station, and providing a transfer air stream to quickly move a cap part into a transfer chute 19.The third (upper) control provides timed discharge airstreams through nozzles 87 to move the finished caps into the discharge chutes 19A.",
"A pulse generator PG is driven by the press crankshaft (not shown) in typical fashion to generate a train of pulses related to the angular position of the crankshaft as it rotates, and these pulses are directed to the system PLC (Programmable Logic Controller).",
"Since the diagram is divided into three functions which occur during a press cycle, the controller PLC is shown in each of the three diagram parts, but in fact one PLC is employed in the control system.",
"While the method herein described, and the form of apparatus for carrying this method into effect, constitute preferred embodiments of this invention, it is to be understood that the invention is not limited to this precise method and form of apparatus, and that changes may be made in either without departing from the scope of the invention, which is defined in the appended claims."
]
] | Patent_10451057 |
[
[
"Diving aid",
"Diving aid for divers, at least comprising a power supply, alarm sound-generating means (3) and activating means (2) for activating the sound-generating means (3), which activating means (2) are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means (6) for attaching to the divers's body or the divers's equipment in an unmistakable orientation.",
"The diving aid according to the invention is preferably designed in the form to a wristwatch."
],
[
"1.Diving aid for reducing the dive stress of divers, in particular pre-dive stress, at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and which device also comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.",
"2.Diving aid according to claim 1, wherein the diving aid also comprises light-signal-generating means that are connected to the activating means.",
"3.Diving aid according to claim 1, wherein the activating means comprise a push-button.",
"4.Diving aid according to claim 1, wherein the activating means comprise an acceleration detector.",
"5.Diving aid according claim 1, wherein the diving aid comprises water detection means by means of which the device can be activated.",
"6.Diving aid according to claim 1, wherein the diving aid is designed in the form of a wristwatch.",
"7.Diving aid according to claim 1, wherein the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.",
"8.Diving aid according to claim 1, wherein the diving aid forms part of an ordinary diving computer."
],
[
"The present invention relates to a diving aid.",
"A major problem encountered by both experienced and inexperienced divers during diving is so-called dive stress, in particular pre-dive stress.",
"This dive stress increases the likelihood of mistakes being made prior to and during diving, mistakes which can have fatal consequences.",
"The chance of a panic situation under water is also increased considerably by this dive stress.",
"In practice, it is found that most divers dive only once or twice a year.",
"These are the divers at greatest risk.",
"It has been found that when such an inexperienced diver is in distress or in danger he has only three seconds in which it is possible to take action.",
"In the first second the distress or danger is perceived, in the second second the diver becomes aware of the problem and his position, and in the third second the diver is in a panic.",
"The most common reaction of such divers is to swim as fast as possible towards the surface, with all the risks this involves.",
"In a panic situation inexperienced divers are found to be incapable of any coordinated movement, for example in order to activate an alarm device.",
"The known alarm devices for use as diving aids are therefore found to be inadequate.",
"In this connection reference is made to FR-A-2,695,747.This French patent application discloses an alarm device for attaching to the wrist.",
"This device comprises two parts that are rotatable relative to each other and can be locked relative to each other by means of a locking pin.",
"This locking pin has to be removed in order to activate the alarm device.",
"FR-A-2,755,023 also discloses an alarm device, which comprises a housing with a battery and sound-generating means.",
"The housing comprises a push-button for activating the alarm device.",
"This alarm device is attached to an eye present on the housing.",
"It has been found that a diver who is in a panic is incapable of removing a locking pin in a controlled manner or locating and pressing a push-button on a trailing device.",
"Divers also very often wear gloves during diving, which makes such actions even more difficult.",
"Furthermore, a fully equipped diver has very limited freedom of movement.",
"There are also alarm devices that are operated by compressed air and are therefore connected to the compressed air system of the diver.",
"A common problem is that the diver is not getting any air and there is a fault in the compressed air system.",
"Such devices are therefore not sufficiently reliable, even if the diver were capable of activating the activating means.",
"In other words, all devices known in the prior art require coordinated movements from the diver.",
"These devices are therefore found to be inadequate in practice.",
"Knowledge of this fact increases both pre-dive stress and dive stress among divers.",
"There is consequently a great demand for a diving aid that can give the diver a relaxed and safe feeling.",
"The object of the present invention is to meet this demand, and to that end the invention provides a diving aid at least comprising a power supply, alarm sound-generating means and activating means for activating the alarm sound-generating means, which activating means are designed in such a way that when the alarm device is in use said activating means can be activated by an uncontrolled shock load, and that the device comprises means for attaching to the diver's body or the diver's equipment in an unmistakable orientation.",
"The diving aid according to the invention is found to be extremely satisfactory, even in a situation where an inexperienced diver panics.",
"It appears that an inexperienced diver in a panic is in fact only capable of making an uncoordinated striking movement with one hand in the direction of the alarm device.",
"For the device according to the invention, this is sufficient to activate the latter and make co-divers aware of the distress or danger.",
"It has been proved in practice that knowledge of the substantially increased safety under water through use of the diving aid according to the invention substantially reduces dive stress even in the case of very experienced divers.",
"It is pointed out that alarm devices that can be activated by an uncontrolled shock load are known for use on dry land.",
"These devices are not, however, suitable for use under water.",
"In this connection; reference is made to GB-A-2,267,373, GB-A-2,295,910 and GB-A-2,316,784.Apart from alarm sound-generating means, other means can, of course, also be used for alerting co-divers.",
"The diving aid also preferably comprises light-signal-generating means, which are connected to the activating means.",
"Under water the speed of sound is many times higher than it is in air.",
"The result of this is that under water the human ear is not capable of determining the direction of sound.",
"It is therefore preferable to use light-signal-generating means, such as, for example, a stroboscopic lamp or a stroboscopic LED.",
"Such lighting means are known in the prior art.",
"Another advantage of using light-generating means is that a diver in distress can be located easily even when diving at night or in murky water.",
"Of course, the advantages also apply to a victim who is unconscious, who can be found more easily in this way.",
"The activating means can be designed in many different ways according to the invention.",
"The activating means preferably comprise a relatively large push-button.",
"The push-button is so large that it can be pressed with certainty by an arbitrary uncontrolled striking movement.",
"The surface of the push-button is advantageously at least 2 cm2.More advantageously, the push-button also projects from the surface of the diving aid.",
"In the case of a large push-button it is advantageous if said button is provided with passages for water, so that the water pressure of the environment under the button can be evened out.",
"This ensures that during diving the diving aid is not activated undesirably by the pressure of the water.",
"In particular, a striking colour, for example red or yellow, is selected for such a push-button.",
"The activating means advantageously comprise an acceleration detector.",
"An example of this is a so-called tilt detector.",
"Such detectors are switches that are known in the prior art and can be activated by a shock load, without the direction of the shock load being important.",
"The diving aid can comprise an ON/OFF button, for switching on the diving aid just before diving, and for switching it off after diving.",
"The diving aid preferably comprises water detection means for switching on the diving aid as soon as it comes into contact with water.",
"This has the advantage that the diving aid is therefore always switched on under water, even if the diver forgets to switch on the diving aid.",
"Such water detection means are known in the prior art.",
"An example of these means is the detection electrodes that are also used on diving computers.",
"In this context the term switching on means putting the diving aid into a state ready for use.",
"In other words, when the diving aid is not switched on operation of the activating means will not result in the generation of an alarm sound.",
"In particular, the diving aid is designed in such a way that it can be worn on the wrist.",
"In practice, it has been found that the wrist is a suitable place for attachment of the diving aid, since in practice said diving aid can be reached easily with the other hand, so that in a panic situation the diving aid can be activated by an uncoordinated movement.",
"It will be clear that the diving aid can likewise be attached to the diver's equipment, provided that this attachment involves an unmistakable orientation, so that the diver can always reach the diving aid.",
"As a particular example of this, the means for attaching the diving aid to the diver's body or the diver's equipment comprise a clip.",
"In a special embodiment the diving aid forms part of an ordinary diving computer.",
"An ordinary diving computer is a device from which, inter alia, the diving depth, the diving time that has elapsed and the like can be read.",
"The invention will be explained in greater detail below with reference to the appended drawing, in which: FIG.",
"1 shows a top view of an embodiment of a diving aid according to the invention; FIG.",
"2 shows a perspective view of the embodiment according to FIG.",
"1; FIG.",
"3 shows a top view of a different embodiment of a diving aid according to the invention; and FIG.",
"4 shows a perspective view of the embodiment according to FIG.",
"3.FIG.",
"1 shows an embodiment of a diving aid according to the invention, which is suitable for attachment around the wrist.",
"The diving aid comprises a housing 1 on which a large push-button 2 and alarm sound-generating means 3 are situated.",
"Reference numeral 5 indicates two water detection electrodes, which serve to activate the diving aid on contact with water.",
"A power supply is not shown in the figures, but it will be clear that in principle any power supply that is suitable for use under water can be used.",
"An example in this case is a power supply for a diving computer or the like.",
"Reference numeral 6 indicates a wristband for attachment of the diving aid to a diver's wrist.",
"Finally, reference numeral 7 indicates a reset button, by means of which the diving aid can be switched off after being activated, for example when the diver in distress or danger has been rescued.",
"For testing the functioning of the diving aid according to the invention above water, the diving aid can be switched on by touching the electrodes with moistened fingertips.",
"It will be clear that the diving aid is certain to be activated when in a panic situation a striking movement with the hand is made in an uncontrolled way in the direction of the diving aid according to the invention.",
"All this has been confirmed by experiments.",
"The alarm sound-generating means in this case are in the form of a piezoelectric sound-generating device.",
"FIG.",
"2 shows the diving aid according to FIG.",
"1 in perspective view.",
"It can be seen clearly in this figure that the large push-button 2 projects slightly from the surface of the device, so that said push-button is easy to press.",
"FIGS.",
"3 and 4 show a different preferred embodiment of a diving aid according to the invention, in which similar parts are indicated by the same reference numerals.",
"Apart from the alarm sound-generating means, this diving aid also comprises a stroboscopic xenon gas discharge lamp 8, for generating a light alarm.",
"When a diver in distress/danger uses this diving aid, he will be able to indicate his position not only by means of an audible sound signal, but also by means of light signals.",
"This means that the diver can be found even more quickly by co-divers."
]
] | Patent_10451113 |
[
[
"Composition comprising an oxidizing and complexing compound",
"The present invention is related to a composition comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"The oxidizing compound can be in the form of an aqueous solution.",
"The complexing compound is for complexing metal ions.",
"Metal ions can be present in the solution or in an external medium being contacted with the solution.",
"The present invention can be used for cleaning a semiconductor substrate."
],
[
"1.A composition comprising an aqueous solution, said aqueous solution comprising an oxidizing compound and a complexing compound, said complexing compound comprising a chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.",
"2.The composition as recited in claim 1, further comprising an alkaline compound.",
"3.The composition as recited in claim 1, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.",
"4.The composition as recited in claim 1, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.",
"5.The composition as recited in claim 1, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4( 1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.",
"6.The composition as recited in claim 1, wherein said oxidizing compound comprises hydrogen peroxide.",
"7.The composition as recited in recited in claim 2, wherein said alkaline compound comprises an inorganic basic compound or an organic basic compound.",
"8.The composition as recited in claim 7, wherein said alkaline compound is selected from the group consisting of ammonia and organic amine compounds.",
"9.The composition as recited in claim 2, wherein an amount of oxidizing compound comprises from 0.001 to 30 weight %.",
"10.The composition as recited in claim 1, wherein an amount of the complexing agent comprises from 0.1 to 1000 ppm of said solution.",
"11.The composition as recited in claim 7, wherein an amount of the inorganic basic compound or the organic basic compound comprises from 0.001 to 30 weight %.",
"12.The composition as recited in claim 1, wherein an amount of said oxidizing compound comprises from 0.001 to 95 weight %.",
"13.A method of treating a semiconductor substrate, wherein said method comprises treating a semiconductor substrate with a composition comprising an aqueous solution, said aqueous solution comprising a complexing compound comprising the chemical formula wherein R1, R2, R3, and R4 are each independently selected from the group consisting of H and an organic side chain.",
"14.The method as recited in claim 13, wherein said composition further comprises an oxidizing compound.",
"15.The method as recited in claim 13, wherein said composition further comprises an alkaline compound.",
"16.The method as recited in claim 13, wherein said organic side chain is selected from the group consisting of an aliphatic side chain, a heterocyclic side chain, and an aromatic side chain.",
"17.The method as recited in claim 13, wherein R3 and R4 each comprise hydrogen and R1 and R2 each comprise a functionalized aliphatic side chain.",
"18.The method as recited in claim 13, wherein said complexing compound is selected from the group consisting of 1,2-diethyl-3-hydroxy-4(1H)-pyridinone, 1-ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1,2-dimethyl-3-hydroxy-4(1H)pyridinone, 1-propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone, 1-propyl-2-methyl-3-hydroxy-4(1H)-pyridinone, 1-(2-carboxyethyl)-2-methyl-3-hydroxy-4(1H)-pyridinone, and 1-(2-carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.",
"19.The method as recited in claim 14, wherein said oxidizing compound comprises hydrogen peroxide.",
"20.The method as recited in claim 15, wherein the alkaline compound comprises an inorganic basic compound or an organic basic compound.",
"21.The method as recited in claim 20, wherein said inorganic basic compound or said organic basic compound is selected from the group consisting of ammonia and organic amine compounds.",
"22.The method as recited in claim 14, wherein an amount of the oxidizing compound comprises from 0.001 to 30 weight %.",
"23.The method as recited in claim 15, wherein an amount of the alkaline compound comprises from 0.001 to 30 weight %.",
"24.The method as recited in claim 13, wherein an amount of said complexing compound comprises from 0.1 ppm to 1000 ppm.",
"25.Use of a composition as recited in claim 1 in a field selected from the group consisting of soil remediation, metal etch bath, denture bleaching, bleaching of fats, bleaching of oils, bleaching of waxes, wastewater treatment, paper pulp bleaching, and textile bleaching.",
"26.The composition as recited in claim 8, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds.",
"27.The method as recited in claim 21, wherein said organic amine is selected from the group consisting of tetraalkylammoniumhydroxide compounds, alkanolamine compounds, choline(hydroxyltrialkylammoniumhydroxide) compounds, and guanidine compounds."
],
[
"<SOH> FIELD OF THE INVENTION <EOH>This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"The oxidizing compound can be in the form of an aqueous solution.",
"The complexing compound is for complexing metal ions.",
"Metal ions can be present in the solution or in an external medium being contacted with the solution.",
"Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.",
"In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.",
"In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.",
"In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.",
"Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.",
"In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.",
"Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.",
"In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.",
"In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.",
"Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.",
"In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.",
"Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"Said composition can further comprise an oxidizing compound.",
"Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.",
"The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.",
"Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.",
"Said solution is particularly suitable for cleaning a semiconductor substrate.",
"Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.",
"In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.",
"In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.",
"In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.",
"In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.",
"In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.",
"Said alkaline compound can comprise an inorganic or organic basic compound.",
"Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.",
"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.",
"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.",
"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF THE INVENTION This invention is situated in the field of products and methods for the stabilizing a composition comprising an oxidizing compound.",
"STATE OF THE ART Chemical solutions comprising an oxidizing compound such as hydrogen peroxide are used in a wide area of applications, for example, as bleaching agent for paper pulp applications, dental bleaching composition or as cleaning agents.",
"Chemical solutions comprising an oxidizing compound have often problems related to the stability of the solution.",
"In pure form, aqueous solution are stable over extended periods of time.",
"However, the presence of certain metal ions in the solution causes decomposition of the oxidizing compound.",
"Consequently, stabilizers to prevent such decomposition should be added.",
"Stabilizers can be e.g.",
"a complexing compound, such that the complexing compound will bind to the metal and consequently, the metal is not available for reaction with the oxidizing compound.",
"Thus, the decomposition of the oxidizing compound is substantially inhibited and the lifetime of the solution is increased.",
"Very stringent specifications must be met by oxidizing solutions for specialized applications such as semiconductor applications or reagent chemicals.",
"An overview of stabilizing oxidizing compound, and more specifically hydrogen peroxide solutions, is given in Kirk-Othmer Encyclopedia of Chemical Technology (4th edition), vol.",
"13 p. 965.U.S.",
"Pat.",
"No.",
"4,239,643 discloses an aqueous peroxide-containing solutions used in bleaching of cellulose fiber, wherein the stability of the peroxide is very greatly increased by inclusion of alkali metal polyphosphate and alkali metal diethylene triamine penta(methylene phosphonate).",
"U.S. Pat.",
"No.",
"3,903,244 describes a hydrogen peroxide concentrate containing up to 50% hydrogen peroxide, 1 to 3% of a soluble amino(methyl phosphonic acid) or a salt thereof, and 0.05 to 0.5% of phenol.",
"The concentrate is useful in the preparation of highly acid metal pickling baths.",
"Stabilizing compounds are useful for a wide area of application, such as soil remediation, metal etch bath, denture bleaching, bleaching of fats, oils, waxes,, wastewater treatment, paper pulp bleaching, textile bleaching, CMP applications or semiconductor applications.",
"In cleaning application, for example in semiconductor application, oxidizing solutions have been investigated extensively.",
"Since the invention of the RCA cleaning by Kern et al.",
"in 1965 (W. Kern and D. A. Pautinen, RCA Review 31, 187, 1970) this cleaning cycle has become the most used for semiconductors.",
"The conventional RCA cleaning consists of two steps: an alkaline solution, the so called SC1 solution and an acidic solution, SC2.The SC1 solution is composed of 1 part ammonia (NH4OH), 1 part hydrogen peroxide (H2O2) and 5 parts ultra pure water (H2O) and is often referred to as APM-cleaning (i.e.",
"Ammonia Peroxide Mixture).",
"Originally it was used to remove organic residues by oxidation.",
"Later it has been proven to be very efficient to remove particles.",
"A drawback of the SC1 solution is that metals precipitate on silicon surfaces; especially aluminum, iron and zinc have been shown to adsorb strongly on the wafer surface (Mertens et al., Proc.",
"of the 8th Internat.",
"Symp.",
"On Silicon Materials Science and Technology PV98-1 (1998)).",
"In addition, especially Fe and Cu are found to catalyze the decomposition reaction of the peroxide (Mertens et al., Proc.",
"of the 5th Internat.",
"Symp.",
"on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997)) leading to a decrease in the bath lifetime.",
"In order to remove the metallic surface contamination, the SC2 solution consisting of 1 part hydrochloric acid, 1 part hydrogen peroxide and 6 parts ultra-pure water is used.",
"However, it is expensive to get hydrochloric acid of sufficient quality for the usage in SC2 solution.",
"There is also a risk of re-contaminating the surface with particles.",
"Problems also occur in spray tools due the corrosive behavior of hydrochloric acid.",
"With the progress in semiconductor manufacturing the requirements concerning particle and metal contamination as well as roughness of the silicon surfaces became more stringent.",
"This led to a number of variations of the RCA clean.",
"The potential problems related to the SC2 and the consideration to reduce process time and equipment by leaving out this acidic step led to the development of single-stage cleaning procedures.",
"This can be done by using chemicals with reduced amount of metallic impurities.",
"For that purpose, advanced purification procedures are established for obtaining ultra-pure water, ammonia and hydrogen peroxide.",
"However, these chemicals are very expensive and the purity is not always assured when they are used in a cleaning bath.",
"Moreover, the cleaning solution is not very robust with respect to metal contamination from the semiconductor substrate and from the hardware.",
"Besides this, having an extra step in the cleaning cycle to remove residual metallic contamination implies extra hardware, e.g.",
"a SC2-tank and a rinse tank need to be used, and more chemicals.",
"Leaving out this extra step will results in a reduction of the hardware cost and a reduction of the amount of chemicals used in the cleaning cycle.",
"EP 528053 describes a method for treating a surface of a substrate with a surface treatment composition.",
"The surface treatment composition comprises a liquid medium containing a complexing agent as a metal deposition preventive.",
"The surface treatment composition is improved by incorporating at least two complexing agents.",
"A first complexing agent is preferably an aromatic hydrocarbon ring with at least an OH or O− group bonded to a carbon atom constituting the ring.",
"A second complexing agent is compound having a donor atom, such as heterocyclic amines.",
"Since this cleaning composition comprises two complexing agents, drawbacks such as higher cost and increased waste treatment are observed.",
"Moreover, aromatic hydrocarbons such as Tiron, Catechol derivatives are hazardous for the environment and for humans.",
"U.S. Pat.",
"No.",
"5,290,361 and U.S. Pat.",
"No.",
"5,302,311 describes an aqueous hydrogen peroxide solution further comprising a complexing compound containing phosphonic acid groups and showing complexing ability.",
"Cleaning solution comprising phosphonic acid goups are not effective in removing or suppressing Al from the substrate.",
"Moreover, enhanced deposition of Cu has been measured.",
"This makes the cleaning solutions less suitable.",
"In the present invention, the problems related to removal of metals as mentioned in the prior art, are avoided.",
"AIMS OF THE INVENTION It is an aim of the invention to provide a stable composition comprising an oxidizing compound and a complexing compound.",
"It is a further aim of the invention to provide a new composition for treating a surface which is stable and provokes less or no metal precipitation on the surface.",
"Another aim of the invention is to provide a new cleaning solution.",
"Another aim of the present invention is to provide an efficient APM-cleaning solution having a good robustness with respect to metal contamination.",
"A further aim is to provide a new single-step method for cleaning semiconductor surfaces.",
"FIGURE CAPTIONS FIG.",
"1 chemical structure of complexing molecules FIG.",
"2 bath age (minutes) as function of the normalized H2O2 concentration, 1=no complexing agent; 30 2=DEHP; 3=10×DEHP; 4=1-EMHP; 5=10×1-EMHP; 6-DMHP and 7=10×DMHP.",
"SUMMARY OF THE INVENTION In a first aspect of this invention, a composition is disclosed comprising an oxidizing compound and a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"The oxidizing compound can be in the form of an aqueous solution.",
"The complexing compound is for complexing metal ions.",
"Metal ions can be present in the solution or in an external medium being contacted with the solution.",
"Depending on the metal ion being complexed, one or more complexing molecules/metal ion are required.",
"In an embodiment of this first aspect, said composition as recited in the first aspect of this invention can further comprise an alkaline compound.",
"In a further embodiment of this first aspect, said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.",
"In a further embodiment of the first aspect of this invention, R3 and R4 can be hydrogen while R1 and R2 can be a functionalized aliphatic side chain.",
"Preferably, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.",
"In a further embodiment of this first aspect, a solution as recited in the first aspect of this invention is disclosed characterized in that said oxidizing compound is hydrogen peroxide.",
"Hydrogen peroxide will be stabilized by the addition of the complexing compound, such that decomposition is substantially inhibited.",
"In a further embodiment of this first aspect of the invention, a composition as disclosed wherein said oxidizing compound is hydrogen peroxide.",
"In a further embodiment of this first aspect, a composition as recited in the first embodiment of the first aspect of the invention is disclosed wherein alkaline compound comprises an inorganic or organic basic compound.",
"Said alkaline compound can be ammonia or an organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.",
"In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.",
"Said semicondcutor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"Said composition can further comprise an oxidizing compound.",
"Said composition can be used for treating a substrate, such that particles are oxidized and metallic contamination is removed.",
"The complexing molecule is for complexing metallic residues being present on the substrate and in the solution.",
"Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.",
"Said solution is particularly suitable for cleaning a semiconductor substrate.",
"Said composition for cleaning a semiconductor substrate can be any composition described in the first aspect of this invention.",
"In an embodiment of this second aspect of this invention, said R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"Said organic side chain can comprise an aliphatic side chain, a heterocyclic side chain or an aromatic side chain.",
"In a further embodiment of this second aspect of the invention, R3 and R4 can be hydrogen while R1 and R1 can be a functionalized aliphatic side chain.",
"In a further embodiment, said complexing compound is one of the group consisting of DEHP, 1-EMHP, 2-EMHP, DMHP, PEPH, PMHP, ECMHP and ECEHP.",
"In another embodiment of the second aspect of the present invention, said oxidizing compound can be hydrogen peroxide.",
"In an embodiment of the second aspect of this invention, said composition can further comprise an alkaline compound.",
"Said alkaline compound can comprise an inorganic or organic basic compound.",
"Said basic compound can be chosen from the group consisting of ammonia and organic amine, preferably an organic amine chosen from the group consisting of tetraalkylammoniumhydroxide, alkanolamine, choline(hydroxyltrialkylammoniumhydroxide) and guanidine compounds.",
"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the concentration of hydrogen peroxide in the solution lies between 0.001 to 30 weight %.",
"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed in which the amount of the complexing agent lies between 0.1 and 1000 ppm of said solution.",
"In an embodiment of the second aspect of this invention, the method as recited in second aspect of the invention is disclosed wherein the amount of the alkaline compound lies between 0.001 and 30 weight %.",
"DETAILED DESCRIPTION OF THE INVENTION In relation to the appended drawings the present invention is described in detail in the sequel.",
"It is apparent however that a person skilled in the art can imagine several other equivalent embodiments or other ways of executing the present invention.",
"In an aspect of this invention, said composition comprises an oxidizing compound and a complexing compound.",
"Said oxidizing compound can be in the form of an aqueous solution.",
"Said complexing agent can have a chemical formula as given in FIG.",
"1.R1, R2, R3 and R4 are independently selected from the group comprising hydrogen (H) or any organic group.",
"R1, R2, R3,or R4 can have a different chemical structure.",
"For the purpose of this invention, said complexing agent as mentioned above is generally referred to as pyridinone.",
"Said organic group can be every possible sequence of C, N, O or S atoms linked to each other by single, double or triple bonds such that the complexing properties of the final complexing agent are assured.",
"Said organic group can be selected from the group comprising aliphatic side chains, heterocycles and aromatic structures.",
"Said organic side chain is every possible sequence of carbon, atoms linked to each other by a single, double or triple bound and optionally characterised by the presence of functional groups linked to the carbon atoms.",
"Functional groups can be alcohol, carboxyl, carbonyl, aldehyde, keton, ether, ester, amine, amide, halogen containing groups.",
"Said heterocycle can be one of the group comprising a crown ether, a cryptant, a calixarene, .",
".",
".",
"In a preferred embodiment, R3 and R4 are H atoms, while R1 and R2 are a methyl, ethyl, (iso)propyl or butyl group.",
"Said methyl, ethyl, (iso) propyl or butyl group can functionalized, e.g.",
"with a carboxyl group (COOH or COO−).",
"Preferably, the complexing molecules as described in FIG.",
"2 are used.",
"DEHP=1,2-Diethyl-3-hydroxy-4(1H)-pyridinone 1-EMHP=1-Ethyl-2-methyl-3-hydroxy-4(1H)-pyridinone 2-EMHP=1-Methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone DMHP=1,2-Dimethyl-3-hydroxy-4(1H)-pyridinone PEPH=1-Propyl-2-ethyl-3-hydroxy-4(1H)-pyridinone PMHP=1-Propyl-2-methyl-3-hydroxy-4(1H)-pyridinone ECMHP=1- (2′Carboxyethyl)-methyl-3-hydroxy-4(1H)-pyridinone ECEHP=1-(2′Carboxyethyl)-2-ethyl-3-hydroxy-4(1H)-pyridinone.",
"Although the addition of the amount of complexing agent in the composition in this invention is not particularly limited, it depends on the degree of metal contamination -and on the kind of other compounds being present in the solution.",
"Furtheron, the amount of complexing agent depends on the specific chemical structure of the complexing compound.",
"The amount of the complexing agent lies 0.01 and 10000 ppm, between 0.1 and 1000 ppm of said composition.",
"For this application, ppm should be understood as parts per million in the composition.",
"The amount of complexing compound depends on the specific complexing compound.",
"Since said complexing agents do not form a 1:1 metal/complexing agent complex, higher concentrations of complexing agents are required, compared to other complexing agents such as EDTA.",
"Typical values for the stoichiometry CA/metal for Fe, Al, Cu and Zn are given in the table: Complex Stability Constants: Fe1 Al2 Cu Zn Fe2 Al2 Cu Zn2 DMHP DMHP DMHP DMHP DEHP DEHP DEHP DEHP [ML]/[M] [L] 15.10 12.20 10.62 7.19 15.2 13.42 10.74 7.70 [ML2]/[ML] 11.51 11.05 8.99 6.34 11.76 11.64 9.07 6.09 [L] [ML3]/[ML2] 9.27 9.37 9.78 8.48 5.12 [L] [MLx]/[M] 35.88 32.62 19.61 13.53 36.8 33.54 19.81 18.91 [L]x 2 DMHP 1-EMHP DEHP [MLx]/[M] 37.2/36.4 37.7 36.8 [L]x PFe 19.4 19.7 1X = 3 2Values have been taken from different authors and determined by different techniques.",
"Said oxidizing compound can be every chemical compound having oxidizing properties.",
"E.g.",
"organic species, metallic compounds, inorganic particles, silicon, etc.",
"can be oxidized.",
"The oxidizing compound is a compound selected from the group comprising hydrogen peroxide or oxidizing anions.",
"The oxidizing anions can be e.g.",
"nitric acid and its salts, nitrate, persulfate, periodate, perbromate, perchlorate, iodate, bromate and chlorate salts of ammonium.",
"Preferably, the oxidizing compound is hydrogen peroxide.",
"The concentration of the oxidizing compound can be, but is not limited hereto, between 0.0001 and 99 weight %, between 0.001 and 90 weight % and preferebly between 0.001 to 30 weight %.",
"In this application, weight % should be understood as the percentage of weight of the specified compound in the composition.",
"Said composition can further comprise an alkaline compound.",
"The alkaline compound or base can be every chemical compound with a pH higher than 7.The alkaline compound can be an organic or inorganic compound.",
"The alkaline compound can be an organic base, ammonia, ammoniumhydroxide, or an alkaline solution containing metal ions such as potassium or sodium.",
"Said organic base can be a quaternary ammonium hydroxide such as tetraalkyl ammonium hydroxide in which the alkyl groups can contain hydroxy- and alkoxy-containing groups with 1 to 4 carbon atoms in the alkyl or alkoxy group.",
"Said organic base can further be an organic amine such as an alkanol amine.",
"Alkanol amines can be 2-aminoethanol, 1-amino 2-propanol, 1-amino 3-propanol.",
"Preferably, the alkaline compounds are tetramethyl ammonium hydroxide, and trimethyl 2-hydroxy ethyl ammonium hydroxide (choline) and ammonium hydroxide.",
"The amount of the alkaline compound lies between 0.0001 and 90 weight %, between 0.001 and 50 weight %, between 0.001 and 30 weight %.",
"Said composiiton can further comprise a surfactant.",
"In a second aspect of this invention, a method for treating a semiconductor substrate is disclosed.",
"Said semiconductor substrate is treated with a composition comprising an aqueous solution of a complexing compound with the chemical formula wherein R1, R2, R3 and R4 are selected from the group consisting of H and any organic side chain.",
"Said composition can further comprise an oxidizing compound.",
"Said composition can be, but is not limited hereto, the composition described in the first aspect of this invention.",
"Said composition is particularly usefull for cleaning a substrate such that particles are oxidized and metallic contamination is removed.",
"The complexing compound is for complexing metals being present on the substrate and in the solution.",
"Additionally, the lifetime of the solution is increased since de decomposition of the oxidizing compound is substantially inhibited.",
"A substrate can be, but is not limited hereto, a substrate such as semiconducting material, glass, quartz, ceramics, metal, plastic, magnetic material, superconductor and the like.",
"Preferably, said substrate is a semiconductor substrate.",
"Semiconductor substrate can be every possible substrate used in semiconductor processing.",
"Said semiconductor substrate can be a substrate selected from the group, but not limited hereto, comprising a substrate made of silicon, germanium, gallium arsenide, indium phosphide, etc.",
"The semiconductor substrate be e.g.",
"the substrates as mentioned above covered entirely op partially with a thin film of e.g.",
"an oxide, a nitride, a metal, a polymeric insulating layer, an anti-reflecting coating, a barrier, a photoresist layer, etc.",
"The present invention is particularly relevant for cleaning or etching a semiconductor substrate of which the surface should be highly clean.",
"When the composition is used for treating a substrate, the weight concentration range of the alkaline compound in the-cleaning solution are typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.",
"For ammonium hydroxide, the weight concentration range of the alkaline compound in the cleaning solution are typically but not limited to 0.001-30%, 0.1-20% and preferably 0.1-5% by weight.",
"For other alkaline compounds, the weight concentration range is equivalent, and function of the strength of the alkaline compound.",
"For peroxide, the weight concentration the hydrogenperoxide is typically but not limited to 0.001-100%, 0.1-20% and preferably 0.1-5% by weight.",
"In the preferred embodiment of this invention, a composition for treating a semiconductor surface comprises ammonium hydroxide, hydrogen peroxide, water (hereafter called APM mictures) and additionally a complexing agent.",
"Said complexing agent is one of the group consisting of the molecules as described in FIG.",
"2.APM-cleaning mixtures comprising a complexing agent according to the present invention are robust with respect to metal contamination coming from the fresh chemicals as well as with respect to metal contamination introduced in the course of its use for cleaning.",
"The robustness of the basic APM process can be improved by the addition of complexing agents which keep the metals in solution and prevent the above mentioned catalysis of the peroxide decomposition.",
"The volume mixing ratio of NH4OH(296)/H2O2(30%)/H2O is typically, but not limited hereto, 0.25/1/5.The cleaning solution is prepared with the amounts as described above and afterwards the semiconductor substrate is treated with the cleaning solution.",
"In the best mode known to the applicant, molecule DMHP is selected and added in the amounts described above.",
"The complexing agent can be added as the pure compound to the cleaning solution.",
"Alternatively, the complexing agent can be dissolved in either water, ammonia or peroxide or a dilution of the two latter chemicals and added as such to the cleaning solution.",
"It is a further aim of the invention to disclose a process for treating a semiconductor substrate comprising the steps of treating said semiconductor substrate with the cleaning solution as described above and drying said semiconductor substrate.",
"An optional step: rinsing said semiconductor substrate can be performed after said treating said semiconductor substrate with the cleaning solution as described above.",
"In the step of treating said semiconductor substrate with said cleaning solution, the semiconductor substrate can be immersed in a bath containing the cleaning solution.",
"Alternatively, the cleaning solution can be dispensed or sprayed onto the semiconductor substrate for instance by using a spray processor.",
"In all cases, the cleaning performance of the solution can be enhanced by using a megasonic transducer.",
"The temperature range for treating the semiconductor substrate with the cleaning solution is typically but not limited to 0-95 degrees Celcius, 10-80 degrees Celcius and preferably between 20-70 degrees Celcius.",
"In the step of drying the semiconductor substrate, several techniques known in the art can be used, e.g.",
"spin-drying, Maragoni-drying, drying techniques using organic vapours.",
"The step of rinsing the semiconductor substrate comprises treating the semiconductor substrate with DI water or treating the semiconductor substrate with a diluted acidic solution or with DI water containing the complexing agent in an amount of 1 to 100000 ppm, 10 to 10000 ppm and by preference 100 to 1000 ppm.",
"It is a further aim of the invention to describe a process for treating a semiconductor substrate comprising the step of: treating said semiconductor substrate with any cleaning solution and/or treating said semiconductor substrate with any rinsing solution Said any cleaning solution can be any cleaning solution, not being limited to the compositions described in this application.",
"Said rinsing solution comprises said complexing agent and water.",
"Said complexing agent can be any complexing agent described in this application.",
"The amount of the complexing agent in the composition can be between 1 and 100000 ppm, 10 and 10000 ppm and by preference between 100 and 1000 ppm.",
"This rinsing solution can also comprise a surfactant in an amount of 0.1 ppm to 10 w %.",
"A surfactant is a surface-active agent comprising a lyophobic group and a lyophilic group.",
"The lyophobic group can be a straight-chain alkyl group or a branched-chain alkyl group (C8-C20), an long-chain (C8-C20) alkyl benzene residue, an alkylnaphtalene residue (C3 and greater-length alkyl groups), high-molecular-weight propylene oxide polymers (polyoxypropylene glycol derivatives), long-chain perfluoroalkyl or polysiloxane groups.",
"Depending upon the lyophilic group, the surfactant can be an anionic, cationic, nonionic or zwitterionic surfactant.",
"Anionic surfactants can be carboxylic acids or carboxylic acid salts (such as sodium and potassium salts of straight-chain fatty acids), sulfonic acids or sulfonic acid salts (such as linear alkylbenzenesulfonates, higher alkylbenzenesulfonates, benzene-, toluene-, xylene- and cumenesulfonates, ligninsulfonates, petroleum sulfonates, N-acyl-n-alkyltaureates, paraffin sulfonates, secondary n-alkanesulfonates, α-olefin sulfonates, sulfosuccinate esters, alkylnaphtalenesulfonates or isethionates), sulfuric acid ester salts (such as sulfated linear primary alcohols, sulfated polyoxyethylenated straight-chain alcohols or sulfated triglyceride oils), phosphoric and polyphosphoric acid esters.",
"Cationic surfactants can be primary amines and their salts, diamines and polyamines and their salts, quaternary ammonium salts (such as tetralkylammonium salts or imidazolinium salts), polyoxyethylenated long-chain amines (RN(CH2CH2O)xH]2), quaternized polyoxyethylenated long-chain amines or amine oxides (such as N-alkyldimethylamine oxides).",
"Nonionic surfactants can be polyoxyethylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long-chain carboxylic acid esters (such as glyceryl and polyglyceryl esters of natural fatty acids, propylene glycol, sorbitol or polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters and polyoxyethylenated fatty acids), alkanolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones or alkylpolyglycosides.",
"Zwitterionic surfactants have both anionic and cationic charges present in the lyophilic portion (such as β-N-alkylaminopropionic acids, N-alkyl-β-iminodipropionic acids, imidazoline carboxylates, N-alkylbetaines, amine oxides, sulfobetaines or sultaines) (M. J. Rosen, Surfactants and Interfacial phenomena, 2nd Edition, John Wiley and Sons, New York, 1989]) No additional alkaline compound should need to be added to the said rinsing solution.",
"The pH range of said rinsing solution can typically be, but not limited to, between 5 and 8.Said Rinse solution can be dispensed or sprayed onto the semiconductor surface as described above.",
"During rinsing the performance can also be enhanced by using a megasonic transducer.",
"The process of treating a semiconductor substrate with a cleaning solution comprising the above mentioned steps can be performed for a certain number of semiconductor substrates.",
"After treating at least one substrate, but preferably after treating more substrates, the composition of the cleaning solution can be modified by e.g.",
"adding extra alkaline compound, adding extra complexing compound, adding oxidizing compound such that the initial composition of the cleaning solution is kept constant as function of the process time.",
"COMPARATIVE EXAMPLES The present invention will be further described using non-limiting examples and drawings.",
"The effectiveness of the new class of complexing agents on the inhibition of the metal catalyzed decomposition of peroxide and the prevention of metal outplating on silicon wafers in metal contaminated APM cleaning solutions is described next.",
"For complexing agents as disclosed in this invention, different model compounds are selected (1-EMHP, 2-EMHP, DMHP, DEHP, PEHP, PMHP, ECMHP and ECEHP) and added to the bath at a concentration of 2.67×10−5 M and at a 10×, 40×, or 50× higher concentration (namely 2.65×10−4 10.6×10−4 and 13.25×10−4 M).",
"A comparison is made with other types of complexing agents containing as functional groups either phosphonic acids, such as diethylene (kleine letter) triamine penta-methylenephosphonic acid (DTPMP), carboxylic acids, such as ethylene diamino tetra acetic acid (EDTA).",
"An overview of the different chemicals used for the experiments is given in Table 1.All experiments were done in a class 1000 clean room environment or better.",
"TABLE 1 Chemicals used for preparation of APM baths.",
"Chemical Vendor Grade H2O2 30 (w/w) % Ashland TB(*) NH4OH 29 (w/w) % Ashland TB(*) EDTA Fluka DTPMP Monsanto Dequest 2060S DMHP Aldrich (*)TB-grade corresponds with a specification of maximal 100 ppt of metal ions in the chemical.",
"Example 1 Metal Outplating from APM Mixtures in Presence of Different Complexing Agents The efficiency of complexing agents to suppress the deposition of metallic contamination onto wafer surfaces was studied.",
"This was done through intentionally spiking well controlled trace amounts of metallic contamination to cleaning solutions.",
"For these metal deposition tests, p-type monitor wafers with a diameter of 150 mm and <100> orientation were used.",
"The wafers were pre-cleaned using IMEC Clean® in an automated Steag wet bench (i.e.",
"SOM+dHF+O3-rinse rendering a perfectly clean hydrophilic surface).",
"The metal deposition experiments were performed in a static quartz tank with a quartz cover plate.",
"This tank was not equipped with a megasonic transducer.",
"APM mixtures were prepared containing 1 w-ppb of different metals of interest with/without the complexing agent added.",
"The metals spiked to the APM bath were added from AAS-standard solutions (Merck).",
"After a bath age of 5 minutes, three wafers were immersed for 10 minutes, rinsed for 10 minutes in an overflow rinse tank and dried with a commercially available Marangoni drier (STEAG).",
"The resulting metal contamination was measured with straight TXRF or VPD-DSE-DC-TXRF (Vapor Phase Decomposition-Droplet Surface Etching-Droplet Collection Total X-Ray Fluorescence).",
"Determination of Al water surface concentration was done using VPD-DC GF-AAS (Graphite Furnace Atomic Absorption Spectroscopy).",
"An overview of the resulting metal surface contamination after dipping a clean wafer in an APM spiked with metals and different complexing agents is given in Table 2.TABLE 2 Measured metal surface concentrations after immersion in metal contaminated 0.25/1/5 APM solution at 20-50° C. with different complexing agents present.",
"Complexing Conc.",
"CA Conc.",
"Mt Al (1010 Fe agent (2.7 × 10−5 M) (w-ppb) at/cm2) (1010 at/cm2) Pre-clean only NA NA 2.34 <DL None 0 0 39.3 ± 6 1.5 ± 0.4 None 0 1 360 ± 21 109 ± 14 EDTA 1 1 NM 25.1 ± 0.1 10 1 272 ± 16 NM 100 1 274 2.78 EDTA + 1 + 2 1 257 ± 2 <DL DTPMP 1-EMHP (1) 1 1 356 ± 32 0.51 ± 0.02 10 1 142 ± 68 0.23 1-EMHP (2) 1 1 292 ± 1 0.20 ± 0.00 10 1 27 ± 33 NA 50 1 0.5 ± 0.2 0.26 2.4 ± 1.4 0.16 2-EMHP 1 1 404 0.35 ± 0.01 10 1 248 ± 69 0.22 50 1 0.8 ± 0.2 0.15 DEHP 1 1 369 ± 0 0.33 ± 0.01 10 1 150 ± 41 0.92 50 1 2.6 ± 0.6 0.15 DMHP 1 1 392 ± 0 0.45 ± 0.01 (Aldrich) 10 1 101 ± 2 0.21 50 1 1.2 ± 0.1 NM DMHP 1 1 387 ± 0 0.40 ± 0.02 10 1 230 ± 5 0.18 PEHP 40 1 4.6 ± 0.2 0.14 ± 0.0 PMHP 40 1 1.13 0.14 ± 0.14 ECMHP 40 1 15.0 ± 0.5 0.08 ± 0.04 ECEHP 40 1 14.2 ± 1.7 0.15 ± 0.05 These data show that the addition of EDTA results in a decrease of the final Fe contamination levels but has no effect on the reduction of Al contamination.",
"The use of DTPMP in a 1 w-ppb metal spiked bath, is very efficient to reduce the final Fe surface concentration but has no effect on suppression of Al deposition.",
"The new class of pyridinone complexing agents are able to reduce the final Fe and Al surface concentration on the wafer.",
"Higher concentrations of complexing agent are needed because the pyridones do not form a 1:1 metal/chelate complex.",
"When using the pyridinones at a concentration of 13.25×10−4 M in the bath, the resulting Fe and Al surface contamination levels are below the values measured on a wafer treated with a clean, i.e.",
"no metals spiked, APM solution.",
"Example 2 Decomposition of Peroxide in APM Cleaning Mixtures in Presence of Trace Metal Contamination and Metal Complexing Agents The effect of the addition of a complexing agent to APM cleaning solutions on the kinetics of the decomposition reaction of H2O2 has been investigated.",
"Well controlled amounts of metallic contamination were added to the cleaning mixture under study.",
"As hydrogen peroxide decomposes, an amount of oxygen gas is liberated following the overall reaction 2 H2O2⇄O2+2 H2O The decay of the total peroxide concentration in the APM mixture can be monitored by measuring the time-dependent increase of the pressure due to the O2-evolution in a dedicated set-up as described by Schmidt.",
"Numerical integration over time yields the actual peroxide concentration in the bath.",
"It is convenient to use peroxide concentrations normalized to its initial value [H2O2]i as [ H 2 O ] n = [ H 2 O 2 ] [ H 2 O 2 ] i Since the decomposition reaction is mainly catalyzed by Fe and in a lesser content Cu (Mertens et al.",
"Proc.",
"of the 5th Internat.",
"Symp.",
"on Cleaning Technology in Semiconductor Device Manufacturing PV97-35 (1997), the decay of peroxide concentration in a metal contaminated bath and in presence of a CA, illustrates the ability of complexing primarily Fe in the APM bath.",
"The decomposition rate as function of bath age is determined in APM mixtures (0.25/1/5 29%NH4OH/30% H2O2/H2O) spiked with 1 w-ppb of the metals of interest with and without different complexing agents.",
"The effect of different additives on the inhibition of the metal catalyzed decomposition reaction of peroxide in APM cleaning mixtures is shown in FIG.",
"2.This graph shows the normalized H2O2 concentration as function of bath age for an APM mixture at 50° C. spiked with different complexing agents and the metals of interest.",
"All complexing agents are found to suppress to some extent the decomposition reaction, at least when the mixture is fresh.",
"For some CA, the suppression action vanishes over time.",
"This may be attributed to the destruction of the complexing agent or more specifically of the metal-complex in the hot APM.",
"The time over which they remain active in complexing metals is summarized in Table 3 and can be used to establish the effective lifetime.",
"In example 1 it was demonstrated that higher CA concentrations have to be used in the cleaning solution.",
"FIG.",
"1 and Table 3 show that with increasing complexing agent concentration, the effective lifetime of the bath can be increased substantially.",
"Increasing the complexing agent concentration even further would result in a more prolonged lifetime.",
"TABLE 3 Effective lifetime of complexing agents in 0.25/1/5 metal spiked APM-cleaning mixtures at 50° C. Conc.",
"CA Effective lifetime Complexing agent (2.7 × 10−5 M) (min) None 0 40 DEHP 1 40 10 120 1-EMHP (1) 1 40 10 50 1-EMHP (2) 1 60 10 90 2-EMHP 1 30 10 40 DMHP FfU 1 40 10 100 DMHP Aldrich 1 20 10 60"
]
] | Patent_10451230 |
[
[
"Level sensor",
"A level sensor (2) comprising a support (6) with a bearing (11) for a lever arm (3) with a float (4) and a guide rail (16) pointing in the direction of the bearing (11).",
"The lever arm (3) has a stirrup (5) made of plastic and a lever wire (7) clipped thereto.",
"The stirrup has two guide elements (12,13) which are arranged next to each other and which engage with the guide rail (16), thereby preventing the dual contact (10) of the stirrup (5) from rising above the slideways (9) of a potentiometer (8) which are disposed on the support (6)."
],
[
"1.A filling-level sensor for generating electrical signals as a function of a pivoting angle of a float-carrying lever arm for a tank of a motor vehicle, comprising: a carrier for fastening to a lateral wall in the tank; a yoke made of plastic and pivotable in a mounting of the carrier; a lever wire holding a float and fastened to the yoke; and a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier, wherein the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at an end facing away from the mounting and which is in engagement with the guide rail.",
"2.The filling-level sensor as claimed in claim 1, wherein the guide rail has a web-shaped configuration and is arranged parallel to the carrier.",
"3.The filling-level sensor as claimed in claim 1, wherein the fork-shaped guide element is arranged at the end of the yoke which faces away from the mounting.",
"4.The filling-level sensor as claimed in claim 1, wherein the yoke is designed to engage around at least part of the carrier in the region of the mounting.",
"5.The filling-level sensor as claimed in claim 1, wherein two fork-shaped guide elements are arranged next to one another.",
"6.The filling-level sensor as claimed in claim 1, further comprising a spring elements prestressing a wiper contact against a wiping track of a potentiometer fastened between the guide elements arranged next to one another.",
"7.The filling-level sensor as claimed in claim 1, wherein the guide rail has a projecting edge.",
"8.The filling-level sensor as claimed in claim 1, wherein the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and in that surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.",
"9.The filling-level sensor as claimed in claim 1, wherein the guide rail is held at its ends by holding parts in front of the carrier."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Prior art filling-level sensors usually have a potentiometer with a wiping track arranged on the carrier and with wiper contacts, fastened to the yoke, for generating the electrical signals.",
"Alternatively, the electrical signals are often also generated by means of a magnetically active position sensor.",
"Magnetically active position sensors mostly have a resistance network which is to be fastened to the carrier and which is switchable by a magnet arranged on the yoke.",
"The tank may be, for example, a fuel tank or a washing-water tank of a windshield cleaning system.",
"The guide rail of the known filling-level sensor is designed as part of the carrier having a plane surface and is oriented away from the mounting.",
"A U-shaped guide element arranged on the yoke engages behind the guide rail.",
"This, in the case of lateral forces acting on the yoke and the lever wire, prevents the yoke from being bent away from the carrier and therefore the wiper contacts from being lifted off from the wiping track or the magnet from being removed from the resistance network.",
"One disadvantage of the known filling-level sensor is that, because of the guide element, the yoke has very large dimensions and is therefore heavy.",
"As a result, the center of gravity of the yoke is at a very great distance from the mounting, and therefore the filling-level sensor is very slow-acting.",
"Consequently, minor variations in the filling level are detected by the potentiometer only with a delay or abruptly.",
"Moreover, because of the high weight of the yoke, the latter requires a large quantity of material."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention is directed a filling-level sensor, with a carrier provided for fastening to a lateral wall in the tank, with a yoke made from plastic and pivotable in the mounting of the carrier, with a lever wire holding the float and fastened to the yoke, and with a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier.",
"The filling-level sensor according to the invention detects a variation in the filling level particularly quickly and is constructed cost-effectively.",
"According to an aspect of the invention, the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at its end facing away from the mounting and engages with a guide rail.",
"By virtue of this design, the yoke has very small dimensions and a particularly low weight.",
"Moreover, the center of gravity of the yoke is arranged very near the mounting, so that the yoke has particularly low inertia.",
"The yoke can therefore follow a variation in the filling level in the tank particularly quickly.",
"Due to the low weight of the yoke, the latter necessitates the use of a correspondingly small amount of material, so that it can be produced particularly cost-effectively.",
"The guide rail could, for example, have a round cross section.",
"However, according to an aspect of the invention, the carrier can be manufactured at a particularly low outlay in one piece with the guide rail from plastic by the injection molding method if the guide rail has a web-shaped configuration and is arranged parallel to the carrier.",
"By virtue of this configuration, the guide rail has a sliding track for the guide element on each of the two sides.",
"The fork-shaped guide element could, for example, be arranged in a middle region of the yoke.",
"However, a contribution to further reducing the dimensions of the yoke is made when the fork-shaped guide element is arranged at that end of the yoke which faces away from the mounting.",
"The yoke is particularly reliably mounted on the carrier axially and can nevertheless be mounted on the carrier very simply when it is designed to engage around at least part of the carrier in the region of the mounting.",
"For mounting, the yoke can simply be pushed over the carrier from the mounting.",
"The yoke is subsequently guided axially in the region of the mounting and on the guide rail.",
"To prevent the yoke from tilting, the guide element may be made particularly wide.",
"However, because of this, the guide element has a high weight and a large bearing surface on the guide rail.",
"The large bearing surface leads to undesirable frictional losses during the pivoting movement of the yoke.",
"According to another aspect of the invention, a tilting of the yoke can be prevented in a simple way by two fork-shaped guide elements arranged next to one another, without the weight of the yoke being appreciably increased thereby.",
"According to another aspect of the invention, the yoke is mounted in a particularly simple way when a spring element prestressing the wiper contact against the wiping track of the potentiometer is fastened between the guide elements arranged next to one another.",
"A deformation of the guide rail could lead to the wiper contact being lifted off from the wiping track or to a rubbing of the guide element.",
"According to another aspect of the invention, the guide rail has very high stability when it has a projecting edge.",
"The filling-level sensor according to the invention is manufactured particularly cost-effectively when the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and when surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.",
"By virtue of this configuration, the carrier can be manufactured, together with the guide rail, in a very simple way from plastic by the injection molding method in an injection mold from which removal can easily take place axially.",
"According to another aspect of the invention, the carrier requires the use of a particularly small amount of material when the guide rail is held at its ends by holding parts in front of the carrier."
],
[
"CLAIM FOR PRIORITY This application claims priority to International Application No.",
"PCT/DE01/04729 which was published in the German language on Jul.",
"4, 2002.TECHNICAL FIELD OF THE INVENTION The invention relates to a filling-level sensor for generating electrical signals as a function of a pivoting angle of a float-carrying lever arm for a tank of a motor vehicle.",
"BACKGROUND OF THE INVENTION Prior art filling-level sensors usually have a potentiometer with a wiping track arranged on the carrier and with wiper contacts, fastened to the yoke, for generating the electrical signals.",
"Alternatively, the electrical signals are often also generated by means of a magnetically active position sensor.",
"Magnetically active position sensors mostly have a resistance network which is to be fastened to the carrier and which is switchable by a magnet arranged on the yoke.",
"The tank may be, for example, a fuel tank or a washing-water tank of a windshield cleaning system.",
"The guide rail of the known filling-level sensor is designed as part of the carrier having a plane surface and is oriented away from the mounting.",
"A U-shaped guide element arranged on the yoke engages behind the guide rail.",
"This, in the case of lateral forces acting on the yoke and the lever wire, prevents the yoke from being bent away from the carrier and therefore the wiper contacts from being lifted off from the wiping track or the magnet from being removed from the resistance network.",
"One disadvantage of the known filling-level sensor is that, because of the guide element, the yoke has very large dimensions and is therefore heavy.",
"As a result, the center of gravity of the yoke is at a very great distance from the mounting, and therefore the filling-level sensor is very slow-acting.",
"Consequently, minor variations in the filling level are detected by the potentiometer only with a delay or abruptly.",
"Moreover, because of the high weight of the yoke, the latter requires a large quantity of material.",
"SUMMARY OF THE INVENTION The invention is directed a filling-level sensor, with a carrier provided for fastening to a lateral wall in the tank, with a yoke made from plastic and pivotable in the mounting of the carrier, with a lever wire holding the float and fastened to the yoke, and with a guide rail, arranged on the carrier, for guiding a guide element of the yoke in an intended axial position in relation to the carrier.",
"The filling-level sensor according to the invention detects a variation in the filling level particularly quickly and is constructed cost-effectively.",
"According to an aspect of the invention, the guide rail is arranged so as to point in the direction of the mounting, and the yoke has a fork-shaped guide element which is open at its end facing away from the mounting and engages with a guide rail.",
"By virtue of this design, the yoke has very small dimensions and a particularly low weight.",
"Moreover, the center of gravity of the yoke is arranged very near the mounting, so that the yoke has particularly low inertia.",
"The yoke can therefore follow a variation in the filling level in the tank particularly quickly.",
"Due to the low weight of the yoke, the latter necessitates the use of a correspondingly small amount of material, so that it can be produced particularly cost-effectively.",
"The guide rail could, for example, have a round cross section.",
"However, according to an aspect of the invention, the carrier can be manufactured at a particularly low outlay in one piece with the guide rail from plastic by the injection molding method if the guide rail has a web-shaped configuration and is arranged parallel to the carrier.",
"By virtue of this configuration, the guide rail has a sliding track for the guide element on each of the two sides.",
"The fork-shaped guide element could, for example, be arranged in a middle region of the yoke.",
"However, a contribution to further reducing the dimensions of the yoke is made when the fork-shaped guide element is arranged at that end of the yoke which faces away from the mounting.",
"The yoke is particularly reliably mounted on the carrier axially and can nevertheless be mounted on the carrier very simply when it is designed to engage around at least part of the carrier in the region of the mounting.",
"For mounting, the yoke can simply be pushed over the carrier from the mounting.",
"The yoke is subsequently guided axially in the region of the mounting and on the guide rail.",
"To prevent the yoke from tilting, the guide element may be made particularly wide.",
"However, because of this, the guide element has a high weight and a large bearing surface on the guide rail.",
"The large bearing surface leads to undesirable frictional losses during the pivoting movement of the yoke.",
"According to another aspect of the invention, a tilting of the yoke can be prevented in a simple way by two fork-shaped guide elements arranged next to one another, without the weight of the yoke being appreciably increased thereby.",
"According to another aspect of the invention, the yoke is mounted in a particularly simple way when a spring element prestressing the wiper contact against the wiping track of the potentiometer is fastened between the guide elements arranged next to one another.",
"A deformation of the guide rail could lead to the wiper contact being lifted off from the wiping track or to a rubbing of the guide element.",
"According to another aspect of the invention, the guide rail has very high stability when it has a projecting edge.",
"The filling-level sensor according to the invention is manufactured particularly cost-effectively when the guide rail is at a greater or equal distance from the carrier at its end facing away from the mounting than at its end facing the mounting, and when surfaces of the carrier and of the guide rail which face one another in each case have a plane configuration.",
"By virtue of this configuration, the carrier can be manufactured, together with the guide rail, in a very simple way from plastic by the injection molding method in an injection mold from which removal can easily take place axially.",
"According to another aspect of the invention, the carrier requires the use of a particularly small amount of material when the guide rail is held at its ends by holding parts in front of the carrier.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a perspective illustration of a filling-level sensor according to the invention, FIG.",
"2 shows a sectional illustration through the filling-level sensor from FIG.",
"1 along line II-II, FIG.",
"3 shows a further embodiment of the filling-level sensor according to the invention in a longitudinal section through a yoke.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG.",
"1 shows a filling-level sensor 2 according to the invention, mounted on a vertical wall 1 within a fuel tank.",
"The filling-level sensor 2 has a float 4 fastened to a lever arm 3.The lever arm 3 is mounted pivotably on a carrier 6 by a yoke 5 made from plastic and has a lever wire 7, fastened to the yoke 5, for holding the float 4.The float 4 follows a fuel level in the fuel tank and at the same time pivots the lever arm 3.The pivoting angle of the lever arm 3 is detected by a potentiometer 8.The potentiometer 8 has two wiping tracks 9 arranged on the carrier 6 and a wiper contact 10 fastened to the yoke 5.The wiper contact 10 is designed as a double contact for bridging the wiping tracks 9.To guide the yoke 5 in an intended axial position in relation to the carrier 6, the yoke 5 engages behind the carrier 6 in the region of a mounting 11.The yoke 5 has, at its end facing away from the mounting 11, two fork-shaped guide elements 12, 13 which are arranged next to one another and which engage with a guide rail 16 fastened laterally to the carrier 6 by holding parts 14, 15.By the yoke 5 being guided in the intended axial position in relation to the carrier 6, the wiper contact 10 is reliably prevented from being lifted off from the wiping tracks 9.FIG.",
"2 shows a filling-level sensor 2 according to the invention from FIG.",
"1 in a sectional illustration along the line can be seen here that the II-II.",
"FIG.",
"2 illustrates that the lever wire 7 forms, in the region of the mounting 11 of the yoke 5, an angled portion 17 functioning as a bearing axle for the lever arm 3.The carrier 6 has a bearing bore 18 comprising two parts 19, 20.A free end of an engaging-around portion 21 of the yoke 5 is arranged between the two parts 19, 20 of the bearing bore.",
"The wiper contact 10 is prestressed against the wiping tracks 9 by a tongue-shaped spring element 22.The tongue-shaped spring element 22 is fastened to the yoke 5 between to two fork-shaped guide elements 12, 13.To increase the stability of the guide rail 16, the latter has two vertically projecting edges 23, 24.FIG.",
"3 shows a further embodiment of the filling-level sensor according to the invention, with a lever arm 26 pivotable on a carrier 25 in a mounting 31.The lever arm 26 has a yoke 28 snapped together with a lever wire 27.The carrier 25, here, has a one-part bearing bore 29, behind which an angled portion 30 of the yoke 28 is engaged.",
"At the end facing away from the mounting 31, the yoke 28 has a fork-shaped guide element 33 which engages with a guide rail 32 of the carrier 25.The guide rail 32 and a region of the carrier 25 which faces the guide rail 32 has plane surfaces 34, 35 arranged parallel to one another."
]
] | Patent_10451269 |
[
[
"Inks for in-mould decoration",
"An energy-core ink or varnish composition is provided for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight, a solvent, wherein the resin comprises a urethane acrylate oligomer having an aromatic or aliphatic polycarbonate backbone.",
"For inks, the composition additionally includes a pigment or dye.",
"For photocure compositions, a photoinitiator is also included.",
"Also provided is an in-mould decoration (IMD) process employing the ink or varnish composition."
],
[
"1.An energy-cure ink or varnish composition for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and optionally and in an amount not exceeding 10% by weight of the composition, a solvent, wherein the energy-curable resin comprises a urethane acrylate oligomer having an aromatic or aliphatic polycarbonate backbone.",
"2.A composition according to claim 1, wherein the composition is an ink and further comprises a pigment or dye.",
"3.A composition according to claim 2, wherein the pigment or dye is present in an amount of from 1 to 40% by weight of the composition.",
"4.A composition according to any preceding claim wherein the composition is a photocure composition and further comprises a photoinitiator.",
"5.A composition according to claim 4, wherein the photoinitiator is present in an amount of from 1 to 14% by weight of the composition.",
"6.A composition according to any preceding claim wherein the polycarbonate backbone is aliphatic.",
"7.A composition according to any preceding claim, wherein the polycarbonate backbone is linear.",
"8.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate oligomer has a molecular weight of from 1,000 to 30,000, preferably from 3,000 to 15,000, and more preferably from 6,500 to 10,000.9.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate component is present in an amount of from 5 to 70% by weight of the composition.",
"10.A composition according to any preceding claim, wherein the polycarbonate-backboned urethane acrylate oligomer is of the general formula (I) or (II): wherein: R2 and R3 are such that OCN—R—NCO (where R=R2 or R3) represents a diisocyanate; Y represents a hydrogen atom or a methyl group; R1 and R4 together with their attached (meth)acrylate group represent the residue of a hydroxy (meth)acrylate functional unsaturated monomer.",
"11.A composition according to any preceding claim, wherein the composition comprises additional acrylate monomer and/or oligomer and the acrylate monomer and/or oligomer is present in an amount of from 0 to 80% by weight of the composition.",
"12.A composition according to claim 11, wherein the acrylate monomer and/or oligomer is monofunctional.",
"13.A composition according to any preceding claim, wherein the composition comprises the reactive monomer N-vinyl-2-pyrrolidone (NVP), in an amount not exceeding 10%, preferably not exceeding 5%, by weight of the composition.",
"14.A composition according to any one of claims 1 to 13, wherein the composition comprises an organic solvent in an amount not exceeding 5% by weight of the composition.",
"15.A composition according to any one of claims 1 to 13, wherein the composition is free of organic solvent.",
"16.A method of in-mould decoration employing an ink or varnish composition as defined in any of the preceding claims.",
"17.A method according to claim 16, wherein the ink is printed onto a polycarbonate or polycarbonate/polyester substrate.",
"18.A method according to claim 16 or claim 17, wherein the ink is printed onto a substrate and the ink on the substrate is subjected to polycarbonate or polycarbonate/polyester blend resin injection to provide a polycarbonate or a polycarbonate/polyester blend backfill adhered to the printed substrate."
],
[
"The present invention relates to a new ink composition for in-mould decorative use and to an in-mould decorative process employing this ink.",
"In its broadest sense, in-mould decorating (IMD) simply means applying a decoration in the course of moulding the decorated part.",
"A particular IMD process referred to as insert moulding or insert IMD has been known for many years, but has seen little practical uses except in the automotive industry, until recently.",
"In insert IMD processes, a film substrate is printed with the desired decoration this is preferably a second surface printing process in which the decoration is printed in reverse on the reverse side of a transparent or translucent (or partially transparent or translucent) film substrate, so that it shows through and is protected by the substrate.",
"Then, in one or more steps, the substrate is, if necessary, formed into shape and further resin or plastics material is injection moulded to give the final product.",
"It will be seen readily that this process can give rise to several difficulties.",
"First, if the printed substrate is to be moulded after printing, the printing ink must have the necessary mechanical properties to wild the strains of moulding.",
"Thus, it must be flexible, and preferably have at least a similar flexibility to that of the substrate, so as to stretch with the substrate as the substrate is moulded.",
"It must also have sufficient adhesion to the substrate and abrasion resistance to withstand any abrading action in the course of moulding.",
"In the final step, where a liquid resin or resin precursor is injected onto the formed decorated substrate, the printing ink must be able to resist the heat, pressure and shear imparted to it by the resin injection, or it has to be protected with an additional layer.",
"Otherwise, the ink will be caused to degrade or smudge from its printed location by the injection process, resulting in so-called “wash-out”.",
"Finally, the ink has to be compatible with the injected resin and in particular must provide a good adhesion with the injected resin so as to prevent delamination of the printed substrate from the resin backfill.",
"One way of solving the problems of avoiding wash-out of the ink and ensuring good adhesive bond strength to the resin backfill has been to provide the printed substrate with an additional coating over the irk which additional coating is typically an aqueous laminating adhesive that is applied over the graphic inks.",
"This coating then serves to bond or laminate an additional sheet of substrate such that the ink is sandwiched between the substrate and the additional sheet of substrate.",
"In this twin-layer construction, the laminated substrate serves to protect the ink against wash-out by the backfill injection and provides good adhesion to the injected resin.",
"However, this construction method requires additional processing steps and material cost.",
"DE-A-19832570 discloses a solvent-based ink system comprising a blend of a polycarbonate and a thermoplastic polyester polyurethane.",
"U.S. Pat.",
"No.",
"5,648,414 discloses a solvent-based ink suitable for insert IMD, containing a polycarbonate based on geminally disubstituted, dihydroxydiphenyl cycloalkanes as binder.",
"One such ink is commercially available as Noriphan™ (ex Proll under licence from Bayer AG).",
"Whilst this solvent-based ink permits a single-layer printed substrate construction to be used in IMD processes, it is associated with certain disadvantages.",
"For example, because it is a solvent-based ink, it is relatively difficult or inconvenient to screen print due to its poor press stability and difficulties in washing up.",
"Also, the printed substrate has been found to be susceptible to curling, and it is often therefore difficult to place the printed substrate in registration in the mould tool prior to injection with the resin backfill.",
"Furthermore, it is essential to ensure that all solvent is completely removed before the printed substrate is formed and injection moulded, in order to prevent wash-out, and blister or bubble formation, leading to delamination.",
"Another disadvantage is that the range of colours available is limited due to the fact that certain pigments can break down the polymer resin.",
"Finally, the need for a solvent in the ink has obvious environmental and health hazard implications.",
"These deficiencies associated with solvent-based systems have led to the development of photocurable ink systems, for example inks curable by UV-energy, for use in IMD processes, which require less or no solvent.",
"However, existing UV-curable inks are not wholly satisfactory, particularly in terms of formability, washout resistance and adhesion to injected resin backfill.",
"It will be appreciated that inks suitable for IMD must have very good adhesion to the substrate, good flexibility and elongation to permit forming with the substrate, good mechanical resistance to abrasion by the moulds, good washout resistance to the resin injection step, and good adhesive bonding to the injected resin backfill.",
"Because UV-curable inks undergo cross-linking, it is far more difficult to achieve this combination of properties in a UV-curable ink than in a solvent-based ink.",
"Although these UV-curable ink systems have eliminated or reduced the solvent, and hence the problems associated with the use of solvent in a solvent-based ink system the need for flexibility and elongation has meant that the inks have tended to be too so* to withstand the mechanical impact of the forming and injection processes.",
"In addition adhesion to the resin backfill was often not satisfactory.",
"Therefore, it was generally still found necessary to provide the printed substrate with a protective or tie coating, in order to ensure adequate resistance to the resin injection process and to provide good bonding to the backfill resin, so as to avoid wash-out, delamination, or both.",
"In addition to boost adhesion to the substrate and avoid delamination at the ink:substrate interface, it was generally still necessary to include aggressive monomers, such as N-vinyl-2-pyrrolidone (NVP), in the ink, which in turn have associated drawbacks, for example they may lead to delamination due to unreacted material, and to health and safety concerns.",
"We have now found, surprisingly, that it is possible to provide an energy-curable composition, preferably a UV-curable ink, suitable for use in IMD processes, that can be printed onto a substrate, that allows the printed substrate to be formed using conventional forming techniques, and backfilled with a resin injection without undergoing wash-out, even in the absence of a protective coating.",
"Furthermore, the ink can provide excellent adhesion to the resin backfill without the need for a tie coating.",
"Because the claimed energy-curable resin composition gives improved adhesion the requirement for NVP is reduced or eliminated.",
"A fisher advantage is that solvent may be significantly reduced or even eliminated from the IMD process.",
"The reduction or removal of solvent or NVP, or both, may lead to further advantages, apart from obvious environmental and health considerations, such as reduced delamination of the final part especially in environmental testing, caused by poor removal or poor full reaction of potentially volatile materials.",
"In addition, we have found that ink compositions according to the present invention can provide a harder, tougher surface, when cured, than conventional insert IMD inks that contain high molecular weight urge acrylate resins, whilst exhibiting excellent flexibility and forming characteristics.",
"It will be appreciated that hardness and toughness are desirable in an insert IMD ink in order to avoid accidental marking or damage caused by forming or moulding tools.",
"Broadly speaking, this has been achieved by using a new urethane acrylate resin having a polycarbonate backbone as carrier for the pigment or dye.",
"Accordingly, in a first aspect, the present invention provides an energy-cure ink or varnish composition for use in an in-mould decoration (IMD) process, comprising an energy-curable resin, additional reactive monomers and/or oligomers and, optionally and in an amount not exceeding 10% by weight of the composition, a solvent, wherein the energy-curable resin comprises a urethane acrylate, oligomer having an aromatic or aliphatic-polycarbonate backbone.",
"In a second aspect, the invention provides a method of in-mould decoration employing the ink or varnish composition.",
"By “energy-curable resin” or “energy-cure compositions”, as used herein, is meant a resin or composition that is curable by exposure to a source of radiation of an appropriate wavelength or intensity, i.e.",
"is photocurable by exposure to a source of electromagnetic radiation of an appropriate wavelength, such as ultraviolet (UV) radiation, or is electron beam (EB) curable by exposure to an electron beam of an appropriate intensity.",
"Preferably, the energy-cure compositions according to the present invention are at least UV-curable.",
"The energy-cure compositions according to the invention contain an energy-curable resin, additional reactive monomers and/or oligomers, additives and, in the case of an ink composition, pigments or dyes.",
"In the case of photocure compositions such as UV-cure compositions, a photoinitiator is also included in the composition.",
"Energy-Curable Resin: The energy-curable resin at least comprises a mono-, di- or trifunctional urethane acrylate oligomer that has a polycarbonate backbone and is obtainable by reaction of a diisocyanate, a hydroxy (meth)acrylate functional unsaturated monomer and a polycarbonate polyol.",
"Preferably, the resin at least comprises a mono- or difunctional urethane acrylate oligomer, as represented by the general formula (I) or (II): wherein: R2 and R3 are such that OCN—R—NCO (where R=R2 or R3) represents the diisocyanate used in the synthesis; Y represents a hydrogen atom or a methyl group; R1 and R4 together with their attached (meth)acrylate group represent the residue of the hydroxy (meth)acrylate functional unsaturated monomer used in the synthesis.",
"Examples of the diisocyanate compounds include, but are not limited to, hexamethylene diisocyanate (HMDI), isophorone diisocyanate (IPDI), 4,4-dicyclohexylmethane diisocyanate (H12MDI), 2,2,4-trimethyl hexamethylene diisocyanate 2,4-tolylene diisocyanate (TDI), 2,6-tolylene diisocyanate (TDI), trimethylhexamethylene diisocyanate (TMDI), diphenylmethane diisocyanate (MDI), tetramethylxylene diisocyanate (TMXDI), and xylene diisocyanate (XDI).",
"A preferred isocyanate is isophorone diisocyanate (IPDI) because of improved resistance and selective reactivities of the isocyanate groups enabling preparation of an ethylenically unsaturated monoisocyanate.",
"The above diisocyanate compounds may be used individually or in combination.",
"Examples of the hydroxy (meth)acrylate functional unsaturated monomers include, but are not limited to, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, 1,4-butanediol monoacrylate and glyceryl diacrylate, trimethylolethane di(meth)acrylate.",
"Suitable polycarbonate polyols are represented by the general formula (III): wherein: R5 and R6 represent the same or different aliphatic or aromatic groups; and n is an integer of 1 to 60.The polycarbonate polyols can be produced through, for example, an ester interchange reaction or alcoholysis of diethyl carbonate or diphenyl carbonate with a polyol, preferably a diol such as an alkylene diol, e.g.",
"1,4-butane diol, 1,6-hexane diol, or an alkylene ether diol.",
"e.g.",
"triethylene glycol, tripropylene glycol.",
"Other suitable diols include 2,2-bis-[4-(2-hydroxypropoxy)phenyl]propane and 1,1-bis-(4-hydroxyphenyl)-3,3,5-trimethyl cyclohexane.",
"Polyols with three or more hydroxyl groups, such as trimethylol propane, glycerine and pentaerythritol, can be incorporated for preparing polycarbonate polyols suitable for synthesising trifunctional polycarbonate urethane acrylates.",
"Mixtures of the polycarbonate polyols may also be utilised in the synthesis.",
"Other polyols such as polyether- or polyester-polyols may also be included, although to achieve the desired IMD properties the backbone should be substantially polycarbonate in nature.",
"The polycarbonate backbone may be aliphatic or aromatic but preferably is aliphatic in nature.",
"It may be linear or branched, and is preferably linear.",
"Suitable polycarbonate backboned urethane acrylates are available as NTX 4711 and NTX 4867 (ex Sartomer).",
"RD2/105.RD2/106.RD3/101, RD3/102, RD4/103 and RD4/104 (ex UCB Chemicals SA), and RXX-01-344 (ex Rahn).",
"We prefer that the polycarbonate urethane acrylate (PCUA) oligomer has a average molecular weight in the range of from 1,000 to 30,000, more preferably from 3,000 to 15,000, still more preferably from 4,000 to 10,000, and in particular from 6,500 to 10,000.For the purposes of providing improved flexibility and elongation, higher molecular weight polycarbonate backbones would be desirable.",
"However, higher molecular weights can have an adverse effect on the viscosity and printability of the ink.",
"Moreover, higher molecular weights tend to produce unduly soft ink coatings, which are more susceptible to marking by mould parts or during handling.",
"Thus, it will be appreciated that the particular molecular weight chosen will represent a compromise between these conflicting factors, on the one hand providing acceptable flexibility and elongation whilst on the other hand, ensuring satisfactory viscosity, printability and hardness.",
"If desired, the flexibility, elongation and hardness properties of the ink can be tailored by blends of monofunctional, difunctional or trifunctional PCUA oligomer.",
"It will be appreciated that the higher degrees of cross-linking afforded by inclusion of di- and trifunctional oligomers will tend to reduce the overall flexibility and elongation of the ink, and increase its hardness.",
"Thus, the energy-curable resin may comprise a mono-, di- or trifunctional polycarbonate urethane acrylate oligomer, or a blend of two or more of mono-, di- and trifunctional polycarbonate urethane acrylate oligomers.",
"Preferably, the energy-curable resin at least comprises a mono- or difunctional polycarbonate urethane acrylate oligomer, or a blend thereof.",
"We prefer that the average functionality of the energy-curable resin is less than 2.5, more preferably less than 2.2, and most preferably less than 2.1.The monofunctional polycarbonate urethane acrylate may be present in the composition in an amount of from 0 to 70% by weight.",
"Difunctional polycarbonate urethane acrylate may be included in an amount from 0 to 50% by weight.",
"Trifunctional polycarbonate urethane acrylate may be included in an amount from 0 to 5% by weight.",
"The total polycarbonate urethane acrylate component is present in an amount of from 5 to 70%, preferably from 20 to 60%, by weight of the composition.",
"Photoinitiators: Preferably the compositions according to the invention are at least photocurable, and most preferably are UV-curable, and accordingly contain a photoinitiator.",
"However, it will be appreciated that EB-cure compositions in accordance with the invention can be formulated without the need for a photoinitiator.",
"For photocure compositions such as UV-cure compositions, a wide range of commercially available photoinitiators can be incorporated to initiate the photocure mechanism.",
"Preferably these photoinitiators have low potential to migration and low volatility, so as to avoid problems such as delamination of the injection moulded part.",
"The photoinitiator package should also have low UV thermal yellowing, important to meet typical heat and environmental cycling tests typically used on the final IMD part.",
"The photoinitiator package is typically present in an amount of from 0.5 to 20% for example from 1 to 14%, by weight of the composition.",
"Suitable photoinitiators include, for example, 2,4,6-trimethylbenzyl diphenyl phosphine oxide (Lucerin™ TPO ex BASF), bis(2,4,6-trimethylbenzoyl)phenyl phosphine oxide (Irgacure 819, ex Ciba Geigy), bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentyl phosphine oxide, ethyl-2,4,6-trimethylbenzoylphenyl-phosphinate (Lucerin™.",
"TPO-L, ex BASF), 2-benzyl-2-dimethylamino-1-(4-morpholinphenyl) butan-1-one (Irgacure™ 369, ex Ciba Geigy), 1-hydroxycyclohexyl acetophenone (Irgacure™ 184, ex Ciba Geigy), iso-propyl thioxanthone (Quantacure™ ITX, ex IBIS or Speedcure™ ITX ex Lambson; 2-chloro thioxanthone (Kayacure™ CTX, ex Nippon Kayaku), oligo[2-hydroxy-2-methyl-1-[4-(1-methylvinyl)phenyl]propanone and 2-hydroxy-2-methyl-1-phenyl-1-propanone (Esacure™ KIP100F, ex Lamberti), methyl benzoyl formate (Genocure MBF ex Rahn), benzophenone, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (Irgacure™ 907, ex Ciba Geigy).",
"Of these, 2,4,6-trimethylbenzoyl diphenyl phosphine oxide (Lucerin™ TPO, ex BASF), bis(2,4,6-trimethylbenzoyl)-phenyl phosphine oxide (Irgacure 819, ex Ciba Geigy), and bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentyl phosphine oxide are preferred photoinitiators.",
"Additional Reactive Monomers and/or Oligomers: Additional reactive energy-cure monomers and/or oligomers may be present in an amount of from 0 to 80%, preferably from 0 to 60%, by weight of the composition.",
"These reactive monomers and/or oligomers are preferably monofunctional.",
"Suitable acrylate monomers or oligomers include, but are not limited to, isobornyl acrylate (IBOA), 2-phenoxy ethyl acrylate (2PEA), 2-(2-ethoxyethoxy) ethyl acrylate (EOEOEA).",
"CTF acrylate, 4-t-butylcyclohexyl acrylate.",
"THF-acrylate, alkoxylated acrylates, diethylene glycol diacrylate, dipropylene glycol diacrylate, 1,6-hexanediol diacrylate, low molecular weight monofunctional urethane acrylates, polyether acrylates, polyester acrylates and low molecular weight epoxy acrylates.",
"Non-acrylated reactive diluents that may be incorporated include, but are not limited to, acryloyl morpholine (ACMO), n-vinylformamide (NVF), n-vinylformamide derivatives and n-vinyl caprolactam (NVC).",
"The compositions of the invention can provide improved adhesion to the substrate and backfill, thereby advantageously allowing the incorporation of N-vinyl-2-pyrrolidone (NVP) to be significantly reduced or even eliminates NVP is conventionally used to boost adhesion to the substrate, but concerns about health and safety, and its effect due to the volatility of unreacted material on delamination, means that it would be advantageous to eliminate, or at least reduce, NVP.",
"Nevertheless, in certain cases it will still be desirable to include monomers such as NVP in the ink and varnish compositions of the invention, to improve ‘key’ to substrate.",
"If used, NVP can be incorporated in an amount not exceeding 30%, preferably not exceeding 15%, more preferably not exceeding 10%, and in particular not exceeding 5%, by weight of the composition.",
"Most preferably, the composition is free of NVP.",
"Additives: If desired, inert or passive resins such as acrylics, styrene acrylates, polyesters, polycarbonates or celluloses may be included in the ink in small amounts, in order to improve the adhesion of the ink coating.",
"However, these inert or passive resins tend to adversely affect the resistance of the ink to injection of the backfill resin, and thus increase the likelihood of wash-out.",
"If included, therefore, we that only a small amount is used, for example an amount not exceeding 10%, and preferably not exceeding 7%, by weight of the composition.",
"Additives such as wetting agents, silicone and non-silicone antifoams may be incorporated to improved print properties such as substrate wetting and flow-out, and may be included in an amount of from 0 to 5%, preferably from 0 to 2%, by weight of the composition.",
"It should be noted that some additives, typically with low molecular weights, may have a tendency to migrate to the print surface in the cured coating.",
"This can affect the IMD properties, and therefore the total additive content should be kept to a minimum and additives with a high migration potential avoided.",
"Where the composition is intended for use as an ink, instead of a varnish, a pigment or dye is included in the composition.",
"Organic and/or inorganic pigments or dyes can be incorporated in an amount of from 0 to 50%, preferably from 0 to 40%, by weight of the composition.",
"The pigments or dyes should be selected to have good resistance to thermal decomposition and change, and resistance to sublimation.",
"Suitable pigments include, but not limited to, titanium dioxide white, zinc sulphide, carbon black, azo diarylide yellow, isoindoline yellow, diarylide orange, quinacridone magneta, diketo-pyrrolo-pyrrol red, copper phthalocyanine blue, copper phthalocyanine green, dioxazine violet, diketometal oxides.",
"Speciality effect pigments, such as metal oxide-coated mica pigments and aluminium metallic pigments, can also be included.",
"Fillers may be included to control the viscosity and rheology of the composition typically to improve printing characteristics, and may be present in an amount of from 0 to 40%, preferably from 0 to 30%, by weight of the composition.",
"Suitable fillers include, but not limited to, calcium carbonate, china clay, aluminium hydrate, talc, barium sulphate, aluminium silicate, and silica.",
"It will be appreciated that the compositions according to the invention will be substantially free of organic solvent.",
"However, small amounts of organic solvent may be included, if needed, in amounts not exceeding 10%, and preferably not exceeding 5%, by weight of the composition.",
"Most preferred is that the composition is free of organic solvent.",
"In-Mould Decorating (IMD) Process: The energy-cure compositions of the invention are particularly suitable for use in IMD processes, and especially in insert IMD or insert moulding processes.",
"The application of the compositions to the substrate may be effected using conventional printing techniques.",
"Preferred techniques include flexographic, lithographic, digital and screen print processes, but other methods may be used as appropriate.",
"Application by screen printing is particularly preferred.",
"Suitable substrate materials onto which the ink may be printed or the varnish applied as the case may be, include print-receptive polyester, polycarbonate, ABS.",
"PMMA, polycarbonate/polyester blends, polycarbonate/ABS blends materials such as those supplied by Bayer AG (Bayfol®, Makrolon®, Marofol®, Bayblend®), GE Structured Products (Lexan®) and Autotype (Autoflex Hiform™, Autoflex XtraForm™).",
"Preferably, the substrate is of a polycarbonate or polycarbonate/polyester blend resin material.",
"Similarly, suitable backfill materials which may be injected onto the printed substrate include the following or blends of the following: polyesters polycarbonate, styrenes ABS and PMMA resin materials.",
"Preferably, the injected backfill is of polycarbonate or a polycarbonate/polyester blend resin material.",
"The invention will be further illustrated by reference to the following non-limiting Examples: EXAMPLES A) Formulations: Example 1 Comparative Solvent-Based IMD Technology A sample of Noriphan™ black HTR952 was obtained (ex Proll) for comparison to UV IMD ink technology.",
"Prints of Noriphan™ were prepared in accordance to the Noriphan™ product data sheet supplied by Proll.",
"The prints were examined for adhesion, surface hardness, print curl, forming, and IMD properties.",
"Example 2 Comparative Standard Flexible Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.",
"Urethane acrylate, Actilane ™ 290 24.0 IBOA, SR506 ex Sartomer 31.8 NVP ex BASF or ISP 12.0 Silicone antifoam 0.7 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Lucerin ™ TPO ex BASF 4.0 Irgacure ™ 184 ex Ciba 3.0 Filler 20.0 Carbon Black 4.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The prints were examined for adhesion, surface hardness, forming, and IMD properties.",
"Example 3 Invention Polycarbonate Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.",
"PCUA, NTX 4867 34.3 IBOA, SR506 ex Sartomer 23.5 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Silicone antifoam 0.7 Lucerin ™ TPO ex BASF 4.0 Quantacure ™ ITX ex IBlS 1.0 NVP ex BASF 12.0 Filler 20.0 Carbon Black 4.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The prints were examined for adhesion, surface hardness, print curl, forming, and IMD properties.",
"Example 4 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.",
"PCUA, NTX 4867 ex Sartomer 36.4 EOEOEA, S256 ex Sartomer 8.7 IBOA, SR506 ex Sartomer 11.9 NVC, ex BASF or ISP 18.1 Polyester urethane acrylate ex Rahn 3.8 UV stabiliser, Genorad ™ 16 ex Rahn 0.4 Lucerin ™ TPO ex BASF 4.7 Irgacure ™ 184 ex Ciba 2.6 Silicone antifoam 0.7 Carbon black 3.6 Filler 9.1 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The prints were examined for adhesion, surface hardness, forming, and IMD properties.",
"Example 5 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.",
"PCUA, NTX 4867 40.0 IBOA, SR506 ex Sartomer 11.8 UV Stabiliser, Genorad ™ 16 ex Rahn 0.5 Silicone antifoam 0.7 Lucerin ™ TPO ex BASF 4.0 Irgacure ™ 184 ex Ciba Geigy 1.0 ACMO ex Rahn 18.0 EOEOEA, SR256 ex Sartomer 8.0 Filler 10.0 Red 6.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The prints were examined for adhesion, surface hardness, forming and IMD properties.",
"Example 6 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.",
"PCUA, RD2/105 ex UCB 25.8 EOEOEA, S256 ex Sartomer 6.0 IBOA, SR506 ex Sartomer 37.2 ACMO ex Rahn 7.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Genocure ™ MBF ex Rahn 2.0 Speedcure ™ ITX ex Lambson 1.0 Silicone antifoam 0.7 Phthalocyanine blue 6.0 Filler 10.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The prints were examined for adhesion, surface hardness, forming, and IMD properties.",
"Example 7 Invention Polycarbonate Urethane Acrylate Technology The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μm) was achieved.",
"PCUA, RXX0] 344 ex Rahn 26.0 IBOA, SR506 ex Sartomer 27.0 NVP ex BASF or ISP 12.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Silicone antifoam 0.7 Titanium dioxide white pigment 30.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The prints were examined for adhesion, surface hardness, forming, and IMD properties.",
"Example 8 Invention Polycarbonate Urethane Acrylate Technology (NVP Free) The following screen ink composition was prepared by first premixing the materials and then grinding the resultant mixture on a triple roll mill until a grind of <12 microns (μM) was achieved.",
"PCUA, RD3/101 ex UCB 40.0 EOEOEA, S256 ex Sartomer 6.0 IBOA, SR506 ex Sartomer 15.0 ACMO ex Rahn 7.0 UV stabiliser, Genorad ™ 16 ex Rahn 0.3 Lucerin ™ TPO ex BASF 4.0 Genocure ™ MBF ex Rahn 2.0 Speedcure ™ ITX ex Lambson 1.0 Silicone antifoam 0.7 Red 6.0 Filler 18.0 Total (weight %) 100.0 Single and multilayer prints of the above screen ink composition were printed through a 150-34 mesh onto a polycarbonate substrate and cured using a medium pressure mercury lamp (80 Wcm−1).",
"The print were examined for adhesion, surface hardness, forming, and IMD properties.",
"B) Experimental Results: 1) Physical Properties TABLE 1 Pencil Hardness Ink system Hardness* Comments Example 1 H Can be marked, but good scratch resistance Example 2 HB Moderate scratch, easily marked Example 3 2H Difficult to scratch, excellent adhesion Example 4 H-2H Difficult to scratch, excellent adhesion Example 5 H-2H Difficult to scratch, excellent adhesion Example 6 H Difficult to scratch, excellent adhesion Example 7 H Difficult to scratch, excellent adhesion Example 8 H Difficult to scratch, excellent adhesion *according to ASTM D3363, BS 3900-E19, ISO 15184 TABLE 2 Cross-Hatch Adhesion Ink system Ranking** Comments Example 1 4-5 No removal Example 2 4 Slight/no removal Example 3 5 No removal Example 4 5 No removal Example 5 5 No removal Example 6 5 No removal Example 7 5 No removal Example 8 5 No removal **1 = poor (total ink removal).",
"5 = excellent (no ink removal) according to BS 3900-E6.ISO 2409 2) Forming Properties The printed substrates were examined for forming properties using the most commonly used techniques (vacuum thermoform.",
"HPF-Niebling, hydroform, matched metal): TABLE 3 Vacuum Thermoform Ink system Ranking*** Comments Example 1 4 Good forming, suitable for medium to deep draw Example 2 3-4 Mould marking can be a problem, inferior at high temps/long cycle times Example 3 4 Suitable for medium to deep draw, improved resistance to mould marking Example 4 4 Suitable for medium to deep draw, improved resistance to mould marking Example 5 4 Suitable for medium to deep draw, improved resistance to mould marking Example 6 4 Suitable for medium to deep draw, improved resistance to mould marking Example 7 4 Suitable for medium to deep draw, improved resistance to mould marking Example 8 4 Suitable for medium to deep draw, improved resistance to mould marking TABLE 4 HPF-Niebling Ink system Ranking*** Comments Example 1 4 Good forming suitable for medium to deep draw Example 2 4 Good forming suitable for medium to deep draw Example 3 4 Good forming suitable for medium to deep draw Example 4 4 Good forming suitable for medium to deep draw Example 5 4 Good forming suitable for medium to deep draw Example 6 4 Good forming suitable for medium to deep draw Example 7 4 Good forming suitable for medium to deep draw Example 8 4 Good forming suitable for medium to deep draw TABLE 5 Hydroform Ink system Ranking*** Comments Example 1 3-4 Moderate forming suitable for medium draw Example 2 4 Good forming suitable for medium to deep draw Example 3 4 Good forming suitable for medium to deep draw Example 4 4 Good forming suitable for medium to deep draw Example 5 4 Good forming suitable for medium to deep draw Example 6 4 Good forming suitable for medium to deep draw Example 7 4 Good forming suitable for medium to deep draw Example 8 4 Good forming suitable for medium to deep draw TABLE 6 Matched Metal Ink system Ranking*** Comments Example 1 5 Superior resistance to marking from mould tool Example 2 2 Composition too soft.",
"marked by mould tool Example 3 5 Superior resistance to marking from mould tool Example 4 5 Slightly softer than Example 3 Example 5 5 Superior resistance to marking from mould tool Example 6 5 Superior resistance to marking from mould tool Example 7 5 Superior resistance to marking from mould tool Example 8 5 Superior resistance to marking from mould tool ***1 = poor (not formable), 5 = excellent (deep draw possible within limits of forming technique) 3) Resistance to Wash-Out The printed substrates were subjected to direct injection with a sprue gate, of a polycarbonate backfill.",
"The degree of ink movement (wash-out) was visually assessed and ranked: TABLE 7 Resistance to PC injection Ink system Ranking**** Comments Example 1 5 No wash-out, but total solvent removal critical Example 2 2 Wash-out, can be improved by use of tie/protective coat e.g.",
"Aqualam ™ (ex Coates) Example 3 5 No wash-out Example 4 5 No wash-out Example 5 5 No wash-out Example 6 5 No wash-out Example 7 5 No wash-out Example 8 4-5 Slight wash-out directly around injection point ****1 = poor (ink wash-out), 5 = excellent (no ink wash-out) 4) Adhesion or Bond to Injected Backfill Resin The printed substrates were subjected to injection of polycarbonate resin using a plaque tool with a wide fan gate injection port.",
"The bond strength between the backfill resin and the printed substrate was assessed by peel strength analysis: TABLE 8 Peel Strength Ink system Typical value (N) Range (N) Example 1 50 50.0-58.0 Example 2 6.7 0.0-8.8 Example 3 51.1 37.8-64.4 Example 4 52.3 34.2-68.0 Example 5 53 33.8-69.2 Example 6 48.8 30.0-60.2 5) Resistance to Heat Ageing and Environmental Cycling Heat ageing and environmental cycling test were undertaken on full IMD parts to ensure that there were no changes to e.g.",
"laminate strength, colouristic properties after the heat/environmental cycles: TABLE 9 Heat Ageing/Environmental Cycling Delamination Ink system test Comments Example 1 Pass Thorough drying critical Example 2 Fail Poor bond strength prior to testing Example 3 Pass No appreciable loss of properties Example 4 Pass No appreciable loss of properties Example 5 Pass No appreciable loss of properties Example 6 Pass No appreciable loss of properties TABLE 10 Heat Ageing/Environmental Cycling Ink system Colouristic test Comments Example 1 Pass — Example 2 Fail — Example 3 Pass No appreciable loss of properties Example 4 Pass No appreciable loss of properties Example 5 Pass No appreciable loss of properties Example 6 Pass No appreciable loss of properties 6) Print Curl Single and multilayer prints of Example 1 and Example 3 were prepared on 125 micron (μm) Bayfol® and assessed for degree of substrate curl or distortion after 24 hours.",
"TABLE 11 Ink film Assessment Number of thickness***** of curl Ink system print layers (μm) (mm) Comment Example 1 1 5 >45 Very poor 2 12 >47 Very poor 3 17 >47 Very poor 4 22 49 Very poor Example 3 1 10 2 — 2 16 2-3 — 3 22 3 — 4 29 3-6 Very slight curl *****Ink film thickness determined using digital micrometer and comparing printed (various number of ink deposits) and unprinted areas of the substrate."
]
] | Patent_10451279 |
[
[
"Map kinase integrin-binding domain",
"Dislosed is the integrin-binding domain of MAP kinase.",
"Interaction between MAP kinase and integrin via this domain activates MAP kinase and initiates cellular activity.",
"Methods of modulating cellular activity by inhibiting direct MAP kinase-integrin binding are provided.",
"These methods have particular use in inhibiting growth of cancer cells."
],
[
"1.A fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.",
"2.A fragment of a MAP kinase wherein the fragment comprises a binding domain of the MAP kinase for an integrin or a partial amino acid sequence of the binding domain that is capable of binding to the integrin and has a length of 100 amino acids or less, or an analog or derivative of the fragment that is capable of binding to the integrin.",
"3.A fragment according to claim 2 wherein the fragment has a length of about 50 amino acids or less.",
"4.A fragment according to claim 3 wherein the fragment has a length of about 40 amino acids or less.",
"5.A fragment according to claim 4 wherein the fragment has a length of about 30 amino acids or less.",
"6.A fragment according to claim 5 wherein the fragment has a length of about 20 amino acids or less.",
"7.A fragment according to claim 2 comprising the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or a contiguous amino acid sequence thereof.",
"8.A fragment according to claim 2 comprising the amino acid sequence PSNLLLNTTCDLKIC or a contiguous amino acid sequence thereof.",
"9.A fragment according to claim 1 wherein the MAP kinase is an ERK or a JNK family member.",
"10.A fragment according to claim 9 wherein the MAP kinase is ERK2.11.A fragment according to claim 1 wherein the integrin is selected from the group consisting of β3, β5 and β6.12.An isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.",
"13.A polypeptide according to claim 12 comprising a core amino acid sequence of a binding domain of the MAP kinase for the integrin.",
"14.A polypeptide according to claim 12 wherein the MAP kinase is a member of the ERK family or the JNK family.",
"15.A polypeptide according to claim 14 wherein the MAP kinase is ERK2.16.A polypeptide according to claim 12 wherein the integrin is selected from the group consisting of β3, β5 and β6.17.A fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.",
"18.A fusion protein according to claim 17 adapted for passing across a cell membrane.",
"19.A fusion protein according to claim 18 further incorporating a carrier moiety for facilitating passage across the cell membrane.",
"20.A fusion protein according to claim 17 wherein the binding moiety comprises a fragment of a MAP kinase consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin or a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.",
"21.A fusion protein according to claim 18 adapted for releasing the binding moiety after passing across the cell membrane.",
"22.An agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or a binding fragment thereof.",
"23.An agent according to claim 22 wherein the binding and carrier moieties are capable of being released from the targeting moiety at the cell for passage of the carrier moiety and the binding moiety across the cell membrane of the cell.",
"24.An agent according to claim 23 wherein the agent incorporates an enzyme cleavage site for being cleaved to thereby release the carrier moiety and the inhibitor moiety at the cell.",
"25.An agent according to claim 24 wherein the enzyme cleavage site is a cleavage site for matrix-metalloproteinase-9 (MMP-9).",
"26.An agent according to claim 22 adapted for release of the inhibitor moiety from the carrier moiety after passage of the inhibitor moiety across the cell membrane of the cell.",
"27.An agent according to claim 22 wherein the targeting moiety is an antibody or a binding fragment of an antibody.",
"28.An agent according to claim 27 wherein the antibody or the binding fragment is specific for an extracellular region of the integrin.",
"29.An agent according to claim 22 wherein the targeting moiety is an integrin receptor targeted peptide for binding to the integrin.",
"30.An agent according to claim 22 wherein the binding moiety is capable of binding to a binding domain of the integrin for the MAP kinase.",
"31.An agent according to claim 30 wherein the binding moiety comprises a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin or a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative of the fragment or polypeptide that is capable of binding to the binding domain of the integrin for the MAP kinase.",
"32.An agent according to claim 22 wherein the binding moiety is an analog of a binding domain of a MAP kinase for an integrin.",
"33.An agent capable of inhibiting binding of a MAP kinase to an integrin, wherein the agent comprises an analog of a binding domain of the MAP kinase for the integrin.",
"34.An agent according to claim 33 wherein the analog comprises a mimetic of the binding domain of the MAP kinase.",
"35.An isolated nucleic acid sequence encoding a fragment as defined in claim 1 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase.",
"36.An isolated nucleic acid sequence encoding a polypeptide as defined in claim 12 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase.",
"37.An isolated nucleic acid sequence encoding a fusion protein as defined in claim 17.38.An isolated antisense nucleic acid sequence complementary to a nucleic acid sequence as defined in claim 35.39.An isolated antisense nucleic acid sequence according to claim 38 wherein the antisense nucleic acid sequence is labelled.",
"40.An oligonucleotide comprising from 14 to 45 contiguous nucleotides of a nucleic acid sequence as defined in claim 35.41.An oligonucleotide comprising from 14 to 45 nucleotides and which is capable of hybridising with a nucleic acid sequence as defined in claim 35.42.A vector incorporating a nucleic acid sequence as defined in claim 35.43.An expression vector incorporating a nucleic acid sequence as defined in claim 35.44.A host cell transformed with a vector as defined in claim 42.45.A host cell transformed according to claim 44 wherein the host cell is selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell and a cancer cell.",
"46.A host cell according to claim 45 wherein the host cell is a colon cancer cell.",
"47.A pharmaceutical composition comprising a fragment as defined in claim 1 or an analog or derivative thereof capable of binding to the binding domain of the integrin for the MAP kinase, together with a pharmaceutically acceptable carrier or diluent.",
"48.A pharmaceutical composition comprising a polypeptide as defined in claim 12 or an analog or derivative thereof capable of binding to the binding domain of the integrin of the MAP kinase, together with a pharmaceutically acceptable carrier or diluent.",
"49.A pharmaceutical composition comprising fusion protein as defined in claim 17 together with a pharmaceutically acceptable carrier or diluent.",
"50.A pharmaceutical composition comprising an agent for inhibiting binding of a MAP kinase with an integrin as defined in claim 22, together with a pharmaceutically acceptable carrier or diluent.",
"51.A pharmaceutical composition comprising a vector as defined in claim 43 together with a pharmaceutically acceptable carrier or diluent.",
"52.A pharmaceutical composition comprising an antisense nucleic acid sequence as defined in claim 38 together with a pharmaceutically acceptable carrier or diluent.",
"53.A pharmaceutical composition comprising an oligonucleotide as defined in claim 40 together with a pharmaceutically acceptable carrier or diluent.",
"54.An antibody specific for a binding domain of a MAP kinase for an integrin, or a binding fragment of the antibody.",
"55.An antibody specific for a fragment as defined in claim 1 or an analog or derivative of the fragment that is capable of binding to the binding domain of the integrin for the MAP kinase.",
"56.An antibody specific for a polypeptide as defined in claim 12 or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin in the MAP kinase.",
"57.An antibody according to claim 54 wherein the antibody is a monoclonal antibody.",
"58.A method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.",
"59.A method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.",
"60.A method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.",
"61.A method according to claim 60 wherein the integrin is selected from the group consisting of β3, β5 and β6.62.A method according to claim 61 wherein the MAP kinase is an ERK family member or a JNK family member.",
"63.A method according to claim 62 wherein the MAP kinase is ERK2.64.An agent identified to be capable of binding to a binding domain of a MAP kinase for an integrin by a method as defined in claim 61.65.A method of isolating an agent from a sample utilizing a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.",
"66.A method according to claim 65 wherein the molecule is a MAP kinase or fragment thereof, or an analog or derivative of a fragment of a MAP kinase.",
"67.A method according to claim 65 wherein the molecule is a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin, or an analog or derivative thereof.",
"68.A method according to claim 65 wherein the molecule is a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative thereof.",
"69.A method according to claim 65 wherein the integrin is selected from the group consisting of β3, β5 and β6.70.A method according to claim 65 wherein the MAP kinase is an ERK family member or a JNK family member.",
"71.A method according to claim 70 wherein the MAP kinase is ERK2.72.An agent isolated from a sample by a method as defined in claim 61.73.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of the MAP kinase for the integrin to thereby cause expression of the MAP kinase to be down-regulated.",
"74.A method according to claim 73 wherein the oligonucleotide is about 50 nucleotides or less in length.",
"75.A method according to claim 74 wherein the oligonucleotide is 45 nucleotides in length or less.",
"76.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of the MAP kinase for the integrin to thereby cause expression of the MAP kinase to be down-regulated, wherein the antisense oligonucleotide is an oligonucleotide as defined in claim 38.77.A method according to claim 73 wherein the cell is transfected with the antisense oligonucleotide or the antisense oligonucleotide is transcribed from a vector introduced into the cell.",
"78.A method according to claim 73 wherein the cell is a cancer cell.",
"79.A method according to claim 73 wherein the MAP kinase is an ERK family member or a JNK family member.",
"80.A method according to claim 79 wherein the MAP kinase is ERK2.81.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.",
"82.A method according to claim 81 wherein the binding domain is mutagenised.",
"83.A method according to claim 81 wherein the MAP kinase is an ERK family member of a JNK family member.",
"84.A method according to claim 81 wherein the MAP kinase is ERK2.85.A method according to claim 81 wherein the integrin subunit is selected from the group consisting of β3, β5 and β6.86.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.",
"87.A method according to claim 86 wherein the agent is a fragment consisting of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin, or an analog or derivative thereof capable of binding to the binding domain of the integrin.",
"88.A method according to claim 86 wherein the agent is a polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase, or an analog or derivative thereof capable of binding to the binding domain of the integrin.",
"89.A method according to claim 86 wherein the agent is a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.",
"90.A method according to claim 86 wherein the agent is an agent comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or a binding fragment thereof.",
"91.A method according to claim 86 wherein the activity of the cell is growth, migration, spreading, invasion, matrix-degrading enzyme secretion, differentiation and/or apoptosis of the cell.",
"92.A method according to claim 86 wherein the cell is a cancer cell.",
"93.A method according to claim 92 wherein the cancer cell is a colon cancer cell.",
"94.A method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"95.A method according to claim 94 wherein the agent comprises an antibody or binding fragment thereof.",
"96.A method according to claim 94 wherein the activity of the cell is growth, migration, spreading, invasion, matrix-degrading enzyme secretion, differentiation and/or apoptosis.",
"97.A method according to claim 94 wherein the cell is a cancer cell.",
"98.A method according to claim 94 wherein the cancer cell is a colon cancer cell.",
"99.A method according to claim 73 comprising treating a mammal in need of such treatment.",
"100.A method according to claim 73 wherein the method comprises treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.",
"101.A method according to claim 100 wherein the cancer is selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.",
"102.A method according to claim 101 wherein the cancer is colon cancer.",
"103.A method of prophylaxis or treatment of cancer in a mammal, comprising: treating the mammal with an effective amount of an antisense nucleic acid for hybridising with a nucleic acid encoding a MAP kinase to cause expression of the MAP kinase to be down-regulated wherein the MAP kinase has a binding domain for an integrin.",
"104.A method according to claim 103 comprising administering an effective amount of the antisense nucleic acid to the mammal.",
"105.A method according to claim 103 comprising administering to the mammal an effective amount of a vector incorporating an nucleic acid sequence complementary to the antisense nucleic acid for generation of the antisense nucleic sequence in vivo.",
"106.A method according to claim 103 wherein the antisense nucleic acid is an antisense oligonucleotide for hybridising with at least part of the sequence of the nucleic acid encoding the binding domain of the MAP kinase.",
"107.A method according to claim 106 wherein the oligonucleotide has a length of 150 nucleotides or less.",
"108.A method according to claim 107 wherein the oligonucleotide has a length of 90 nucleotides or less.",
"109.A method according to claim 108 wherein the oligonucleotide has a length of 50 nucleotides or less.",
"110.A method according to claim 109 wherein the oligonucleotide has a length of from 15 to 30 nucleotides.",
"111.A method according to claim 103 wherein the integrin subunit is selected from the group consisting of β3, β5 and β6.112.A method according to claim 103 wherein the cancer is colon cancer.",
"113.A fragment according to claim 9 wherein the integrin is selected from the group consisting of β3, β5 and β6.114.A polypeptide according to claim 15 wherein the integrin is selected from the group consisting of β3, β5 and β6.115.A vector incorporating an oligonucleotide as defined in claim 40.116.An expression vector incorporating an oligonucleotide as defined in claim 41 for being expressed in a cell.",
"117.A host cell transformed with a vector as defined in claim 43.118.A pharmaceutical composition comprising an antisense nucleic acid sequence as defined in claim 39 together with a pharmaceutically acceptable carrier or diluent.",
"119.A pharmaceutical composition comprising an oligonucleotide as defined in claim 41 together with a pharmaceutically acceptable carrier or diluent.",
"120.An antibody according to claim 55 wherein the antibody is a monoclonal antibody.",
"121.An antibody according to claim 56 wherein the antibody is a monoclonal antibody.",
"122.A method according to claim 69 wherein the MAP kinase is an ERK family member or a JNK family member.",
"123.A method according to claim 86 comprising treating a mammal in need of such treatment.",
"124.A method according to claim 86 wherein the method comprises treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Colorectal cancer is the commonest internal malignancy affecting men and women in Australia.",
"About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990.In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.",
"Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer.",
"Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India.",
"Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand.",
"Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.",
"Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body.",
"Survival rates are significantly improved where the disease is detected and treated early.",
"The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors.",
"Recognition of the genetic component of colorectal cancer is growing.",
"Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult.",
"There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer.",
"FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.",
"However, in the vast majority of patients the aetiology of large bowel cancer remains unknown.",
"Most colon cancer arises within pre-existing benign precursor lesions or adenomas.",
"Adenomas are classified by histological architecture as tubular, tubulovillous or villous.",
"Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia.",
"Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.",
"Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers.",
"For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp.",
"Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise.",
"The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes.",
"This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.",
"Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites.",
"Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels.",
"It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases.",
"Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers.",
"These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.",
"Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.)",
"In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin.",
"It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.",
"Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins.",
"Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis.",
"Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.",
"The integrin family comprises 17 different α and 8β subunits and the αβ combinations are subsumed under 3 subfamilies.",
"Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.",
"In the β1 subfamily, the β1 chain combines with any one of nine a chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor.",
"For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin.",
"In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).",
"The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin.",
"This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains.",
"The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994).",
"All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa.",
"Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48.These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.",
"The α-subunits have a molecular mass ranging from 150-200 kDa.",
"They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.",
"The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992).",
"In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992).",
"At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992).",
"These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits.",
"The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.",
"Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca 2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes.",
"In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995).",
"Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.",
"The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992).",
"The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5.The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).",
"The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension.",
"The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation.",
"However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).",
"The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999).",
"What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996).",
"Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).",
"Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).",
"The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear.",
"However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).",
"Integrins can signal through the cell membrane in either direction.",
"The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999).",
"Outside-in signalling can be roughly divided into two descriptive categories.",
"The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus.",
"Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g.",
"focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P 1 P 2 ) synthesis.",
"The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998).",
"In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.",
"MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane.",
"The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g.",
"Raf-1) (Garrington and Johnson, 1999).",
"Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms.",
"ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991).",
"These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90 rsk ) cytoskeletal proteins (such as microtubule-associated phospholipase A 2 ), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).",
"ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).",
"MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases.",
"The FAK pathway is activated by most integrins.",
"In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996).",
"It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997).",
"It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997).",
"The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997).",
"The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited.",
"In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999).",
"Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.",
"While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs.",
"For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).",
"Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc.",
"For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999).",
"Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.",
"The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question.",
"Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997).",
"In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).",
"Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains.",
"Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998).",
"A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995).",
"More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).",
"Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling.",
"For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995).",
"Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits.",
"When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996).",
"Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans).",
"The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996).",
"An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).",
"Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors.",
"For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury.",
"In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997).",
"Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation.",
"Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>Broadly stated, the present invention relates to the surprising finding that members of the mitogen activated protein (MAP) kinase family can associate with the cytoplasmic domain of an integrin molecule.",
"It is believed that no member of the MAP kinase family has previously been found to directly associate with any integrin.",
"The identification of this functional relationship permits the rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.",
"In an aspect of the present invention there is provided an agent capable of binding to a binding domain of an integrin for a MAP kinase.",
"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase or a homolog, analog, variant or derivative of the polypeptide.",
"The polypeptide may consist of or comprise the binding domain of the MAP kinase to which the integrin binds or sufficient core amino acid sequence of the binding domain of the MAP kinase to enable binding of the polypeptide to the integrin.",
"Preferably, the polypeptide will be a fragment of a MAP kinase.",
"Most preferably, the polypeptide will comprise the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC or a region or regions of such sequences.",
"Accordingly, in a further aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment is capable of binding with an integrin, or a homolog, analog, variant or derivative of the fragment.",
"In another aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.",
"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.",
"Preferably, a polypeptide or fragment of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.",
"Typically, the length will be between about 5 to about 30 amino acids.",
"In yet another aspect of the present invention there is provided a fusion protein a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.",
"Preferably, the binding moiety will comprise a fragment or polypeptide of the invention.",
"Most preferably, the fusion protein will incorporate a carrier moiety for facilitating passage across the cell membrane.",
"The carrier moiety may be penetratin.",
"In still another aspect of the present invention there is provided an agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or an binding fragment thereof.",
"In another aspect of the present invention there is provided a MAP kinase or a fragment thereof with a mutagenised binding domain for an integrin or in which the binding domain is deleted, or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.",
"In another aspect of the present invention there is provided an isolated nucleic acid sequence encoding a fragment or polypeptide of the invention or a homolog, analog, variant or derivative of the fragment or polypeptide.",
"In another aspect of the invention there is provided a nucleic acid sequence coding for a MAP kinase or fragment thereof having a mutagenised binding domain for an integrin or in which the binding domain is deleted or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.",
"In a still further aspect there is provided an isolated nucleic acid sequence encoding a fusion protein or agent of the invention.",
"There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences of the invention.",
"Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides.",
"Sense oligonucleotides coding for all or part of the binding domain of a MAP kinase for an integrin, or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes.",
"A nucleic acid primer or probe of the invention may be labelled with a suitable reporter molecule for enabling detection of hybridisation of the primer or probe to a target nucleic acid sequence.",
"In yet another aspect of the present invention there is provided a vector incorporating a nucleic acid sequence of the invention.",
"Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.",
"In a further aspect of the present invention there is provided a host cell transformed with a vector of the invention.",
"Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell.",
"Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.",
"In yet another aspect of the present invention there is provided an antibody generated with the use of a polypeptide, fragment, agent or fusion protein of the invention.",
"In a still further aspect of the invention there is provided an antibody and binding fragment thereof capable of binding to a binding domain of a MAP kinase for an integrin.",
"The antibody may be a polyclonal or monoclonal antibody.",
"Preferably, the antibody is a monoclonal antibody.",
"In still another aspect of the present invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.",
"In yet another aspect of the invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.",
"In another aspect of the present invention there is provided a method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.",
"Preferably, a polypeptide or fragment of the invention comprising or consisting of the binding domain of the MAP kinase or core amino acid sequence of the binding domain or a homolog, analog, variant or derivative of the polypeptide or fragment or core amino acid sequence is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the MAP kinase.",
"Most preferably, the polypeptide or fragment will comprise or consist of the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC, core amino acid sequence of such sequences capable of binding to an integrin, or a homolog variant, analog or derivative of such sequences.",
"Testing or assaying of an agent for ability to bind to the binding domain of the MAP kinase for the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or prior to the addition of the integrin.",
"Rather than using the MAP kinase, a polypeptide, fragment of a MAP kinase or other molecule capable of binding to the binding site on the integrin for the MAP kinase may be used in such testing or assaying.",
"Similarly, rather than utilising an intact integrin, a polypeptide or integrin fragment comprising or incorporating the binding domain of the integrin for the MAP kinase, or other molecule capable of binding with the binding domain on the MAP kinase for the integrin, may be utilised.",
"In still another aspect of the present invention there is provided a method of isolating an agent from a sample utilising a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.",
"The molecule may be the MAP kinase, or a fusion protein, a polypeptide, or a fragment of the invention to which the integrin is capable of binding, or for instance a homolog, analog, variant or derivative of such a polypeptide or fragment.",
"In yet another aspect of the present invention there is provided the agent so isolated.",
"An agent of the invention will usually be provided in the form of a pharmaceutical composition.",
"Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an agent of the invention capable of binding to a binding domain of an integrin for a MAP kinase, and a pharmaceutically acceptable carrier or diluent.",
"The agent may or not be proteinaceous in nature.",
"Preferably, the agent will comprise a fusion protein, or polypeptide.",
"Most preferably, the agent will be coupled to a carrier molecule for facilitating entry of the agent into a cell.",
"In a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier or diluent.",
"Preferably, the nucleic acid sequence is incorporated into a vector as described herein.",
"Alternatively, the nucleic acid sequence may be joined to a carrier molecule such as penetratin for facilitating entry of the nucleic acid sequence into a target cell.",
"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, for being expressed by the cell and having a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.",
"In still another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.",
"In yet another aspect of the present invention there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide is capable of binding to a binding domain of an integrin for a MAP kinase.",
"In still another aspect of the invention there is provided a method of modulating activity of a cell, comprising causing the expression of a MAP kinase to which an integrin is able to bind to be down-regulated.",
"Preferably, down-regulation of the expression of the MAP kinase is achieved using an antisense nucleic acid sequence that inhibits expression of gene coding for the MAP kinase.",
"The antisense nucleic acid sequence may be administered to the cell or generated within the cell.",
"Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence.",
"Preferably, the antisense nucleic acid sequence will specifically hybridise with sense nucleic acid sequence coding for at least part of the binding domain of the MAP kinase for the integrin and/or intron sequence between such coding sequence.",
"Accordingly, in another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of a MAP kinase for an integrin to thereby cause expression of the MAP kinase to be down-regulated.",
"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.",
"In a further aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"In yet another aspect of the invention there is provided a method of modulating the activity of a cell, comprising: contacting the cell with an effective amount of a fusion protein, antibody or other molecule of the invention capable of binding to the binding domain of an integrin for a MAP kinase.",
"Preferably, modulation of the activity of a cell by a method as described herein will comprise treating a mammal in need of such treatment.",
"Accordingly, in a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a MAP kinase or a fragment thereof or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for an integrin or in which the binding domain has been deleted and wherein the nucleic acid sequence is capable of being expressed.",
"In another aspect of the invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid capable of causing the expression of a MAP kinase to which an integrin is able to bind to be down regulated.",
"In a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.",
"In still another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence for expression of a polypeptide or a fragment of a MAP kinase within the cell, wherein the polypeptide or fragment is capable of binding to a binding domain of an integrin for the MAP kinase to thereby modulate the cellular activity.",
"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of an integrin for a MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.",
"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"In another aspect of there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein the disease or condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase and wherein the agent comprises a polypeptide or fragment of the invention or a homolog, variant, analog or derivative of such a polypeptide or fragment, and the method comprises administering an effective amount of the agent to the mammal.",
"In yet another aspect of the present invention there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase wherein the agent is a fusion protein of the invention.",
"In another aspect there is provided use of a nucleic acid of the invention in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.",
"In a still further aspect of the invention there is provided the use of an agent capable of binding to a binding domain of an integrin for a MAP kinase in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable, wherein the agent is other than an antibody or binding fragment thereof.",
"The agent may for instance be a polypeptide, fragment or the like as described herein.",
"In another aspect of the present invention there is provided the use of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"In another aspect there is provided the use of fusion protein of the invention in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.",
"Methods as described above will typically comprise treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.",
"The cancer may for instance be selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.",
"In still another aspect of the present invention there is provided a method of causing an activity of a cell to be upregulated, comprising: contacting the cell with an effective amount of a nucleic acid capable of being expressed within the cell and coding for an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.",
"In a still further aspect of the invention there is provided a method of causing an activity of a cell to be upregulated, comprising contacting the cell with an effective amount of an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.",
"Typically, the agent will be a polypeptide or fragment of an integrin comprising or consisting of the binding domain of an integrin for a MAP kinase or comprising or consisting of the core amino acid sequence of the binding domain directly involved in the binding to the MAP kinase, or a homolog, variant, analog or derivative of such polypeptide or fragment.",
"Preferably, the polypeptide or fragment will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.",
"Typically, the length will be between about 5 to about 30 amino acids.",
"Most preferably, the polypeptide will comprise or consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.",
"Up regulation of cellular activity is particularly desirable for instance in wound healing, re-endothelialisation within natural or artificial blood vessels, nerve growth and for instance induction of labour.",
"The cellular activity desired to be modulated will typically but not exclusively, be cell growth or proliferation.",
"Indeed, any activity mediated by MAP kinase signalling is expressly included within the scope of the invention.",
"The cell may be any cell type in which functional activity arising from signalling mediated by a MAP kinase may occur.",
"Preferably, the cell will be an epithelial cell and usually, a neoplastic cell.",
"Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase.",
"Preferably, the MAP kinase is ERK2 or JNK-1.Most preferably, the MAP kinase is ERK2.The mammal may be any mammal treatable with a method of the invention.",
"For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being.",
"Preferably, the mammal will be a human being.",
"As will be understood, modulation of cellular activity within the context of the invention may be up regulation or down regulation of cellular activity.",
"Reference to “down modulation” or “down-regulating” or the like should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell.",
"Conversely, reference to “up regulation” or like terms should be understood to include enhancing or increasing one or more aspects of the activity of the cell.",
"In the broadest sense, the term “integrin” unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit.",
"Usually, the integrin will be a member of the αv subfamily.",
"Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6.Most preferably, the integrin comprises β6.By “binding domain” is meant the minimum length of contiguous amino acid sequence required for binding.",
"By “core amino acid sequence” is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction.",
"Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.",
"The term “homolog” is to be taken to mean a molecule that has amino acid sequence similarity.",
"The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.",
"The sequences are considered homologous at a position if the amino acids at that position are the same.",
"Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about 50% or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.",
"Homology with a binding domain may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.",
"The term “analog” is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog.",
"An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function.",
"By “structural” similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function.",
"Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function.",
"Indeed, it is not necessary that an analog have amino acid sequence homology, and an analog may not be proteinaceous at all.",
"An analog may for instance be a mimetic of a molecule.",
"By the term “variant” is meant an isoform, an allelic variant of a gene or region thereof, a naturally occurring mutant form of a gene or region thereof, or a polypeptide or fragment having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function.",
"This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids.",
"Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed.",
"A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an amino acid sequence incorporating the desired amino acid changes which possibility is well within the capability of the skilled addressee.",
"Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.",
"By conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (eg.",
"structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.",
"Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added.",
"A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.",
"By the term “derivative” is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule.",
"A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like.",
"A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.",
"The term “polypeptide” is used interchangeably herein with “peptide” and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.",
"The term “neoplastic cell” is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell.",
"“Growth” is to be taken in its broadest sense and includes proliferation of the cell.",
"In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.",
"Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.",
"The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings."
],
[
"FIELD OF THE INVENTION The present invention relates to a method of modulating cell activity mediated by a mitogen activated protein kinase (MAP) and more particularly, modulation of the growth and/or the proliferation of a cell such as a neoplastic cell.",
"In particular, the invention finds use inter alia in applications requiring modulation of cell activity.",
"The present invention also provides agents for use in the above.",
"BACKGROUND OF THE INVENTION Colorectal cancer is the commonest internal malignancy affecting men and women in Australia.",
"About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990.In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.",
"Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer.",
"Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India.",
"Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand.",
"Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.",
"Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body.",
"Survival rates are significantly improved where the disease is detected and treated early.",
"The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors.",
"Recognition of the genetic component of colorectal cancer is growing.",
"Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult.",
"There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer.",
"FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.",
"However, in the vast majority of patients the aetiology of large bowel cancer remains unknown.",
"Most colon cancer arises within pre-existing benign precursor lesions or adenomas.",
"Adenomas are classified by histological architecture as tubular, tubulovillous or villous.",
"Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia.",
"Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.",
"Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers.",
"For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp.",
"Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise.",
"The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes.",
"This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.",
"Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites.",
"Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels.",
"It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases.",
"Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers.",
"These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.",
"Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.)",
"In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin.",
"It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.",
"Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins.",
"Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis.",
"Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.",
"The integrin family comprises 17 different α and 8β subunits and the αβ combinations are subsumed under 3 subfamilies.",
"Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.",
"In the β1 subfamily, the β1 chain combines with any one of nine a chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor.",
"For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin.",
"In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).",
"The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin.",
"This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains.",
"The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994).",
"All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa.",
"Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48.These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.",
"The α-subunits have a molecular mass ranging from 150-200 kDa.",
"They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.",
"The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992).",
"In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992).",
"At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992).",
"These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits.",
"The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.",
"Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes.",
"In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995).",
"Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.",
"The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992).",
"The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5.The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).",
"The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension.",
"The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation.",
"However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).",
"The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999).",
"What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996).",
"Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).",
"Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).",
"The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear.",
"However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).",
"Integrins can signal through the cell membrane in either direction.",
"The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999).",
"Outside-in signalling can be roughly divided into two descriptive categories.",
"The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus.",
"Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g.",
"focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P1P2) synthesis.",
"The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998).",
"In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.",
"MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane.",
"The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g.",
"Raf-1) (Garrington and Johnson, 1999).",
"Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms.",
"ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991).",
"These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90rsk) cytoskeletal proteins (such as microtubule-associated phospholipase A2), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).",
"ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).",
"MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases.",
"The FAK pathway is activated by most integrins.",
"In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996).",
"It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997).",
"It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997).",
"The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997).",
"The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited.",
"In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999).",
"Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.",
"While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs.",
"For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).",
"Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc.",
"For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999).",
"Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.",
"The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question.",
"Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997).",
"In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).",
"Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains.",
"Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998).",
"A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995).",
"More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).",
"Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling.",
"For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995).",
"Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits.",
"When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996).",
"Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans).",
"The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996).",
"An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).",
"Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors.",
"For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury.",
"In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997).",
"Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation.",
"Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin.",
"SUMMARY OF THE INVENTION Broadly stated, the present invention relates to the surprising finding that members of the mitogen activated protein (MAP) kinase family can associate with the cytoplasmic domain of an integrin molecule.",
"It is believed that no member of the MAP kinase family has previously been found to directly associate with any integrin.",
"The identification of this functional relationship permits the rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.",
"In an aspect of the present invention there is provided an agent capable of binding to a binding domain of an integrin for a MAP kinase.",
"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase or a homolog, analog, variant or derivative of the polypeptide.",
"The polypeptide may consist of or comprise the binding domain of the MAP kinase to which the integrin binds or sufficient core amino acid sequence of the binding domain of the MAP kinase to enable binding of the polypeptide to the integrin.",
"Preferably, the polypeptide will be a fragment of a MAP kinase.",
"Most preferably, the polypeptide will comprise the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC or a region or regions of such sequences.",
"Accordingly, in a further aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment is capable of binding with an integrin, or a homolog, analog, variant or derivative of the fragment.",
"In another aspect of the present invention there is provided a fragment of a MAP kinase wherein the fragment consists of a binding domain of the MAP kinase for an integrin, or an analog or derivative thereof capable of binding to the integrin.",
"In another aspect of the present invention there is provided an isolated or purified polypeptide capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin, or an analog or derivative of the polypeptide that is capable of binding to the binding domain of the integrin, wherein the polypeptide is other than a MAP kinase or a fragment of a MAP kinase.",
"Preferably, a polypeptide or fragment of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.",
"Typically, the length will be between about 5 to about 30 amino acids.",
"In yet another aspect of the present invention there is provided a fusion protein a fusion protein incorporating a binding moiety capable of binding to a binding domain of an integrin for a MAP kinase and thereby inhibiting binding of the MAP kinase to the integrin.",
"Preferably, the binding moiety will comprise a fragment or polypeptide of the invention.",
"Most preferably, the fusion protein will incorporate a carrier moiety for facilitating passage across the cell membrane.",
"The carrier moiety may be penetratin.",
"In still another aspect of the present invention there is provided an agent for inhibiting binding of a MAP kinase to an integrin, comprising: a targeting moiety for targeting a cell expressing the integrin; a binding moiety for binding to a binding domain of the integrin for the MAP kinase to thereby inhibit the binding of the MAP kinase to the integrin; and a carrier moiety for facilitating passage of the binding moiety across the cell membrane of the cell; wherein the binding moiety is other than an antibody or an binding fragment thereof.",
"In another aspect of the present invention there is provided a MAP kinase or a fragment thereof with a mutagenised binding domain for an integrin or in which the binding domain is deleted, or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.",
"In another aspect of the present invention there is provided an isolated nucleic acid sequence encoding a fragment or polypeptide of the invention or a homolog, analog, variant or derivative of the fragment or polypeptide.",
"In another aspect of the invention there is provided a nucleic acid sequence coding for a MAP kinase or fragment thereof having a mutagenised binding domain for an integrin or in which the binding domain is deleted or a homolog, analog or variant of the MAP kinase or fragment, wherein capability to bind with the integrin is thereby reduced.",
"In a still further aspect there is provided an isolated nucleic acid sequence encoding a fusion protein or agent of the invention.",
"There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences of the invention.",
"Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides.",
"Sense oligonucleotides coding for all or part of the binding domain of a MAP kinase for an integrin, or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes.",
"A nucleic acid primer or probe of the invention may be labelled with a suitable reporter molecule for enabling detection of hybridisation of the primer or probe to a target nucleic acid sequence.",
"In yet another aspect of the present invention there is provided a vector incorporating a nucleic acid sequence of the invention.",
"Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.",
"In a further aspect of the present invention there is provided a host cell transformed with a vector of the invention.",
"Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell.",
"Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.",
"In yet another aspect of the present invention there is provided an antibody generated with the use of a polypeptide, fragment, agent or fusion protein of the invention.",
"In a still further aspect of the invention there is provided an antibody and binding fragment thereof capable of binding to a binding domain of a MAP kinase for an integrin.",
"The antibody may be a polyclonal or monoclonal antibody.",
"Preferably, the antibody is a monoclonal antibody.",
"In still another aspect of the present invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if any said agent is capable of binding to the binding domain of the MAP kinase on the basis of the testing.",
"In yet another aspect of the invention there is provided a method of screening for an agent capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing a number of agents for ability to bind to the MAP kinase; (b) selecting an agent or agents identified as being able to bind to the MAP kinase on the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents is capable of binding to the binding domain of the MAP kinase for the integrin.",
"In another aspect of the present invention there is provided a method of evaluating whether an agent is capable of binding to a binding domain of a MAP kinase for an integrin, comprising: (a) testing the agent for ability to bind to the binding domain of the MAP kinase for the integrin; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.",
"Preferably, a polypeptide or fragment of the invention comprising or consisting of the binding domain of the MAP kinase or core amino acid sequence of the binding domain or a homolog, analog, variant or derivative of the polypeptide or fragment or core amino acid sequence is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the MAP kinase.",
"Most preferably, the polypeptide or fragment will comprise or consist of the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC, core amino acid sequence of such sequences capable of binding to an integrin, or a homolog variant, analog or derivative of such sequences.",
"Testing or assaying of an agent for ability to bind to the binding domain of the MAP kinase for the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or prior to the addition of the integrin.",
"Rather than using the MAP kinase, a polypeptide, fragment of a MAP kinase or other molecule capable of binding to the binding site on the integrin for the MAP kinase may be used in such testing or assaying.",
"Similarly, rather than utilising an intact integrin, a polypeptide or integrin fragment comprising or incorporating the binding domain of the integrin for the MAP kinase, or other molecule capable of binding with the binding domain on the MAP kinase for the integrin, may be utilised.",
"In still another aspect of the present invention there is provided a method of isolating an agent from a sample utilising a molecule immobilised on a solid support and which is capable of binding to a binding domain of an integrin for a MAP kinase, comprising: (a) contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the agent to the molecule; (b) eluting the agent from the solid support; and (c) collecting the eluted agent; wherein the molecule is other than an antibody or binding fragment of an antibody.",
"The molecule may be the MAP kinase, or a fusion protein, a polypeptide, or a fragment of the invention to which the integrin is capable of binding, or for instance a homolog, analog, variant or derivative of such a polypeptide or fragment.",
"In yet another aspect of the present invention there is provided the agent so isolated.",
"An agent of the invention will usually be provided in the form of a pharmaceutical composition.",
"Accordingly, in another aspect of the present invention there is provided a pharmaceutical composition comprising an agent of the invention capable of binding to a binding domain of an integrin for a MAP kinase, and a pharmaceutically acceptable carrier or diluent.",
"The agent may or not be proteinaceous in nature.",
"Preferably, the agent will comprise a fusion protein, or polypeptide.",
"Most preferably, the agent will be coupled to a carrier molecule for facilitating entry of the agent into a cell.",
"In a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier or diluent.",
"Preferably, the nucleic acid sequence is incorporated into a vector as described herein.",
"Alternatively, the nucleic acid sequence may be joined to a carrier molecule such as penetratin for facilitating entry of the nucleic acid sequence into a target cell.",
"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with an expression vector incorporating a nucleic acid sequence encoding a MAP kinase of a fragment thereof, or a homolog, analog or variant of the MAP kinase or fragment, for being expressed by the cell and having a mutagenised binding domain for the integrin or in which the binding domain has been deleted whereby binding to the integrin is inhibited or prevented.",
"In still another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: transfecting the cell with a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.",
"In yet another aspect of the present invention there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide is capable of binding to a binding domain of an integrin for a MAP kinase.",
"In still another aspect of the invention there is provided a method of modulating activity of a cell, comprising causing the expression of a MAP kinase to which an integrin is able to bind to be down-regulated.",
"Preferably, down-regulation of the expression of the MAP kinase is achieved using an antisense nucleic acid sequence that inhibits expression of gene coding for the MAP kinase.",
"The antisense nucleic acid sequence may be administered to the cell or generated within the cell.",
"Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence.",
"Preferably, the antisense nucleic acid sequence will specifically hybridise with sense nucleic acid sequence coding for at least part of the binding domain of the MAP kinase for the integrin and/or intron sequence between such coding sequence.",
"Accordingly, in another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an antisense oligonucleotide for hybridising with at least part of a nucleic acid sequence coding for the binding domain of a MAP kinase for an integrin to thereby cause expression of the MAP kinase to be down-regulated.",
"In another aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the integrin for the MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.",
"In a further aspect of the present invention there is provided a method of modulating activity of a cell expressing a MAP kinase with a binding domain for an integrin, comprising: treating the cell with an effective amount of an agent capable of binding to the binding domain of the MAP kinase, wherein the agent is other than an agent comprising a binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"In yet another aspect of the invention there is provided a method of modulating the activity of a cell, comprising: contacting the cell with an effective amount of a fusion protein, antibody or other molecule of the invention capable of binding to the binding domain of an integrin for a MAP kinase.",
"Preferably, modulation of the activity of a cell by a method as described herein will comprise treating a mammal in need of such treatment.",
"Accordingly, in a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a MAP kinase or a fragment thereof or a homolog, analog or variant of the MAP kinase or fragment, with a mutagenised binding domain for an integrin or in which the binding domain has been deleted and wherein the nucleic acid sequence is capable of being expressed.",
"In another aspect of the invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid capable of causing the expression of a MAP kinase to which an integrin is able to bind to be down regulated.",
"In a further aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal a nucleic acid sequence encoding a fragment of a MAP kinase, or a homolog, analog or variant of the fragment, for being expressed by the cell and capable of binding to the binding domain of an integrin for the MAP kinase.",
"In still another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence for expression of a polypeptide or a fragment of a MAP kinase within the cell, wherein the polypeptide or fragment is capable of binding to a binding domain of an integrin for the MAP kinase to thereby modulate the cellular activity.",
"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of an integrin for a MAP kinase, wherein the agent is other than an antibody or a binding fragment thereof.",
"In another aspect of the present invention there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cellular activity is desirable, comprising: administering to the mammal an effective amount of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"In another aspect of there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein the disease or condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase and wherein the agent comprises a polypeptide or fragment of the invention or a homolog, variant, analog or derivative of such a polypeptide or fragment, and the method comprises administering an effective amount of the agent to the mammal.",
"In yet another aspect of the present invention there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent capable of binding to a binding domain of an integrin for a MAP kinase wherein the agent is a fusion protein of the invention.",
"In another aspect there is provided use of a nucleic acid of the invention in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.",
"In a still further aspect of the invention there is provided the use of an agent capable of binding to a binding domain of an integrin for a MAP kinase in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable, wherein the agent is other than an antibody or binding fragment thereof.",
"The agent may for instance be a polypeptide, fragment or the like as described herein.",
"In another aspect of the present invention there is provided the use of an agent capable of binding to the binding domain of a MAP kinase for an integrin wherein the agent is other than an agent comprising the binding domain of the integrin for the MAP kinase or an analog or derivative thereof.",
"In another aspect there is provided the use of fusion protein of the invention in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition in which modulation of cellular activity is desirable.",
"Methods as described above will typically comprise treatment or prophylaxis of cancer or a condition associated with a predisposition to cancer.",
"The cancer may for instance be selected from the group consisting of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.",
"In still another aspect of the present invention there is provided a method of causing an activity of a cell to be upregulated, comprising: contacting the cell with an effective amount of a nucleic acid capable of being expressed within the cell and coding for an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.",
"In a still further aspect of the invention there is provided a method of causing an activity of a cell to be upregulated, comprising contacting the cell with an effective amount of an agent capable of binding to a binding domain of a MAP kinase for an integrin or a binding domain of the integrin for the MAP kinase.",
"Typically, the agent will be a polypeptide or fragment of an integrin comprising or consisting of the binding domain of an integrin for a MAP kinase or comprising or consisting of the core amino acid sequence of the binding domain directly involved in the binding to the MAP kinase, or a homolog, variant, analog or derivative of such polypeptide or fragment.",
"Preferably, the polypeptide or fragment will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and more usually about 40 amino acids or less.",
"Typically, the length will be between about 5 to about 30 amino acids.",
"Most preferably, the polypeptide will comprise or consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.",
"Up regulation of cellular activity is particularly desirable for instance in wound healing, re-endothelialisation within natural or artificial blood vessels, nerve growth and for instance induction of labour.",
"The cellular activity desired to be modulated will typically but not exclusively, be cell growth or proliferation.",
"Indeed, any activity mediated by MAP kinase signalling is expressly included within the scope of the invention.",
"The cell may be any cell type in which functional activity arising from signalling mediated by a MAP kinase may occur.",
"Preferably, the cell will be an epithelial cell and usually, a neoplastic cell.",
"Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase.",
"Preferably, the MAP kinase is ERK2 or JNK-1.Most preferably, the MAP kinase is ERK2.The mammal may be any mammal treatable with a method of the invention.",
"For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being.",
"Preferably, the mammal will be a human being.",
"As will be understood, modulation of cellular activity within the context of the invention may be up regulation or down regulation of cellular activity.",
"Reference to “down modulation” or “down-regulating” or the like should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell.",
"Conversely, reference to “up regulation” or like terms should be understood to include enhancing or increasing one or more aspects of the activity of the cell.",
"In the broadest sense, the term “integrin” unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit.",
"Usually, the integrin will be a member of the αv subfamily.",
"Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6.Most preferably, the integrin comprises β6.By “binding domain” is meant the minimum length of contiguous amino acid sequence required for binding.",
"By “core amino acid sequence” is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction.",
"Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.",
"The term “homolog” is to be taken to mean a molecule that has amino acid sequence similarity.",
"The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.",
"The sequences are considered homologous at a position if the amino acids at that position are the same.",
"Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about 50% or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.",
"Homology with a binding domain may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.",
"The term “analog” is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog.",
"An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function.",
"By “structural” similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function.",
"Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function.",
"Indeed, it is not necessary that an analog have amino acid sequence homology, and an analog may not be proteinaceous at all.",
"An analog may for instance be a mimetic of a molecule.",
"By the term “variant” is meant an isoform, an allelic variant of a gene or region thereof, a naturally occurring mutant form of a gene or region thereof, or a polypeptide or fragment having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function.",
"This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids.",
"Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed.",
"A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an amino acid sequence incorporating the desired amino acid changes which possibility is well within the capability of the skilled addressee.",
"Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.",
"By conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (eg.",
"structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.",
"Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added.",
"A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.",
"By the term “derivative” is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule.",
"A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like.",
"A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.",
"The term “polypeptide” is used interchangeably herein with “peptide” and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.",
"The term “neoplastic cell” is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell.",
"“Growth” is to be taken in its broadest sense and includes proliferation of the cell.",
"In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.",
"Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.",
"The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings.",
"BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS FIG.",
"1: Surface biotinylation and immunoprecipitation of integrin subunits β5 and β6 in HT29 colon cancer cells stably transfected with either vector alone (mock transfectants) or antisense β6 construct; FIG.",
"2: Amplification of β6 and glyceraldehyde dehydrogenase (GAPDHu) mRNA: Ethidium-stained agarose gels with amplification products following RT-PCR from total RNA extracted from transfected HT29 and WiDr cell lines.",
"Equal amounts of PCR product obtained from RT-PCR reactions were loaded on each lane and the β6 (141 basepairs) and GAPDH (216 basepairs) bands are indicated (WT, wild-type; S, sense β6; A/S, antisense β6; mock, vector alone); FIG.",
"3: Non-transfected HT29 cells (wild) and cells transfected with vector alone (mock), sense β6 and antisense β6 analysed by FACScan for expression of the β6 subunit.",
"White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-β6); FIG.",
"4: WiDr cells transfected with vector alone (mock) or antisense β6 and analysed by FACScan for expression of the β6 subunit.",
"White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-β6); FIG.",
"5: Tumor cell proliferation in vitro assessed by (3H)-thymidine uptake for WiDr wild-type cells and transfectants (mock and antisense β6) cultured on plastic for the times indicated.",
"FIG.",
"6: Tumor cell proliferation in vitro assessed by (3H)-thymidine uptake for HT29 wild-type cells and transfectants (mock, sense β6 and antisense β6) cultured on plastic for the times indicated.",
"FIG.",
"7: Tumour growth after 6 weeks following subcutaneous inoculation of 106 viable cells of HT29 mock (vector alone) and antisense β6 transfectants; FIG.",
"8: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of clone 1 from WiDr mock (vector alone) and antisense β6 transfectants; FIG.",
"9: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of HT29 mock (vector alone) and antisense β6 transfectants; FIG.",
"10: β6-associated ERK identified by immunoprecipitations of integrin subunits from SW480 β6 transfectants.",
"(Top) Cell lysates were immunoprecipitated with mAbs QE2E5 (anti-β1), L230 (anti-αv), P1F6 (anti-β5), R6G9 (anti-β6), or matched isotype control Abs (IgG1 and IgG2A), and (bottom) blotted with anti-ERK1/2 mAb, SC-1647, which recognises total ERK (phosphorylated and non-phosphorylated).",
"Purified, non-phosphorylated ERK2 is shown in the left hand lane; FIGS.",
"11(A) and 11(B): (A) Western blotting: equal protein loads of cell lysates from one representative clone each from WiDr mock and antisense β6 blotted with anti-ERK mAb (SC-1647) against total ERK.",
"Purified non-phosphorylated ERK2 is shown in the left hand lane.",
"(B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of the cell lysates in (A) probed with anti-ERK mAb (E10).",
"Purified phosphorylated ERK2 is shown in the left hand lane; FIG.",
"12: β6-bound ERK shown for the high and low SW480 β6-expressing clones by probing β6 immunoprecipitates with anti-ERK mAb (E10) against phosphorylated forms of ERK1/2.Purified, phosphorylated ERK2 is shown in the left hand lane; FIGS.",
"13(A) and 13(B): (A) Surface biotinylation of WiDr wild-type cells and β6 immunodepletion of the cell lysates by three successive rounds of β6 and β5 immunoprecipitations using mAb R6G9 and P1F6, respectively or control mAb (IgG2A).",
"The β6 and partner αv bands are arrowed.",
"(B) β6-immunodepleted WiDr cell lysates after 3 successive rounds of β6-immunoprecipitations probed with anti-ERK mAb SC-1647 recognising both phosphorylated and non-phosphorylated forms of ERK1/2 and compared with non-β6 immunodepleted lysates and control lysates sequentially immunoprecipitated 3 times with either isotype matched control mAb (IgG2A) or mAb P1F6 (anti-β5); FIG.",
"14: Non-transformed (HaCaT) and Ras-transformed (HaRas) human keratinocytes: β6 immunoprecipitation and ERK western blots probed with monoclonal antibody E10 (against Phosphorylated ERK 1/2) and monoclonal antibody SC1647 (against total ERK 1/2), respectively.",
"FIGS.",
"15(A) and 15(B): (A) Depletion of β6 with sequential rounds of ERK immunodepletion of cell lysates using anti-ERK mAb (SC-1647).",
"(B) MAP kinase activity in cell lysates before and after 5 rounds of sequential β6 immunodepletion from SW480 β6 and SW480 mock transfectants (full grey and hatched bars, respectively).",
"MAP kinase activity is shown as the mean of three independent experiments.",
"The reduction in MAP kinase activity following β6 immunodepletion was highly significant (P≦0.005, students T test).",
"FIG.",
"16: Shows the amino acid sequence of the cytoplasmic domain of the β6 subunit as well as the amino acid sequences for the β1 to β3 subunits, respectively; FIG.",
"17: Graph showing binding of non-phosphorylated ERK2 (GST.ERK2) to overlapping fragments corresponding to different regions of the cytoplasmic domain of the β6 subunit; FIG.",
"18: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic domain and the peptide fragments indicated in FIG.",
"16 over a range of concentrations of ERK2; FIG.",
"19: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic domain and fragments thereof; FIG.",
"20: Graph showing binding of ERK2 (GST.ERK2) to a 15 mer fragment of the β6 cytoplasmic domain and which has the amino acid sequence RSKAKWQTGTNPLYR; and FIG.",
"21: Shows regions of the cytoplasmic domain of the β6 subunit corresponding to synthesised fragments thereof evaluated for capacity to be bound by ERK2.FIG.",
"22: Graph showing assay results for ERK2 (GST.ERK) binding to synthesised 10 mer fragments identified in FIG.",
"21.FIG.",
"23: Graph showing binding of ERK2 (thrombin cleaved) to synthesised peptide having the amino acid sequence RSKAKNPLYR compared to the 15 mer RSKAKWQTGTNPLYR fragment of the cytoplasmic domain of the β6 subunit.",
"FIG.",
"24: Graph showing binding of JNK-1 to the β6 cytoplasmic domain.",
"FIG.",
"25: Location of β6 Δ746-764, β6(770t) and β6(777t) deletions in the cytoplasmic domain of the β6 subunit.",
"FIG.",
"26: SW480 cells transfected with wild-type full length coding sequence for β6 or β6 Δ746-764 deletion mutant analysed by FACScan for expression of wild-type or mutant β6.White and black histograms represent cells stained in the absence and presence of the integrin subunit, respectively.",
"FIGS.",
"27(A) and 27(B): (A) Western blotting: equal protein loads of cell lyates from SW480 cells expressing wild-type β6 or β6 Δ746-764 deletion mutant.",
"(B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of cell lysates (A) probed with anti-ERK mAb (E10).",
"FIG.",
"28: Proliferation of HT29 colon cancer cells cultured for 48 hours and treated with penetratin, the fragment of β6 cytoplasmic domain having amino acid sequence RSKAKWQTGTNPLYR alone or the fragment coupled to penetratin for the final 24 hours of the incubation period.",
"FIG.",
"29: Proliferation of SW480 cells expressing wild-type β6 cultured on plastic for 48 hours and treated with penetratin, the RSKAKWQTGTNPLYR peptide alone or the peptide coupled to penetratin for the final 24 hours of the incubation period.",
"FIGS.",
"30(A) to 29 (C): (A) SW480 cells cultured with control additive for 24 hours; (B) SW480 cells cultured with penetratin for 24 hours; (C) SW480 cells cultured with RSKAKWQTGTNPLYR bound to penetratin for 24 hours.",
"FIGS.",
"31(A) and 31 (B): (A) SW480 mock (−β6) and SW480 β6 transfectants (+β6) cultured in presence of the RSKAKWQTGTNPLYR peptide coupled to penetratin.",
"(B) Photomicrographs of cells shown in (A) cultured in the presence/absence of the peptide penetratin complex.",
"FIGS.",
"32(A) to 32(C): (A) SW480 cells cultured in a 3-dimensional collagen type I matrix photographed in the gel at the end of 10 days.",
"(B) The SW480 cells following dissolution of the collagen with collagenase.",
"(C) Graph showing numbers of colonies of the SW480 cells having a diameter exceeding 200μ.",
"FIGS.",
"33(A) and 33(B): Graphs showing inhibition of proliferation of SW480 cells expressing full length wild-type β6 in the presence of RSKAKWQTGTNPLYR peptide bound to penetratin.",
"FIG.",
"34: Graph showing binding of ERK2 to RSKAKWQTGTNPLYR peptide and peptides corresponding to regions of the cytoplasmic domain of β3 and β5.FIG.",
"35(A) to 35(C): (A) Immunoprecipitations of β5 and β6 subunits followed by immunoblotting with anti-biotin antibody.",
"αv/β5/β6 bands are shown for the SW480 cell line: wild-type cells, mock transfectants (vector alone) or cells expressing either full length β6 or a Δ746-764 deletion mutant.",
"(B) Indentical amounts of precipitated proteins shown in (A) were immunoblotted with anti-ERK mAb (E10) against phosphorylated ERK1/2.Purified phosphorylated ERK2 is shown in the left hand lane.",
"(C) Comparative binding of ERK2 (1 μm) to synthetic peptides comprising either RSKAKWQTGTNPLYR or peptide sequences derived from β5 and β1 cytoplasmic domains that correspond to the binding domain on β6 for ERK.",
"FIG.",
"36: In vitro phosphorylation of GST-Elk-1 by immunoprecipitated ERK from cell lysates prepared from human umbilical vein endothelial cells (HUVECs) cultured in the absence/presence of epidermal growth factor (EGF) after a 24 hour culture period in serum-free and growth factor free medium.",
"FIGS.",
"37 and 38: Overlapping peptides derived from ERK2 sequence tested for ability to inhibit the β6-ERK2 interaction.",
"FIG.",
"39: Inhibition of β6-ERK2 interaction using ERK2 peptide HRDLKPSNLLLNTTCDLK (0.25 μm) and β6 cytoplasmic tail peptide.",
"FIGS.",
"40(A) to 40(D): Illustrate a dose-dependent wound repair process for peptide-agonist complexed with penetratin as a delivery vehicle.",
"FIG.",
"41: Nucleotide and amino acid sequence for ERK2.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION The distribution of β6 integrin subunit within various tissues has been assessed by both in situ hybridisation and immunostaining and reported in the art.",
"For instance, β6 mRNA in adult primate tissues was detected only in epithelial cells and at very low or undetectable levels in most normal tissues (Breuss et al, 1993).",
"High-level expression of β6 has been observed in secretory endometrial glands while low-level expression was detected in the ductal epithelia of salivary gland, mammary gland and epididymis, in gall and urinary bladder, and in the digestive tract.",
"Immunostaining data has also shown that β6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumourigenesis and epithelial repair (Breuss et al, 1993; 1995).",
"During development of the kidney, lung and skin, β6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin.",
"In contrast, high level expression of β6 has been observed in several types of carcinoma.",
"For example, β6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, and often focally localised at the infiltrating edges of tumour cell islands (Breuss et al, 1995; Thomas et al, 1997).",
"Moreover, expression of the β6 subunit has been observed in renal cell carcinoma and testicular tumour cell lines (Takiuchi et al, 1994) and 50% of lung cancers have been shown to express this subunit (Smythe et al, 1995).",
"Recent studies have shown that αvβ6 is a major fibronectin-binding receptor in colorectal cancer (Agrez et al, 1996).",
"Moreover, normal colonic epithelium from cancer patients does not express αvβ6 in immunostaining studies, and as with squamous cell carcinomas from the oral mucosa (Thomas et al, 1997) maximal β6 expression in colon cancer has been observed at the invading edges of tumour cell islands (Agrez et al, 1996).",
"Importantly, heterologous expression of the β6 subunit in colon cancer cells lacking constitutive expression of this receptor has been shown to stimulate tumour cell proliferation in vitro and tumour growth in athymic immune-deficient mice, and this growth-promoting effect is mediated through the 11 amino acid C-terminal cytoplasmic extension unique to the β6 subunit (Agrez et al, 1994).",
"These findings suggest that one mechanism of the enhanced growth effect involves induced secretion of matrix-degrading enzymes as has been previously suggested for invasive melanoma cells.",
"Support for this arises from findings that the proliferative capacity of colon cancer cells within 3-dimensional collagen matrices is inversely related to the density of the extracellular matrix (Agrez, 1989), and the observation that induced expression of αVβ6 in colon cancer cells markedly enhances gelatinase B secretion (Agrez et al, 1999).",
"Indeed, the β6 subunit is widely observed in cancers of various origins (Breuss et al, 1995).",
"As described above, β6 is detected in at least 50% of bowel cancer tumours.",
"Others have reported its presence in oropharyngeal cancers where it is also present and strongly expressed in the invading margins of the cancer cell islands as is commonly found in bowel cancer.",
"In the oropharyngeal mucosa, no β6 is observed in the normal lining cells of the mouth but in both primary and metastatic tumours from the oropharyngeal mucosa, strong β6 expression is seen which does not correlate with degree of differentiation and in particular, is restricted to the basal layer of epithelial cells.",
"In colon cancer, β6 expression is similarly maximal at the advancing edges of tumour cell islands (Agrez et al, 1996).",
"Hence, modulation of MAP kinase interaction with the β6 subunit in epithelial cells is of interest in the prophylaxis or treatment of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, female and male breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.",
"In terms of prophylactic use in relation to cancer, modulation of the MAP kinase β6 interaction and specifically down regulation of the interaction may find application in protecting against ultraviolet-induced skin cancer, lung cancer in smokers, cancer of the gut where polyps are present as in polyposis coli or other inheritable disease where a pre-disposition to the development of polyps exists, breast cancer in high risk patients with a familial history of breast cancer or otherwise identified as carrying known mutations of the breast cancer susceptibility gene BRCA1 or BRCA2 associated with breast cancer, transitional cell cancers arising from bladder papillomas, and cancer of the cervix in individuals deemed to be at high risk.",
"Expression of β6 is also up-regulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing.",
"αvβ6 is not expressed in normal epithelium (Jones et al, 1997).",
"However, following experimental wounding, αv appears to switch its heterodimeric association from β5 to β6 subunit during re-epithelialisation.",
"At day 3 after wounding, β6 is absent but then appears around the perimeter of the basal cells of the migrating epidermis (Clark et al, 1996).",
"By day 14 after wounding, when re-epithelialisation is complete, all suprabasalar cells overlying the wound express β6 but not β5.In human mucosal wounds, maximal expression of β6 has been observed relatively late when epithelial sheets are fused and granulation tissue is present (Haapasalmi et al, 1996).",
"Furthermore, those investigators observed maximal expression of tenascin with αvβ6 expression.",
"Interestingly, freshly isolated keratinocytes have not been found to express β6 but begin to express this after subculturing (Haapasalmi et al, 1996).",
"Moreover, TGF-β1 has been shown to induce the de novo expression of αvβ6 at the cell surface on keratinocytes (Zambruno et al, 1995).",
"This is particularly relevant in view of the recent observation that αvβ6 binds and activates latent TGF-β1 which may be a means of locally regulating TGF-β1 function in vivo during tissue response to injury (Munger et al, 1999).",
"In contrast to persistent αvβ6 expression observed in colon cancer cells, new expression of αvβ6 in migrating keratinocytes is down-regulated to undetectable levels once re-epithelialisation is complete (Happasalami et al, 1996; Cass et al, 1998).",
"While in the normal unwounded skin, just as in other normal epithelia, αvβ6 expression is absent indicating that this MAP kinase activation pathway is normally suppressed.",
"However, alterations in cell shape trigger biochemical signalling by MAP kinases in human keratinocytes and it is likely that alterations of skin mechanics in vivo during wound formation and healing trigger physiological responses via the MAP kinase pathway (Krippenberg et al, 2000).",
"In addition, gelatinase B secretion by keratinocytes which facilitates the process of cell migration during normal re-epithelialisation is MAP kinase-dependent (McCawley et al, 1999).",
"As outlined above, MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane which regulate cell proliferation, differentiation, migration and invasion.",
"In particular, extracellular signal-regulated kinases (ERKs) become activated upon integrin ligation and, thereby, regulate cell migration possibly by initiating matrix degrading enzyme secretion (Klemke et al, 1997) such as gelatinase B. Gelatinase B production has been shown to be dependent on endogenous ERK signalling in growth factor-stimulated human kertinocytes (McCawley et al, 1999; Zeigler et all, 1999).",
"Aside from integrins, growth factors such as foetal calf serum or epidermal growth factor (EGF) have been shown to stimulate ERK activity via the Ras-Raf-Mek-MAPk cascade resulting in directed cell migration through a porous membrane (Giehl et al, 2000).",
"There is also abundant data indicating that the MAP kinase cascade plays a major role in alterations of cell shape and migration for a range of normal cell types.",
"For example, the MAP kinase signalling pathway has been shown to mediate neurite outgrowth by cerebellar neurons (Schmid et al, 2000) implying a role for MAP kinase in nerve repair, while growth factor-induced migration of endothelial cells has been shown to be dependant on MAP kinase signalling and this dependency for new blood vessel formation (angiogenesis) on sustained ERK activity is regulated by the ligation state of both growth factor receptors and integrins (Eliceiri et al, 1998).",
"In addition, ERK activation has been demonstrated to be essential for up-regulation of vascular endothelial growth factor (VEGF) in other cell types (Jung et al, 1999), and induction of endothelial tube formation by human umbilical vein endothelial cells (HUVECs) dispersed within a 3-dimensional collagen gel has been shown to be dependent on MAP kinase activation (Ilan et al, 1998).",
"Growth factors and their receptors are also known to play important roles in tissue repair and ulcer healing.",
"For example, EGF accelerates gastroduodenal ulcer healing by stimulating cell proliferation, and this event is regulated through the Ras/Raf/MAP kinase pathway (Tarnawski and Jones, 1998).",
"Moreover, in the healing process of gastric ulcers, ERKs have been shown to translocate to cell nuclei with activation of the c-fos gene which triggers cell proliferation (Tarnawski et al, 1998).",
"Cutaneous wound re-epithelialisation and keratinocyte migration requires ordered gene expression and the fibroblast growth factor (FGF)-inducible response element (FiRE) required for migrating (but no proliferating) keratinocytes has been shown to be induced by treatment of cells with okadaic acid, the inhibitor of protein phosphatases which inactivate ERKs (Jaakkola et al, 1998).",
"The MAP kinase cascade is also be implicated in events relating to parturition.",
"For example, induction of labour is commonly achieved with oxytocin and oxytocin induction of prostaglandin synthesis has been shown to be MAP kinase-dependent (Strakova et al, 1998).",
"In fact, oxytocin causes rapid activation of MAP kinases in both human and rat puerperal uterine myometrial cultured cells (Nohara et al, 1996).",
"Moreover, MAP kinase inhibition has been shown to inhibit prostaglandin-induced uterine contraction in animal models (Ohmichi et al, 1997).",
"In addition, gelatinase B has been identified in amniotic fluid association with normal labour (Vadillo-Ortega et al, 1996) and gelatinase B production has been shown to be dependent on endogenous ERK signalling in other cell types such as keratinocytes (Zeigler et al, 1999; McCawley et al, 1999).",
"Taken together, these date suggest that MAP kinase signalling may be an important regulator of parturition.",
"αvβ6 expression is also upregulated in type II alveolar epithelial cells during lung injury caused by injection of live bacteria and αvβ6 mRNA is induced within 5 hours of acute injury (Breuss et al, 1995).",
"Interestingly, αvβ6 has been shown to be expressed on proximal airway epithelial cells in 50% of smokers undergoing lung resection (Weinacker et al, 1995).",
"Just as in human keratinocytes, in primary cultures of human airway epithelial cells, TGF-β1 has been shown to dramatically increase expression of αvβ6 without affecting surface expression of any other integrin.",
"Moreover, epidermal growth factor (EGF) also induces αvβ6 in these cells, and the effect of both growth factors on β6 expression has been shown to be additive (Wang et al, 1996).",
"αvβ6 expression has also been observed in adult lungs and kidneys at focal sites of sub-clinical inflammation as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys (Breuss et al, 1995).",
"Taken together, this data and studies outlined above indicate that αvβ6 has a role in inflammation and affects for instance cell spreading, migration and growth during re-organisation of epithelial in development, tissue repair and neoplasia.",
"Hence, the application of agents, on either unwounded epithelial tissues (such as before surgery) or upon wounded epithelium, which up-relate MAP kinase activity may enhance cell migration and wound re-epithelialisation and thereby, hasten the healing process.",
"Such agents may be based upon the binding domain on β6 for ERK2 and act therefore as β6 agonists (in the absence of this integrin) leading to MAP kinase activation.",
"Examples of the application of such agents may include cell migration in wound healing, re-endothelialisation within natural or artificial blood vessels, or renewed nerve outgrowth following injury or disease.",
"In addition, use of such agonists may for instance be useful in promoting induction of labour.",
"The epithelial restricted integrin subunit β6 is shown herein to interact with at least MAP kinases ERK2 and JNK-1, and its down-regulation dramatically suppresses growth of colon cancer.",
"Hence, therapeutic strategies to inhibit growth and proliferation of colon cancer and growth of other malignant cancer cells by switching off either the permissive β6 integrin and/or inhibiting the β6 MAP kinase interaction are of particular interest.",
"In particular, the fact that β6 expression may be significantly upregulated on tumour cells compared to normal cells offers the potential for tumour cell specificity substantially without impairment of normal cellular function.",
"Down-regulation of the functional activity of an integrin or MAP kinase may be achieved by preventing or down-regulating the expression of the integrin molecule or MAP kinase or by inhibiting the signalling function of the integrin or MAP kinase.",
"Gene therapy is one strategy for treating cancers of different types.",
"The use of recombinant adenoviruses has for instance been utilised for restoring expression of polypeptide encoded by a wild-type p53 tumour suppressor gene (Bookstein et al, 1996), and adenoviral vectors for expression of wild-type p53 have been shown to suppress growth of human colon cancers by as much as 60% in animal models following intra-tumoural injection of recombinant virus (Spitz et al, 1996).",
"Rather than replacing a defective gene with one encoding a polypeptide the expression of which restores normal function of the cell as in the above examples, the possibility exists to achieve down regulation of cancer cells such as colon cells by introducing a gene that codes for an integrin subunit in which the binding domain for a MAP kinase has been rendered defective by mutagenesis, or in which the binding domain has been wholly or partially deleted, to thereby achieve down regulation through the inhibition of the MAP kinase integrin interaction.",
"The defective integrin subunit will nevertheless usually be able to associate with its normal partner integrin subunit and be expressed on the cell membrane.",
"Preferably, the defective integrin subunit will be expressed at a higher level than the corresponding wild-type integrin subunit such that down regulation is achieved by a dominant negative effect.",
"Alternatively, such therapy may involve the introduction and expression of a nucleic acid sequence that encodes a fragment or truncated form of an integrin subunit that excludes the binding domain for the MAP kinase or in which the binding domain has been partially or wholly deleted or otherwise mutagenised so as to be defective.",
"Similarly, a gene coding for a MAP kinase or a nucleic acid sequence coding for a fragment of a MAP kinase comprising the binding domain for an integrin may also be modified such that the binding domain of the MAP kinase is mutagenised or deleted.",
"Another option is to introduce a nucleic acid sequence encoding a polypeptide capable of binding to the binding domain on the integrin for the MAP kinase or to the binding site on the MAP kinase for the integrin upon being expressed within the cell to thereby inhibit intracellular binding of the MAP kinase to the integrin and thereby achieve down regulation of cellular activity.",
"A gene or nucleic acid sequence encoding an integrin subunit or MAP kinase may also be modified such that although the encoded binding domain for integrin or the MAP kinase as the case may be remains unaltered, the amino acid sequence of a region distant from the binding domain, or the amino acid sequence of either one or both regions immediately flanking the binding domain, is altered to achieve a change in the three dimensional conformation of the integrin subunit or MAP kinase such that the MAP kinase integrin binding interaction is inhibited.",
"The gene or nucleic acid sequence may be altered to achieve the desired outcome by the deletion, insertion or substitution of one or more nucleotides such that the corresponding amino acid sequence is modified to the extent that binding of the MAP kinase to the integrin is inhibited.",
"Inhibition in this context may be partial or total inhibition.",
"The gene or nucleic acid sequence can be introduced into a cell in an appropriate expression vector for expression of the gene or nucleic acid sequence extrachromosomally or more preferably, for facilitating integration of the gene or nucleic acid sequence into genomic DNA by heterologous or homologous recombination events.",
"In another strategy, down-regulation of the expression of an integrin subunit such as β6 and hence an integrin heterodimer such as αvβ6 or the down regulation of expression of a MAP kinase such as ERK2, is achieved using antisense nucleic acid sequences (e.g.",
"oligonucleotides) for inhibiting expression of the subunit or MAP kinase.",
"Typically, this will involve expression of a nucleic acid construct incorporating all or part of a coding region of the gene for the integrin subunit or MAP kinase inserted in the reverse orientation resulting in the synthesis of antisense RNA which inhibits translation of mRNA encoding the integrin subunit or MAP kinase by hybridisation of the antisense RNA to the mRNA.",
"More broadly, a method of down regulating an activity of a cell by inhibiting expression of an intact integrin subunit or a MAP kinase may comprise contacting the cell with a first nucleic acid molecule that is capable of interacting with a target nucleic acid sequence coding for the subunit or MAP kinase, or which first nucleic acid molecule is capable of being transcribed to a nucleic acid molecule capable of interacting with the target sequence whereby the interaction of the first nucleic acid molecule with the target sequence inhibits expression of the integrin subunit or MAP kinase.",
"Reference to the first nucleic acid molecule is to be understood as a reference to any nucleic acid molecule which directly or indirectly facilitates reduction, inhibition or other form of down regulation of the expression of the integrin molecule or MAP kinase.",
"Nucleic acid molecules which fall within the scope of this definition include antisense sequences administered to a cell and antisense sequences generated in situ which have sufficient complementarity with target sequence such as mRNA coding for the integrin subunit or MAP kinase or for instance, a transcription regulatory sequence controlling transcription of the gene coding for the integrin subunit or MAP kinase, to thereby be capable of hybridising with the target sequence and inhibit the expression of the integrin subunit or MAP kinase.",
"The first nucleic acid molecule may also be a ribozyme capable for instance, specifically binding to that region of nucleic acid encoding the binding domain of an integrin subunit or MAP kinase or the binding domain of a MAP kinase for an integrin, and cleaving the nucleic acid.",
"Typically, an antisense strategy in accordance with the present invention will be directed toward inhibition of an intact integrin subunit to thereby down regulate cell activity via down regulation of interaction of the integrin with a MAP kinase.",
"On a priori grounds, targeting the expression of the β6 subunit in malignant cells such as in colon cancer by means of adenoviral-mediated antisense therapy is preferred because down-regulating β6 by means of a non-adenoviral approach may increase cell surface expression of the β5 subunit.",
"The relevance to such therapy is that the vitronectin-binding integrin αvβ5 promotes adenovirus internalisation (Thomas et al, 1993).",
"Given that abundant αvβ5 is always present on the surface of colon cancer cells for example, a secondary benefit of inhibiting β6 expression during the course of therapy may be the concomitant rise of β5 in cells already transduced with antisense β6 nucleic acid.",
"This is likely to facilitate further virus uptake into such cells since the amount of integrin αvβ5 present has been shown to be closely related to levels of gene expression following adenovirus-mediated gene transfer (Takayama et al, 1998).",
"Typically, an antisense nucleic acid sequence will hybridise with all or part of that region of the target sense nucleic acid encoding the binding domain of an integrin subunit or the corresponding binding domain of a MAP kinase.",
"An antisense nucleic sequence may for instance be capable of hybridising to exon and/or intron sequences of pre-mRNA.",
"Preferably, an antisense nucleic acid sequence will be designed for specifically hybridising to mature mRNA in which intron sequences have been spliced out by normal cellular processing events.",
"It is not necessary that an antisense nucleic acid sequence have total complementarity with its target sequence only that substantial complementarity exists for specificity and to allow hybridisation under cellular conditions.",
"Preferably, an antisense nucleic sequence will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably, about 90% or 95% or greater.",
"Preferred antisense sequences are oligonucleotides wherein the complementary sense nucleotides encode for about 50 amino acids or less, preferably about 35 or 30 amino acids or less, more preferably less than about 25 or 20 amino acids, most preferably about 15 amino acids or less and usually between about 5 to about 15 amino acids.",
"Antisense nucleic acid sequences may be generated in vivo by transcription of a suitable expression vector within a cell transformed with the vector, or ex vivo and then be introduced into a target cell to effect down regulation of the MAP kinase integrin interaction.",
"Antisense sequences will desirably be designed to be resistant to endogenous exonucleases and/or endonucleases to provide in vivo stability in target cells.",
"Modification to the phosphate backbone, sugar moieties or nucleic acid bases may also be made to enhance uptake by cells or for instance solubility, and all such modifications are expressly encompassed.",
"Such modifications include modification of the phosphodiester linkages between sugar moieties, the utilisation of synthetic nucleotides and substituted sugar moieties, linkage to liphophilic moieties and the such like as described in U.S. Pat.",
"No.",
"5,877,309.Methods for the construction of oligonucleotides for use in antisense therapy have previously been described (see Van der Krol et al, 1998 Biotechniques 6:958-976; and Stein et al, 1998 Cancer Res 48:2659-2668; Bachman et al, 1998, J. Mol.",
"Med.",
"76:126-132).",
"Any means able to achieve the introduction of a gene or a nucleic acid into a target cell may be used.",
"Gene transfer methods known in the art include viral and non-viral transfer methods.",
"Suitable virus into which appropriate viral expression vectors may be packaged for delivery to target cells include adenovirus (Berkner, 1992; Gorziglia and Kapikian, 1992); vaccinia virus (Moss, 1992); retroviruses of avian (Petropoulos et al, 1992); murine (Miller, 1992) and human origin (Shimada et al, 1991); herpes viruses including Herpes Simplex Virus (HSV) and EBV (Margolskee, 1992; Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1997; Freese et al, 1990); papovaviruses such as SV40 (Madzak et al, 1992), adeno-associated virus (Muzyczka, 1992); BCG and poliovirus.",
"Particularly preferred virus are replication deficient recombinant adenovirus (eg.",
"He et al, 1998).",
"Engineered virus may be administered locally or systemically to achieve delivery of the gene or nucleic acid sequence of interest into a target cell.",
"Gene transfer methods as described above may be used in the provision of transgenic animals for studying in vivo the effect of for instance, modifying the binding site of an integrin for a MAP kinase or the binding domain of the MAP kinase for the intergrin.",
"A transgenic animal is one with cells that contain heterologous nucleic acid as a result of the deliberate introduction of the nucleic acid.",
"The nucleic acid may be introduced indirectly by viral transfer as indicated above or directly by microinjection into a pronucleus of a fertilised egg prior to transfer of the egg to a surrogate mother for development to term.",
"Alternatively, a gene or nucleic acid sequence of interest may be introduced into an embryonal stem (ES) cell in culture and the transformed cell injected into a recipient blastocyst which is then transferred to a surrogate mother for development to term.",
"Techniques for generation of transgenic animals are for instance described in U.S. Pat.",
"No.",
"4,873,191; Van der Putten, 1985; Thompson et al, 1989 and Lo, 1983.A transgenic animal homozygous for a transferred gene for instance may be obtained by the mating of animals heterozygous for the gene as will be appreciated.",
"Both animal cells expressing a heterologous gene or nucleic acid sequence and transgenic animals in which a particular gene has been mutagenised by homologous recombination may be provided.",
"A transgenic animal may for instance be a mouse, rat, hamster or pig.",
"Typically, the transgenic animal will be a mouse.",
"Agents for modulating the functional activity of a cell arising from the interaction of a MAP kinase like ERK2 or JNK-1 with a integrin include antagonists and inhibitors capable of associating with the integrin or MAP kinase to thereby inhibit the MAP kinase and integrin interaction.",
"Antagonists and inhibitors include those agents that act by binding the integrin or MAP kinase adjacent to the relevant binding domain and stearically hindering the interaction of the MAP kinase with the integrin, as well as allostearic inhibitors that distort the binding domain upon associating with the integrin.",
"Additional agents include agonists of a MAP kinase or an integrin, that are capable of binding to the pertinent binding domain involved in the MAP kinase integrin interaction.",
"A binding domain involved in the MAP kinase integrin interaction may be identified and characterised using protocols and techniques described herein.",
"Specifically, a binding domain may be localised by assessing the capacity of respective overlapping peptide fragments of the cytoplasmic domain of an integrin subunit to bind with a MAP kinase or correspondingly, assessing the capacity of overlapping peptide fragments of the MAP kinase to bind with an integrin.",
"The specific amino acid sequence which constitute the binding domain may then be determined utilising progressively smaller peptide fragments of a fragment observed to interact with the MAP kinase or the integrin.",
"In particular, test peptides are readily synthesised to a desired length involving deletion of an amino acid or amino acids from either or both ends of the larger amino acid sequence, and tested for their ability to associate with the MAP kinase or integrin.",
"This process is repeated until the minimum length peptide capable of binding with the MAP kinase or integrin substantially without compromising the optimum observed level of binding is identified.",
"The amino acids that play an active role in the MAP kinase integrin interaction may be achieved with the use of further synthesised test peptides in which one or more amino acids of the sequence are deleted or substituted with a different amino acid or amino acids to determine the effect on the ability of the peptide of associate with the MAP kinase or integrin.",
"Typically, substitution mutagenesis will involve substitution of selected ones of the amino acid sequence with alanine or other relatively neutrally charged amino acid.",
"By deletion is meant deletion of one or more of the amino acids between the N-terminal and C-terminal amino acid residues of the identified amino acid sequence.",
"Nucleotide and amino acid sequence data for the β6 integrin subunit for instance is found in Sheppard et al, 1990.The amino acid sequence for β6 is also set out in SEQ ID NO: 1.The nucleotide and amino acid sequence for ERK2 may be found in Boulton et al, 1991 and is set out in FIG.",
"39.Reference to such published data allows the ready design of peptide fragments of a MAP kinase or an integrin subunit cytoplasmic domain for use in the identification and localisation of the binding domain for the MAP kinase or the integrin as is applicable, and the identification of the corresponding nucleic acid sequence encoding such peptide fragments as well as the amino acid sequence of the binding domain.",
"Localisation and characterisation of a binding domain for a MAP kinase or integrin enables the design of agents for binding to the binding domain for modulation of cell activity either for down regulating the functional activity of the integrin or the MAP kinase and more particularly the MAP kinase integrin binding interaction, or as agonists of the MAP kinase or integrin.",
"This will typically involve determining the physical properties of the binding domain such as size and charge distribution, and the tertiary structure of the binding domain.",
"Specifically, at least the region of the integrin containing the binding domain for the MAP kinase or the region of the MAP kinase containing the binding domain for the integrin is modelled taking into account the stereochemistry and physical properties of the binding domain such as size and charge distribution as well as its three dimensional structure as determined using x-ray chrstallography, nuclear magnetic resonance and/or commercially available computer modelling software.",
"Such modelling techniques are well known in the art.",
"In a variation of this approach, the modelling will take into account the interaction of the relevant binding domain with the MAP kinase or the integrin as the case may be such that any change in conformation arising from the interaction may be taken in to account in the design of an agent.",
"Modelling flanking regions adjacent the binding domain also allows the design of agents for associating with such flanking regions but which are nevertheless capable of inhibiting the MAP kinase integrin interaction either by stearic hindrance or by distorting the conformation of the binding domain of the MAP kinase or integrin (eg.",
"allostearic inhibitors).",
"The design of a mimetic of the binding domain of interest will usually involve selecting or deriving a template molecule onto which chemical groups are grafted to provide required physical and chemical characteristics.",
"The selection of template molecule and chemical groups is based on ease of synthesis, likely pharmacological acceptability, risk of or potential for degradation in vivo, stability and maintenance of biological activity upon administration.",
"Pharmacological acceptability and the like are also taken into consideration in the design of other agent types.",
"In order to constrain a polypeptide or other agent in a three dimensional conformation required for binding, it may be synthesised with side chain structures or otherwise be incorporated into a molecule with a known stable structure in vivo.",
"In particular, a polypeptide or the like may be incorporated into an amino acid sequence at least part of which folds into a β-pleated sheet or helical structure such as an α-helix (eg.",
"see Dedhar et al., 1997).",
"A polypeptide or other agent may also be cyclised to provide enhanced rigidity and thereby stability in vivo.",
"Various methods for cyclising peptides, fusion proteins or the like are known (eg.",
"Schiller et al., 1985).",
"For example, a synthetic peptide incorporating two cysteine residues distanced from each other along the peptide may be cyclised by the oxidation of the thiol groups of the residues to form a disulfide bridge between them.",
"Cyclisation may also be achieved by the formation of a peptide bond between the N-terminal and C-terminal amino acids of a synthetic peptide or for instance through the formation of a bond between the positively charged amino group on the side chain of a lysine residue and the negatively charged carboxyl group on the side chain of a glutamine acid residue.",
"As will be understood, the position of the various amino acid residues between which such bonds are formed will determine the size of the cycle.",
"Variation of cycle size for optimisation of binding affinity may be achieved by synthesising peptides in which the position of amino acids for achieving cyclisation has been altered.",
"The formation of direct chemical bonds between amino acids or the use of any suitable linker to achieve cyclisation is also well within the scope of the skilled addressee.",
"Further strategies for identifying possible agents include large scale screening techniques as are known to the skilled addressee.",
"For example, peptide library technology provides an efficient way of testing a vast number of potential agents.",
"Such libraries and their use are well known.",
"Prospective agents identified may be then further evaluated in suitable activity, competitive and other immunoassays.",
"A method of screening for an agent or evaluating whether an agent is capable of binding to a MAP kinase or an integrin, and in particular the binding domain of a MAP kinase for an integrin or the binding domain of an integrin for a MAP kinase, and thereby inhibiting the MAP kinase integrin interaction or acting as a MAP kinase or integrin agonist, may for instance involve utilising the agent in an assay whereby the agent has the opportunity of binding to the MAP kinase or the integrin in the presence of the integrin or the MAP kinase as the case may be or prior to the addition of the integrin or the MAP kinase, and determining whether inhibition of binding of the MAP kinase to the integrin results.",
"An alternate screening method may for instance involve selecting a test agent capable of binding with the integrin or MAP kinase, measuring cellular activity in the presence of the test agent, and comparing that activity with cellular activity in the absence of the test agent.",
"Cellular activity may be assessed by cell growth as indicated by [3H]-thymidine uptake or other measurement of cellular activity.",
"As will be understood, a difference in observed functional activity in the presence of the test agent is indicative of the modulating effect provided by the test agent.",
"It will be understood that the integrin in the context of such assays may be an integrin subunit or polypeptide or fragment incorporating the binding domain of the integrin for the MAP kinase, or a homolog, analog, variant or derivative of such a molecule to which the MAP kinase is capable of binding.",
"Similarly, a MAP kinase in this context may be an intact MAP kinase or a fragment thereof incorporating the binding domain for the integrin, or a homolog, analog, variant or derivative of such a molecule that is capable of binding with the binding domain of the integrin.",
"In addition, determination of whether an agent is capable of binding to the binding domain of an integrin or MAP kinase may be achieved using a polypeptide or fragment as described herein consisting of the binding domain or a core amino acid sequence of the binding domain that directly participates in the binding interaction, or analogs or the like of such molecules.",
"It is not necessary that an agent be proteinaceous in character and indeed, mimetics may be prepared which may not be a polypeptide at all but which nevertheless possess the capability of binding with the integrin or MAP kinase.",
"Polypeptides including fusion proteins and fragments of a MAP kinase comprising the binding domain for an integrin or fragments of an integrin subunit comprising the binding domain for a MAP kinase, or which incorporate sufficient core amino acid sequence of the binding domain of the integrin or MAP kinase for binding by the MAP kinase or integrin are contemplated herein.",
"Typically, a polypeptide will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and generally, less than about 40 amino acids.",
"Preferably, the length will be from between about 5 to about 30 amino acids, and more preferably from between about 10 amino acids and about 25 amino acids.",
"Preferably, a polypeptide will comprise or incorporate the amino acid sequence RSKAKWQTGTNPLYR, more usually the amino acid sequence RSKAKNPLYR, or one or both of sequences RSKAK and NPLYR.",
"Alternatively, a polypeptide may have the amino acid sequence HRDLKPSNLLLNTTCDLKICDFGLAR or PSNLLLNTTCDLKIC.",
"Polypeptides and fusion proteins or the like may be synthesised or produced using conventional recombinant techniques.",
"Nucleic acid encoding a fusion protein may for instance be provided via the joining together of separate DNA fragments encoding peptides or polypeptides having desired three dimensional conformations and/or other characteristics by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini as appropriate, and ligation of cohesive ends prior to insertion of the resultant chimeric sequence into a suitable expression vector.",
"Alternatively, PCR amplification of DNA fragments can be utilised employing primers which give rise to amplicons with complementary termini which can be subsequently ligated together (eg.",
"see Current Protocols in Molecular Biology.",
"John Wiley & Sons, 1992).",
"Nucleic acid sequences encoding for the polypeptides of the invention, mutagenised integrin subunits and the like as described herein are also encompassed as are the respective complementary antisense nucleic acid sequences.",
"Sense oligonucleotides encoding for the binding site of an integrin subunit or MAP kinase or a partial amino acid sequence of a binding domain, and the complementary antisense oligonucleotides are particularly suitable for use as primers in polymerase chain reaction (PCR) amplification methods or as probes for detection of the presence the respective target nucleic acid sequences with which they hybridise such as in Southern blotting protocols, or in affinity chromatography purification of the target nucleic acid sequence.",
"Probes may be labelled with for instance commonly used isotopes such as P32, fluorophores, chemiluminescent agents and enzymes (see eg.",
"Essential Molecular Biology.",
"A Practical Approach Vol.",
"II, Oxford University Press, 1993; Current Protocols in Molecular Biology, Ausubel FM., John Wiley & Sons Inc., 1998).",
"The choice of a label will vary depending on the degree of sensitivity required, ease of conjugation with the probe, safety and other factors.",
"Oligonucleotides for use as probes or primers will usually have a length of less than about 60 nucleotides, usually less than about 50 or 40 nucleotides preferably, between about 14 and about 30 nucleotides, and more preferably, between about 14 and about 25 nucleotides.",
"While it is desirable that a primer or probe has 100% complementarity with its target sequence, oligonucleotides may be designed with less complementarity but which nevertheless hybridise with the target sequence.",
"Typically, a primer or probe will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably about 90% or 95%, or greater.",
"A probe will generally be designed for being capable of hybridising with its target nucleic acid sequence under moderate or high stringency wash conditions.",
"Moderate stringency wash conditions are for example those that employ 0.2×SSC (0.015M NaCl/0.0015M sodium citrate)/0.1% SDS (sodium dodecylsulfate) wash buffer at 42° C. High stringency wash conditions employ for instance, 0.1×SSC wash buffer at 68° C. Generally, the content of purine relative to the content of pyrimidine nucleotides in the region of target nucleic acid of interest will be taken into account in the design of such primers and probes as will be their length in accordance with well accepted principles known in the art.",
"In addition, the present invention provides vectors incorporating nucleic acid sequences of the invention.",
"The term “vector” is to be taken to mean a nucleic acid molecule capable of facilitating the transport of a nucleic acid sequence inserted therein into a cell and includes expression vectors and cloning vectors.",
"Suitable expression vectors include plasmids and cosmids capable of expression of a DNA (eg.",
"genomic DNA or cDNA) insert.",
"An expression vector will typically include transcriptional regulatory control sequences to which the inserted nucleic acid sequence is operably linked.",
"By “operably linked” is meant the nucleic acid insert is linked to the transcriptional regulatory control sequences for permitting transcription of the inserted sequence without a shift in the reading frame of the insert.",
"Such transcriptional regulatory control sequences include promotors for facilitating binding of RNA polymerase to initiate transcription, expression control elements for enabling binding of ribosomes to transcribed mRNA, and enhancers for modulating promotor activity.",
"A promotor may be a tissue specific promotor which facilitates transcription of the nucleic acid insert only in specific cell lineages and not in other cell types or only to a relatively low level in such other cell types.",
"The design of an expression vector will depend on the host cell to be transfected, the mode of transfection and the desired level of transcription of the nucleic acid insert.",
"Numerous expression vectors suitable for transfection of prokaryotic (eg.",
"bacterial) or eukaryotic (eg yeast, insect or mammalian cells) are known in the art.",
"Expression vectors suitable for transfection of eukaryotic cells include pSV2neo, pEF.PGK.puro, pTk2, pRc/CNV, pcDNAI/neo, non-replicating adenoviral shuttle vectors incorporating the polyadenylation site and elongation factor 1-α promotor and pAdEasy based expression vectors most preferably incorporating a cytomegalovirus (CMV) promotor (eg.",
"see He et al, 1998).",
"For expression in insect cells, baculovirus expression vectors may be utilised examples of which include pVL based vectors such as pVL1392, and pVL941, and pAcUW based vectors such as pAcUW1.Viral expression vectors are particularly preferred.",
"Typical cloning vectors incorporate an origin of replication (ori) for permitting efficient replication of the vector, a reporter or marker gene for enabling selection of host cells transformed with the vector, and restriction enzyme cleavage sites for facilitating the insertion and subsequent excision of the nucleic acid sequence of interest.",
"Preferably, the cloning vector has a polylinker sequence incorporating an array of restriction sites.",
"The marker gene may be drug-resistance gene (eg.",
"Ampr for ampicillin resistance), a gene encoding an enzyme such as chloramphenicol acetyltransferase (CAT), β-lactamase, adenosine deaminase (ADA), aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), or for instance β-galactosidase encoded by the E. coli lacZ gene (LacZ′).",
"Yeast reporter genes include imidazole glycerolphosphate dehydratase (HIS3), N-(5′-phosphoribosyl)-anthranilate isomerase (TRP1) and β-isopropylmalate dehydrogenase (LEU2).",
"As will be appreciated, expression vectors of the invention may also incorporate such marker genes.",
"Cloning vectors include cloning vectors for mammalian, yeast and insect cells.",
"Particular vectors that may find application include pBR322 based vectors and pUC vectors such as pUC118 and pUC119.Suitable expression and cloning vectors are for instance described in Molecular Cloning.",
"A Laboratory Manual., Sambrook et al., 2nd Ed.",
"Cold Spring Harbour Laboratory., 1989.Host cells suitable for being transformed by vectors of the invention include bacteria such as E. coli, Bacillus such as B. subtilis, Streptomyces and Pseudomonas bacterial strains, yeast such as Sacchromyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells.",
"Host cells may be cultured in a suitable culture medium and under conditions for facilitating expression of nucleic acid sequences of the invention or replication of cloning vectors, prior to purification from the host cells, and/or supernatants as the case may be using standard purification techniques.",
"Rather than utilising viral mediated transfection of cells, nucleic acid sequences and other molecules of the invention may also be delivered to a cell in vitro or in vivo by liposome mediated transfection.",
"The liposomes may carry targeting molecules for maximising delivery of the agent or agents contained therein to specific cell types of interest.",
"Such targeting molecules may be for instance antibodies, ligands or cell surface receptors for facilitating fusion of liposomes to the specific cells of interest.",
"Agents may also be intracellularly delivered in vitro using conventional cold or heat shock techniques or for instance, calcium phosphate coprecipitation or electroporation protocols.",
"Yet another strategy is to design the agent to have the inherent ability to pass across the lipid bilayer of a cell.",
"A particularly preferred way of achieving intracellular delivery of an agent is to use “carrier peptides” which have the ability to deliver macro-molecules across cell membranes in an energy-independent manner (Prociantz, 1996).",
"Indeed, such peptides provide the possibility of both testing potential agents in cell culture without drastically altering cell membrane integrity and of delivering agents in vivo.",
"Carrier peptides that are known in the art include penetratins and variants thereof (Derossi et al, 1994, 1996), human immunodeficiency virus Tat derived peptide (Prociantz, 1996), and transportan derived peptide (Pooga et al.",
"1998).",
"Indeed, carrier peptides have been successfully used to facilitate internalisation of mimetics of Src homology 2 binding sites, and peptides which inhibit protein kinase C mediated axon development and CD44 (hyaluronate receptor) dependent migration (Theodore et al, 1995; Williams et al, 1997; Peck Isacke, 1998; Derossi et al, 1998).",
"Specific targetting to β6-expressing cancer cells may also be achieved by coupling humanised anti-β6 antibody to carrier molecules such as penetratin coupled to an agent capable of inhibiting binding of a MAP kinase with an integrin expressed by the cell or down regulation of the expression of the integrin.",
"Coupling may for instance be by a peptide bond or disulfide bridge.",
"Given that β6 expression enhances effective proteolysis at the cell surface by matrix metalloproteinase-9 (Agrez et al, 1999), such targetting approaches may include engineering an MMP-9 cleavage site between the antibody and the carrier peptide penetratin to facilitate internalisation of the pentratin-agent complex.",
"Another approach may employ coupling the penetratin-agent complex to β6 integrin receptor-targetted peptides, targetted for binding to the extracellular β6 domain by virtue of their DLXXL sequence.",
"For example, a ligand recognition motif for αVβ6 integrin, RTDLDSLRTYTL (Kraft et al, 1999) may be used in conjunction with or without an engineered MMP-9 cleavage site to release the penetratin-agent complex at the cell surface.",
"Further protocol for targetting nucleic acids to cells by targetting integrins is described in Bachmann et al, 1998.The toxicity profile of an agent of the invention may be tested on normal and malignant cells by evaluation of cell morphology, trypan-blue exclusion, assessment of apoptosis and cell proliferation studies (eg cell counts, 3H-thymidine uptake and MTT assay).",
"Agents of the invention may be co-administered with one or more other compounds or drugs.",
"For example, an agent or agents may be administered in combination with antisense therapy or chemotherapeutic drugs.",
"Alternatively, an agent may be administered in conjunction with antisense therapy and/or chemotherapeutic drugs.",
"By “co-administered” is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes.",
"By “sequential” administration is meant a time difference of between the administration of the agents, drugs and other therapies which can be administered in any order.",
"The time difference may range from very short times up to hours or for instance days or weeks.",
"The agent or agents will typically be formulated into pharmaceutical composition incorporating pharmaceutically acceptable carriers, diluents and/or excipients for administration to the intended subject.",
"Pharmaceutical forms include sterile aqueous solutions suitable for injection, (where the agent or agents is water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.",
"Such injectable compositions will be fluid to the extent that the syringability exists and typically, will be stable to allow for storage after manufacture.",
"The carrier may be a solvent or dispersion medium containing one or more of ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils, and suitable mixtures thereof.",
"Fluidity may be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.",
"Sterile injectable solutions will typically be prepared by incorporating the active agents in the desired amount in the appropriate solvent with various other components enumerated above, prior to sterilising the solution by filtration.",
"Generally, dispersions will be prepared by incorporating the sterile active agents into a sterile vehicle which contains the dispersion medium and other components.",
"In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active agent plus any additional desired ingredient from previously sterile filtered solutions thereof.",
"For oral administration, the active agents may be formulated into any orally acceptable carrier deemed suitable.",
"In particular, the active ingredient may be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule.",
"Alternatively, it may be incorporated directly into food.",
"Moreover, an active agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and the like.",
"Such compositions will generally contain at least about 1% by weight of the active agent or agents.",
"The percentage may of course be varied and may conveniently be between about 5 to about 80% w/w of the composition or preparation.",
"As will be appreciated, the amount of active agent or agents in such compositions will be such that a suitable effective dosage will be delivered to the subject taking into account the proposed mode of administration.",
"Preferred oral compositions according to the invention will contain between about 0.1 μg and 2000 mg of each active agent, respectively.",
"Active agents may also be formulated into topically acceptable carriers conventionally used for forming creams, lotions, ointments and the like for internal or external application.",
"Topical formulations may be applied to a site to be treated by dressings and the like impregnated with the formulation.",
"Typically, a composition of the invention will incorporate one or more preservatives such as parabens, chlorobutanol, phenol, sorbic acid, and thimersal.",
"In many cases, a composition may furthermore include isotonic agents such as sugars or sodium chloride.",
"In addition, prolonged absorption of the composition may be brought about by the use in the compositions of agents for delaying absorption such as aluminium monosterate and gelatin.",
"Tablets, troches, pills, capsules and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium sterate; a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, orange or cherry flavouring.",
"When the dosage unit form is a capsule, it may contain in addition to one or more of the above ingredients a liquid carrier.",
"Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit.",
"For instance, tablets, pills or capsules may be coated with shellac, sugars or both.",
"In addition, an active agent may be incorporated into any suitable sustained-release preparation or formulation.",
"Pharmaceutically acceptable carriers, diluents and/or excipients include any suitable conventionally known solvents, dispersion media and isotonic preparations or solutions.",
"Use of such ingredients and media for pharmaceutically active substances is well known.",
"Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in therapeutic and prophylactic compositions is contemplated.",
"Supplementary active ingredients can also be incorporated into the compositions if desired.",
"It is particularly preferred to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.",
"Dosage unit form as used herein is to be taken to mean physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic or prophylactic effect in association with the relevant carrier, diluent and/or excipient.",
"A unit dosage formed will generally contain each active agent in amounts ranging from about 0.5 μg to about 2000 mg/ml of carrier respectively.",
"A pharmaceutical composition may also comprise vectors capable of transfecting target cells where the vector carries a nucleic acid molecule for modulating functional activity or expression of an integrin or MAP kinase.",
"The vector may for instance, be packaged into a suitable virus for delivery of the vector into target cells as described above.",
"The dosage of an active agent will depend on a number of factors including whether the agent is to be administered for prophylactic or therapeutic use, the condition for which the agent is intended to be administered, the severity of the condition, the age of the subject, and related factors including weight and general health of the subject as may be determined by the physician or attendant in accordance with accepted principles.",
"Indeed, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the subjects response.",
"Similarly, frequency of administration may be determined in the same way that is, by continuously monitoring the subjects response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.",
"The route of administration of a pharmaceutical composition will again depend on the nature of the condition for which the composition is to be administered.",
"Suitable routes of administration include but are not limited to respiritoraly, intratracheally, nasopharyngeally, intraveneously, intraperitonealy, subcutaneously, intracranialy, intradermially, intramuscularly, intraoccularly, intrathecally, intranasally, by infusion, orally, rectally, via IV group patch, topically and by implant.",
"With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply a tumour or particular organs to be treated.",
"Agents may also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into tumour tissue.",
"The production of antibodies and monoclonal antibodies is well established in the art (eg.",
"see Antibodies, A Laboratory Manual.",
"Harlow & Lane Eds.",
"Cold Spring Harbour Press, 1988).",
"For polyclonal antibodies, a mammal such as a sheep or rat for instance is immunised with a polypeptide of the invention and antisera subsequently isolated prior to purification of the antibodies therefrom by standard affinity chromatography techniques such as Sepharose-Protein A chromatography.",
"Desirably, the mammal is periodically challenged with the relevant antigen to establish and/or maintain high antibody titre.",
"To produce monoclonal antibodies, B lymphocytes can be isolated from the immunised mammal and fused with immortalising cells (eg.",
"myeloma cells) by standard somatic cell fusion techniques (eg.",
"utilising polyethylene glycol) to produce hybridoma cells (Kohler and Milstein, 1975; see also Handbook of Experimental Immunology, Weir et al Eds.",
"Blackwell Scientific Publications.",
"4th Ed.",
"1986).",
"The resulting hybridoma cells may then be screened for production of antibodies specific for the peptide by an enzyme linked immunosorbant assay (ELISA) or other immunoassay.",
"Conventionally used methods for preparing monoclonal antibodies include those involving the use of Epstein-Barr virus (Cole et al.",
"Monoclonal Antibodies and Cancer Therapy, Allen R. Liss Inc. pp.",
"77-96, 1985).",
"The term “antibody” or “antibodies” as used herein is to be taken to include within its scope entire intact antibodies as well as binding fragments thereof such as Fab and (Fab′)2 fragments which may be obtained by papain or pepsin proteolytic cleavage, respectively.",
"An antibody of the invention may be labelled for enabling detection of antibody binding in immunoassays including competitive inhibition assays.",
"A “label” may be any molecule which by its nature is capable of providing or causing the production of an analytically identifiable signal which allows the detection of an antibody and antigen complex.",
"Such detection may be qualitative or quantitative.",
"An antibody can for instance be labelled with radioisotopes including 32p, 125I or 131I, an enzyme, a fluorescent label, chemiluminescent molecule or for instance an affinity label such as biotin, avidin, streptavidin and the like.",
"An enzyme can be conjugated with an antibody by means of coupling agents such as gluteraldehyde, carbodiimides, or for instance periodate although a wide variety of conjugation techniques exist.",
"Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase amongst others.",
"Detection utilising enzymes is achieved with use of a suitable substrate for the selected enzyme.",
"The substrate is generally chosen for the production upon hydrolysis of a detectable colour change.",
"However, fluorogenic substrates may also be used which yield a fluorescence product rather than a chromogen.",
"Suitable fluorescent labels are those capable of being conjugated to an antibody substantially without altering the binding capacity of the antibody and include fluorescein, phycoerythrin (PE) and rhodamine which emit light at a characteristic wavelength in the colour range following illumination with light at a different wavelength.",
"Methods for labelling of antibodies can be found in Current Protocols in Molecular Biology.",
"Ausubel FM., John Wiley & Sons Inc. Immunoassays in which antibodies of the invention may be utilised include radioimmunoassays (RIA) and ELISA (eg., see Handbook of Experimental Immunology, Weir et al., Vol.",
"1-4, Blackwell Scientific Publications 4th Edition, 1986).",
"Such assays include those in which a target antigen is detected by direct binding with a labelled antibody, and those in which the target antigen is bound by a first antibody, typically immobilised on a solid substrate (eg., a microtitre tissue culture plate formed from a suitable plastics material such as polystyrene or the like) and a labelled second antibody specific for the first antibody used to form an antigen-first antibody-second antibody complex that is detected by a signal emitted by the label.",
"Sandwich techniques in which the antigen is immobilised by an antibody for presentation to a labelled second antibody specific for the antigen are also well known.",
"An antibody can be bound to a solid substrate covalently utilising commonly used amide or ester linkers, or by adsorption.",
"Optimal concentrations of antibodies, temperatures, incubation times and other assay conditions can be determined by the skilled addressee with reference to conventional assay methodology and the application of routine experimentation.",
"Antibodies and other molecules of the invention including polypeptides and oligonucleotides as described herein when bound to a solid support can be used in affinity chromatography for the purification of a binding partner for which they are specific.",
"In particular, a polypeptide either alone or as a fusion protein comprising or consisting of the binding domain for a MAP kinase for instance can be utilised in the purification, isolation or concentration of an integrin or fragment thereof or other molecule capable of binding to the binding domain of the MAP kinase for the integrin.",
"It may also be used for assaying levels of the integrin for instance in cell extracts.",
"Similarly, an antibody or other such molecule as described herein that specifically binds to the binding domain of a MAP kinase has use in the purification of the MAP kinase or fragments thereof incorporating a binding domain for the integrin from a relatively crude preparation or mixture.",
"Suitable solid supports include agarose, sepharose and other commercially available supports such as beads formed from latex, polystyrene, polypropylene, dextran, glass or synthetic resins, typically packed in an affinity column through which the relevant sample containing the binding partner is passed at a pH and conditions (eg., low salt concentration) under which the binding partner becomes bound by the antibody, polypeptide or other such molecule.",
"The column is then washed utilising a suitable buffer whereby the binding partner is retained bound on the column, prior to being eluted therefrom utilising a suitable elution buffer (eg., with a higher salt concentration and at an altered pH, typically pH 2.5 or pH 11) that facilitates the release of the binding partner from the affinity column, and collected.",
"Protocols for affinity chromatography are described in Current Protocols in Molecular Biology-Ausubel FM., John Wiley & Sons Inc. Buffers and conditions utilised for the purification, isolation or concentration of a binding partner will vary depending on the affinity the antibody, polypeptide or the like for the binding partner.",
"A kit for use in assays as described herein may include one or more of an antibody, polypeptide, vector, nucleic acid or other molecule of the invention.",
"The kit may also comprise one or more other reagents such as washing solutions, dilution buffers and the like together with instructions for use.",
"The antibody or other molecule of the invention may or may not be labelled, bound to a solid support or be coupled with another molecule.",
"Particularly preferred kits are those provided for use in affinity chromotography or RIA, ELISA or other type of immunoassay.",
"The present invention will be described herein after with reference to a number of examples.",
"EXAMPLE 1 Anti-integrin antibodies and antibodies against the β1 subunit have been shown to inhibit proliferation of retinal pigment epithelial cells (Hergott et al, 1993).",
"In endothelial cells, inhibition of cell-matrix interactions by anti-integrin antibodies specifically against α2β1 converts the cells from a proliferative to a differentiated phenotype (Gamble et al, 1993).",
"In another study, synthetic peptides containing the integrin recognition sequence arginine-glycine-aspartate (RGD) have been shown to inhibit tumour cell invasion in vitro and tumour metastases from melanoma in an animal model (Humphries et; al, 1986; Gehlsen et al, 1988).",
"In colon cancer, the contribution made by the α5β1 receptor to regulation of growth appears to be ligand (fibronectin)-dependent.",
"For example, induced expression of α5β1 in human colon cancer cells constitutively lacking this integrin has been shown to result in decreased tumour cell proliferation in vitro (Varner et al, 1995).",
"Interestingly, when the appropriate ligand was present, cell proliferation was restored, indicating that the unoccupied receptor mediated a negative growth signal in these cells (Varner et al, 1995).",
"Moreover, induction of α5β1 expression was associated with a marked reduction of tumourigenicity in immune-deficient mice.",
"Failure to ligate all of the tumour cell α5β1 molecules with sufficient murine fibronectin most likely accounts for the in vivo tumour suppression in these studies (Varner et al, 1995).",
"In the present study the effect of down-regulating αvβ6 expression on colon cancer growth was examined.",
"1.1 Methods 1.1.1 Generation of sense and antisense β6 constructs in pEF.PGK.puro vector.",
"For β6 antisense constructs, β6 cDNA was excised from the vector pcDNA1neo β6 (Weinacker et al, 1994) using the restriction enzymes SnaB1 and Xba1.This produced a 5′ overhang (Xba1) and a 3′ blunt end (SnaB1).",
"The 5′ overhang was blunted with Klenow (Promega) prior to ligation.",
"pEF.PGK.puro vector (a gift from D. Huang, the Walter & Eliza Hall Institute, Melbourne Australia) was cut with EcoRV which produced blunt ends.",
"The pEF.puro vector was de-phosphorylated using calf intestinal alkaline phosphatase (CIAP, Promega) and the β6 cDNA ligated overnight into pEF.puro using T4 DNA ligase (Promega) at a ratio of vector:insert of 1:5.After ligation all the reaction mix was used for transformation into competent JM109 cells and the cells plated onto LB plates containing ampicillin.",
"After overnight incubation at 37° C., colonies were selected, the plasmid DNA extracted by microprepping, and the DNA cut with BstE11 to confirm the antisense orientation of β6.Digestion with BstE11 produced the expected two fragments, one of 6.0 basepairs and the other 3.5 basepairs.",
"To provide β6 sense constructs, β6 cDNA was also excised from the pcDNA1neo b6 vector using the restriction enzymes Snab1 and Xba1 as described above.",
"The pEF.puro vector was then cut with Xba1 and EcoRV and the insert was ligated in sense direction into the pEF.PGK.puro vector without having to blunt the insert.",
"The ligation reaction was prepared at a ratio of vector:insert of 1:1.Digestion with BstE11 produced two expected fragments, one of 7.5 basepairs and the other 2.0 basepairs.",
"1.1.2 Transfection of WiDr and HT29 Colon Cancer Cells The human colon cancer cell lines, WiDr and HT29 were obtained from the American Type Culture Collection (ATCC), Rockville, Md., USA and maintained as monolayers in standard medium comprising Dulbecco's Modified Eagle's medium (DMEM; 4.5 gm/litre of glucose) with 10% heat-inactivated foetal bovine serum (FBS) supplemented with HEPES and penicillin/streptomycin.",
"WiDr and HT29 cell lines constitutively express αvβ6.Stable transfectants of WiDr and HT29 cells expressing β6 in sense and antisense direction were generated using lipofectamine and puromycin as the selection antibiotic.",
"One stable antisense β6 transfectant from HT29 cells and three stable clones from WiDr cells were successfully established for use in all experiments.",
"Mock transfectants using vector alone were also generated as controls.",
"Initial killing curves performed using a range of concentrations of puromycin established that WiDr and HT29 cells could be stably transfected with puromycin concentrations of 1.0 μg and 2.5 μg/ml, respectively.",
"1.1.3 Assessment of β6 Expression in the Transfected Cell Lines β6 expression was assessed by means of FACScan analyses of parent cell lines and clones generated therefrom by means of limiting-dilution experiments.",
"Stability of the transfectants was confirmed regularly by repeated FACScan analyses, and surface biotinylation and immunoprecipitation as described below.",
"1.1.4 FACScan Analyses Monolayer cultures of cell lines were harvested with trypsin/EDTA and then blocked with goat serum at 4° C. for 10 min.",
"Cells were washed once with PBS, incubated with primary antibody against integrin subunits for 20 min at 4° C. and then washed twice with PBS.",
"Cells were then stained with secondary antibody conjugated with phycoerythrin for 20 min at 4° C., washed twice with PBS and resuspended in 0.5 ml PBS prior to FACScan analysis (Becton Dickinson, Rutherford, N.J., USA).",
"1.1.5 Integrin Immunoprecipitation Cells were harvested with trypsin/EDTA, the trypsin neutralised with standard culture medium, and the cell pellets washed once with cold PBS.",
"Cell pellets were then exposed to biotin-CNHS-ester (Sigma) in biotinylation buffer (10 mM sodium borate, 150 mM sodium chloride, pH 8.8) for 30 mins at 4° C. with continuous slow mixing.",
"Cell pellets were then centrifuged at 4° C., washed twice with cold PBS and exposed to lysis buffer (containing 100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS and 0.1% NP-40 at pH 7.4 and containing 1 mM phenylmethylsulfonyl fluoride (PMSF)) for 30 min at 4° C. Lysates were then clarified by ultracentrifugation (10,000 g) for 30 min and the protein content measured using the BCA protein assay reagent kit.",
"Lysates containing equal protein amounts were pre-cleared with rabbit anti-mouse (RAM) immunoglobulin coupled to Sepharose-4B beads for 12 hrs.",
"Immunoprecipitations were carried out indirectly using RAM-Sepharose 4B and analysed by 7.5% SDS-PAGE under non-reducing conditions.",
"1.1.6 Reverse Transcriptase-PCR (RT-PCR) mRNA levels for β6 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR).",
"Total RNA was extracted from cell cultures using the commercial TriPure isolation reagent based on the method of Chomozynski and Sacchi (1987).",
"0.4-2 μg of RNA was used to prepared cDNA by reverse transcription.",
"Briefly, a reaction mixture in a final volume of 40 μl containing 8 μl of 5×RT reaction buffer (250 mM Tris, 15 mM MgCl2, 375 mM KCL, pH 8.3), 8 μl of 2.5 mM of each dNTP, 4 μl of 100 mM DTT, 40U of an Rnase inhibitor, Rnasin (Promega, Madison, Wis., USA), 0.5 μg of random hexamers (Promega) and 200U of Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Promega) were mixed and incubated at 38° C. for a minimum of 90 min.",
"The reaction was stopped by heating at 95° C. for 5 min and cDNA stored at 4° C. until PCR.",
"2-5 μl of this cDNA was combined with 5 μl of 10×PCR buffer (100 mM Tris, 500 mM KCl, 15 mM MgCl2, pH8.3) 8 μl of 1.25 mM dNTP each and 1.25 μl of 20 μM of both forward and reverse primers.",
"The forward primer sequence was 5′AGGATAGTTCTGTTTCCTGC3′ and the reverse primer sequence 5′ATCATAGGAATATTTGGAGG3′.",
"The reaction was initiated by 2.5U of Taq polymerase in a final volume of 50 μl.",
"After an initial 5 min incubation at 94° C., 30 cycles of amplification were performed under the following conditions: 94° C. 1 min, 54° C. 1 min and 72° C. for 1 min.",
"The reaction was stopped by incubating at 72° C. for 10 min.",
"To verify that equal amounts of RT product from cells were subjected to PCR amplification, the same amounts of cDNA were amplified for the “house-keeping” gene GAPDH using specific primers.",
"The same reaction conditions were used except that the annealing temperature was changed to 48° C. and PCR amplification performed for 35 cycles.",
"1.2 Results 1.2.1 αvβ6 Expression in HT29 and WiDr Transfected Cell Lines Transfection of the colon cancer cell lines HT29 and WiDr with the β6 gene construct in an antisense orientation resulted in a marked reduction of β6 expression at the transcript level and on the cell surface as shown in FIGS.",
"1 to 4.Transfection of cells with β6 in the sense orientation did not enhance β6 surface expression.",
"However, a consequence of down-regulation of the β6 subunit in antisense transfectants was a marked increase in surface expression of the β5 subunit.",
"The changes in surface expression of β6 and β5 subunits noted on FACScan analyses of antisense β6 transfectants was confirmed by surface-labelling cells with biotin and immunoprecipitating integrin subunits with either anti-β6 mAb (R6G9) or anti-β5 mAb (P1F6).",
"1.2.2 Effect of Suppression of αvβ6 Expression on Cell Binding to Fibronectin The major substrate for αvβ6 is fibronectin and to investigate the effect that reduction in β6 surface expression in antisense β6 transfectants might have on cell-matrix adhesion, WiDr and HT29 antisense β6 transfectants were seeded on fibronectin in adhesion assays.",
"Since both cell lines can adhere to fibronectin through members of the β1 integrin subfamily (either α3β1 or α5β1) αvβ6-mediated adhesion to fibronectin was assessed in the absence/presence of blocking anti-β1 antibody.",
"The cell-adhesion assays were performed in wells of non-tissue culture-treated polystyrene 96-well flat bottom microtitre plates (Nunc, Roskilde, Denmark).",
"Culture wells were coated with fibronectin, washed with phosphate-buffered saline (PBS) and then blocked with 0.5% bovine serum albumin (Sigma) in PBS for 1 hr at 37° C. Harvested cells were seeded at a density of 105 cells/well for HT29 and 1.5×105 cells/well for WiDr cells in 200 μl of standard DMEM which lacked FBS but contained 0.5% bovine serum albumin.",
"To block adhesion, cells were incubated with anti-β1 blocking antibodies for 15 min at 4° C. before plating.",
"The plates were centrifuged (top side up) at 10×g for 5 min, then incubated for 1 hr at 37° C. in humidified 5% carbon dioxide.",
"Non-adherent cells were removed by centrifugation top side down at 48×g for 5 min.",
"The attached cells were fixed and stained with 0.5% crystal violet (in 20% methanol and 1% formaldehyde) and the wells washed with phosphate-buffered saline.",
"The relative number of cells in each well was evaluated by measuring the absorbance at 595 nm in a Microplate Reader (Bio-Rad).",
"With reduction in cell surface expression of β6, β1-independent adhesion of cells to fibronectin was reduced compared with mock transfectants (containing vector alone) which express surface β6 at similar levels to wild-type cells.",
"The further addition of blocking anti-αv antibody completely prevented binding to fibronectin.",
"1.2.3 Effect of Suppression of αvβ6 Expression on Tumour Cell Proliferation and Tumour Growth In Vivo To investigate the effect of diminished αvβ6 surface expression on cell proliferation in vitro, WiDr and HT29 antisense β6 transfectants were seeded as monolayers in 96-well microtitre culture plates (5,000 viable cells per well) in standard culture medium containing the puromycin selection antibiotic.",
"Cells were pulsed with 1 μCi (3H)-thymidine (Amersham) per well for the last 24 hours of each experiment before automated harvesting and measurement of radioactivity.",
"WiDr wild-type, mock and antisense β6 transfectants, and HT29 wild-type, mock, sense β6 and antisense β6 transfectants were harvested second daily during a six-day culture period.",
"A marked increase in thymidine incorporation was observed for WiDR and HT29 cells expressing normal levels of αvβ6 compared with antisense β6 transfectants (see FIGS.",
"5 and 6).",
"1.2.4 Effect of Suppression of αvβ6 Expression on Tumour Formation The ability of HT29 antisense β6 transfactants to form tumours in immune-deficient mice was assessed.",
"BALB/C female athymic mice (8 weeks of age purchased from the Animal Resource Centre, Perth, Western Australia) were maintained under pathogen-free conditions and fed standard mouse chow and water ad lib.",
"The mice were divided into groups of ten each and all mice within each group inoculated with a single cell line.",
"Cells used were WiDr mock (transfected with vector alone) and antisense β6 transfected clones (clones 1-3) and HT29 mock, sense and antisense β6 cell lines.",
"Mice received subcutaneous flank injections of 106 viable tumour cells suspended in 0.2 ml of standard DMEM culture medium.",
"Animal weights and tumour sizes (breadth and length as measured with calipers) were recorded weekly.",
"Six weeks following the last injection, visible subcutaneous tumours were excised, weighed and fixed in 4% formalin.",
"At the time of euthanasia, all internal organs were routinely inspected for presence of metastases.",
"Tumour growth after six weeks following inoculation of HT29 mock and antisense β6 transfectants is shown in FIG.",
"7.Measurements of tumour growth for WiDr and HT29 cells are shown in FIGS.",
"8 and 9, respectively.",
"Similar tumour growth profiles for mock and antisense β6 WiDr clones 2 and 3 each inoculated into 10 mice are not shown.",
"Of a total of 40 mice injected with cells expressing antisense β6 (HT29-10 mice and WiDr cells lines, 3 clones-30 mice) tumours completely disappeared in 93% of animals.",
"In the remaining 3 animals tumour sizes diminished to 1 mm2 in size during the six week period compared with tumours at least 15 mm2 in size from cells expressing normal levels of β6.To confirm the presence of tumour xenografts histologically at one week following subcutaneous inoculation of mock and antisense β6 transfectants, tumour nodules were excised, fixed in formalin and stained with haematoxylin and eosin.",
"1.2.5 Effect of Suppression of αvβ6 Expression on Gelatinase B Secretion Serum-free tumour-conditioned medium was collected from each of the 3 WiDr mock and antisense β6 clones and concentrated ×44 for measurement of gelatinase B using the Biotrak MMP-9 activity assay system (Amersharn Pharmacia Biotech, Uppsala Sweden).",
"Down-regulation of αvβ6 expression resulted in a marked reduction in gelatinase B secretion.",
"1.3 Discussion of Results Induced expression of β6 in Chinese hamster ovary (CHO) cells has been shown to result in decreased surface expression of the β5 integrin subunit which also partners αv (Weinacker et al, 1994).",
"The concept of integrin switching depends on the availability of the promiscuous αv partner subunit.",
"In the present study, the reverse was observed.",
"As a consequence of down-regulation of β6 in colon cancer cells which constitutively express αvβ6, β5 surface expression increased, most likely secondary to increased availability of the αv subunit partner.",
"Heterologous expression of αvβ6 in colon cancer cells has previously been reported to enhance tumour growth in immune-deficient mice (Agrez et al, 1994).",
"Suppression of αvβ6 expression in the present study was shown to result in nearly complete disappearance of tumours in 93% of animals following subcutaneous inoculation of tumour cells.",
"Moreover, in the remaining 7% of animals, a 95% reduction in tumour size was observed over a six week period compared with large tumours seen in all animals injected with cells in which αvβ6 expression had not been perturbed.",
"Similar findings have been described with loss of the classical vitronectin receptor αvβ3 in melanoma.",
"For example, in experimental animal models, the loss of αvβ3 expression in melanoma cells has been shown to lead to reduced in vivo proliferation which is restored upon re-expression of the receptor (Felding-Habermann et al, 1992).",
"Although the mechanisms involved in αvβ6-mediated tumour growth remain to be elucidated, the present in vitro data show that loss of β6 expression is associated with decreased proliferative capacity of the cells.",
"Taken together with the marked reduction in gelatinase B secretion seen for colon cancer cells transfected with antisense β6, the findings reported in the present study suggest that intracellular signalling pathways activated via the integrin αvβ6 play a major role in promoting progression of this tumour type.",
"EXAMPLE 2 The association of αvβ6 expression and MAP kinase activity were evaluated using WiDr, HT29 and SW480 cell lines.",
"2.1 Methods 2.1.1 SW480 Colon Cancer β6 Transfectants Stable transfectants of SW480 colon cancer cells (ATCC) expressing gene constructs of either wild-type or mutant forms of the β6 integrin subunit or the expression plasmid only (pcDNA1neo) have been previously described (Agrez et al, 1994).",
"The transfected SW480 cell lines were maintained in standard medium supplemented with the neomycin analogue G418.Stable transfectants of WiDr and HT29 cells expressing wild-type β6 in antisense orientation were generated as described in Example 1.1.2 and maintained in standard medium supplemented with puromycin.",
"2.1.1 MAP Kinase Assay.",
"Cultures of WiDr and HT29 mock and antisense β6 transfectants were established by seeding 1×106 cells/5 ml of culture medium in 25 cm2 tissue culture flasks.",
"Cells were incubated at 37° C. in humidified CO2 for 24 hours before serum starvation in serum-free medium for the next 16 hours.",
"Foetal calf serum was then added to a final concentration of 10% for 30 mins before MAP kinase assays were performed.",
"Before each experiment the cells were washed twice with PBS, resuspended in extraction buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM orthovanadate, 1 mM PMSF, 4 μg/ml aprotonin, 2 μg/ml leupeptin and 1 μg/ml pepstatin, pH 7.4) and sonicated at a setting of 7, using a Soniprep 150 watt ultrasonic disintegrator for a total of 90 seconds in three 30 second pulses with an interval of 30 seconds between each pulse.",
"Cellular debris was removed by centrifugation at 900 g for 10 min at 4° C. The assay was performed on equal cell numbers using a MAP kinase assay system (Amersham Pharmacia Biotech, Uppsala Sweden).",
"The ability of cells to transfer phosphate from [γ32P]-ATP to a synthetic peptide that contains specifically a p42/p44 MAP kinase phosphorylation site was measured as described in the manufacturer's instructions.",
"[32P]-labelled peptides were spotted onto PEI-cellulose paper, unbound radioactivity was washed with 75 mM phosphoric acid and bound [32P]-labelled peptides were measured by liquid scintillation counting.",
"Protein estimation was performed on each cell lysate used and enzyme activity calculated as described in the manufacturer's instructions.",
"Where MEK inhibitors, PD98059 and U0126 were used, cells were cultured as described above and the inhibitors, at a final concentration of 40 μM, were added one hour before the addition of serum to the medium.",
"2.1.2 Western Blotting To detect the MAP kinases ERK1/2, cells were lysed in lysis buffer containing 100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS and 0.5% NP-40 at pH7.4, supplemented with enzyme inhibitors (1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/ml pepstatin A, 2.5 μg/ml aprotonin, 1 mM benzamidine, 1 μg/ml leupeptin).",
"Lysates were clarified by ultracentrifugation and equal protein loads electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions.",
"Electrophoresed proteins were transferred to nitrocellulose membranes (Biotrace NT, Gelman Sciences, Ann Arbor, Mich.) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS) for 2 hours at a constant voltage of 40 volts in a Transfer Blot Cell (Bio-Rad).",
"Membranes were blocked with 5% casein for 1 hr at room temperature and probed with monoclonal anti-ERK antibodies (E10 which recognises only phosphorylated ERK1/2 (New England BioLabs) and SC-1647 which recognises total ERKs, non-phosphorylated and phosphorylated (Santa Cruz Biotechnology).",
"In some experiments, membranes were probed with polyclonal anti-ERK antibody (New England BioLabs) which also recognises total ERKs.",
"Membranes were then washed three times in Tris buffered saline, containing 0.1% Tween and incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit antibody.",
"Blots were visualised by the enhanced chemi-luminescence detection system according to the manufacturer's instructions (Du Pont).",
"2.1.3 Integrin β-Subunit Immunoprecipitations Tumour cells were harvested and divided into two equal aliquots based on cell counts.",
"One aliquot was surface biotinylated and the cells lysed for integrin immunoprecipitations as described in Example 1.1.5, with the exception that three sequential rounds of immuno-precipitation were performed to deplete the lysates of integrin subunits β5 and β6.Lysis buffer was the same as that used for Western blotting.",
"The other aliquot, intended for subsequent ERK2 immunoblotting to examine the effect of integrin immunodepletion on ERKs was not biotinylated but lysed immediately and divided into three samples each at a protein concentration of 1 mg/ml.",
"Three sequential rounds of immunoprecipitation were performed against anti-β6 monoclonal antibody, R6G9 (using isotype matched antibody, IgG2A, and anti-β5 monoclonal antibody, P1F6, in control immunoprecipitations).",
"The integrin-depleted lysates were electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membranes.",
"Membranes were blocked with casein as for Western blotting and probed with anti-ERK monoclonal antibodies E10 and SC-1647.In parallel experiments, the sequentially immunoprecipitated β6 subunit bound to rabbit anti-mouse (RAM) coupled Sepharose B4 beads was also electrophoresed in 10% SDS-PAGE under non-reducing conditions, transferred to nitrocellulose membranes, and the membranes probed with anti-ERK monoclonal antibody E10 which recognises only phosphorylated forms of ERK1/2.2.3 Results 2.3.1 Effect of Serum on MAP Kinase Activity The effect of MEK (MAP kinase) inhibitors UO126 and PD98059 on MAP kinase activity in WiDr and HT29 wild-type cells was tested in the absence/presence of serum.",
"Adherent cell monolayers on plastic were grown for 24 hrs in standard culture medium, then washed three times in PBS followed by 16 hrs in culture under serum-free conditions.",
"Serum was then added for 30 min and MAP kinase activity assessed before and after addition of serum.",
"The addition of serum markedly stimulated MAP kinase activity for both cell lines and was inhibitable by both MEK inhibitors.",
"2.3.2 Effect of Altered β6 Expression on MAP Kinase Activity Following Serum Stimulation.",
"In these experiments, WiDr transfectants (3 mock and 3 antisense β6 clones), HT29 transfectants (mock and antisense β6) and SW480 β6 transfectants (mock and sense β6) were serum-starved for 16 hrs followed by 30 mins exposure to serum.",
"Increased expression of αvβ6 was associated with a marked increase in MAP kinase activity upon serum stimulation compared with cells lacking αvβ6 altogether (SW480 mock).",
"Cells in which β6 expression had been down-regulated (antisense β6 transfectants) exhibited suppressed MAP kinase activity.",
"Induced expression of β6 in the non-β6-expressing colon cancer cell line SW480 resulted in a three fold increase in serum-dependent MAP kinase activity.",
"2.3.3 αvβ6 Binds an Extracellular Signal-Related Kinase (ERK).",
"A highly surprising finding arising from phage display screening which underpins the present work is that the integrin β6 cytoplasmic domain binds a MAP kinase.",
"Use of phage display to screen a γgt11 cDNA colon cancer cell library with the β6 cytoplasmic domain as bait yielded a clone which coded for ERK2 at the 3′ end with 100% nucleotide sequence identity (across 393 bases) to the published sequence of ERK2 (Boulton et al, 1991).",
"This putative association was investigated further.",
"Integrin immunoprecipitations were performed on equal protein loads of tumour cell lysates and the transferred proteins blotted with antibodies recognising phosphorylated and non-phosphorylated forms of ERK1/2.The specificity of this interaction was examined in integrin immunoprecipitations against β1, β5, β6 and αv integrin subunits.",
"As shown in FIG.",
"10, the anti-ERK mAb′SC-1647 identified a β6-specific band migrating at the position of purified ERK2 protein.",
"The integrin-associated ERK band was identified only in immunoprecipitations of the β6 subunit and its partner αv and not in immunoprecipitations of the β1/β5 subunits or in immunoprecipitations using isotype-matched control antibodies.",
"2.3.4 Effect of Altered β6 Expression on Total Cellular ERK and β6-Bound ERK.",
"Equal protein loads from one representative clone each from WiDr mock and antisense β6 transfectants were electrophoresed, transferred to nitrocellulose and blotted with anti-ERK mAb (SC-1647) as shown in FIG.",
"11(A).",
"β6 immuno-precipitates from equal protein loads of the WiDr mock and antisense β6 clones were electrophoresed, transferred and probed with anti-ERK mAb (E10) as shown in FIG.",
"11(B).",
"As indicated, suppression of β6 expression resulted in a reduction of both total cellular and phosphorylated integrin-associated ERK compared with WiDr mock transfectants.",
"Similarly, β6 immunoprecipitations from SW480 β6 transfected clones expressing high and low levels of β6 (confirmed by FACScan and β6 immunoprecipitations) showed parallel changes in β6-bound phosphorylated ERK (FIG.",
"12).",
"2.3.5 Effect of β6 Immunodepletion on Total Cellular ERK and β6-Bound ERK WiDr wild-type cells were surface biotinylated and the cell lysates immunodepleted of β6 in three rounds of sequential immunoprecipitation using anti-β6 mAb (R6G9) resulting in a marked loss of β6 from the lysates as shown in FIG.",
"13(A).",
"β6-immunodepleted lysates were then transferred and blotted with anti-ERK1/2 antibody (SC-1647), which recognises both phosphorylated and non-phosphorylated forms of ERK1/2.As shown in FIG.",
"13(B), following three rounds of β6 immunodepletion, levels of ERK1/2 in β6-depleted lysates compared with non-immunodepleted lysates, were markedly reduced suggesting a significant contribution of β6-bound ERK to total cellular ERK.",
"In contrast, immunodepletion of β5 by three successive rounds of immunoprecipitation with mAb P1F6 or isotype-matched control antibody IgG2A did not result in any reduction of cellular ERK levels compared with non-immunodepleted cell lysates.",
"2.3.6 Effect of ERK Immunodepletion on β6-Bound ERK Cell lysates from WiDr wild-type cells were immunodepleted of ERK1/2 by means of sequential immunoprecipitations using the anti-ERK mAb, SC-1647.ERK-immunodepleted cell lysates probed with either E10 or SC-1647 mAbs contained markedly less ERKs.",
"To examine the effect of ERK-immunodepletion on β6-bound ERK, the ERK-immunodepleted cell lysates were immunoprecipitated with anti-β6 mAb (R6G9) and the β6-immunoprecipitates probed with anti-ERK mAb (E10 recognising phosphorylated ERK1/2).",
"ERK immunodepletion effectively reduced levels of β6-bound ERK.",
"2.3.7 Effect of Preventing Cell Attachment on αvβ6-Bound ERK SW480 mock and β6 expressing transfected cells were grown to 80-90% confluency.",
"Cells were washed twice with PBS and harvested after trypsinization.",
"Cells were divided into three equal portions (approximately 4×106/batch).",
"The first group was plated on a 75 cm2 plastic flask in normal 10% serum containing medium (attached cells) while the other two groups were plated on 0.3% agarose underlay in serum free medium (non-attached cells).",
"Cells were allowed to grow for 24 hours at 37° C., after which in one group of 0.3% agarose underlay, 15 ml of serum-free medium was added while in the other an equal volume of 10% serum-containing medium was added for 30 mins.",
"Cells were collected washed with PBS and lysed in cell lysis buffer (100 mM Tris-HCl pH-7.5, 150 mM NaCl, 1 mM CaCl2, 1% Triton, 0.1% SDS, 0.1% Np-40, 1 mM vanadate, 1 μg/ml pepstatin, 1 mM PMSF, 5 μg/ml aprotonin and 1 μg/ml of leupeptin).",
"10 μl (10 μg of protein) was used for the analyses of cell lysates after adding equal volumes of non-reducing Laemmli buffer.",
"For SW480-β6-transfected cells the rest of the cell lysate was used for immunoprecipitation of αvβ6 integrin.",
"Cell lysates and β6 immunoprecipitates were subjected to western blotting and probed with E10 monoclonal antibody (recognising phosphorylated ERK1/2).",
"Cell lysates from the non-attached cells were found to require serum factors to maintain phosphorylation of total cellular ERK.",
"In contrast, non-attached SW480 β6 colon cancer cells do not require serum factors to maintain the activation phosphorylation) state of β6-bound ERK.",
"2.3.8 Effect of PP2A Phosphatase on αvβ6-Bound ERK In experiments to examine the effect of protein phosphatase 2A (PP2A) on β6-bound ERK, SW480 β6-transfected cells were sonicated in buffer comprising 5 mM Tric-HCl (pH 8.0), 10 mM MgCl2 and 0.01 mM EGTA together with enzyme inhibitors, and cell lysates from both serum-starved and serum-induced cells treated with 0.5 units of PP2A (Promega) at 30° C. for 10 minutes.",
"The reaction mixture was stopped by addition of equal volumes of non-reducing Laemmli sample buffer.",
"In parallel, PP2A-treated cell lysates were immunoprecipitated with mAb R6G9 (anti-β6) followed by Western blot with anti-ERK mAb E10.MAP kinase activity assays were performed according to the manufacturer's instructions (Amersham Pharmacia Biotec) using γ32P-ATP.",
"Exposure of cell lysates prepared from serum-supplemented and serum-starved cells to the PP2A catalytic subunit resulted in dephosphorylation of total ERK.",
"In contrast, dephosphorylation of β6-bound ERK was not observed in β6 immunoprecipitates prepared from serum-supplemented or serum-starved cells β6 Bound ERK may therefore serve to maintain adjacent growth factor receptors in an activated state and thereby alter their sensitivity to exogenous co-factors.",
"2.3.9 Inhibition of MAP Kinase Activity Inhibits Secretion of Gelatinase B. SW480 β6 transfectants were cultured under serum-free conditions for 48 hours in the absence/presence of the MEK inhibitor PD98059 (40 μM) or DMSO (vehicle control).",
"Tumour-conditioned medium was assayed for gelatinase B by analysis of equal protein loads in a gelatin zymogram.",
"Inhibition of MAP kinase activity by the MEK inhibitor reduced gelatinase B secretion compared with controls.",
"2.3.10 β6-ERK2 Association in HaCaT and HaRas Cell Lines β6 immunoprecipitates were prepared from human kerotinocyte cell lines (HaCaT and HaRas were obtained from Prof N. Fusenig, The German Cancer Research Institute, Heidelberg, Germany) using mAb R6G9 (anti-β6) and the immunoprecipitates probed with mab E10 (against phosphorylated ERK 1/2).",
"FIG.",
"14 shows that ERK2 associates with β6 in both of HaCat and HaRas cells.",
"2.4 Discussion of Results The MAP kinase pathway has been shown to be important in experimental tumour metastases (Mansour et al, 1994) and recent data implicate MAP kinases in tumour growth and invasiveness of colon cancer cells (Sebolt-Leopold et al, 1999).",
"In the present study, up-/down-regulation of β6 expression in various colon cancer cell lines was shown to enhance/suppress respectively, MAP kinase activity.",
"The presence of serum induced a three-fold increase in MAP kinase activity above that observed for serum-starved β6-expressing cells.",
"In contrast, only a one-fold increase in serum-dependent MAP kinase activity was observed for cells in which β6 had been down-regulated consequent upon transfection with antisense β6.Moreover, induced expression of β6 in the non-β6-expressing colon cancer cell line SW480, was associated with a three-fold increase in serum-dependent MAP kinase activity.",
"Serum contains a mixture of growth factors raising the possibility that one role for αvβ6 in colon cancer cells is to lower the threshold for activation of MAP kinase signalling pathways at times when the supply of serum-containing growth factors is limited.",
"The ERK band co-immunoprecipitated with β6 migrated either at or 1-2 kD higher than the mobility of purified phosphorylated ERK2 depending on the acrylamide concentration used in SDS-PAGE showing that the kinase does indeed, associate with the β6 subunit.",
"Slight differences in mobility of the β6-associated ERK band compared with the pure ERK protein could arise if β6-bound ERK is hyper-phosphorylated and/or exists in an altered conformation consequent upon its association with β6.ERK bound to β6 may also be an alternatively spliced variant of ERK2 causing it to migrate differently.",
"Finally, the purified ERK2 protein is derived from mouse which differs slightly from human ERK2 by being two amino acid residues shorter and also containing a single amino acid substitution.",
"In the present study, increased/decreased β6 expression in colon cancer cell lines was associated with increased/decreased β6-bound ERK, respectively.",
"In addition, immunodepletion experiments suggest that β6-bound ERK makes a substantial contribution to total phosphorylated ERK within the cell and overall MAP kinase activity (see FIGS.",
"15(A) and 15(B)).",
"The observation that β6-mediated colon cancer growth in vitro is inhibitable by a matrix metalloproteinase inhibitor (Agrez et al, 1999) suggests that the increased gelatinase B secretion by β6-expressing colon cancer cells contributes to tumour progression.",
"Taken together with the finding that inhibition of MAP kinase activity by the MEK inhibitor PD98059 diminished gelatinase B secretion in β6-expressing cells, it seems that activation of MAP kinase signalling plays a role, at least in part, in β6-mediated induction of gelatinase B secretion.",
"EXAMPLE 3 3.1 Identification of the Binding Domain on the β6 Subunit Cytoplasmic Tail Domain for ERK2 Peptide fragments corresponding to regions of the cytoplasmic tail domain of the β6 subunit were screened in an enzyme-linked immunosorbent assay (ELISA) for binding with ERK2.The 52 amino acid long β6 cytoplasmic tail is shown in FIG.",
"16 as are the amino acid sequences for the cytoplasmic domains of the β1 to β3 subunits.",
"In particular, four synthetic peptides designated fragment 1 to fragment 4 were prepared and biotinylated at the N-terminal end of each, respectively (Auspep Pty Ltd, Melbourne Australia).",
"The region of the β6 tail to which each corresponds is indicated in FIG.",
"16 and set out below.",
"Fragment 1: HDRKEVAKFEAERSKAKWQTGT Fragment 2: RSKAKWQTGTNPLYRGSTST Fragment 3: NPLYRGSTSTFKNVTYKHRE Fragment 4: FKNVTYKHREKQKVDLSTDS The fragments overlap by 10 amino acids and are each 20 amino acids long with the exception of the fragment 1 with a length of 22 amino acids.",
"Fragment 4 was synthesised with a terminal serine rather than a cysteine as found in wild-type β6 to avoid formation of a disulfide bridge between peptides.",
"Overlapping biotinylated fragments 1 to 4 were coated onto streptavidin coated polystyrene plates (Pierce, Rockford Ill. USA, Cat No.",
"15125) and the ELISA performed substantially according to manufacturers instructions.",
"Briefly, wells are washed with 3×200 μl of wash buffer (TBS, 0.1% BSA, 0.05% Tween 20 or SuperBlock™ blocking buffer in TBS, Pierce, Prod.",
"No.",
"37535) prior to addition of biotinylated peptides (100 μl) and incubation for 1 hr at room temperature to allow for capture of the peptides on the plate.",
"Following another washing step, peptides were overlayed with GST-ERK, ERK (or JNK-1) alone at a volume of 100 μl per well, and the plates incubated for a further 1 hr at room temperature before removal of any unbound ERK by further washing.",
"Binding of ERK to the peptides is detected using 100 μl anti-ERK1/2 mAb SC1647 (Santa Cruz) as the primary antibody at a dilution of 1:700 (isotype matched antibody IgG2b is used as a control).",
"This is followed by another washing step and addition of 100 μl rabbit anti-mouse antibody (Biorad) conjugated to alkaline phosphatase at a concentration of 1:1000 for 30 minutes again at room temperature.",
"A 10 aliquot of detection reagent (alkaline phosphatase detection kit-Biorad) is then introduced into each well after a final washing step and allowed to react for 15-30 minutes at room temperature in the dark before absorbance is measured at 405 nm.",
"All dilutions of peptides, MAP kinase and antibodies were performed using the wash buffer.",
"GST-ERK is a fusion protein consisting of ERK coupled to glutathione-S-transferase and purified from host cells transfected with pGEX vector.",
"As shown in FIG.",
"17, significant binding of non-phosphorylated GST.ERK2 (0.25 μg/100 μl) to peptide fragment 2 was observed (1 μg/100 μl) while only negligible or low level binding for the other fragments was found.",
"Significant binding of non-phosphorylated ERK2 to both fragment 2 and β6 cytoplasmic tail peptide compared to fragments 1, 3 and 4 over a range of concentrations of ERK2 was also observed (see FIG.",
"18).",
"Similar results were observed using a range of concentrations of the peptide fragments as shown in FIG.",
"19.To further localise the binding domain on the cytoplasmic tail of the β6 subunit, progressively shorter peptides from the region of the β6 cytoplasmic tail corresponding to peptide fragment 2 were synthesised, biotinylated and the capacity to associate or otherwise bind to ERK2 assessed as described above.",
"The binding of GST.ERK2 to a 15 mer test peptide (seq.",
"4) having the amino acid sequence RSKAKWQTGTNPLYR and a 10 mer test peptide having the sequence RSKAKWQTGT is shown in FIG.",
"20 compared to fragment 2 over a range of concentrations of the peptides.",
"As can be seen, no reduction in binding to the seq.",
"4 peptide compared to fragment 2 was found.",
"Binding of ERK2 to the seq.",
"3 peptide was substantially less than that observed for seq.",
"4.A number of 10 mer biotinylated peptides corresponding to regions of fragment 2 or fragment 3 were then tested.",
"The amino acid sequence for each peptide is as follows and their location in the β6 cytoplasmic domain is indicated in FIG.",
"21.10(1): NPLYRGSTST 10(2): WQTGTNPLYR 10(3): KFEAERSKAK The results are set out in FIG.",
"22 and show that GST.ERK binding to the 10 mer peptides is substantially reduced compared to binding to the seq.",
"4 peptide suggesting that opposite end regions of seq.",
"4 participate in the binding of ERK2.Comparable binding of ERK2 to seq.",
"4 was found using a further 10 mer peptide identified as 10(4) in which amino acid sequence WQTGT of seq.",
"4 is omitted indicating that WQTGT is a linker sequence that does not participate directly in the binding of ERK to seq.",
"4.Negligible binding of ERK2 to the 5 mer peptide RSKAK was observed as shown in FIG.",
"23.ERK2 cleaved from GST-ERK2 by thrombin was used in this assay.",
"Results (not shown) indicate that greater than a 3 fold increase in assay sensitivity can be achieved using thrombin cleaved ERK2 rather than GST-ERK2.EXAMPLE 4 4.1 MAP Kinase JNK-1 Binds to the Cytoplasmic Tail Domain of β6 In view of the observation that ERK2 associates with the cytoplasmic tail of the β6 subunit, the MAP kinase JNK-1 was tested to evaluate whether it also could associate with the cytoplasmic tail of β6.Briefly, 0.05-1.5 μm/100 μl of JNK-1 (Santa Cruz) was aliquoted into wells of a 96 well culture place containing increasing concentrations of the β6 cytoplasmic domain tail peptide used in Example 3.For comparison purposes, non-phosphorylated GST-ERK2 (0.05-1.5 μm/100 μl) was aliquoted into wells containing β6 cytoplasmic tail peptide or peptide fragments 1 or 2, respectively.",
"Binding of JNK-1 was detected using mouse anti-JNK-1 mAb SC 474-G (Santa Cruz) and HRP-conjugated goat anti-mouse antibody.",
"Absorbance was read at 405 nm and the results are shown in FIG.",
"24.Significant binding of JNK-1 to the β6 cytoplasmic tail peptide was found.",
"EXAMPLE 5 5.1 Evaluation of Ability of ERK2 to Bind to β6 Δ746-764 Deletion Mutant.",
"To examine the role of the amino acid sequence RSKAKWQTGTNPLYR in the β6 cytoplasmic domain in situ, a β6 deletion construct lacking the coding sequence for AERSKAKWOTGTNPLYRG was transfected into colon cancer cell line SW480 which does not constitutively express the α0Vβ6 integrin using the calcium phosphate method previously described for transfections into this cell line (Agrez et al, 1994).",
"The location of the β6 Δ746-764 deletion is indicated in FIG.",
"25.Construction of the β6 Δ746-764 deletion mutant in the vector pcDNA1neo and failure of the expressed receptor to localise to focal adhesions in Chinese hamster ovary cells has been reported (Cone et al, 1994).",
"Facscan analysis revealed comparable levels of surface expression of mutant β6 to that seen for the full length wild-type receptor (see FIG.",
"26).",
"Equal protein loads of cell lysates prepared from SW480 cells were immunoprecipitated with either anti-β6 monoclonal antibody (mAb R6G9) or matched isotype control antibody.",
"Surface biotinylation prior to immunoprecipitation confirmed equal surface expression of mutant and wild-type β6 (see FIG.",
"27(A).",
"Aliquots of the immunoprecipitates were electrophoresed and transferred to nitrocellulose for Western blotting using monoclonal antibody E10 which recognises ERK1/2.As seen in FIG.",
"27(B), loss of the RSKAKWQTGTNPLYR sequence in the β6 cytoplasmic domain reduced levels of β6-bound ERK by greater than approximately 75% of that observed for the wild type receptor.",
"EXAMPLE 6 6.1 Growth Inhibition Study.",
"HT29 and SW480 β6-expressing colon cancer cell lines were seeded into wells of 96-well microtitre plates (Nunclon) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, glutamine, Hepes, and antibiotics.",
"Seeding cell densities were 3×103 cells per triplicate well for each condition tested and after 24 hours incubation of cell cultures in 5% CO2, 100% humidity at 37° C., the culture medium was exchanged for serum-free DMEM medium supplemented with insulin, transferrin, selenous acid, hydrocortisone, non-essential amino acids, glutamine, Hepes and antibiotics containing either peptide RSKAKWQTGTNPLYR alone or penetratin-peptide complex at a concentration of 10 μm for HT29 cells or 30 μm for SW480 β6-expressing cells.",
"Cell cultures were incubated for a further 24 hours following which cultures were photographed (Kodak Techpan Film at 100 ASA setting) and the experiments terminated by addition of the cell proliferation reagent WST-1 (Boehringer Mannheim) to monitor effects of the peptide on cell growth.",
"The cell proliferation reagent WST-1 is designed to be used for the non-radioactive, spectrophotometric quantification of cell growth and viability in proliferation and chemosensitivity assays.",
"The colourmetric assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells.",
"Specifically, at the termination of experiments, 30 μl of WST-1 was added to 270 μl culture medium volume in each microtitre well and the colour change quantitated in an ELISA plate reader by measuring absorbance of the formazan product at 450 mn (using a reference wavelength of more than 600 nm).",
"The mean absorbance readings from triplicate wells (±standard error of the means) after subtraction of background control wells (culture medium without cells) was determined.",
"Only the carrier penetratin-peptide complex was effective in inhibiting cell proliferation in contrast to either peptide or penetratin alone as shown in FIGS.",
"28 and 29 indicating that the penetratin-peptide complex was internalised by both the HT29 and SW480 cells resulting in the observed suppression of colon cancer growth.",
"Photographs of the SW480 cells treated with penetratin alone or the penetratin-peptide complex are shown in FIG.",
"30(A) to (C).",
"EXAMPLE 7 7.1 Growth Inhibition in SW480 Mock and SW480 β6 Transfectants.",
"SW480 mock and SW480 β6 transfectants were cultured in DMEM medium supplemented with 1% foetal bovine serum in the presence of 20 μM seq.",
"4 coupled to penetratin.",
"Percentage inhibition was assessed by the WST-1 colorimetric dehydrogenase assay described in Example 6.The percentage inhibition of growth observed for the −β6 and +β6 expressing cells was 17% and 50%, respectively as indicated in FIG.",
"31A.",
"The −β6 and +β cultured cells are shown in FIG.",
"31B.",
"EXAMPLE 8 The proliferation of SW480 cells expressing a β6 Δ746-764 deletion mutant was compared with non-β6 expressing SW480 cells and SW480 cells expressing full length wild-type β6.Cells were cultured for 10 days within a 3-dimensional collagen type I matrix.",
"Collagen gels were prepared as bilayers (upper layer cell-containing and lower layer minus cells) in 24 well culture plates as previously described (Agrez, 1989; Agrez, 1994) except for the use of 5% foetal bovine serum as the supplement for DMEM.",
"Colonies were photographed in the gel FIG.",
"32(A) at 10 days (bar represents 200μ) and following dissolution of the collagen with collagenase FIG.",
"32(B) prior to visual colony counting of all colonies exceeding 200μ in diameter within each well FIG.",
"32(C).",
"As can be seen, significant proliferation of the SW480 cells expressing the full length wild-type β6 was observed compared to the non-β6 expressing cells and cells expressing the β6 Δ746-764 deletion mutant.",
"EXAMPLE 9 Growth inhibition of SW480 cells expressing full length wild-type β6 exposed to seq.4 coupled to penetratin or RSKAKWQTGTNPLYR peptide coupled to penetratin (5, 10, 20, 30 μM in DMEM minus foetal bovine serum) but which peptide contained alanine substitutions at the four positions indicated was assessed.",
"As shown in FIG.",
"33(A) and FIG.",
"33(B), progressive inhibition of proliferation in a dose-response manner was observed for the seq.4 penetratin complex compared with the alanine substituted peptide-penetratin complex which was without effect at all doses tested.",
"EXAMPLE 10 Binding of ERK2 to the seq.4 peptide (RSKAKWQTGTNPLYR) was compared with peptides corresponding to regions of the cytoplasmic domain of integrin subunits β1, β2, β3 and β5.The amino acid sequences for those peptides is shown below: β3 KFEKEKMNAKWDTGENPIYK β2 KEKLKSQWNNDNPLFK β3 RARAKWDTANNPLYK β5 RSRARYEMASNPLYR As shown in FIG.",
"34, significant binding of ERK2 to the seq.",
"4 peptide was observed.",
"Binding of ERK2 to the β5 and β3 peptides was also found.",
"The results have been corrected for non-specific binding and indicate a hierarchy of binding of ERK2 to integrin subunits.",
"EXAMPLE 11 Angiogenesis, the growth of new blood vessels, plays a role in diverse disease states including progression of cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, atherosclerosis, pregnancy and endometriosis.",
"For examples, in cancers, the ability of microscopic tumour deposits to enlarge beyond 1-2 mms in diameter is dependent upon tumour-induced angiogenesis or sprouting of new host-derived blood vessels to nourish enlarging tumour cell nodules.",
"The mitogen activated protein (MAP) kinase signalling pathway is critical for endothelial cell proliferation and new blood vessel formation.",
"Moreover, a relationship exists between extracellular signal-regulated (ERK) kinase 1/2 activation and vascular endothelial growth factor (VEGF) expression with each activating the other (Gupta et al, 1999; Yu & Sato, 1999: Pages et al, 2000).",
"Two growth factor-dependant pathways of angiogenisis have now been shown to exist defined by their dependency on the distinct vascular integrins αvβ3 and αvβ5 (Friedlander et al, 1995: Eliceiri and Cheresh, 1999).",
"It is noteworthy that the vascular system lacks αvβ6 expression (Breuss et al, 1993).",
"Antibodies against αvβ5 and inhibition of protein kinase C signalling have both been shown to block VEGF-induced angiogenesis (Freidlander et al, 1995).",
"The importance of αvβ5 in neovascularisation is further highlighted by the observation that β3 integrin-deficient mice exhibit normal blood vessel development in contrast to αv-deficient mice which implicates other beta integrin partners such as β5 (Eliceiri and Cheresh, 1999).",
"11.1 The β5 Integrin Subunit Binds ERK in Cells that Either Lack β6 or Express β6 Lacking the Binding Site for ERK2 The SW480 cell line expresses abundant αvβ5 but only minimal levels of αvβ3 and lacks αvβ1 and αvβ6.SW480 cells provide a model, therefore, with which to test whether the β5 integrin subunit can co-immunoprecipitate with ERK either in the absence of constitutive β6 expression or in the presence of β6 that lacks the binding site for ERK2 (RSKAKWQTGTNPLYR).",
"To evaluate this, cell lysates containing equal protein loads were prepared from SW480 wild-type cells and SW480 cells transfected with either vector alone (mock transfectants), wild type β6 or the β6 deletion mutant Δ746-764 (lacking the sequence EAERSKAKWQTGTNPLYRG, Cone et al, 1994).",
"The lysates were immunoprecipitated with mAbs R6G9 (anti-β6), P1F6 (anti-β5) or murine IgG control antibody (mlgG).",
"Western blotting of the immunoprecipitates with mAb E10 recognising phosphorylated ERK1 and 2 revealed no β5-bound ERK in the SW480 β6 transfectants expressing wild-type β6 in contrast to β5-associated ERK observed for the cell lines which either lack β6 or express the deletion mutant lacking the ERK2 binding sequence (FIGS.",
"35A and 35B).",
"To determine whether β5 can bind ERK2 in vitro, a 15-mer peptide fragment derived from the β5 cytoplasmic domain corresponding to the ERK binding domain on β6 was tested for its ability to bind ERK2 in an ELISA.",
"In vitro MAP kinase activity assays were performed using a non-radioactive assay kit (Cell Signalling Technology, Beverley, Mass.)",
"which measures phosphorylation of the ERK1 and 2 substrate Elk-1 presented as a fusion protein comprising the Elk-1 codons 307-428 coupled to GST.",
"In the assay, ERK kinase is first immunoprecipitated from complete or integrin-immunodepleted soluble cell lysates and relative enzyme activity determined by the in vitro phosphorylation of GST-Elk-1.Dose-dependant binding of the β5 fragment (761RSRARYEMASNPLYR775) to ERK2 was observed as shown in FIG.",
"35C although less avidly than the 15-mer β6 peptide.",
"In contrast, a synthetic peptide derived from the cytoplasmic domain of the β1 subunit (765KFEKEKMNAKWDTGENPIYK784) which corresponds to the ERK binding domain on β6 failed to bind ERK2 (FIG.",
"35C).",
"11.2 The Increase in Cytosolic MAP Kinase Activity Upon Growth-Factor Stimulation is All Accounted for by β5-Bound ERK in Human Umbilical Vein Endothelial Cells that Lack β6 To determine the amount of ERK 1/2 activity associated with β5 following the activation of the MAP kinase pathway, the activity of ERK1/2 was compared in non-β5 immunodepleted lysates with that in β5 immunodepleted lysates derived from human umbilical vein endothelial cells (HUVECs) before and after stimulation with epidermal growth factor (EGF).",
"Sequential immunodepletion of the β5 integrin subunit from cell lysates prepared from HUVECs results in total loss of all epidermal growth factor-induced MAP kinase (ERK 1/2) activity as determined by an Elk-1 phosphorylation assay (FIG.",
"36).",
"Briefly, HUVECs were cultured in the absence/presence of 50 ng/ml epidermal growth factor (EGF) for 10 minutes after a prior 24 hour culture period in serum-free and growth factor-free medium.",
"Bands show MAP kinase activity from immunodepleted cell lysates (anti-β5 or control IgG or anti-β6 immunodepletions, five rounds each) prepared from HUVECs before and after EGF stimulation.",
"Non-phosphorylated and phosphorylated Elk-1 controls are shown in the left hand two lanes.",
"These results demonstrate that in β5-expression endothelial cells, the rise in cytosolic MAP kinase activity above basal levels upon EGF stimulation is completely accounted for by β5-bound ERK.",
"The findings also indicate that a hierarchy of integrin-ERK2 interactions exists within cells specified by β6 over β5 in β6-expressing epithelial cells and β5 over other β integrins in endothelial cells.",
"Given the critical requirement of ERK1/2 activity in the process or angiogenesis and responsiveness of endothelial cells to various growth factors these findings show, for the first time, how the αvβ5 integrin may regulate angiogenesis through maintaining activated ERK at the plasma membrane.",
"Accordingly, targeting the binding site on β5 for ERK2 or the binding site on ERK2 for the integrin offers a means of inhibiting new blood vessel formation.",
"Moreover, the findings allow for anti-angiogenic therapies to benefit patients with a range of disorders including not only cancer, but also diabetic retinopathy, arthritis and other inflammatory conditions.",
"EXAMPLE 12 The β6 integrin immunodepletion/ERK immunoblotting experiments together with the antisense nucleic acid strategies reported herein show that the β6 integrin subunit contributes substantially to the total MAP kinase activity with for instance, colon cancer cells.",
"Moreover, the fact β6-bound phosphorylated ERK2 is reduced by more than 75% in colon cancer cells lacking constitutive β6 expression stably transfected with a β6 deletion mutant in which the precise ERK2 binding domain is lacking, and that tumour growth is also reduced significantly compared with β6-expressing cells, shows that ERK2 plays a significant role in promoting cancer growth.",
"Accordingly, interruption of the β6-MAP kinase pathway has important implications not only for control of advanced colon cancer, but epithelial cancers in general given the wide expression of this integrin in other cancer types such as oropharyngeal, breast and ovarian cancer.",
"Hence, identification of the ERK2 binding sequence to β6 facilitates the development of mimetics which interrupt the β6-MAP kinase growth-promoting axis in cancer.",
"12.1 ELISA Methodology for Determination of β6-Binding Domain in ERK2.To determine the β6-binding domain(s) of ERK2, a biotinylated β6 cytoplasmic tail peptide spanning the cytoplasmic tail of β6 was incubated with a soluble ERK2 peptide fragment in a total incubation volume of 20 μl phosphate buffered saline (PBS).",
"The mixture was stood for 45 minutes at room temperature (RT).",
"Soluble ERK2 in PBS was then added and the solution left for a further 30 minutes.",
"Following incubation the reaction volume was made to 400 μl with carbonate buffer (0.015 M Na2CO3 and 0.035M NaHCO3 at pH 9.6) and 100 μl of this mixture added to each of 4 wells of a polystyrene ELISA plate (Nunc, Maxicorp).",
"ERK2/peptides were allowed to attach to the plastic substrate for 18 to 20 hours at RT in the dark.",
"After immobilisation the overlying buffer was flicked from the wells and excess solid surface blocked with casein (0.5% skim milk) in PBS for 60 minutes at RT.",
"Wells were washed (×3) with was buffer (PBS plus 0.5% skim milk powder, 0.05% Tween-20) prior to incubation with the anti-β6 antibody SC-6632 (Santa Cruz) diluted 1:500 v/v in wash buffer (2 wells) or a control antibody (2 wells).",
"Incubation with the primary antibody was carried out for 30 minutes at RT.",
"The ELISA plate was flicked to remove the antibody mixture before washing 3 times with wash buffer.",
"Wells were then incubated with anti-species alkaline phosphatase (AP) conjugated antibody (1:2000 v/v in wash buffer) for 30 minutes at RT, flicked and washed as above.",
"Specific binding events were quantitatively determined using a colourimetric substrate system by measuring absorbency at 405 mm (Bio-Rad, AP substrate kit).",
"12.2 Identification of β6-Binding Domain of ERK2.In a preliminary study, ERK peptides were probed in small groups to identify a possible ERK2 competing peptide that binds with the β6 cytoplasmic tail peptide.",
"Overlapping ERK2 peptides 1 to 14 comprising the full length ERK2 sequence (360 amino acids) were utilised, and an inhibitory effect on β6-ERK2 binding was identified within the group comprising peptides 7, 8, and 9 (data not shown).",
"The amino acid sequences for the peptides and position within ERK2 are shown below.",
"1.MAAAAAAGAGPEMVRGQVFDVGPRYTNLSY 2.YTNLSYIGEGAYGMVCSAYDNVNKVRVAIK 3.VRVAIKKISPFEHQTYCQRTLREIKILLRF 4.KILLRFRHENIIGINDIIRAPTIEQMKDVY 5.QMKDVYIVQDLMETDLYKLLKTQHLSNDHI 6.LSNDHICYFLYQILRGLKYIHSANVLHRDLK 7.HRDLKPSNLLLNTTCDLKICDFGLAR 8.DFGLARVADPDHDHTGFLTEYVATRWYRAPEIMLNSKGY 9.NSKGYTKSIDIWSVGCILAEMLSNRPIFPG 10.PIFPGKHYLDQLNHILGILGSPSQEDLNCI 11.DLNGIINLKA RNYLLSLPHKNKVPWNRLFP 12.NRLFPNADSKALDLLDKMLTFNPHKRIEVE 13.RIEVEQALAHPYLEQYYDPSDEPIAEAPFK 14.EAPFKFDMELDDLPKEKLKELIFEETARFQPGYRS Individual overlapping peptides of the ERK2 sequence were then tested at equimolar concentrations for the ability to inhibit the β6-ERK interaction.",
"Only peptide 7 (HRDLKPSNLLLNTTCDLKICDFGLAR) was found to be effective (see FIG.",
"37).",
"Repeat testing using adjacent ERK2 overlapping peptides as controls yielded the same result (see FIG.",
"38).",
"Importantly, β6 bound to ERK2 does not interfere with ERK2 binding to the well, thereby confirming the validity of the ERK2 peptide competition assay.",
"To optimise the assay, β6 cytoplasmic tail peptide was incubated at increasing concentrations with ERK2.Maximal β6 detected when bound to ERK was found to be at a concentration of 0.25 μM (data not shown).",
"β6 cytoplasmic tail peptide (0.25 μM) was then either pre-incubated with ERK peptides 2, 7, 8 or 10 in the competition assay or directly with ERK2 (positive control).",
"An inhibition of 83% for β6-ERK2 binding was again demonstrated for sequence 7 (see FIG.",
"39).",
"To demonstrate that neither β6 alone or ERK2 peptide fragments 2, 7, 8 and 10 interfered with ERK2 binding to the plate, parallel samples of each condition identified in FIG.",
"38 were probed with anti-ERK2 antibody.",
"None of the peptide or β6 coincubations with ERK2 interfered with ERK binding to the ELISA plate.",
"EXAMPLE 13 13.1 Induction of Wound Healing Response Keratinocytes in vivo in unwounded skin do not express β6 integrin but do so after sub-culturing in vitro.",
"Hence, to avoid the conflicting presence of integrin αvβ6, the epithelial cell line SW480 which lacks αvβ6 expression was chosen in order to examine the effect of peptide β6 agonists on cell migration following artificial wounding in vitro.",
"The wound assay was performed by seeding 2×105 cells into 24 well (1.6 cm diameter) tissue culture plates in Dulbecco's modified Eagles medium (DMEM) supplemented by 10% foetal calf serum, HEPES, glutamine and antibotics.",
"The confluent cell monolayers were streaked with the tip of a plastic pipette in a cross-wire fashion, and loose non-adherent cells washed off gently with medium before replacing the culture medium with DMEM supplemented with 1% foetal calf serum.",
"In addition, RSKAKWQTGTNPLYR peptide ligated to penetratin (carrier peptide) by means of cysteine Cys (Npys) bonding was added to each well at a final concentration in the range of 1.25-10 μM.",
"Cell migration across the wounded (streaked) area was photographed (Techpan, Kodachrome, ASA100) after a further 48 hours in culture.",
"As shown in FIGS.",
"40A to 40D, a dose-dependent wound repair process (enhanced cell migration) was observed for the peptide-agonist complexed with the intracellular delivery vehicle penetratin.",
"The conditions utilised in the assay were as follows: A: no peptide-penetratin complex added, B: 2.5 μM peptide-penetratin complex added, C: 5 μM peptide-penetratin complex added, D: 10 μM peptide-penetratin complex added.",
"Cell migration was not stimulated by either the peptide-agonist uncoupled to penetratin or penetratin alone (not shown).",
"The bar length shown in FIG.",
"40D represents 80 μM in length.",
"13.2 Discussion The activity of ERK2 is tightly regulated.",
"Dual phosphorylation events, one on residue 185 (Y) and one on residue 183 (T) are required for ERK2 activation (Zhang et al, 1995).",
"In unphosphorylated ERK2, Y185 is found largely buried within the molecule whereas T183 is exposed.",
"In particular, the phosphorylation lip of ERK2 consists of 15 amino residues from residue 173 (D) to residue 187 (A) namely DPDHDHTGFLTEYVA.",
"This sequence is near the mouth of the active site (Zhang et al, 1995).",
"The 26 mer peptide fragment of ERK2 (peptide 7) found to inhibit binding of β6 cytoplasmic domain to ERK2 stretches from amino acid residue 145 (H) to residue 170 (R).",
"This inhibitory binding sequence lies adjacent to the phosphorylation lip of ERK2.The reason for low stability of the phosphorylation lip in ERK2 is uncertain, but may be due to the unusual cluster of three positively charged amino acids namely residue 146 (R), 170 (R) and 201 (K) (Zhang et al, 1995).",
"Residues 146(R) and 170(R) of ERK2, both of which are included within peptide 7, appear to be conserved among many serine/threonine specific kinases.",
"For conserved amino acid residue 146 (R) to bind to a phosphate ion, it must lose its interaction with the backbone of the loop defined by amino acid residues 199 to 205 of ERK2 adjacent to the phosphorylation lip (residues 173 (D) to 187 (A)).",
"Accordingly, β6 may bind to the 15 mer PSNLLLNTTCDLKIC within peptide 7 and break the interaction between residue 146 (R) and the backbone loop allowing ERK to be phosphorylated on residue 183 (T) and 185 (Y).",
"Although the present invention has been described hereinbefore with reference to a number of preferred embodiments, the skilled addressee will understand that numerous variations and modifications are possible without departing from the scope of the invention.",
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]
] | Patent_10451291 |
[
[
"Modified tachykinin receptors",
"The invention provides a modified tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups.",
"The receptor exhibits similar ligand binding characteristics to the wild type receptor but is incapable or substantially incapable of initiating an endogenous signal.",
"Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G protein, whereby there is substantially no transduction of ligand binding signals to the cell, The invention also includes fragments of the receptor containing the modified DRY sequence, and polynucleotides that encode the modified receptor as aforesaid.",
"Therapeutic and diagnostic uses for the receptor are disclosed."
],
[
"1.A mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with 5 intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but exhibiting substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding 10 signals to the cell; or a fragment of said receptor containing said modified DRY sequence; or an isolated protein or polypeptide containing an amino acid sequence at least 80% identical to the above sequence; or a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; or an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.",
"2.A non-human mammalian receptor according to claim 1.3.A rat or mouse receptor according to claim 1.4.A human receptor according to claim 1.5.A mutant NIL-1 receptor according to claim 1.6.A mutant NK-2 receptor according to claim 1.7.A mutant NK-3 receptor according to claim 1.8.A receptor according to claim 1, wherein the replacement amino acids are selected from G and A.",
"9.The receptor of claim 8, wherein DRY is replaced by GGA.",
"10.A tachykinin receptor of SEQ ID No5, or a receptor having at least 80% amino acid identity with the receptor of SEQ ID No5, and that is capable of binding to substance P but is substantially incapable of initiating its endogenous signal, or a fragment of said receptor.",
"11.An isolated cell membrane incorporating a tachykinin receptor as defined in claim 1.12.Any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a tachykinin receptor as claimed in claim 1; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.",
"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.",
"13.Any of the following: (a) an isolated nucleic acid molecule having the nucleotide sequence of SEQ ID No6; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.",
"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.",
"14.A method for producing a receptor protein having an amino acid sequenceas defined in claim 1, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing, in an a culture medium, a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying, from said culture medium or from said host cell, the thus-produced receptor protein.",
"15.A pharmaceutical composition comprising an effective amount of a modified tachykinin ligand as defined in claim 1 or a nucleic acid sequence encoding said ligand and a pharmaceutically and pharmacologically acceptable carrier.",
"16.Use of a modified tachykinin receptor as defined in claim 1 in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; 17.A method for the treatment or prevention of a condition associated with over-expression or inappropriate expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tachykinin ligand as defined in claim 1.18.A method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as defined in claim 1; (b) adding test sample to a solution containing labeled tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled ligand mixture from step (b) to allow binding of said ligand and test sample to the modified tachykinin receptor; (d) optionally, separating the non-bound labeled ligand from the labeled ligand bound to the modified tachykinin receptor; and, if desired, (e) measuring the amount of labeled ligand that is bound to the modified tachykinin receptor.",
"19.Use of a modified tachykinin receptor as defined in claim 1 as a substitute in an assay to identify and/or evaluate entities that bind to the wild type tachykinin receptor.",
"20.Use of a modified tachykinin receptor as defined in claim 1 as a substitute in an assay in order to determine the concentration of ligand in body fluids in patients with arthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinal disorders and diseases of the GI tract.",
"21.An assay procedure comprising the following steps: (a) providing a cell line is provided that expresses a modified tachykinin receptor as defined in claim 1; (b) labeling the cell line; (c) adding the test sample and labeled cells to a matrix binding SP or other ligand; (d) incubating the labeled cells, test sample and matrix-bound SP or other ligand to allow binding of SP or other ligand and test sample to the expressed modified tachykinin receptor; (e) separating the labelled non-bound cells from the SP or other ligand bound cells; and, if desired, (f) measuring the amount of labelled cells containing the modified tachykinin receptor that has bound to SF or other ligand.",
"22.Use of a modified tachykinin receptor as defined in claim 1 in protein therapy to reduce the effects of an excess of or inappropriately produced endogenous ligand.",
"23.A method for treatment of a patient in need thereof, which comprises administering to said patient a composition in the form of an aerosol that comprises a modified tachykinin receptor as defined in claim 1.24.A method for gene therapy treatment of a patient in need thereof, which comprises administering to said patient a nucleic acid sequence, virus or plasmid encoding a modified tachykinin receptor as defined in claim 1."
],
[
"<SOH> BACKGROUND TO THE INVENTION <EOH>Tachykinins are important in the mediation of many physiological and pathological processes including inflammation, pain, migraine, headache and allergy induced asthma.",
"They belong to an evolutionary conserved family of peptide neurotransmitters that have an established role in neurotransmission.",
"They share the C-terminal sequence Phe-Xaa-Gly-Leu-Met-NH 2 (SEQ ID NO 12) in which Xaa represents a hydrophobic residue.",
"That sequence is characteristic of tachykinins and believed to be mainly responsible for their biological activity at neurokinin receptors.",
"Mammalian tachykinins include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) which exert their effects by binding to specific receptors.",
"SP is the most prominent member of the tachykinergic system and is released from sensory nerve endings throughout the body.",
"Its amino acid sequence is: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 (SEQ ID N o 1) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"The amino acid sequence for NKA is: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2 (SEQ ID N o 2) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"The amino acid sequence for NKB is: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH 2 (SEQ ID N o 3) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"SP has been implicated in the pathology of numerous diseases.",
"For example, it has been shown to be involved in the transmission of pain, in conditions associated with vasodilation, smooth muscle contraction, bronchoconstriction, activation of the immune system and neurogenic inflammation.",
"It has been implicated in migraine, as well as in disorders of the central nervous system, such as anxiety and schizophrenia; in respiratory and inflammatory diseases, such as asthma and rheumatoid arthritis; and in gastrointestinal (GI) disorders and diseases of the GI tract, such as ulcerative colitis and Crohn's disease.",
"Tachykinin receptors include NK-1, NK-2 and NK-3 and are membrane proteins of the super-family of guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR).",
"They have extra-cellular binding sites that have preferential affinities for the ligands SP, NKA and NKB respectively.",
"Like other G-protein coupled receptors, they possess an extra-cellular N-terminus, an intra-cellular C-terminus and seven trans-membrane (TM) α-helices of 20-30 amino acids connected by first, second and third extra-cellular loops and by first, second and third intra-cellular or cytoplasmic loops.",
"The greatest homology is found in the membrane-spanning α-helices of trans-membrane domains TM1-TM7 while the N- and C-termini show greater diversity between the three types of neurokinin receptor.",
"Cloned NK-1 receptors have been reported, including those for the Rana catesbeina , (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Mus musculus , (Sundelin et al, Eur.",
"J. Biochem.",
"203: 625-631 (1992)), Rattus norvegicus , (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) and Yokota et al, J. Biol.",
"Chem., 264: 17649-17652 (1989)), Cavia porcellus , (Gorbulev et al., Biochem.",
"Biophys.",
"Acta 1131: 99-102 (1992)), and Homo sapiens (human), (Takeda et al, Biochem.",
"Biophys.",
"Res.",
"Comm.",
"179: 1232-1240 (1991) and Fong et al U.S. Pat.",
"No.",
"5,525,712 and U.S. Pat.",
"No.",
"5,584,886).",
"Cloned rat and bovine neurokinin-2 receptors have been reported (Y. Sasi et al., Biochem.",
"Biophys.",
"Res.",
"Comm., 165: 695 (1989), and Y. Masu, et al., Nature 329: 836 (1987)).",
"Cloned rat neurokinin-3 receptor has also been reported (R. Shigemoto, et al., J. Biol.",
"Chem., 265:623 (1990)).",
"All three receptors share the signal transduction mechanisms of a G-protein coupled receptor.",
"The receptor is in an OFF state when no ligand is present, but, is triggered into an ON state when an agonist ligand binds to the receptor.",
"The G-protein when in its ON state triggers a downstream signaling pathway or signal cascade.",
"The G-protein comprises α-, β- and γ-units that are bound together in the OFF state, the α-subunit then having GDP bound to it.",
"In the ON state GTP replaces the GDP bound to the α-subunit.",
"The α-subunit becomes dissociated from the β- and γ-subunits and becomes available for activating the signal cascade.",
"After a short period, the GTP becomes hydrolyzed to GDP and the G-protein returns to its non activated OFF state.",
"Hydrolysis provides a negative feedback mechanism that ensures that the G protein is only in its activated ON state for a short period.",
"Various studies have been undertaken, involving different G-protein receptors, to determine how the various regions of the protein structure affect intra-cellular coupling of the receptor to the G-protein and consequential transduction of ligand binding signals to the cell.",
"G-protein receptors have a well-conserved sequence in the second intracellular loop where the loop joins the third trans-membrane domain that is known as the DRY sequence.",
"It has the residues in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"5′-DRYXXV(P)XXPL-3′ (SEQ ID N o 4) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"in which L represents Leu, Ile, Val, Met or Phe and X represents any amino acid.",
"It has been suggested that the DRY sequence contributes to the efficient binding and activation of G-proteins.",
"Fraser et al., Proc.",
"Natl Acad Sci.",
"USA, 85: 5478-5482 (1988) report a change of Asp to Asn at position 130 of the human β-adrenergic receptor (i.e.",
"the D of the DRY sequence) resulting in human β-adrenergic receptor that exhibits high affinity binding of agonist whilst being unable to interact effectively with G-protein.",
"However, a second paper from the same laboratory reports that the previous very high agonist binding efficiency in human β-adrenergic receptor mutated at position 130, upon which the above mentioned conclusion had been based, had not been reproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)).",
"In a review article Savarese and Fraser said that this locus may be important for coupling to some, but not all, G-proteins ( Biochem J., 283: 1-19 (1992)).",
"Moro et al made mutants of the Hm1 muscarinic cholinergic receptor with changes towards the 5′-end of the DRY sequence and found that replacing L at position 131 with A gave the strongest reduction in coupling efficiency ( J. Biol.",
"Chem.",
"268: 22273-22278 (1993)).",
"Subsequently, Shibata et al made a mutant of the angiotensin II receptor type I in which DRY at positions 125-127 is replaced by GGA and M at position 134 is replaced by A, resulting in uncoupling of the mutant A receptor from G-proteins ( Biochem.",
"Biophys.",
"Res.",
"Com.",
"218: 383-389 (1996)).",
"The authors concluded that DRY sequence as a whole including the final lipophilic amino acid L serves as a general site for G-protein coupling but they did not go on to consider what effects might be obtained by change confined to the DRY portion of the sequence.",
"Comparing the binding affinities of the two known isoforms of the human NK-1 receptor shows the importance of the cytoplasmic tail.",
"The long form (407 amino acids) and short form (311 amino acids) differ in the length of the C-terminus.",
"The long form has similar substance P binding characteristics to the rat NK-1 receptor, while the short form of the receptor has an apparent substance P binding affinity 10-fold less than the rat NK-1 receptor.",
"Furthermore, studies on these and other receptors have shown that the effect of mutations is unpredictable and specific to certain families of receptors.",
"Hence, a change in one G-protein coupling family will not necessarily have the same effect on another.",
"Other attempts to distinguish protein binding and signaling effects bear this out."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but being incapable or substantially incapable of initiating an endogenous signal.",
"Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding signals to the cell.",
"The way in which tachykinin receptors attach to cell membranes, the trans-membrane domains extra-cellular and cytoplasmic loops and the place where the DRY sequence referred to above occurs are apparent by inspection of FIG.",
"5 of the accompanying drawings.",
"The invention further provides any of the following: a fragment of said mutant tachykinin receptor containing said modified DRY sequence; an isolated protein or polypeptide containing an amino acid sequence at least 95% identical to the above sequence; a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; and an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.",
"The invention also comprises an isolated cell membrane in which a modified tachykinin receptor as aforesaid is incorporated as membrane protein.",
"Such cell membrane material finds utility for research and in particular for screening for therapeutically useful compounds as described below.",
"The invention yet further provides any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a modified tachykinin receptor as aforesaid; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.",
"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.",
"The invention also provides a method for producing one of the amino acid sequences described herein and especially the receptor protein defined by SEQ ID N o 5, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing in a culture medium a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying from said culture medium or from said host cells the thus-produced receptor protein.",
"In step (d) of the above method, the receptor protein may be obtained either from the culture medium and/or by lysing the host cell, for example by sonication or osmotic shock.",
"The invention yet further provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as aforesaid; (b) adding test sample to a solution containing labeled SP or other tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separating the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.",
"The invention further provides: (a) the use of a modified tachykinin receptor according to the invention in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; (b) the use of such a modified tachykinin receptor in therapy; (c) a method for the treatment or prevention of a condition associated with over-expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tackykinin ligand as described above; (d) a composition comprising a modified tachykinin ligand as described above in association with a pharmaceutically and pharmacologically acceptable carrier therefor; and (e) a use, method or composition according to any one of (a) to (d) above, in which the modified tachykinin ligand becomes generated in vivo from a nucleic acid sequence encoding such a ligand."
],
[
"FIELD OF THE INVENTION The present invention relates to a nucleic acid sequence encoding a tachykinin receptor, the tachykinin receptor encoded by said sequence, methods for its preparation and its use in therapy and screening.",
"In particular, the invention relates to a modification to tachykinin receptor proteins that gives rise to unexpected and useful properties.",
"BACKGROUND TO THE INVENTION Tachykinins are important in the mediation of many physiological and pathological processes including inflammation, pain, migraine, headache and allergy induced asthma.",
"They belong to an evolutionary conserved family of peptide neurotransmitters that have an established role in neurotransmission.",
"They share the C-terminal sequence Phe-Xaa-Gly-Leu-Met-NH2 (SEQ ID NO 12) in which Xaa represents a hydrophobic residue.",
"That sequence is characteristic of tachykinins and believed to be mainly responsible for their biological activity at neurokinin receptors.",
"Mammalian tachykinins include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) which exert their effects by binding to specific receptors.",
"SP is the most prominent member of the tachykinergic system and is released from sensory nerve endings throughout the body.",
"Its amino acid sequence is: H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SEQ ID No1) The amino acid sequence for NKA is: H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (SEQ ID No2) The amino acid sequence for NKB is: H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 (SEQ ID No3) SP has been implicated in the pathology of numerous diseases.",
"For example, it has been shown to be involved in the transmission of pain, in conditions associated with vasodilation, smooth muscle contraction, bronchoconstriction, activation of the immune system and neurogenic inflammation.",
"It has been implicated in migraine, as well as in disorders of the central nervous system, such as anxiety and schizophrenia; in respiratory and inflammatory diseases, such as asthma and rheumatoid arthritis; and in gastrointestinal (GI) disorders and diseases of the GI tract, such as ulcerative colitis and Crohn's disease.",
"Tachykinin receptors include NK-1, NK-2 and NK-3 and are membrane proteins of the super-family of guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR).",
"They have extra-cellular binding sites that have preferential affinities for the ligands SP, NKA and NKB respectively.",
"Like other G-protein coupled receptors, they possess an extra-cellular N-terminus, an intra-cellular C-terminus and seven trans-membrane (TM) α-helices of 20-30 amino acids connected by first, second and third extra-cellular loops and by first, second and third intra-cellular or cytoplasmic loops.",
"The greatest homology is found in the membrane-spanning α-helices of trans-membrane domains TM1-TM7 while the N- and C-termini show greater diversity between the three types of neurokinin receptor.",
"Cloned NK-1 receptors have been reported, including those for the Rana catesbeina, (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Mus musculus, (Sundelin et al, Eur.",
"J. Biochem.",
"203: 625-631 (1992)), Rattus norvegicus, (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) and Yokota et al, J. Biol.",
"Chem., 264: 17649-17652 (1989)), Cavia porcellus, (Gorbulev et al., Biochem.",
"Biophys.",
"Acta 1131: 99-102 (1992)), and Homo sapiens (human), (Takeda et al, Biochem.",
"Biophys.",
"Res.",
"Comm.",
"179: 1232-1240 (1991) and Fong et al U.S. Pat.",
"No.",
"5,525,712 and U.S. Pat.",
"No.",
"5,584,886).",
"Cloned rat and bovine neurokinin-2 receptors have been reported (Y. Sasi et al., Biochem.",
"Biophys.",
"Res.",
"Comm., 165: 695 (1989), and Y. Masu, et al., Nature 329: 836 (1987)).",
"Cloned rat neurokinin-3 receptor has also been reported (R. Shigemoto, et al., J. Biol.",
"Chem., 265:623 (1990)).",
"All three receptors share the signal transduction mechanisms of a G-protein coupled receptor.",
"The receptor is in an OFF state when no ligand is present, but, is triggered into an ON state when an agonist ligand binds to the receptor.",
"The G-protein when in its ON state triggers a downstream signaling pathway or signal cascade.",
"The G-protein comprises α-, β- and γ-units that are bound together in the OFF state, the α-subunit then having GDP bound to it.",
"In the ON state GTP replaces the GDP bound to the α-subunit.",
"The α-subunit becomes dissociated from the β- and γ-subunits and becomes available for activating the signal cascade.",
"After a short period, the GTP becomes hydrolyzed to GDP and the G-protein returns to its non activated OFF state.",
"Hydrolysis provides a negative feedback mechanism that ensures that the G protein is only in its activated ON state for a short period.",
"Various studies have been undertaken, involving different G-protein receptors, to determine how the various regions of the protein structure affect intra-cellular coupling of the receptor to the G-protein and consequential transduction of ligand binding signals to the cell.",
"G-protein receptors have a well-conserved sequence in the second intracellular loop where the loop joins the third trans-membrane domain that is known as the DRY sequence.",
"It has the residues 5′-DRYXXV(P)XXPL-3′ (SEQ ID No4) in which L represents Leu, Ile, Val, Met or Phe and X represents any amino acid.",
"It has been suggested that the DRY sequence contributes to the efficient binding and activation of G-proteins.",
"Fraser et al., Proc.",
"Natl Acad Sci.",
"USA, 85: 5478-5482 (1988) report a change of Asp to Asn at position 130 of the human β-adrenergic receptor (i.e.",
"the D of the DRY sequence) resulting in human β-adrenergic receptor that exhibits high affinity binding of agonist whilst being unable to interact effectively with G-protein.",
"However, a second paper from the same laboratory reports that the previous very high agonist binding efficiency in human β-adrenergic receptor mutated at position 130, upon which the above mentioned conclusion had been based, had not been reproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)).",
"In a review article Savarese and Fraser said that this locus may be important for coupling to some, but not all, G-proteins (Biochem J., 283: 1-19 (1992)).",
"Moro et al made mutants of the Hm1 muscarinic cholinergic receptor with changes towards the 5′-end of the DRY sequence and found that replacing L at position 131 with A gave the strongest reduction in coupling efficiency (J. Biol.",
"Chem.",
"268: 22273-22278 (1993)).",
"Subsequently, Shibata et al made a mutant of the angiotensin II receptor type I in which DRY at positions 125-127 is replaced by GGA and M at position 134 is replaced by A, resulting in uncoupling of the mutant A receptor from G-proteins (Biochem.",
"Biophys.",
"Res.",
"Com.",
"218: 383-389 (1996)).",
"The authors concluded that DRY sequence as a whole including the final lipophilic amino acid L serves as a general site for G-protein coupling but they did not go on to consider what effects might be obtained by change confined to the DRY portion of the sequence.",
"Comparing the binding affinities of the two known isoforms of the human NK-1 receptor shows the importance of the cytoplasmic tail.",
"The long form (407 amino acids) and short form (311 amino acids) differ in the length of the C-terminus.",
"The long form has similar substance P binding characteristics to the rat NK-1 receptor, while the short form of the receptor has an apparent substance P binding affinity 10-fold less than the rat NK-1 receptor.",
"Furthermore, studies on these and other receptors have shown that the effect of mutations is unpredictable and specific to certain families of receptors.",
"Hence, a change in one G-protein coupling family will not necessarily have the same effect on another.",
"Other attempts to distinguish protein binding and signaling effects bear this out.",
"SUMMARY OF THE INVENTION The invention provides a mutant tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups, said receptor exhibiting similar ligand binding characteristics to the wild type receptor but being incapable or substantially incapable of initiating an endogenous signal.",
"Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G-protein, whereby there is substantially no transduction of ligand binding signals to the cell.",
"The way in which tachykinin receptors attach to cell membranes, the trans-membrane domains extra-cellular and cytoplasmic loops and the place where the DRY sequence referred to above occurs are apparent by inspection of FIG.",
"5 of the accompanying drawings.",
"The invention further provides any of the following: a fragment of said mutant tachykinin receptor containing said modified DRY sequence; an isolated protein or polypeptide containing an amino acid sequence at least 95% identical to the above sequence; a variant thereof with sequential amino acid deletions from either the C terminus or the N-terminus; and an allelic variant, heterospecific homologue or biologically active proteolytic or other fragment thereof containing said modified DRY sequence.",
"The invention also comprises an isolated cell membrane in which a modified tachykinin receptor as aforesaid is incorporated as membrane protein.",
"Such cell membrane material finds utility for research and in particular for screening for therapeutically useful compounds as described below.",
"The invention yet further provides any of the following: (a) an isolated nucleic acid molecule comprising a polynucleotide that encodes a modified tachykinin receptor as aforesaid; (b) an isolated nucleic acid molecule comprising a sequence that is hybridizable to the above sequence; (c) a gene which is the result of extending the above sequence or any sequence that is hybridizable to the above sequence; (d) a sequence or gene that is functionally equivalent to the above sequence or to a gene that is an extension of the above sequence, i.e.",
"that is not identical to the sequence or gene referred to but functions biologically as equivalent to the sequence or gene referred to, including any allelic variants and heterospecific mammalian homologues, including artificial or recombinant sequences created from cDNA or genomic DNA; (e) a recombinant vector comprising the above gene sequence; and (f) a host cell transformed with the vector.",
"The invention also provides a method for producing one of the amino acid sequences described herein and especially the receptor protein defined by SEQ ID No5, which method comprises the steps of: (a) inserting said nucleic acid sequence into an appropriate vector; (b) culturing in a culture medium a host cell previously transformed or transfected with the recombinant vector of step (a); (c) harvesting cells containing the receptor protein obtained from step (b); and (d) separating or purifying from said culture medium or from said host cells the thus-produced receptor protein.",
"In step (d) of the above method, the receptor protein may be obtained either from the culture medium and/or by lysing the host cell, for example by sonication or osmotic shock.",
"The invention yet further provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing a modified tachykinin receptor as aforesaid; (b) adding test sample to a solution containing labeled SP or other tachykinin ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separating the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.",
"The invention further provides: (a) the use of a modified tachykinin receptor according to the invention in the preparation of a medicament for the treatment or prophylaxis of a condition associated with substance P or other tachykinin (neurokinin) receptor-binding ligand; (b) the use of such a modified tachykinin receptor in therapy; (c) a method for the treatment or prevention of a condition associated with over-expression of an endogenous tachykinin ligand, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of such a modified tackykinin ligand as described above; (d) a composition comprising a modified tachykinin ligand as described above in association with a pharmaceutically and pharmacologically acceptable carrier therefor; and (e) a use, method or composition according to any one of (a) to (d) above, in which the modified tachykinin ligand becomes generated in vivo from a nucleic acid sequence encoding such a ligand.",
"BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be further described with reference to the accompanying drawings, in which: FIG.",
"1: describes a protein sequence, being a translation of the sequence shown in FIG.",
"1 and being the sequence of a modified human NK-1 receptor according to the invention (hNK-1Rv1).",
"FIG.",
"2: is a consensus cDNA sequence encoding a modified human NK-1 receptor according to the invention.",
"FIG.",
"3: is a bar chart showing the results of Calcium imaging experiments on NK-1 wild type receptor and the modified receptor of FIG.",
"1 (hNK-1Rv1), and wild type receptor antisense transfected COS cells.",
"FIG.",
"4: shows the sequence alignments between NK-1 and other NK receptors in various species in the region of the DRY motif in TM3, demonstrating high conservancy (In the Figure, NK-1 sequences 1-4 provide SEQ ID NO 13, NK-1 sequence 5 provides SEQ ID NO 14, NK-2 sequences 1-2 and 4-7 provide SEQ ID NO 15, NK-2 sequence 3 provides SEQ ID NO 16 and NK-3 sequences 1-3 provide SEQ ID NO 17).",
"FIG.",
"5: is a diagram showing the nNK-1Rv1 receptor and portions of the cell membrane into which it is incorporated (SEQ ID NO 18; in addition to the DRY motif, L223 is changed to I; this change is believed to be inconsequential).",
"DESCRIPTION OF PREFERRED EMBODIMENTS Definitions Definitions for a number of terms used in this specification are given below: Alleles or allelic sequences mean alternative forms of the receptor genes referred to above resulting from one or more variations in the nucleic acid sequence, resulting in altered mRNAs and proteins or polypeptides Functional equivalence when used in relation to gene sequences or amino acid sequences covers sequences that are not identical to the sequence referred to but function biologically or chemically as equivalents of the disclosed sequence.",
"Incapable of initiating an endogenous signal in the present context means that, within experimental error (±5% of control), the modified NKR does not result in conversion of GTP to GDP in the cell or a recombinant construct; and/or does not evoke a calcium flux therein; and/or does not evoke other components of the signal transduction pathway.",
"Isolated, when used in relation to a polynucleotide sequence, means such a sequence that has been removed from its natural environment, i.e.",
"from the organism in which it occurs in nature and/or from genes that are immediately contiguous (one at the 5′ end and the other at the 3′ end) in the naturally occurring genome of the organism from which it is derived.",
"Isolated when used in relation to a cell membrane refers to the membrane as a discrete entity substantially separate from cytoplasmic material.",
"Operably linked refers to a linkage of polynucleotide elements in a functional relationship.",
"For instance, a promoter or an enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.",
"More specifically, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides neither results in the introduction of a frame-shift mutation nor interferes with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.",
"Stringent hybridisation conditions is a recognized term in the art and for a given nucleic acid sequence refers to those conditions which permit hybridisation of that sequence to its complementary sequence and not to a substantially different sequence.",
"It generally implies at least about 97% identity between the sequences.",
"Protein and Nucleic Acid Sequences In the above mentioned mutant receptors, amino acids for modifying the DRY sequence that are non-polar and have the correct hydrophilic/lipophilic balance include glycine and alanine which are preferred.",
"However, the inventors also envisage the use of other non polar amino acids.",
"In a preferred aspect, the invention provides a nucleic acid sequence as shown in FIG.",
"2 [SEQ ID No6], which encodes the consensus amino acid sequence for the modified hNK-1v or a sequence that hybridizes thereto under stringent hybridization conditions.",
"The invention also provides a preferred hNK-1v receptor sequence shown in FIG.",
"1 [SEQ ID No5].",
"The invention further provides a nucleic acid sequence encoding a receptor protein having at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the hNK-1Rv1 encoded by the nucleic acid sequence of SEQ ID No6, and which encodes protein that can bind to SP, but cannot initiate its endogenous signal, or encodes a peptide fragment thereof according to sequence (d), or encodes a sequence complementary thereto according to sequence (e), as defined hereinabove.",
"The invention also provides a protein that: (a) has at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the hNK-1Rv1 protein having the amino acid sequence of SEQ ID No5; and (b) can bind to SP or to a peptide fragment thereof, or to a sequence complementary thereto, but cannot initiate its endogenous signal to G protein.",
"The hNK-1Rv1 variant of the NK-1 receptor shown in SEQ ID No5 has similar ligand binding characteristics to the wild type receptor, but is deficient in cell signaling capabilities, as demonstrated by the results of tests described in Examples 4 and 5.Some mammalian receptors may exhibit overall homology with the hNK-1 receptor of less than 80% (for example, rat NK-2 receptor has about 48% homology to rat NK-1 receptor), but are nevertheless included within the scope of this invention in view of their conservancy in the intracellular DRY region of TM3.Accordingly, the present invention provides a polypeptide having as low as 40% overall amino acid identity with the hNK-1Rv1 protein, but having at least 75%, such as at least 80%, preferably 90%, more preferably 95%, and most preferably 98% amino acid identity with the TM3 intracellular loop of the bNK-1Rv1 protein in the vicinity of the DRY motif, and having the properties previously specified.",
"In view of the high level of conservancy demonstrated by other NK receptors in the DRY region of TM3 intracellular loop, and also the high level of conservancy observed in that region between human and other species, the present invention also provides a nucleotide sequence that encodes a modified neurokinin receptor, wherein the modification is or includes substitution of the DRY motif within the intracellular loop of TM3, namely, Asp129, Arg130 and Tyr131, by Gly, Gly and Ala, respectively, for example wherein the receptor is a modified NK-2 or modified NK-3 mammalian receptor.",
"Vectors The invention further provides a vector (e.g.",
"a plasmid or virus) comprising a nucleic acid encoding the modified tachykinin receptor, especially the hNK-1Rv1 receptor or any other modified hNK-1R of this invention.",
"A recombinant vector of the invention comprises an expression vector comprising a nucleic acid sequence encoding the modified NK-R, especially the hNK-1Rv1, polypeptide.",
"One suitable vector for the expression of a human variant NK-1 receptor of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell-lines.",
"Expression requires that appropriate signals are provided in the vector, said signals including various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.",
"The regulatory sequences of the expression vectors are operably linked to the nucleic acid encoding the modified tachykinin receptor, especially the human NK-1 variant receptor.",
"Generally, recombinant expression vectors include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.",
"The heterologous structural sequence is assembled in an appropriate frame with the translation, initiation and termination sequences, and preferably a leader sequence capable of directing sequences of the translated protein into the periplasmic space or the extra-cellular medium.",
"Where the vector is adapted for transfecting and expressing desired sequences in eukaryotic host cells, preferred vectors comprise an origin of replication from the desired host, a suitable promoter and an enhancer, and also any necessary ribosome binding sites, polyadenylation site, transcriptional termination sequences, and optionally 5′-flanking non-transcribed sequences.",
"Suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids have to be expressed.",
"A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression, or may be endogenous to the native polynucleotide containing the coding sequence to be expressed.",
"Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.",
"A recombinant vector of the invention may be used to amplify a polynucleotide derived from the nucleic acid sequence encoding the modified NKR, especially the hNK-1Rv1 polypeptide that has been inserted in a suitable host cell, this polynucleotide being amplified every time the recombinant vector replicates.",
"DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, and polyadenylation sites may be used to provide the require non-transcribed genetic elements.",
"The suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids are to be expressed.",
"A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively it can be endogenous to the native polynucleotide containing the coding sequence to be expressed.",
"Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.",
"Preferred bacterial promoters are the LacI, LacZ, T3 or T7 bacteriophage RNA polymerase promoters, the lambda PR, PL and trp promoters (EP 0 036 776), the polyhedrin promotor, or the p10 protein promoter from baculovirus (Kit Novagen; Smith et al., 1983); O'Reilly et al., 1992, Baculovirus expression vectors: A Laboratory Manual.",
"W.H.",
"Freeman and Co., New York).",
"Preferred selectable marker genes contained in the expression recombinant vectors of the invention for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, TRP1 for S. cerevisiae or tetracyclin, rifampicin or ampicillin resistance in E. coli, or Levan saccharase for mycobacteria, this latter marker being a negative selection marker.",
"Preferred bacterial vectors of the invention are listed hereafter as illustrative but not limitative examples: pQE70, pQE60, pQE-9 (Qiagen), pD10, fephagescript, psiX174, p.Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (Qiagen).",
"Preferred bacteriophage recombinant vectors of the invention are P1 bacteriophage vectors such as described by Sternberg N. L. (1992;1994).",
"A suitable vector for the expression of hNK-1vRa polypeptide of the invention or a fragment thereof, is a baculovirus vector that can be propagated in insect cells and in insect cell-lines.",
"A specific suitable host vector system is the pVL 1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from spodoptera frugiperda.",
"The recombinant expression vectors of the invention may also be derived from an adenovirus such as those described by Feldman and Steg.",
"(1996) or Ohno et al.",
"(1994).",
"Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type two or five (Ad 2 or Ad 5) or an adenovirus of animal origin (French Patent Application noFR 93 05 954).",
"Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, murine sarcoma virus, and Ross Sarcoma Virus.",
"Other preferred retroviral 5 vectors are those described in Roth et al.",
"(1996), in PCT Application WO 93/25 234, in PCT Application WO 94/06920, and also in Roux et al.",
"(1989), Julan et al.",
"(1992) and Nada et al.",
"(1991).",
"Yet, another viral vector system that is contemplated by the invention consists in the adeno associated virus (AAV) such as those described by Flotte et al.",
"(1992), Samulski et al.",
"(1989) and McLaughlin et al.",
"(1996).",
"Expression Systems For expression of the modified neurokinin receptors, the invention provides host cells transformed (prokaryotic cells) or transfected (eukaryotic cells) with such a vector; and any cell, or live organism, including a non-human mammal, that has been genetically engineered to produce such a polypeptide, said cell or live organism incorporating expressibly therein a nucleic acid sequence according to this invention.",
"Heterologous expression systems may be used to express cloned NK-1 receptor, NK-2 receptor and NK-3 receptor, including human and non-human mammalian variants thereof.",
"The choice of expression system depends on a number of factors including stability of protein expression, post translational modification and required yield.",
"However, as a general rule, the more complex the organism the lower the yield of expressed receptor, but the greater the likelihood that the receptor will be in its native conformation.",
"Several expression hosts are commonly available Prokaryotic host cells, e.g.",
"Escherichia coli DH5-α (see Sambrook et al., for a comprehensive guide to gene expression in E. coli).",
"Yeasts, e.g.",
"Pichia pastoris.",
"The feasibility of expressing NK-1 receptor in yeast has been demonstrated by Arkinstall et al (1995, FEBS 275 183-187) who successfully expressed therein the closely related receptor, human NK-2.Large-scale production of a human G-protein coupled receptor in yeast was demonstrated by Sizemann et al (1996, Receptors and Channels, Vol 4, 197-203).",
"In brief, a protocol to express a functional NK-1 receptor in yeast can be represented by: (1) splicing the NK-1 cDNA into a yeast expression vector; (2) Transforming this vector into yeast; and (3) Selecting for yeast containing the NK-1 cDNA and expression of the NK-1 cDNA.",
"Eukaryotic host cells, e.g.",
"insect cells, non-mammalian vertebrate cells and mammalian cells.",
"Non-mammalian vertebrate cell lines include Xenopus (frog) oocytes.",
"Other cell lines which are contemplated in this invention include HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL 1650; NoCRL 1651), Sf-9 cells (ATCC NoCRL 1711), C127 cells (ATCC NoCRL-1804), 3T3 cells (ATCC NoCRL-6361), CHO cells (ATCC NoCCL-61), human kidney 293 cells (ATCC No 45504; NoCRL-1573) and BHK (ECACC No84100 501; No84111301); PC12 (ATCC No CRL-1721), NT2, SHSY5Y (ATCC No CRL-2266), NG108 (ECACC No88112302) and F11, SK—N—SH (ATCC No CRL-HTB-11), SK—N—BE(2) (ATCC No CRL-2271), IMR-32 (ATCC No CCL-127).",
"A preferred system to which the gene of the invention can be expressed are cell lines such as COS cells, 3T3 cells, HeLa cells, 292 cells and CHO cells.",
"A preferred system for the efficient expression of hNK-1vR involves the use of CHO and COS cell lines.",
"The gene can be expressed through an endogenous promoter of native CHO or COS, or through an exogenous promoter.",
"Suitable exogenous promoters include such as SV40 and CMV, or perhaps a eucaryotic promoter such as the tetracycline promoter.",
"The preferred promoter being CMV.",
"In some instances, it may be required to tag e.g.",
"a human NK-1 variant receptor prior to purification.",
"The tag is then, in most instances, encoded into the nucleotide sequence that is needed to express the polypeptide.",
"Examples of such tags include, but are not limited to sequences encoding C-myc, FLAG, a sequence of histidine residues, haemaglutin A, V5, Xpress or GST.",
"Most of these tags can be incorporated directly into the sequence, for instance through PCR amplification by incorporating the appropriate coding sequence in one of the PCR amplification primers.",
"However, the tag can also be introduced by other means, such as by covalent binding of the appropriate nucleic acid sequence encoding the tag moiety, such as GST, with the 3′ or 5′ end of the nucleic acid sequence encoding the polypeptide sequence.",
"Purification of the NK variant receptor may, in the case of the use of a histidine tag, then be carried out by passage onto a nickel or copper affinity chromatography column, such as a Ni NTA column.",
"The polypeptide thus produced may optionally be further characterized, for example by binding onto an immuno-affinity chromatography column on which polyclonal or monoclonal antibodies directed to the NK variant receptor have been previously immobilised.",
"For some purposes it may be useful to provide mammals e.g.",
"mice, rats or guinea-pigs in which in which modified human or non-human mammalian tackykinin receptors, e.g.",
"NK-1v receptors, are present.",
"Transgenic rats, mice and other mammalian cells may be produced by generating a targeting vector and transfecting the cells to be cultured e.g.",
"using the gene targeting services of, e.g.",
"DNX Transgenic Sciences.",
"In the case of transgenic mammals, stem cells are transfected, cultured to blastocytes and introduced into the uterus of a female mammal.",
"Because they have in their cell membranes tachykinin receptors that bind to endogenous ligands such as SP but are incapable, or substantially incapable, of initiating their endogenous signal, the animals are useful, in research into the effects of these ligands and the developments of transduction inhibitors, especially because they can indicate the behaviour of the animal the presence of endogenous ligand but in the absence of a transduced signal and in the absence of antagonist, and can therefore provide a true control.",
"(Silver, Lee M. Title: Mouse genetics—concepts and applications Publisher: New York; Oxford University Press 1995); P De-Felipe-C et al;Nature.",
"1998 Mar.",
"26; 392(6674): 394-7).",
"Assays The present invention further provides a modified tachykinin receptor, such as a human NK-1v receptor, used as a substitute in an assay to identify and evaluate entities that bind to the wild type tachykinin receptor.",
"The invention also includes human NK-1v receptor used as a substitute in an assay in order to determine the concentration of substance P in body fluids in patients with arthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinal disorders and diseases of the GI tract, like ulcerative colitis and Crohn's disease.",
"As previously mentioned three tachykinin receptors have been identified, referred to as NK-1, NK-2 and NK-3 and they have respective endogenous lignads SP, NKA and NKB.",
"Although each tachykinin has a preferred ligand, each receptor has the ability to interact with the other tachykinin ligands, and the pathology of cross-ligand binding in disease states is poorly understood.",
"Study of a system where NK-1, NK-2 or NK-3 receptor response either singularly or in tandem has been diminished in vitro or in vivo could significantly aid understanding of the role of tachykinin receptors and their ligands in disease states.",
"Screening Methods The invention also provides a method for screening for therapeutically active compounds, said method comprising the following steps: (a) providing a cell line expressing the modified tachykinin receptor, e.g.",
"the human NK-1v receptor; (b) adding test sample to a solution containing labeled SP or other ligand and the cell line from step (a); (c) incubating the cell line, test sample and labeled SP or other ligand mixture from step (b) to allow binding of SP or other ligand and test sample to the modified tachykinin receptor; (d) optionally separate the non-bound labeled SP or other ligand from the labeled SP or other ligand bound to the modified tachykinin receptor; and, if desired, (e) measuring the amount of labeled SP or other ligand that is bound to the modified tachykinin receptor.",
"Preferably, the assay involves COS-7 cell lines.",
"Cell membranes containing the modified tachykinin receptor, e.g.",
"the human NK-1v receptor, can be used instead of whole cells.",
"The SP or other tachykinin ligand e.g.",
"NKA or NKB may be labeled by any method known in the screening art, e.g.",
"by a radioactive label, such as 125I, or by a fluorescent label.",
"In certain circumstances, (e.g.",
"fluorescence polarization assays), bound and unbound SP or other tachykinin ligand do not have to be separated to quantify the amount of SP or other ligand bound to the receptor.",
"Alternatively, the SP or other ligand may be bound to a matrix, and labelled cells may be used to quantify the binding of a modified tachykinin receptor, such as human NK-1 receptor, to the SP.",
"In this case, the assay procedure may follow the steps set out below: (a) a cell line is provided that expresses a modified tachykinin receptor, such as the human NK-1v receptor; (b) the cell line is labeled; (c) the test sample and labeled cells are added to a matrix binding SP or other ligand; (d) the labeled cells, test sample and matrix-bound SP or other ligand are incubated to allow binding of SP or other ligand and test sample to the expressed modified tachykinin receptor; (e) the labeled non-bound cells are separated from the SP or other ligand bound cells; and, if desired, (f) the amount of labeled cells containing the modified tachykinin receptor, such as the human NK-1v receptor, that has bound to SP or other ligand is measured.",
"In order to provide transgenic animals or cell lines for use in assays, which animals and/or cell lines comprise a sequence as described herein, general methods are known and may be adapted accordingly.",
"Different types of vectors including modified retroviruses, adenovirus, adeno-associated virus, herpes virus and plasmid DNA have been proposed as vehicles to introduce foreign genetic material into cells or tissues.",
"Protein Therapy The invention also encompasses modified tachykinin receptors, especially human NK-1v receptor, for use in protein therapy to reduce the effects of, or an excess of endogenous ligand.",
"Protein therapy can be used for the suppression of the action of SP in interstitial fluid of the lungs.",
"For example, a purified preparation of a modified tachykinin receptor, such as human NK-1v receptor, (eg a liquid or powder carrier formulation) can be directly administered to the airways.",
"Once in the airways, the tachykinin variant receptor can interact and bind to, for example, SP molecules.",
"This has the effect reducing the amount of SP available for the endogenous NK receptor.",
"Likewise, a modified tachykinin receptor, such as the human NK-1v receptor, can be directly introduced to body cavities such as joints and interstitial lung space where SP is present.",
"The variant receptor has the capability to bind SP, therefor reducing the amount of SP available to interact with the wild type receptor and causing down-regulation of the SP cellular response.",
"The invention provides a modified NK receptor, such as a human NK-1v receptor, for use in removing or suppressing SP in body fluids, e.g.",
"the interstitial fluid of the lungs and fluid in the cavities of joints.",
"A purified preparation of a modified NK receptor, such as human NK-1v receptor, (e.g.",
"a liquid carrier formulation) may be administered directly to a joint.",
"Once in the fluid- filled joint cavity, the NK variant receptor interacts and binds to SP molecules thereby reducing the amount of SP available to activate the endogenous NK receptor.",
"The hNK-1v receptor can reduce the effect of excess or inappropriately expressed SP in patients with pain associated with migraine, neuralgia, diabetic, peripheral, AIDS-related and chemotherapy-induced neuropathy, and neuropathies of diverse origin; anxiety and anxiety disorders, such as panic disorder, phobias and obsessive-compulsive behavior; schizophrenia; asthma; rheumatoid arthritis; and in gastrointestinal disorders and diseases of the GI tract, for example ulcerative colitis and Crohn's disease.",
"Other conditions or disease states that can be treated, ameliorated or prevented include: psychosomatic and psycho-immunological disorders; attention deficit disorder; pre-menstrual (PMT or PMS) or late luteal phase syndrome; mania or hypomania; aggressive behavior disorders; emesis, including motion sickness, migraine-induced sickness and that arising from chemotherapy; postherpetic neuralgia; depression; inflammation; eating disorders, such as obesity, bulimia nervosa and compulsive eating disorders; cognitive disorders, such as dementia and amnestic disorders; movement disorders, such as dyskinesias, akinetic-rigid syndromes, Gilles de la Tourette syndrome, tremor, or dystonia; schizophrenic disorders; substance abuse disorders; bipolar disorder; sexual dysfunction, including impotence; stress; alteration of circadian rhythmicity; Alzheimer's disease; bladder disorders; hypertension; angina; ischaemia; multiple sclerosis; chronic obstructive lung disease; scleroderma; CNS disorders; and other conditions where excess tachykinin peptides such as SP are involved.",
"Furthermore, the inventors believe that modification to the DRY motif of NK-2 receptor and NK-3 receptor in a similar manner to that described for the NK-1 receptor above, would allow for the production of modified tachykinin receptors which could be used to remove their specific ligands from body fluids.",
"Several factors need to be taken into account in protein therapies.",
"These include: solublisation, maintenance of activity and stability of the protein, delivery and dose.",
"The first three points are closely related.",
"Proteins are large relative to conventional drugs and their biological activity is dependent on their primary, secondary, tertiary and in some instances quaternary structure being maintained.",
"They often have labile bonds and numerous chemically reactive groups in their side chains.",
"Disruptions of their structure by denaturation or aggregation can lead to loss of activity or increase in immunogenicity.",
"One of the key problems in devising effective formulations for biologically active proteins is to find a formulation that is both stable and biologically active.",
"The route of administration of the protein also plays a significant role in formulation.",
"Microsphere formulations can be used for injection, aid in the maintenance of stability and activity of the protein, and offer the possibility of slow release formulations.",
"Solid large powder formulations are most suitable for use in aerosol and topical treatments.",
"Putney and Burke (Nature Biotechnology 1998: 153-157) outline various methods for producing microspheres.",
"One such method is the atomization-freezing process.",
"In this encapsulation method, the micronised solid protein is suspended in biodegradable polymers of DL-lactic co-glycolic acid (PLGA) solution that is then atomised using sonication or air-atomisation.",
"This produces droplets that are then frozen in liquid nitrogen.",
"Addition of ethanol at <−40° C., in which both the protein and the PLGA are insoluble, extracts the organic solvent from the micro-spheres.",
"This process is further described in U.S. Pat.",
"No.",
"5,019,400.An alternative polymer for use in the process is methylene chloride polymer,to encapsulate growth hormone see Johnson et al, Nature Medicine 2: 795-799 (1996).",
"The following method can be used to incorporate modified NKR, such as human NK-1v receptor, post-purification into microspheres: (a) concentrate the purified, active microsphere modified NKR, such as human NK-1v receptor to >100 mg/ml in the presence of stabilisers; (b) add PLGA or methylene chloride polymer solution and mix; then (c) atomise the frozen suspension in liquid nitrogen to fix the microspheres and extract with ethanol.",
"Microspheres thus produced should have a diameter in the μM order.",
"The invention further provides a method for treatment of a patient in need thereof, which comprises administering to said patient a composition in the form of an aerosol that comprises a modified tachykinin receptor as described above, e.g.",
"an hNK-1Rv receptor.",
"SP or other ligand such as NKA or NKB can also be removed directly from biological fluids using the modified tachykinin receptors described above.",
"For example, purified human NK-1v receptor could be bound to a suitable matrix.",
"Biological fluids containing SP can be passed over the matrix, so that the SP preferentially binds to matrix while other components of the fluid do not and SP becomes preferentially removed from the biological fluid.",
"Nucleic Acid Therapy The invention further provides a method for gene therapy treatment of a patient in need thereof, which comprises administering to said patient a nucleic acid sequence, virus or plasmid encoding a modified tachykinin receptor as described above, e.g.",
"an hNK-1Rv receptor.",
"Different types of vector including modified retroviruses, adenovirus, adeno-associated virus, herpes virus and plasmid DNA have been proposed as vehicles for introducing foreign genetic material into the cells or tissues of patients, and can in principle be used to introduce the modified tachykinin receptor nucleic acid sequences referred to above.",
"The appropriate strategy for administering the nucleic acid sequence depends on the target tissue, disease state and longevity of the proposed therapy.",
"Furthermore, down regulation of the effect SP on a cellular system could be achieved by expressing the hNK-1v receptor in cells other than those expressing native NK-1.The expression of SP in these cells (which are in the proximity of cells expressing native NK-1 receptor) could have the effect of “moping” up SP and reducing the available SP for the native receptor to interact with.",
"Introduction of a modified NK receptor, such as a human NK-1v receptor, (by way of direct introduction of protein or introduction of an expressible gene encoding this protein) into the outer membrane of cells expressing the wild type receptor gives rise to competition between the wild type and variant receptors for available SP.",
"The variant receptor competes with wild type receptor for binding of available SP and decreases the amount of SP available for the wild type receptor.",
"As the NK-1v receptor is unable to transduce a signal upon SP binding, the result is a down-regulation of the action of the wild type receptor.",
"In the treatment of lung tissue, researchers at Stanford University Medical Center, California have initiated a trial of gene therapy for cystic fibrosis in which the active material is delivered to the lungs by aerosol.",
"The active material consists of a version of the cystic fibrosis trans-membrane conductance regulator gene packed into an adeno-associated virus (AAV) shell.",
"A similar route can be adopted for delivering the hNK-1v receptor to the lungs of patients where the activation of native hNK-1 receptors by receptor ligands requires to be down-regulated.",
"A strategy for down-regulating the action between SP and human NK-1v receptor in lung tissue can be adopted which is similar to the strategy proposed for gene therapy in cystic fibrosis.",
"The same technique of packaging genes into AAV and infection of the lung tissue with aerosol AAV can be used to introduce hNK-1v.",
"Samulski et al (University of North Carolina, Chapel Hill, N.C. 27599, USA) market a vector which can be used to package human NK-1v receptor gene into AAV.",
"Alternatively, a plasmid containing the human NK-1v receptor (pCMVNK-1v) may be used directly in gene therapy.",
"The plasmid may be prepared as a lipid:DNA complex and administered to the lungs as an aerosol, see Pillai et al., Pharm-Res 15(11): 1743-7 (1998), McDonald et al., Pharm-Res 1998 15(5): 671-9(1998).",
"This strategy can also be used with an NK-1 receptor promoter.",
"A pre-requisite for the use of gene therapy for humans is the ability to make sufficient pharmaceutical grade plasmid DNA.",
"This problem has been addressed by Prazeres et al in TIBTECH 17: 169-74 (1999).",
"Both rAAV and lipid:DNA complex may be administered to the lungs via nebulisers, e.g.",
"airjet nebulisers.",
"Methods for administration may generally follow the teachings of McDonald et al, Pharm Res 15(5): 671-9 (1998); Yonemitsu et al, Gene Therapy 4(7): 631-8 (1997); McDonald et al, Human Gene Therapy, 8(4): 411-22 (1997); Bellon et al, Human Gene Therapy 8(1): 15-25 (1997); and Niven, Critical Review of Therapeutic Drug Carrier Systems 12(2-3): 151-231 (1995).",
"Both viral and plasmid therapeutic compositions can also be delivered to other target sites such as joints and nervous tissue by infusion and injection.",
"EXAMPLES Embodiments of the invention will now be described in the following Examples Example 1 Preparation of a Modified Human NK-1 Receptor (NK-1v) The starting material used was a plasmid pRc/CMV (Invitrogen Co) containing a cDNA clone encoding the human NK-1 receptor.",
"The clone encoded 407 amino acids and was flanked by a Nco I site at the 5′ end (around the ATG start codon) and a Xba I site at the 3′ end following the stop codon.",
"Human NK-1v receptor was made from the above plasmid using a PCR-based strategy that mutated the DRY motif (Asp129, Arg130, Tyr131) at the end of third transmembrane helix to GGA.",
"Portions of the cDNA clone in the above plasmid were amplified in two separate PCR reactions using Pfu DNA polymerase (Stratagene), 20 cycles of PCR using 1 min at 94° C., 1 min at 55° C., 4 min at 72° C., followed by 10 mins at 72° C., 50 pmol of each primer, 50 ng of plasmid DNA, 100 μl reaction volume.",
"The first PCR reaction used the following primers to produce a predicted product of 473 bp: (SEQ ID No7) 5′ primer, sense-A: 5′-AAC TAG AGA ACC CAC TGC TTA-3′ (SEQ ID No8) 3′ primer, anti-sense-A: 5′-GCC ATA GCG CCG CCA AAA GCC ACA GCC GT-3′ The second PCR reaction used the following primers to produce a predicted product of 888 bp: (SEQ ID No9) 5′ primer, sense-B: 5′-TTT GGC GGC GCT ATG GCT ATC ATA CAT CC-3′ (SEQ ID No10) 3′ primer, anti-sense-B: 5′-AGC TCT AGC ATT TAG GTG ACA-3′.",
"Primers anti-sense-A and sense-B contained 17 bases of overlapping complementary sequence at the 5′ ends of each to allow annealing of the two products to form the full length mutated receptor.",
"All the above primers were custom synthesized by Perkin-Elmer.",
"The two PCR products were gel-purified (QIAEX, Qiagen) and 50 ng of each purified product was added to a 100 μl Pfu PCR reaction and three rounds of PCR performed without primers to allow the two overlapping regions of the PCR products to anneal and extend.",
"50 ng of the flanking primers (sense-A and anti-sense-B) were added and 20 cycles of conventional PCR performed.",
"The resulting full-length product was purified, cloned into pBluescript (Stratagene) and fully sequenced to confirm the mutations had been successfully made.",
"The clone was modified by site-directed mutagenesis using a Clontech Transformer kit to remove an internal Nco I restriction site using the oligonucleotide sense-C also from Perkin-Elmer.",
"Sense C: 5′-CGC GGA GGC TTC TAT GGC TGC AT-3′ (SEQ ID No11) The resulting cDNA was excised from the parental vector, spliced into a mammalian expression vector, pCMV3.1 (Invitrogen).",
"This cDNA was in an orientation which allowed expression of the hNK-1vR polypeptide.",
"Stock plasmid was prepared by amplification in E. coli and the plasmid DNA subsequently purified using a Qiagen endonuclease free DNA preparation kit, and the fidelity of the construct confirmed by DNA sequencing.",
"The nucleic acid sequence of the cDNA is SEQ ID No6 (FIG.",
"2) and the translated protein sequence is SEQ ID No5 (FIG.",
"1).",
"Similarly control (negative control) constructs were prepared whereby wild type and variant HNK-1R cDNA was spliced into the vector in an opposite configuration to that described above.",
"A further control (positive control) was prepared by ligation of the parental wild type hNK-1R cDNA into the vector in a manner which would allow hNK-1R polypeptide to be expressed.",
"Example 2 COS-7 Cells that Transiently Express Human NK-1v Receptor COS-7 cells were grown in DMEM culture medium supplemented with 10% foetal calf serum and 2mM glutamine and maintained under an atmosphere of 5% CO2.Cells were passaged at approximately 70% confluence by reseeding to a concentration of approximately 10% confluence per 175 cm2 flask.",
"COS-7 cells were harvested and prepared for electroporation in Equibio electroporation buffer.",
"Cells (5×106 cells) were electroporated at room temperature in a 4 mm gap cuvette in a final volume of 800 μl containing 30 μg of transforming plasmid DNA, with 250 volts, 1500 μF at infinite resistance using an Equibio EasyJect plus electroporator.",
"Transformed COS-7 cells were cultured for 2-3 days in a 175cm2 flask prior to assay.",
"Alternatively cells (approximately 5-50 ul of transfected cells) were cultured in 6 well culture dishes containing 22mm diameter cover slips.",
"These cover slips coated in cells were used for imaging experiments designed to investigate changes in the concentration of intracellular free calcium ([Ca2+]i).",
"Cover slips were prepared by first immersing the cover slips in approximately 70% ethanol and then quickly passing through a gas flame to sterilize.",
"Cover slips were then allowed to air dry in a culture hood prior to placing in the bottom of a culture well of a standard six well culture dish.",
"Cells were then cultured as described above.",
"Example 3 COS-7 Cell Membranes Containing Human NK-1v Receptors The transfected COS-7 cells of Example 2 were harvested by treatment with Versene.",
"(Gibco BRL).",
"Versene was used, as it allows detachment of the cells from the flask without substantial perturbation to cell membrane proteins, then, the cells were washed once by re-suspending in assay buffer (50 mM Tris HCl pH 7.4, 3 mM MnCl2, 0.02% BSA, 40 μg/ml bacitracin, 2 μg/ml chymostatin, 2 μM phosphoramidon, 4 μg/ml leupeptin) and centrifuged at 1000 g for 5 min.",
"Cells were re-suspended in 5 ml of the assay buffer and a cell count performed prior to lysing cells, using a Brinkman polytron at setting 6 for 15 seconds.",
"The homogenate was centrifuged at 20000 g for 10 minutes.",
"Membrane pellets were re-suspended in assay buffer and stored frozen as 0.5-1 ml aliquots until required for use.",
"Example 4 Ligand Binding to Receptors in Cell Membranes COS-7 cells were transiently transfected with pCMV3.1 (Invitrogen) containing either parental wild type human NK-1 receptor cDNA or the variant human NK-1 receptor cDNA of Example 1, and cell membranes were prepared 2-3 days post-transfection using the procedure of Example 3.Radioligand binding studies were performed using [125I]BH substance P to label NK-1 receptors.",
"Non-specific binding was defined by the NK-1 receptor-selective agonist [Sar9, Met(O2)11]substance P (Bachem) at a final concentration of 1 μM.",
"On the day when they were required, the membrane suspensions were thawed and diluted as appropriate with assay buffer and incubated with varying concentrations of [125I]Bolton-Hunter Substance P (0.05-3 nM, from Amersham Life Sciences) for 50 minutes at 21° C. Saturation analyses were performed to determine the affinity constants and maximum binding capacity for each receptor, by incubating membranes with increasing concentrations of the radioligand, in the presence and absence of [Sar9, Met(O2)11]substance P. Reactions were terminated by rapid filtration under vacuum, onto GFC filters pre-soaked with 0.2% polyethylenimine.",
"[125I]BH substance P bound with high affinity to both wild type and mutant receptors (Kd values of 2.218±1.107 nM (n=3) and 1.446 nM (n=2) respectively).",
"Unpredictably, there appeared to be no significant difference between the dissociation constant and the maximum binding capacity of the wild type and variant NK-1 receptors, indicating that the mutation does not affect agonist binding at the NK-1 receptor.",
"The maximum binding capacity can be directly compared, as shown in Table 1: TABLE 1 Wild Type Variant HNK-1 (n = 3) hNK-1 (n = 2) Kd (nM) 2.218 + − 1.1107 1.446 Bmax 2.201 3.000 Example 5 Analysis of Intra-Cell Receptor Coupling in COS-7 Cells by Changes in Intra-Cellular Free Calcium Concentration Coverslips containing cells were prepared and maintained as described.",
"Cultured cells were washed twice in a Krebs-Hepes extra-cellular medium buffer (EM, composition in mM: NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.2, KH2PO4 1.2, Hepes 10, glucose 11 and BSA 0.1%, (pH 7.2 at 20° C., (Rossant, et al Endocrinology, 140, 1525-1536), and then loaded with Fura-2 (Rossant, et al Endocrinology, 140, 1525-1536) by incubation for 3 h at 20° C. with EM containing Fura-2-AM (2 μM, Molecular Probes).",
"This procedure enables the cells to load with Fura-2-AM, which becomes hydrolysed to the free acid form once inside the intact cells.",
"After loading, coverslips were mounted into imaging chambers and perfused with EM to remove extra-cellular Fura-2-AM and to allow hydrolysis of intracellular Fura-2-AM to occur.",
"Measurements of changes in the free [Ca2+]i in individual cells were made from the fluorescence ratio (excitations 340 nm/380 nm, emission >510 nm) using a Spectral Wizard monochromator, cooled integrating CCD camera and a dedicated suite of software (Merlin, Life Sciences Resources, Cambridge, UK).",
"Data are expressed as ratio units 340/380.Genes encoding wild type sense, wild type anti-sense and variant NK-1 receptors described above were transiently transfected into COS-7 (monkey kidney) cell lines.",
"Two to three days post-transfection, cells were loaded with FURA-2-AM dye (whose fluorescence is [Ca2+]i dependent) and challenged with substance P. The [Ca2+]i was followed by monitoring the change in light emitted (>510 nm) when excitation light of either 340 nm or 380 nm was sequentially used to illuminate the loaded cells and expressed as a ratio (340 nm:380 nm).",
"Cells challenged with UTP (which increases [Ca2+]i in non-transfected cells) caused a [Ca2+]i response indicating the presence of a functional G-protein Ca2+ linked pathway.",
"Results plotted in FIG.",
"3 indicate that cells expressing wild type NK-1 receptor responded to substance P over a 0.01-1000 nM range.",
"Cells expressing wild type anti-sense and variant NK-1 receptor failed to invoke a Ca2+ response with substance P challenge of 1 nM.",
"The effect of treatment with varying concentrations of substance-P or with UTP (3μM) for a 1 min time period on the average change in 340:380 ratio levels of COS 7 cultures expressing either wild type or modified NK-1 receptors (FIG.",
"3, n>50 cells for each treatment group (or on the percentage of COS cells demonstrating a measurable response (Xb) is shown in FIG.",
"3)."
]
] | Patent_10451304 |
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